Scientific Information Concerning the Issue of Whether a Prion Is a
“Pest”

under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA)

(2/17/10)

I.  Introduction and Background

This paper reviews currently available scientific information pertaining
to the issue of whether a prion (“proteinaceous infectious
particles”) should be considered to be a “pest” as defined by the
Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA).  The
scientific information in this review has been considered by the EPA
work group that developed the Notice of Proposed Rulemaking (NPRM) that
proposes to establish a prion as a pest under FIFRA.  At EPA’s
request, the FIFRA Scientific Advisory Panel (SAP) also reviewed this
document and provided comments (USEPA 2009), and EPA has incorporated
many of the SAP’s suggested changes and additional information into
this document.

FIFRA provides EPA the authority to decide what entities are considered
to be a “pest” under FIFRA.  The sections that are potentially
relevant to prions include:

FIFRA Section 2(t):   A pest is “… (2) any other form of terrestrial
or aquatic plant or animal life or virus, bacteria or other
micro-organism…which the Administrator declares to be a pest under
section 25(c)(1).”

FIFRA Section 25(c)(1):   “The Administrator, after notice and
opportunity for hearing, is authorized–(1) to declare a pest any form
of plant or animal life … which is injurious to health or the
environment.”

In September 2003, the EPA’s Office of Prevention, Pesticides, and
Toxic Substances (EPA/OPPTS) decided that a prion should be considered
to be a “pest” under FIFRA and that products intended to inactivate
prions (i.e., “prion products”) should be regulated under FIFRA
(USEPA 2004).  The Agency’s rationale may be summarized as the
following key points:

Prions are unquestionably injurious to human and animal health.

Prions share many characteristics of other deleterious microorganisms.

 Prions share enough characteristics of “other micro-organism” or
“form of life” to fall within the scope of FIFRA section 2(t)(2).
They originate in living organisms, replicate within host organisms, and
can be transmitted to other hosts.

If prion products were not regulated under FIFRA, EPA could not provide
assurance that such products would be effective or safe.

It appears that the intent of Congress in the 1972 FIFRA was that EPA
should regulate products used against deleterious forms of life share
the characteristics of viruses and other microorganisms.

	In order to provide sound, scientific input into the issue of whether a
prion may be considered to be a “pest” under FIFRA, EPA has examined
the available scientific information relevant to the question.  This
document principally addresses the scientific evidence.  Legal and
policy considerations that factor into the Agency’s decision to issue
a NPRM will be described in the Preamble to the NPRM.

II.  The Science of Prions

Description

The leading theory on the causative agent for transmissible spongiform
encephalopathies (TSE) is that the agent is composed of an abnormal form
of a nucleic acid-free, replicating protein (Prusiner et al. 1982;
Bolton et al. 1982; Brown et al. 1990a), now called a prion.  The term
“prion” (Prusiner 1982) makes a distinction between molecular
properties of the novel, proteinaceous infectious particles that cause
scrapie disease and molecular properties of particles, such as viruses,
plasmids and viroids that feature a core of nucleic acid.  Fueled by the
catastrophic outbreak of mad cow disease, or bovine spongiform
encephalitis (BSE), originating in Essex County, England (1986; Wells et
al., 1987), research to characterize the precise nature of prions and
resolve how they cause the diseases known as transmissible spongiform
encephalopathies (TSE) led to Prusiner’s award of a Nobel Prize in
1997.  In the absence of mechanistic evidence, the assumption prior to
this body of work was that kuru in humans and scrapie in sheep and goats
were caused by unconventional viruses, virus-like agents, or slow
viruses because the time interval between exposure and clinical disease
was years or decades (Gajdusek, 1977; Prusiner et al., 1982).  While a
number of scientists have suggested that an unknown virus or co-factor
is the causative agent of TSEs (Manuelidis, 2007; Manuelidis et al.
2007), no such virus or co-factor has been isolated after 40 years of
searching.  Instead, a novel infectious particle containing no nucleic
acid was suggested by Alper et al. (1966; 1967) and subsequently a
protein-only (i.e., free of non-protein moieties) infectious agent was
suggested by Griffith (1967) and Brown et al. (1990a).   This
protein-only hypothesis is now generally accepted by strong weight of
evidence, especially Prusiner’s hypothesis of a novel proteinaceous
infectious agent – the prion (Prusiner et al. 1982; Bolton et al.
1982).  In particular, experimental transmission of TSE diseases by
prion protein synthesized or propagated in cell free systems (Legname et
al. 2004; Castilla et al. 2005; Deleault et al., 2007) is accepted by
many as definitive proof of the prion hypothesis.  Though prions are now
generally accepted as the agent responsible for TSE diseases, the
precise structural properties of the prion protein responsible for
infectivity and the chain of events that produce neural degeneration and
fatal TSEs (e.g., Barron et al. 2007), and what – if any - cofactors
might be featured in the mechanisms of disease, remain unknown.  As a
result of the novelty of the prion hypothesis and controversy
surrounding it as the agent for infectious diseases, the prion protein
and its isoforms have been studied with unusual intensity; yet the
physiological function of the normal protein and the role of the protein
in producing neurodegeneration remain obscure (review Aguzzi et al.
2008).  Highlights of prion research are presented in section II of this
paper.

Numerous breakthroughs have been made in the past several years.  As
detailed below (B. Key Characteristics), the normal isoform of the prion
protein (PrP) is now known to be encoded by the PRNP gene and highly
expressed in neurons on the cell membrane.  The prion protein possess a
unique property, so that when the normal α-helical cellular protein is
converted to certain misfolded, β-sheet rich conformations (Soto, 2006)
by unknown events, the resultant protein aggregates bypasses the normal
proteosomal degradation pathway.  This process typically produces the
non-amyloid or amyloid deposits featured in the TSE diseases, and
acquires the property of TSE infectivity.  The clinical features of the
TSE disease produced vary somewhat depending on the species (e.g., cow,
sheep, human, etc.) and TSE strain.  Experimental intervention to
restore proteosomal degradation of the misfolded protein conformation
“cured” prion infectivity in cultured cells (Webb et al. 2007).  The
property of infectivity appears to reside in the β-sheet-rich
conformation (Müller et al. 2007), which is the focal point of numerous
ongoing investigations to define precisely the structural determinants
of infectivity.   In humans, genetic variation is common at the PRNP
gene codon for amino acid 129, resulting in high frequencies of amino
acid substitutions (129M, 129V) in the translated prion protein.
However, inheritance of identical codons from both parents results in
prion protein homozygosity (129 MM or 129 VV), which produces a cellular
pool of identical prion proteins that are predisposed to misfolding and
aggregation through homologous interaction.  In contrast, inheritance of
two different codons results in prion protein heterozygosity (129 M/V),
which produces a cellular pool of polymorphic proteins that resist
misfolding (Mead et al. 2003).  In addition to mutations in the PRNP
protein coding region, genetic mutations in the PRNP promoter region are
thought to modulate prion protein expression levels and susceptibility
to TSEs, as in the case of BSE (Haase et al. 2007).  What distinguishes
prion diseases from other neurodegenerative diseases featuring
accumulations of protein plaques (such as Alzheimer’s, Parkinson’s,
and ALS) is that misfolded PrP polypeptides recruit and convert normal
PrP polypeptides to adopt the misfolded infectious conformation;
however, a low level of prion-like transmission of altered protein may
occur in other amyloid plaque diseases (Aguzzi, 2009), and “prinoid”
behavior may be a feature of other protein aggregating neuropathologies.
 For example, experimental inflammatory amyloidosis in mice is
accelerated when the mice are also administered purified amyloid fibrils
(Lundmark et al. 2002).  Similar seeding was observed in mice
administered extracts from human Alzhemier’s brain (Meyer-Luehmann et
al. 2006).  These misfolded β sheet proteins produce aggregate
particles (commonly described as plaques), which are linked to cell
death in neurons, and produce a number of fatal spongiform
encephalopathies in humans, livestock and game animals (Brown, 2005).

In summary, normal cellular protein is synthesized and eventually
degraded through normal metabolic processes, whereas misfolding of the
protein produces a conformational shape change that is prone to
formation of prion aggregates.  These changes account for the resistance
of prion particles to proteosomal degradation and, consequently, for the
slow accumulation of amyloid deposits or brain plaques, which are often
observed in some TSEs.   Furthermore, this slow progression of plaque
formation and the prion diseases accounts for early speculation that the
causative agent might be an unknown slow-virus.   It is now clear that
the transmissible encephalopathies (e.g., kuru, scrapie, BSE) feature a
novel disease mechanism.  In the prion hypothesis of disease, it is
thought that prion protein polypeptides in the misfolded,
aggregation-prone isoform convert polypeptides in the normal cellular
isoform to adopt the same misfolded conformation--a process that is now
often described as recruitment and conversion.  While researchers
continue to describe prions as infectious protein particles, as defined
by Prusiner (1982), it is important to note that this usage no longer
necessarily refers to an unknown, slow virus, but alternatively to
describe this novel process of recruitment and conversion.

Since reaching a peak in 1992, the sharp decline in new outbreaks of BSE
indicates that the epidemic in the UK appears to have effectively ended
in cows (Brown, 2001).  However, the potential for long, unresolved
dormancy of prion diseases in humans is a matter of great public concern
(Prusiner et al. 1982; Carrell 2004; Enserink 2005), with the risk that
prion disease in humans resultant from the BSE epidemic, such as the
corresponding peak of vCJD in 2000, might persist in human populations
long after the consumption of contaminated food ended.  Also, surgical
tools contaminated with prions are extraordinarily difficult to
decontaminate (Brown et al. 1990, 2000 and 2005; WHO 1999).

Key Characteristics

		1.  The prion protein and terminology

In its original description, the purified prion particles were
characterized as comprised of a single protein, roughly 27,000 to 30,000
daltons in molecular weight, and resistant to proteinase K digestion.
In particular, enrichment of the protein correlated with the titer of
the infectious agent (Bolton et al. 1982) and failed to exhibit the
spectra indicative of the presence of nucleic acids (Prusiner et al.
1984).  Through the work of more than two decades, it is now believed
that the infectious isoform of the prion protein is a misfolded isoform
of a normal cell membrane protein, and various terminologies have
appeared as numerous researchers have joined the investigations.  The
normal cellular isoform of the protein is typically designated PrPC, and
the infectious isoform in scrapie is PrPSc.  Because a variety of
isoforms produce different transmissible spongiform encephalopathies,
PrPd has been introduced recently to connote any disease isoform (Brown,
2005).  Also, PrPres has been used to connote a PrPd polypeptide
fragment that features the resistance to proteinase K digestion (Brown,
2005).  Other less common usages also appear in the literature.  More
recently, PrPTSE has been used generically for the infectious
conformation of the transcripts of any prion strain, which lends
simplicity to communicating about the homologous prion proteins of
different human and animal species (WHO 2006).  As used in this paper,
“prion” refers to an abnormal, misfolded, infectious isoform of the
prion protein, whereas “prion protein” is an all inclusive phrase
that can refer to any normal or misfolded isoforms.

		2.  Current Status of Dr. Stanley Prusiner’s (1982) protein only
hypothesis

 Amyloid polymerization of recombinant prion protein produced in
bacteria has been shown to accompany the acquisition of prion
infectivity (Legname et al. 2004; Colby et al. 2007) that is
characteristic of the TSEs.  Fibrils consisting of recMoPrP(89-230) were
inoculated intracerebrally into transgenic (Tg) mice expressing
MoPrP(89-231).  Between 380 and 660 days after inoculation of mice with
these transgenic fibrils, all mice developed neuropathology, and their
brain tissue, in turn, was infectious to other mice.  Regarding the
mechanisms of infectivity intrinsic to Prusiner’s prion hypothesis,
the amino acid sequence of the human prion protein, for example, has
been extensively probed and two cationic domains (PrP19-30  and
PrP100-111)  have been shown to be capable of homologous binding between
PrPC and PrPSc, which would account for the principle of recruitment of
PrPC and conversion to infectious PrPSc (Lau et al. 2007).  By using
guanidine hydrochloride as a denaturant and copper binding, apparent
reductions in prion infectivity have been shown to be partly reversible,
which supports the concept that the property of infectivity resides with
the fibrillar, protease resistant conformation (McKenzie et al. 1998).

Large quantities of infectious PrPres can be produced in vitro by
seeding the normal PrPC isoform with small amounts of PrPres, followed
by protein misfolding cyclic amplification in a cell free system
(Castilla et al. 2005; 2008).  Hamsters inoculated with PrPres produced
in vitro developed scrapie disease and died after about 170 days.

Significant recent work by Lindquist (e.g., Tessier and Lindquist 2007;
Alberti et al. 2009) uses yeast prion protein and recombinant DNA models
to resolve the sequence requirements and mechanisms of prion protein
misfolding and – most importantly – the recognition elements
required for “prionogenic” infectivity.  The publication of this
work has been widely acclaimed in a series of news features that
accompanied publication of Tessier and Lindquist (2007), which is
regarded by many as a breakthrough paper that proves the prion
hypothesis (Surewicz 2007; Jonietz 2007; Scientific American 2007).

		3.  Normal Prion Protein

The infectious prion protein is any one of several isoforms of an
otherwise normal cell membrane protein (Stahl et al. 1990), encoded by a
highly conserved gene, variously designated as PRNP, prnp or Prnp
(Sakaguchi 2005) located on chromosome 20 in humans and on chromosome 2
in mice (Oesch et al. 1985; Basler et al. 1986).  In one view (see
following paragraph), a low level of sequence homology may be evidence
that the Prnp gene has been conserved across species from yeast to
primates (Westaway and Prusiner 1986) and may share an ancestral origin
by divergence with a related SPRN-like gene that codes for another brain
protein known as Shadoo (Premzl et al. 2003; 2004).  The neuronal
protein Shadoo may have neuroprotective functions similar to those of
PrPC.  Shadoo is downregulated in prion disease, possibly indicating a
companion role in the mechanism of prion infections (Watts et al. 2007).
 The prion gene family also includes Prnd which encodes Doppel, a
protein with striking sequence similarity to PrPC.  Doppel does not
possess the conversion and recruitment properties of the prion protein
but is neurotoxic, causing cerebellar degeneration when overexpressed in
the brain (Whyte et al. 2003; Moore et al. 2001).  Across the
vertebrates, amino acid sequence homology between the PrP proteins of
frog, turtle, chicken and mammal is conserved at a rate of about 30%,
which is sufficient to preserve the same molecular architecture of
mammalian PrPC (Harris et al. 1991; Calzolai et al. 2005).

Another view is that the similarity of DNA sequences from yeast to
vertebrate prion protein genes is an evolutionary convergence in the
yeast and an example of non-Mendelian inheritance of phenotypes
benefiting yeast adaptations to diverse environments (Alberti et al.
2009).  Based on the limited understanding of prion protein genes across
a comprehensive array of living representative species, the
interpretation of homologous descent with modification (Westaway and
Prusiner, 1986) of a homologous gene from yeast to vertebrates, or the
interpretation of convergent evolution in the yeast to conserve
phenotypic variation (Alberti et al. 2009), are both plausible – but
opposite – interpretations.

While attention has been focused on the disease producing isoforms, it
appears that the normal isoform of PrPC is a copper binding
metalloprotein (Brown DR 1997), and may function as an antioxidant.  As
noted above (McKenzie et al. 1998), copper binding status may also be an
important cofactor in conversion of PrPC and stability of the PrPSc
infectious conformation.  Disruption of normal PrPC function in vivo by
binding with monoclonal antibodies produces rapid cell death in neurons
(Solforosi et al. 2004), which is consistent with indications that PrPC
functions include neuroprotection and that disruption of these normal
functions produces neuronal cell death.  Doppel-induced disease can be
rescued by coexpression of wild-type PrPC, attributing antioxidant
properties to the cellular prion protein (Wong et al. 2001).

Similar to the aggregates of neurotoxic proteins found in other
neurodegenerative diseases (review Taylor et al. 2002), the neurotoxic
PrP isoforms are produced by misfolding and aggregation of -rich
isomers and accumulation of plaques in neurons.  Homozygous PRNP gene
codons for prion protein 129 methionine (Met, M) or 129 valine (Val,V)
predispose PrP to recruitment and misfolding, so that in kuru, for
example, homozygotes have an earlier onset of disease following
cannibalism of contaminated central nervous system (CNS) tissues (Mead
et al. 2003).  Heterozygosity at codon 129 confers some resistance to
prion disease, probably by inhibiting homologous protein interactions
(Palmer et al. 1991).

Because of the apparent link between misfolded isoforms of PrP and the
production of fatal TSEs, the PrP has largely been studied in the brain.
 While most abundantly expressed in the brain, PrPC has been detected in
the liver, kidney, heart and skeletal muscle, among other tissues
(Bosque et al. 2002).  New evidence indicates the normal isoform of the
protein has important functions in non-neural tissues.  Zhang et al.
(2006) show that the prion protein is a biomarker for hematopoietic stem
cells when it is normally expressed on the cell surface.   When bone
marrow is depleted of prion protein expressing stem cells, the capacity
to regenerate is also lost.  This evidence indicates normal prion
protein expression is required for bone marrow stem cells to regenerate.

		4.  Neurotoxic mechanism

	The critical question in the prion hypothesis is how misfolding of an
otherwise normal cellular protein leads to neuronal cell death,
transmission to other neuronal cells leading to fatal brain
neurodegeneration, and infectivity when prion-laden material is
transmitted to others. Ongoing research activity is focusing both on the
biochemistry of the prion proteins and related polypeptide domains
featured in the initiating, misfolding events and on the long cascade of
intermediate cellular events leading to clinical neuronal degeneration
and infectivity.

	One recent study (Jackson et al. 2009) supports the core tenets of the
prion hypothesis by showing that a single amino acid change in PrP is
sufficient to induce a distinct neurodegenerative disease and
spontaneous generation of prion infectivity.  The intermediate steps
linking the amino acid change to neurodegeneration and infectivity will
no doubt remain speculative until the neurotoxicity pathway is resolved
in detail.  However, numerous breakthroughs have been made in the past
several years regarding associations between the accumulation of prion
protein aggregates and neuronal cell death, and the weight of these
cumulative studies hints at the major features of the neurotoxicity
pathway and infectivity of prions.

	Removal of misfolded and damaged proteins is a normal part of cellular
metabolism; however, misfolded prion protein accumulates in cellular
aggregates.  These aggregates are thought to result from failure of the
ubiquitin-proteasome pathway to adequately degrade misfolded protein,
which is a feature prion diseases share with other neurodegenerative
diseases, such as ALS, Parkinson’s disease and Alzheimer’s disease
(Goldberg 2007).  In the case of the TSE diseases, the misfolded prion
protein conformation does not appear to be intrinsically neurotoxic, and
the precise mechanisms and intermediate steps of neurotoxicity remain
unresolved in detail.  As evidenced in several prominent studies, it
appears that the pathology of scrapie prion in neurons may feature
disrupting the function of the normal cell surface protein, and
development of prion disease may require the presence of normal PrP
expression and the continued conversion of PrPC to PrPSc (Brandner et
al. 1996; Mallucci et al 2003).  In these studies, it is thought that
uncoupling normal PrP from the cell surface also uncouples the presence
of PrPSc from the progression of disease.  Key steps in resolving the
mechanisms of the prion diseases include:

her words, the β-sheet rich prion isoform is not intrinsically
neurotoxic.

Depletion of PrPC in neurons during established infection arrests
disease progression, even though extraneuronal PrPSc continues to
accumulate (Mallucci et al. 2003).  Transgenic mice programmed to
disable the PRNP gene in neurons at 12 weeks of age were inoculated with
PrPSc several weeks before PRNP inactivation.  Within days after the
PRNP gene was inactivated, the mice became depleted of PrPC and remained
alive and disease free a year later.  Inoculated control mice lacking
the transgenic downregulation of PRNP all died.

PrPC protein is normally anchored to the neuronal cell membrane by a
glycosylphosphatidylinositol (GPI) linkage.  Disabling the GPI linkage
in transgenic mice does not prevent the accumulation of PrPSc plaques in
inoculated mice, but does lower the prion titer and alters the
development of scrapie disease (Chesebro et al. 2005).

That the relationships between prion aggregation, plaque accumulation,
infectivity and spongiform degeneration remain incompletely understood
is shown by seemingly inconsistent evidence that the formation of
oligomers (or small PrP aggregates) may be an intermediate step in
neuronal degeneration (Simoneau et al. 2007) although in some
circumstances plaque accumulation can occur in the absence of
infectivity and degeneration (Piccardo et al. 2007).

While the mechanisms of prion neurotoxicity and infectivity will likely
remain intense areas of scientific research and debate for some time,
the weight of evidence remains pointed to the prion as the agent
initiating disease and infectivity.

TSE Diseases

	Prion diseases are neurodegenerative diseases also referred to as
transmissible spongiform encephalopathies (TSEs) due to the large
vacuoles (Figure 1) that are seen upon post mortem examination of the
cortex and cerebellum from affected individuals (Collinge 2001). These
diseases are seen in both humans and animals (Table 1).  Scrapie is the
most widely known TSE naturally occurring in sheep; it was first
recognized some 200 years ago in Europe, and now present worldwide
(Collinge 2001).  More recently, other types of TSE have been recognized
such as chronic wasting disease (CWD) in deer, moose and elk (Williams
and Young, 1980), bovine spongiform encephalopathy (BSE--mad cow
disease) (Wells et al. 1987), feline spongiform encephalopathy (FSE)
(Wyatt et al. 1991), and transmissible mink encephalopathy (TME)
(Hartsough and Burger, 1965; Burger and Hartsough, 1965; Marsh 1992).

	In humans, prion based diseases are generally thought to include
Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker (GSS)
disease, fatal familial insomnia (FFI) and kuru.  They are further
categorized into three etiological categories: sporadic, inherited or
acquired (Collinge 2001, Wickner et al. 2004; Brown 2005).  The
incidence of CJD worldwide is about one per million (Collinge 2001), and
most cases appear to be sporadic.  Sporadic CJD is characterized by a
rapid dementia occurring in individuals between 45 and 75 years of age.
Death can result as early as 2-3 months from onset (Collinge 2001).
Other symptoms include fatigue, insomnia, depression, weight loss,
headaches, ataxia, blindness and random pain sensations. On the other
hand, variant CJD (vCJD) appears to be acquired from exposure by
ingesting prion contaminated beef.  No mutations have been found with
sporadic or acquired prion disease in humans.  In contrast, over 20
distinct mutations of the prion protein gene (Prnp) have been found in
inherited cases.  Fifteen percent of human prion diseases are inherited
(Collinge 2001).

	Kuru represents the earliest known example of human acquired prion
disease.  Kuru is thought to have occurred in Fore natives of Papua New
Guinea in the early 1900’s when a case of sporadic CJD became
entrenched in the population following cannibalistic rituals within the
population.  Because of the different roles adult men and women and
children played in Fore communities, kuru was prevalent among women and
children until the practice of cannibalism ended in the 1950s (Fischer
and Fischer 1961; Gajdusek (1977); Alpers 1987 as cited in Collinge
2001).

 	Kuru – human

Bovine spongiform encephalopathy - (cow)

 	Scrapie - (sheep)

Figure 1.  Pathology of Prion Disease in Humans and Animals

Table 1. Prion Diseases in Humans and Animals

Human	Animal

Spongiform

Creutzfeldt-Jacob disease (CJD); variant CJD (vCJD)

Kuru

fatal familial insomnia

	Spongiform

Scrapie – sheep

Bovine spongiform encephalopathy

Transmissible mink encephalopathy

Chronic wasting disease of mule deer, elk, and moose

Feline spongiform encephalopathy

Encephalopathies in zoo animals*

Gerstmann-Sträussler-Scheinker disease (GSS)

	Variant CJD

	* ankole, Arabian orynx, bison, cheetah, eland, gemsbok, greater kudu,
nyala, ocelot, puma, Scimitar horned oryx, and tiger (Jeffrey and Wells
1988).

History of TSE Diseases

	Though sporadic CJD (sCJD) occurs spontaneously in the human
population, the disease is rare and occurs at the rate of about one per
million people per year.  Prion-related disease epidemics tend to result
from acquired TSEs.  It is now known that the first prion-related
epidemic disease in humans was documented in 1957 in Papua New Guinea
among native tribes known to practice ritualistic cannibalism (Zigas and
Gadjusek 1957).  The disease was called ”kuru” and was closely
related to Cruetzfeldt Jacob Disease (CJD) although the prion hypothesis
was not known at that time.  Kuru represents the earliest known example
of human acquired prion disease.  Kuru is thought to have occurred in
Fore natives of Papua New Guinea in the early 1900’s when a case of
sporadic CJD became entrenched in the population following cannibalistic
rituals within the population.  Because of the different roles adult men
and women and children played in Fore communities, kuru was prevalent
among women and children until the practice of cannibalism ended in the
1950s (Fischer and Fischer 1961; Gajdusek (1977); Alpers 1987 as cited
in Collinge 2001).

The first major outbreak of transmissible encephalopathies having
catastrophic public consequences linked to prions was not diagnosed
until 1985 when mad cow disease (BSE) first appeared in Great Britain.
By 1990, 14,324 cases of BSE were confirmed in British cattle, out of an
estimated population of 10 million cattle. In 1992 and 1993, more than
1,000 cases of BSE were reported each week, and in 1995, at the height
of the epidemic, there were approximately 146,000 accumulated cases of
BSE.  Other European countries also reported cases of BSE at that time
including Switzerland (200 cases), Ireland (120 cases), Portugal (30
cases) and France (13 cases).  As the practice of including animal parts
in cattle feed was stopped (Feed ban 1988; Specified Bovine Offals ban
1990; Meat and Bone Meal ban 1996), the epidemic waned and feed security
was assured 1996 (Brown et al. 2001; Univ. of Wisconsin 2004).  However,
since the introduction of monitoring programs to detect BSE in dead and
slaughtered cattle, numerous other countries have also found their first
cases of BSE: Austria, Canada, Czech Republic, Finland, Germany, Greece,
Israel, Italy, Japan, Poland, Slovakia, Slovenia, Spain and the USA.
Small numbers of cases have also been reported in the Falkland Islands
(Islas Malvinas) and Oman, but solely in animals imported from Britain
(WHO 2006).

	In 1995 and 1996, a cluster of cases of vCJD was diagnosed in humans in
Britain.  The link between BSE and vCJD was announced publicly on March
20, 1996.  By the end of 2003, 145 cases of vCJD had appeared in Great
Britain.  Since then, the annual number of new vCJD cases has declined,
from a high of 28 in 2000 to 1 in 2008
(http://www.cjd.ed.ac.uk/cjdq56.pdf). 1The first case of BSE disease
reported in the United States was on December 23, 2003 on a dairy farm
in the state of Washington in a Holstein purchased from Canada.  As a
result, 4,000 cattle on the ranch were destroyed.  A second case of BSE
was detected in Texas in November 2004 in a cow of unknown origin and
born before the U.S. ban on the use of mammalian protein in ruminant
feed (diagnosis final in June 2005)
(http://www.cfsan.fda.gov/~comm/bsefaq.html).  The third and most recent
case of BSE was discovered in Alabama (  HYPERLINK
"http://www.aphis.usda.gov/newsroom/hot_issues/bse/downloads/EPI_Final5-
2-06.pdf"
http://www.aphis.usda.gov/newsroom/hot_issues/bse/downloads/EPI_Final5-2
-06.pdf ; Heaton et al., 2008).

To date,   HYPERLINK "three"  three  cases of vCJD have been reported in
the Unites States.  The first and second patients (dates of onset 2001
and 2005, respectively) were both born in the United Kingdom and resided
there during the defined period of risk for transmitted TSEs from cattle
(1980-1996).  A third US case of vCJD (onset and diagnosis, 2006) is
likely to have originated in Saudi Arabia where the patient was born,
raised and resided until 2005.  None of the patients had medical
histories to which transmission could be attributed (  HYPERLINK
"http://www.cdc.gov/ ncidod/"  http://www.cdc.gov/ ncidod/
dvrd/vcjd/factsheet_nvcjd.htm).  However, there is no epidemiological
evidence to suggest that any of these vCJD patients acquired the disease
in the United States.  They are presumed to have been infected in the
United Kingdom or Saudi Arabia, respectively.

	Chronic Wasting Disease (CWD) was first recognized as a clinical
"wasting" syndrome in 1967 in captive mule deer in a wildlife research
facility in Colorado.  It was identified as a TSE in 1978.  In 1981, CWD
was detected in a wild elk and in 1985 it was detected in a wild mule
deer in Colorado; shortly thereafter it was shown to be present as an
endemic focus in wild deer, elk and moose in northeastern Colorado and
southeastern Wyoming.  Since nationwide wildlife surveillance was
instituted in 1997, CWD has been identified in wild deer and elk in
several additional states: Illinois, Kansas, Nebraska, New Mexico, New
York, South Dakota, Utah, West Virginia and Wisconsin (www.aphis.usda).
However, numbers of positive animals remain low.  For example, in 2002,
only 302 samples were positive out of 91,636 samples tested (a
prevalence of 0.3%) (www.aphis.usda). Within endemic areas, the
prevalence of CWD has been estimated at <1% in elk and <1% to 15% in
mule deer (Williams et al. 2002).  Despite these low endemic levels,
models suggest that epidemics of CWD could lead to local extinctions of
infected deer populations although it is not known how the disease might
become epidemic (Miller et al. 2000).  There is no evidence of human
cases of prion disease linked with CWD.

	Scrapie, a neurological disease of sheep, has been present in Europe
since the early 18th century.  Affected sheep were observed rubbing or
“scraping” their coat against a tree or a fence post as if they
itched.  The disease is generally fatal after a long incubation period.
It is estimated that scrapie costs the U.S. sheep industry $20 million
per year in direct losses (USDA, 2000).  There are no known human cases
associated with scrapie infected sheep.  Scrapie-infected offal in
cattle feed may have been the source of BSE.

	Approximately 100 cases of feline spongiform encephalopathy (FSE) have
been reported in Great Britain and Europe during the BSE epidemic in the
early to mid 1990's (Cornell Feline Health Center 2004).  British cats
were believed to have gotten the disease by eating BSE-contaminated
commercial cat food and butcher scraps.  The number of new cases in
domestic cats dropped off once measures were put into place to prevent
materials potentially contaminated with BSE prions from entering the
food chain.  No new cases have been reported since 1999.  No cases in
U.S. cats have ever been reported (Cornell Feline Health Center 2006).
Britain also reported cases in captive exotic cats including nine cases
in cheetahs (three were diagnosed abroad but originated in Britain),
three in pumas, three in ocelots, two in tigers and two in lions
(Cornell Feline Health Center, 2004).  Outbreaks of transmissible mink
encephalopathy (TME) are rare occurrences (Marsh and Bessen, 1993),
possibly associated with a transmission of a rare TSE of cattle to mink
(Liberski et al., 2009).

Transmission and Potential Routes of Exposure

	TSEs can be transmitted naturally and experimentally.  The most
efficient route of experimental transmission is intracerebral injection
of infectious brain tissue (Weissman et al. 2002a).  Intraocular,
intraspinal, intravenous, intramuscular, intraperitoneal, subcutaneous
injections, scarification, intralingual, intranasal, intrasciated and
oral administration have also been shown to be effective methods of
transmission in the laboratory setting.  Experimental transmission via
the dental route has also been reported (Ingrosso et al. 1999).
Consumption of contaminated food is generally thought to explain the
natural pathogenesis of scrapie in sheep, the spread of kuru, and the
human acquisition of vCJD from BSE-contaminated beef; however, the
precise details about the fate of  infectious prions in association with
other food constituents as they pass through the alimentary canal and
cross the gut mucosa to produce CNS disease remains elusive (Jeffrey et
al. 2006; Rose et al. 2006; Austbø et al. 2007; Scherbet et al. 2007;
Solomon et al. 2007).

	Natural transmission routes of prions vary among TSEs.  Transmission of
scrapie among sheep and goats is not completely understood.  The scrapie
agent is believed to originate from the placentas of infected sheep and
goats (Pattison et al. 1972) although scrapie-contaminated feces have
also been proposed (Weissmann et al. 2002b).  Both scenarios represent
direct horizontal transmission.  Some studies suggest that indirect,
horizontal transmission occurs via the oral route, such as from
contaminated environments (Palsson 1979) or by prionuria (Shanked et
al., 2001; Ligios et al. 2007).  Vertical transmission (mother to
offspring) of scrapie may be possible, but probably not significant.  It
is thought that a more likely route of transmission involves an early
horizontal, post-natal event (Andreoletti et al. 2002).

	Transmission of CWD among deer and elk is not completely understood.
Both direct and indirect horizontal transmissions are known to occur via
contact with infected animals and environments contaminated with excreta
or decomposed carcasses of infected animals (Miller et al. 2004).  In
this regard, prion-contaminated material in soil remains infectious for
years (Brown and Gajdusek 1991; Seidel et al. 2007) and may serve as a
reservoir of disease.  Purified PrPSc protein has been shown to adhere
to soil minerals and remain infectious (Johnson et al. 2006; Ma et al.
2007), and this soil binding may actually enhance infectivity (Johnson
et al. 2007), possibly by a mechanism comparable to the recognized
durability and seemingly enhanced infectivity of prions bound to
surgical steel instrument surfaces (Zobeley et al. 1999; Flechig et al.
2001; Peretz et al. 2006).   It is also perceived that, as in the case
of BSE (above), if  any maternal transmission of CWD occurs, it has not
yet been documented and would be at very low levels (Miller and
Williams, 2003; Wells and Wilesmith, 2004).  A recent study confirms
transmission of CWD in deer via blood and saliva, raising caution
regarding contact with body fluids from infected animals (Mathiason et
al. 2006).  Currently, there is no evidence of a link between CWD and
unusual cases of CJD in humans, though data are limited and more studies
are necessary (Belay et al. 2004).

	Several animal TSEs are associated with consumption of contaminated
feed.  BSE represents a large-scale common source epidemic transmitted
by the feeding of BSE prion-contaminated meat and bone meal to cattle
(Wilesmith et al. 1991). At that time, meat and bone meal were prepared
from the offal of cattle, sheep, pigs and chicken and fed to cattle as a
high protein nutritional supplement.  [Note: In April 2009, the U.S.
Food and Drug Administration imposed a feed ban rule which prevents this
practice.]  Given the weight of focus on contaminated meat and bone meal
as the source of the BSE epidemic in the UK, the discovery in Alabama of
a diseased animal with the BSE-linked 211K mutation (E211K) prompted
Heaton et al. (2008) to survey over 6,000 US cattle, including five
commercial beef processing plants   Given the absence of any other
observed instances of the gene allele for 211K in this survey and the
size of the sample, they estimated that any additional atypical BSE
cases in the U.S. with K211 would be vanishingly low.  Similarly, in
cats, feline spongiform encephalopathy (FSE) has been linked with
consumption of commercial cat food and butcher scraps contaminated with
BSE, and transmissible mink encephalopathy (TME) is associated with the
feeding of tissue from scrapie-infected sheep or some form of
TSE-infected cattle (Chesebro, 2003).

	Human TSEs are associated with a variety of transmission routes.  Most
cases of CJD are classified as sporadic and are likely to result from a
somatic mutation, the spontaneous conversion of PrPC to PrPSc, or
reduced clearance of low levels of PrPSc that may normally be present
(Peretz et al. 2001).  However, CJD can be transmitted by exposure to
contaminated surgical instruments or electroencephalogram electrodes, or
exposure to infectious brain, pituitary, or ocular tissue or associated
products via organ transplantation or other medical procedures (Blattler
2002).

	Both kuru and vCJD are caused by ingestion of contaminated food.  As
described earlier, Kuru, the TSE of New Guinea natives, is associated
with consumption of infected human tissue during cannibalistic rituals.
Exposure is also believed to have occurred through abrasions of
oropharyngeal or conjunctival mucous membranes, as well as via open
wounds on the hands (Gajdusek, 1977).  vCJD is associated with
consumption of BSE-contaminated beef products (Will et al. 1996,
Collinge et al. 1996, Bruce et al. 1997, Will et al. 1999).
Additionally, two cases of transmission of vCJD via blood transfusion
have also been reported (one case manifested clinically and the second
was identified at autopsy following death from an unrelated cause)
(Llewelyn et al. 2004, Peden et al. 2004), and at the present time it is
likely these numbers will continue to rise as surveillance continues (UK
National Prion Clinic).

	Prions have presented a major public health problem in the UK with the
emergence of BSE.  Through a major effort in the UK and other countries,
this disease appears to be under control.  However, because so little is
understood about prions, they will remain an important public health
issue for the foreseeable future, particularly since it is not clear if
measures taken so far will adequately prevent future occurrences.  In
addition, scientists do not completely understand several aspects of
prion diseases, or whether effective treatments might be found through
understanding of the misfolding and infectivity of the fibrillar
structure (Karpuj et al. 2007; Supattapone et al. 1999, 2001).  Finally,
the epidemic of CWD in deer and elk populations in the Western USA
continues to spread and has certainly entered states east of the
Mississippi, but the implications of this disease for humans remains
unknown.

Rapid advances in recombinant and cell free technologies are attacking
the most puzzling question about the prion diseases (Dong et al. 2007;
Müller et al. 2007; Tessier and Lindquist 2007): Why are they
transmissible whereas other neural protein misfolding diseases such as
Alzheimer’s and similar amyloidoses are not?  In addition to known
hereditary prion diseases, it is an especially worrisome health problem
that normal prion protein can also misfold under conditions that have
not been defined to produce spontaneous TSE disease.  For example, the
UK commission report concluded that the catastrophic epidemic of BSE
(1986 – 2000) began with the spontaneous emergence of an abnormal
prion, followed by using contaminated bone meal to feed other cattle.
The resultant intense surveillance now shows low level BSE in other
countries – including three recent cases in the USA.

In summary, prions are a persistent problem but the extent to which
these diseases will pose a public health threat in the future is
unclear.

Existing Occupational Safety Procedures

	 Because TSEs are transmissible to humans by various routes (as
described above), standard protective measures have been put in place to
prevent or minimize occupational exposure to human or animal tissues or
waste materials contaminated or potentially contaminated with prions.
These measures are pertinent to health-care and veterinary care
providers, mortuary workers and laboratory workers, all of whom may
handle or be exposed to contaminated blood, tissues or waste materials.
Standard protective procedures include administrative and engineering
controls, special work practices, personal hygiene measures, personal
protective clothing and decontamination methods.

	The Centers for Disease Control and Prevention (CDC) has published two
documents that provide guidance for health-care workers pertaining to
environmental infection control generally and to “special pathogens”
(including prions) specifically.  Guidelines for Handwashing and
Hospital Environmental Control (Garner and Favero, 1986; Bolyard et al.
1998: CDC/HICPAC 2003) encompasses general infection control, with
emphasis on the handling of air, water and environmental surfaces;
environmental sampling; and disposal of regulated and unregulated
medical waste.  Guidelines for Environmental Infection Control in
Health-Care Facilities (CDC 2003) provides extensive, detailed guidance
on all aspects of infection control in health-care facilities and
specifically addresses Creutzfeldt-Jakob Disease (CJD) in patient-care
areas.  This guidance notes that a limited number of cases result from
direct exposure to prion-containing material (usually central nervous
system tissue or pituitary hormones) acquired as a result of health care
(i.e., iatrogenic cases).  Six documented iatrogenic cases were
associated with neurosurgical instruments and devices that introduced
residual contamination directly to the recipient’s brain.  Because
there is no evidence to suggest that either CJD or vCJD has been
transmitted from environmental surfaces (e.g., during housekeeping
activities), CDC states that routine procedures are adequate for
cleaning and disinfection of a CJD patient’s room.  However, the CDC
recommends that hospitals identify patients with known or suspected CJD
and implement prion-specific infection-control measures for the
operating room and for instrument reprocessing.  Such measures should
include:

Use of disposable, impermeable coverings during autopsies and
neurosurgeries to minimize surface contamination

Cleaning and decontamination of surfaces that have been contaminated
with central nervous system tissue or cerebral spinal fluid (see
decontamination methods described in next section).

	In 2000, the World Health Organization issued its guidance document,
WHO Infection Control Guidelines for Transmissible Spongiform
Encephalopathies (WHO 2000), which focuses specifically on prion
diseases.  In this document, WHO designated central nervous system
tissues and fluids as “high-infectivity,” which should be managed
more conservatively than other diseases.  Key recommendations include
the following:

Covering patient treatment surfaces with disposable non-permeable covers
and uncovered surfaces are to be decontaminated.

Clinicians involved in patient handling are to wear single-use disposal
coverings (mask or visor, goggles, cut-resistant gloves and
liquid-repellent aprons over plastic gowns).

Treatments are to be conducted with equipment and tools that either are
dedicated to a single patient, or are disposed after a single use.

Instruments that have contacted high-infectivity tissues are to be
disposed by incineration, as are all single-use coverings and tools.

Reusable instruments are to be kept moist until cleaning and
decontamination; this measure is needed to prevent drying of tissues or
fluids thereon.

Low- and high-infectivity instruments are to be stored separately
between and during uses.

Bulk fluids are to be decontaminated or absorbed into sawdust, then
incinerated.

Contaminated medical wastes are to be incinerated, as are the disposable
materials.

All incineration is to be conducted in hazardous-waste facilities.

	The WHO document provides similar specific guidelines for mortuary and
laboratory workers who may contact contaminated materials, and for
decontamination of clinical or laboratory workers after occupational
exposures.  Safety recommendations are provided also for the transport
of contaminated specimens by air.

	With respect to veterinary laboratories, the American Association of
Veterinary Laboratory Diagnosticians (AAVLD) has issued recommendations
aimed at reducing potential risks to laboratory personnel posed by TSE
agents in animal tissues and waste materials (AAVLD 2004).  The AAVLD
recommends:

Using standard BSL2 safety precautions such as restricted access to
laboratories, protective clothing and facial protection, special care
with sharp instruments, and avoidance of aerosols.

Using numerous specific protective measures for necropsy laboratories,
histology laboratories and laboratories handling fresh tissues from TSE
suspect animals, such as extensive protective clothing, screened drains
to trap tissue fragments, use of appropriate disinfectants to inactivate
TSE agents on surfaces and non-disposable items, and disposal of
contaminated tissues by certain methods.

Decontamination Methods

	A number of decontamination methods have been developed and tested for
their ability to inactivate prions on inanimate surfaces and on objects
that may have contacted infected animal or human tissues.  This
development and testing has been especially spurred by the successful
migration of BSE into the human population during the mad cow epidemic
(UK 1986-2000) and by the compelling need for methods to decontaminate
environmental surfaces and instruments in veterinary facilities,
research laboratories, health care settings and other settings in which
prion-contamination is a possibility .

	The infectious conformation of prion proteins is extraordinarily
durable, and the measures necessary to destroy such prion proteins are
extreme compared to decontamination methods for other biological
pathogens, including bacterial spores.  In particular, prions bind
tightly to stainless steel, resist elution by exhaustive washing, and
remain highly infectious after formaldehyde treatment (Zobeley et al.
1999), which accounts for historical reports of transmission of CJD by
surgical instruments (Bernoulli et al. 1977).

	Some of the currently recommended decontamination methods include
treatment with

1-2 N NaOH or NaOCl  (2%,or 20,000 ppm chlorine) for one hour followed
by heat in a gravity displacement autoclave at 121º C for one hour,
autoclaving at 134º C  for 1 hr (or up to 5 hrs as in Peretz et al.
2006), or complete incineration (850º C or higher as in Brown et al.
2004).  General descriptions of these procedures are found in several
advisory reports, such as those of the CDC (1999), WHO (2000), AAVLD
(2004), Canadian Food Inspection Agency (2005) and MSU (2005).
However, these recommended decontamination methods are based on limited
academic research, and a number of issues bring into question whether
these methods can demonstrate a disinfectant’s ability to totally
inactivate prions on inanimate surfaces and objects.

The recommended prion-inactivation methods have not been tested using a
validated efficacy test method, and such a method does not yet exist.
Currently, EPA requires that disinfectants and sterilants be tested
using specific, validated efficacy test methods before EPA will register
such products, and such test methods are available (e.g., AOAC Use
Dilution Test).  However, no one has formally validated any of the
prion-inactivation efficacy test methods used to date, so it is
difficult to know whether such efficacy test methods provide consistent,
reliable data.

The risk of transmissible infectivity persists at prion titers far below
the limits of detection using analytical methods, such as gel
electrophoresis and Western blot assay (Yamamoto et al. 2001; Solassol
et al. 2006).  For example, Gregori et al. (2003) found that assaying
for surviving prion infectivity in hamsters was 1000 fold more sensitive
than visual detection by Western blot assay.  Consequently, adequate
confirmation in an animal model is required to confirm effective
elimination of all prion infectivity.

What constitutes an adequate animal test model for determining the
prion-inactivating efficacy of chemicals is uncertain.  Although the
Syrian hamster (Marsh and Kimberlin 1975) and mouse (Chandler 1961) have
a long history of utility in prion research, the life span of these
laboratory animals is relatively short, whereas the time interval from
prion exposure to the development of clinical disease can span decades
of time, as in the human prion diseases, for example.  In experimental
models, it is known that as the titer of infective material decreases,
the incubation time for the development of clinical disease in test
animals increases (Jackson et al. 2005; McDonnell et al. 2005; Solassol
et al. 2006).  Hence, the life span of the test animal may be too short
to detect residual prion infectivity at very low titers, as might be
expected with cleaning procedures that have been largely, but not
entirely, effective.  Ideally, efficacy studies should include positive
controls to determine the relationships between prion titer, infectivity
and the life span of the test animal.  Otherwise, the absence of
clinical signs during the short lifespan of the animal model might be
mistaken as evidence of successful elimination of infectivity.  Attempts
to accelerate the progression of infectivity by using synthetic
mammalian prions (Legname et al. 2004), as well as PrP over-expressing
cell lines (Solassol et al. 2004) and animals (for example, Peretz et
al. 2006) potentially point to ways to solve the lifespan problem, but
these approaches create new uncertainties by using animals already
predisposed to overexpression of host or foreign prion strains.

In addition to the common use of two species of test animals--hamster
and mouse--many different strains of prions are employed in
decontamination research.  These experimental variables might account
for the discrepant results for Environ LpH obtained by Ernst and Race
(1993) and Race and Raymond (2004), using Syrian hamsters as the test
model, with those of Jackson et al. (2005), using mice.

There are important barriers to the transmission of prion diseases
between animal species and humans, and the degree of transmission of
prion diseases from animals to humans varies greatly.  For example,
experimental studies using human and elk prion genes expressed in a
transgenic mouse indicate that elk chronic wasting disease may not
transmit readily to humans (Kong et al. 2005).  On the other hand, the
BSE epidemic in the UK (1986-2000) demonstrates that BSE can cross the
species barrier and become infectious to humans as vCJD (Brown 2001).
Since there are no systematic studies of comparative infectivity of
prion strains across different animal species and humans, it is not
clear how precisely one can extrapolate human risk from efficacy studies
performed in various animal species.  Specifically, some of the strains
used in efficacy studies may pose little or no risk to humans.
Conversely, it seems possible that some prion strains dangerous to
humans may not be testable in lab animals commonly used in efficacy
studies.  Finally, imperfect decontamination procedures may have
consequences for risks to humans that vary from inconsequential to very
significant, depending on the strain of prion being inactivated.  For
example, sheep scrapie is clinically unknown in humans and so the risks
to humans resulting from imperfect decontamination of scrapie would be
expected to be negligible compared to the risks from imperfect
inactivation of BSE prions on surfaces to which humans are exposed.

Similarly, comparisons of the prion proteins of different species in a
common animal model indicate there may be profound differences in the
prions of different species with regard to their stability and
resistance to inactivation during decontamination procedures.  For
example, Peretz et al. (2006) compared the resistance of hamster scrapie
and human CJD prions in transgenic mice expressing either hamster PrP or
a chimeric mouse-human PrP transgene and found that human sCJD prion is
100,000 fold more difficult to inactivate than hamster Sc237 prion.
Preliminary additional studies indicate that the cow BSE prion may be
even more resistant to inactivation than the human CJD prion (Giles et
al. 2006; 2008 in press).  This approach and the results obtained by the
direct comparison of different prion strains in common test models
indicate that decontamination procedures tested with rodent prions may
not be effective when tested with more durable human and cow prions.

Accordingly, although a number of academic studies have demonstrated
that certain chemical and/or physical decontamination methods provide
some assurance of reduction of prion infectivity, current test methods
cannot demonstrate that a disinfectant can totally inactivate prions or
TSE agents on inanimate surfaces and objects.

The Science of Prions As It Relates to the Definitions of “Pest”

	The Introduction to this paper described two sections of FIFRA that
define or give the EPA Administrator the authority to define the term
“pest.”  This section addresses the extent to which the available
scientific information on prions summarized in Section II relates to the
issue of whether prions fit within the definition of “pest”
specified in FIFRA Sections 2(t) or  25(c)(1).

	A.   Definitions of “Microorganism”

	The definition of “pest” in FIFRA Section 2(t) has two parts.  The
first part is whether the pest occurs in “circumstances that make it
deleterious to man or the environment.”  Based on the available
information described in Section II., prions clearly are deleterious to
humans and animals.  Exposure to prions may lead to infectious
acquisition of irreversible and fatal TSE diseases.

	The second part of the definition of “pest” relevant to prions
revolves around whether prions are “any other form of … virus,
bacteria or other microorganism….”  Prions are neither viruses nor
bacteria, as discussed in the preceding sections, so if prions are to be
considered pests under FIFRA they would have to be “other
micro-organisms.”   Below, two approaches have been taken to the
determination of prions as “other micro-organisms.”  In the first
case, prions are examined in the context of common dictionary
definitions of what constitutes a microorganism.  In the second case,
prions are reviewed in the context of Koch’s postulates for microbial
disease agents.

1.  Dictionary definitions of microorganisms.  Although there is no
single, definitive definition of “microorganism,” the definition in
the Oxford Dictionary of Biology (Fourth Edition, 2000) is
representative.  The Oxford Dictionary of Biology defines
“microorganism” as “Any organism that can be observed only with
the aid of a microscope.  Microorganisms include bacteria, viruses,
protists (including certain algae), and fungi.”  “Organism” is
further defined by the Oxford Dictionary as “An individual living
system, such as an animal, plant, or microorganism, that is capable of
reproduction, growth, and maintenance.”  These latter terms are
defined by Dorland’s Medical Dictionary as follows:

Reproduction—the sexual or asexual process by which organisms generate
others of the same kind

Growth—the normal process of increase in size of an organism by
accretion of tissue similar to that originally present

Maintenance—the process of holding a stable, steady state over a long
period

Therefore, whether prions fall within the above definition of
“microorganism” as it is generally used by biologists and the
medical community arguably depends on whether they exhibit reproduction,
growth and maintenance.

	However, this interpretation of “organism” is at odds with the
Oxford Dictionary of Biology’s definition for “microorganism,”
inasmuch as that definition includes viruses as microorganisms, even
though they do not have all of the listed characteristics of an
“organism”, as discussed in more detail below.

2. Koch’s postulates.  As indicated above, the properties that make
prions infectious agents of disease overlap with common characteristics
of microorganisms that cause disease (i.e., pathogens).  In 1884, Robert
Koch and Friederich Loeffler developed a list of general guidelines
that, if met, would indicate that a microorganism would likely cause
disease (i.e., be a pathogen).  Koch later published these
characteristics as four “postulates” (Koch 1893).  While a number of
infectious agents have been identified that do not meet Koch’s
postulates, those postulates have historical importance in the field of
microbiology (Jacomo et al. 2006).  Following are Koch’s postulates:

(1)  The microorganism must be found in abundance in all organisms
suffering from the disease, but should not be found in healthy animals.

(2)  The microorganism must be isolated from a diseased organism and
grown in pure   HYPERLINK "http://en.wikipedia.org/wiki/Cell_culture" \o
"Cell culture"  culture .

(3)  The cultured microorganism should cause disease when introduced
into a healthy organism.

(4)  The microorganism must be re-isolated from the inoculated, diseased
experimental host and identified as being identical to the original
specific causative agent.

	Regarding the first of these postulates, it is clear that all clinical
cases of TSEs feature measurable quantities of prions in the CNS
tissues; however, it is equally clear that prions can be detected in
CNS, blood and perhaps in other tissues in sub-clinical cases of disease
(section II. E., for example).  This is also the case for other diseases
having asymptomatic manifestations or asymptomatic carriers, which Koch
himself observed when he discovered asymptomatic carriers of   HYPERLINK
"http://en.wikipedia.org/wiki/Cholera" \o "Cholera"  cholera  and
HYPERLINK "http://en.wikipedia.org/wiki/Typhoid_fever" \o "Typhoid
fever"  typhoid fever .  Regarding the 2nd postulate, prions can be
isolated from diseased CNS (and other tissues).  Prions can be grown in
pure culture, in the sense that the disease producing conformation of
normal cellular prion protein can be amplified by repetitive cycles of
seeding existing, normal PrPC with prion isoforms, such as in the
cell-free protein misfolding cyclic amplification systems described by
Castilla et al. (2005; 2008; sections  II.A. and II.B.2. above).
However, this is not the same as the genetic reproduction that is
ordinarily used to satisfy Koch’s second postulate.

The 3rd and 4th postulates are satisfied by the numerous demonstrations
that prions enriched from diseased animals or synthesized de novo in
cell-free systems, are both infectious and produce disease (see II.A.2,
above).  Thus, the mechanisms of prion disease and infectivity generally
conform with Koch’s postulates for the microbial basis of disease.

	B.  Definitions of “Life”

	FIFRA Section 25(c)(1) authorizes the EPA to declare “a pest any form
of plant or animal life…which is injurious to health or the
environment.”  Because prions are already known to be injurious to
human and animal health, the remaining issue is whether prions are a
“form of life.”

	Dorland’s Medical Dictionary (2004), defines “life” as:

“  HYPERLINK ""   The aggregate of vital phenomena; a certain peculiar
stimulated condition of organized matter; that obscure principle whereby
organized beings are peculiarly endowed with certain powers and
functions not associated with inorganic matter. Generally, living things
share, in varying degrees, the following characteristics: organization,
irritability, movement, growth, reproduction, and adaptation.”

	This definition adds four more characteristics to the characteristics
beyond those included in the definition of “microorganism” and
“organism” and addressed in the preceding section (i.e.,
reproduction, growth, metabolism).  These additional characteristics
that generally are shared, to varying degrees, by living things are:

Organization—the act or process of being organized.

Irritability—the property of living organisms that allows them to
respond to stimuli.

Movement—the act or process of changing of place or position.

Adaptation—adjustment to environmental conditions by modification of
an organism to make it more fit for existence.

C.  Whether Prions Are Microorganisms or Alive

Given the unique attributes that make prions an infectious agent, and
given the imprecise nature of what constitutes a form of “life” or a
“microorganism,” there are at least two different ways to view the
issue of whether prions may be classified as either alive or a
microorganism.  One can focus on the biochemical properties of prions as
a type of protein molecule and argue that prions are neither alive nor a
microorganism.  Alternatively, one can focus on the attributes that make
prions infectious agents (such as in Koch’s postulates above) and
argue that prions should be classified as microorganisms.  These two
approaches are outlined below.

1)  Prions are not microorganisms or alive:

From the perspective of protein chemistry, the research studies
summarized in Section II indicate that a prion protein (whether normal
or misfolded) does not have all of the  characteristics of life or of a
microorganism.  A protein is a molecule and joins the lipids,
carbohydrates and nucleic acids as the basic molecules from which
microorganisms and the cells of multicellular organisms are made.
Regarding key properties of microorganisms (reproduction, growth and
maintenance), the prion protein is a normal cellular protein constituent
in vertebrate species that is transcribed by a homologous gene conserved
in the vertebrate species.  The individual molecule of prion protein
cannot reproduce, grow or perform self-maintenance in the same way that
microorganisms do.  When a prion protein becomes misfolded, it converts
other normal prion protein molecules to adopt the same misfolded
conformation through recruitment and conversion, which is analogous to
the way crystals grow.  Thus, a misfolded prion protein cannot reproduce
itself; it can only induce a copy of its shape in other prion proteins.
All other infectious agents—algae, fungi, bacteria, viruses and
viroids—produce infectious diseases by infecting and propagating their
numbers within the host species, which is accomplished through genetic
reproduction and the propagation of new generations from genes comprised
of DNA or RNA.  Unlike these other infectious agents, the aggregated
particles of a prion protein are devoid of any genetic element and have
no mechanism for genetic reproduction.  The property of infectivity is
limited to that of misfolded prion protein converting other, existing,
homologous protein molecules to adopt by copying the template of the
misfolded conformation.  There is neither synthesis of new prion protein
nor of new genetic material.  From this perspective, prions would not be
considered microorganisms.

Examining the six properties of “life” identified in the Dorland
definition cited above, one could argue that the only property of living
things that prions share is organization.  With respect to reproduction,
growth and maintenance, prions cannot carry out these functions via
conventional mechanisms due to their lack of genetic material.  With
respect to irritability and movement, prions are not capable of reacting
to stimuli or initiating movement on their own as most living organisms
are.  Further, with respect to adaptation, prions cannot genetically
adapt to their environment or pass along their characteristics because
they contain no DNA.  Finally, individual prion particles cannot modify
themselves to make themselves “more fit for existence” although
prions can adapt or evolve non-genetically, as described in the next
section.

Applying the definitions of “microorganism” and “life” narrowly,
therefore, one could argue that prions are neither microorganisms nor
alive.

2)  Prions are microorganisms and/or alive:

By focusing on the properties of prions that make them infectious to
humans and animals, and distinct from conventional protein toxins, one
can argue that prions are microorganisms and/or are alive.  The terms
“microorganism” and “life” have previously been expanded to
accommodate newly identified infectious agents, and because the
behavior, study and control of prions have more in common with the
behavior, study and control of infectious microorganisms than of
conventional chemical toxicants, it is practical and reasonable to
characterize them as infectious microorganisms.

For example, the Oxford Dictionary of Biology’s definition for
“microorganism” cited above includes viruses as microorganisms even
though viruses are not considered to be alive, and do not have all of
the characteristics of a microorganism as listed above.  Viruses do not
grow, have no metabolism, and cannot reproduce by themselves, although
they do replicate themselves by injecting their DNA into host cells and
taking over the host cell’s reproductive machinery.  Nevertheless,
viruses are often grouped with bacteria and other entities that meet the
entire microorganism criteria described above.  In the absence of a more
general term that expressly includes viruses, viroids and other
infectious agents that do not meet all the criteria of microorganisms,
this broader use of the term “microorganism” is common among
professionals in the biological, medical and public health sciences.

Prions are distinguishable from viruses in their lack of DNA and RNA,
but the International Committee on Taxonomy of Viruses nevertheless
considers prions to be within its scope, and includes taxonomy of
various prions in its Index of Viruses (  HYPERLINK
"http://www.ncbi.nlm.nih.gov/ICTVdb/Ictv/fs_prion.htm"
http://www.ncbi.nlm.nih.gov/ICTVdb/Ictv/fs_prion.htm ). However,
possession of such genetic material is not among the characteristics of
“microorganism” discussed above.  Prions have been characterized as
non-Mendelian elements of phenotypic inheritance (Alberti et al. 2009)
because they are capable of transmitting structurally and functionally
distinct states of the protein molecule.  Recognizing that prions are
the agents of TSEs, Miller (1999) classifies prions as “Subviral
Agents,” and Büchen-Osmond (2003) lists prions in the table of
viruses infecting humans with the notation that prions have the unique
property of a self-replicating infectious protein with no nucleic acid.

Regarding whether prions are a form of life, prions show a number of
characteristics of living organisms.  First and foremost, prions are
infectious agents that cause disease in humans in a manner very similar
to pathogenic viruses and bacteria.  Specifically, prions convert normal
prion proteins in animal nervous system tissues to abnormal,
dysfunctional prion proteins (additional prions) over a long period of
time, leading to neurological disease and death.  Unlike conventional
chemical toxicants, prions successfully replicate themselves, increase
in number, and spread from one host organism to another, much as
pathogenic viruses and bacteria.  Prions move within an organism, move
to other organisms, and move through the environment, and these
movements are closely analogous to the movements of viruses and
bacteria.  While movement of prions appears to be passive rather than
active, such as by motile mechanisms, that is also true for many
organisms universally regarded as living.  Finally, prions can adapt or
evolve (non-genetically) in two ways: (a) when injected into a new host,
the introduced prions (e.g., BSE prions introduced into humans) can
recruit and convert the new host’s prion protein into new prions
(e.g., variant CJD prions) and (b) over time, a prion strain can change
(through a process similar to natural selection) its characteristics
such as method of transmission and incubation period.  Based on these
points, prions could be considered to have some of the characteristics
of “life.”

The FIFRA SAP has also weighed in on the issue of whether prions are
microorganisms or a form of life.  Specifically, the SAP stated:

A majority of the Panel concluded that prions can be included in the
category of “other microorganisms.”  A minority of Panel members
contended on scientific basis that prions lack the significant features
typical of microorganisms. (USEPA 2009)

Several reasons the SAP articulated in support of its majority position
included:

Research spanning the last 50 years leaves no doubt that the agents
which cause prion disease can reproduce, adapt, grow and move within the
host.

Replication of prions has been amply demonstrated by the fact that they
can be titered and that animals infected with a low level of prion
infectivity eventually accumulate exponentially higher levels of
infectivity.  This can only occur if prions can replicate themselves.

As demonstrated by the isolation and careful characterization of
multiple mouse and hamster-adapted sheep scrapie strains, prions can
adapt to new species.

Peripheral inoculation of TSE infectivity eventually leads to
infectivity in the brain, something that can only occur if the
infectious agent moves within the host.

Prions most definitely exhibit a stable steady state over a long period
of time (maintenance).  Established stocks of TSE infectivity can be
maintained for decades with no change in either their titer or their
biological characteristics.

Prions are environmental pathogens that infect mammals, replicate
virus-like, and cause disease according to Koch’s postulates.

The SAP also stated the following conclusion concerning whether prions
are “alive.”

A majority of the Panel agreed that once revised to incorporate
revisions suggested by the Panel the White paper will adequately
identify and summarize the scientific literature concerning prions and
that prions are “other microorganisms” exhibiting some features of
living entities. (USEPA 2009)

Applying the definitions of “microorganism” and “life” broadly,
therefore, one could argue that prions are microorganisms.

	D.  Summary/Conclusion

	With respect to the issue of whether prions are “microorganisms” or
are “alive,” EPA recognizes the possibility of at least two
different points of view: (1) A narrower interpretation of current
definitions that prions are not microorganisms or alive, and (2) a
broader interpretation of current definitions that prions are a unique
type of microorganism and/or are alive.   In either case, prions are
infectious and do produce disease, and in doing so, they generally
adhere to Koch’s postulates for the causal relationships of microbial
diseases. EPA also acknowledges the FIFRA SAP’s position that prions
should be considered to be microorganisms and alive, and is taking this
recommendation into account in formulating a regulatory position on
whether prions should be considered to be a “pest” under FIFRA.

REFERENCES

References to Section I.

U.S. Environmental Protection Agency.  2004.  Considerations of Prions
as a Pest under FIFRA. Memorandum to The Record from Susan B. Hazen,
Principal Deputy Assistant Administrator, Office of Prevention,
Pesticides, and Toxic Substances.  April 29, 2004.

U.S. Environmental Protection Agency.  2009.   Transmittal of Meeting
Minutes of the FIFRA Scientific Advisory Panel Meeting Held March 31 -
April 1, 2009 on Scientific Issues Associated with Designating a Prion
as a ‘Pest’ under the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA), and Related Efficacy Test Methods.  Memorandum
from Myrta R. Christian, Designated Federal Official, FIFRA Scientific
Advisory Panel, to Debbie Edwards, Director, Office of Pesticide
Programs.  June 29, 2009.

References to Sections II.A. and B.

Aguzzi A, Baumann F, and Bremer J. 2008.  The prion’s elusive reason
for being.  Annu. Rev. Neurosci. 31:439-477.

Aguzzi A. 2009.  Beyond the prion princicple.  Nature 459:924-925.

Alberti S, Halfmann R, king O, Kapila A, and Lindquist S. 2009.  A
systematic survey identifies prions and illuminates sequence features of
prionogenic proteins.  Cell 137:146-158.

Alper T.  1966. The exceptionally small size of the scrapie agent.
Biochem. Biophys. Res. Com.  22:278-284.

Alper T, Cramp, WA, Haig DA, and Clarke MC.  1967. Does the agent of
scrapie replicate without nucleic acid?  Nature 214:764-766.

Austbø L, Espenes A, Olsaker I, Press CMcL, Skretting G.  2007.
Increased PrP mRNA expression in lymphoid follicles of the ileal
Peyer’s patch of sheep experimentally exposed to the scrapie agent.
J. Gen. Virol. 88:2083-2090.

Barron RM, Campbell SL, King D, Bellon A, Chapman KE, Williamson RA, and
Manson JC.  2007.  High titers of TSE infectivity associated with
extremely low levels of PrPSc in vivo.  J. Biol. Chem. 282(49):
35878-35886.  Epub 10-8-07.

Basler K, Oesch M, Scott M., Westaway D, Walchli  M, Groth DF, McKinley
MP, Prusiner SB, and Weissmann C.  1986. Scrapie and cellular PrP
isoforms are encoded by the same chromosomal gene.  Cell 46: 417-428.

Bolton  DC, McKinley MP, and Prusiner, SB. 1982.  Identification of a
protein that purifies with the scrapie prion.  Science 218:1309-1311.

Brandner S, Isenmann S, Raeber A, Fischer M, Sailer A, Kobayashi Y,
Marino S, Weissmann C, and Aguzzi A.  1996. Normal host prion protein
necessary for scrapie-induced neurotoxxicity.  Nature 379: 339-343.

Brown DR (Editor).  2005. Neurodegeneration and Prion Disease. Springer,
New Youk.

Brown DR, Qin K, Herms JW, Madlung A, Manson J, Strome R, Fraser PE,
Kruck T, von Bohlen A, Schulz-Schaeffer W, Giese A, Westaway D,
Kretzschmar H.  1997. The cellular prion protein binds copper in vivo.
Nature 390:684-687.

Brown P. Bovine spongiform encephalopathy and variant Creutzfeldt_jacob
diseae.  2001. BMJ 322: 841-844.

Brown P, Liberski PP, Wolff A, and Gajdusek DC.  1990a. Conservation of
infectivity in purified fibrillary extracts of scrapie-infected hamster
brain after sequential enzymatic digestion or polyacrylamide gel
electrophoresis.  PNAS 87:7240-7244.

Brown P, Liberski PP, Wolff A, and Gajdusek DC. 1990b.  Resistance of
scrapie infectivity to steam autoclaving after formaldehyde fixation and
limited survival after ashing at 360° C: practical and theoretical
implications.  J. Infect. Dis. 161: 467-472.

Brown P, Rau EH, Johnson BK, Bacote AE, Gibbs Jr. CJ, and Gajdusek DC.
2000. New studies on the heat resistance of hamster-adapted scrapie
agent: threshold survival after ashing at 600° C suggests an inorganic
template of replication.  PNAS 97: 3418-3421.

Brown SA, Merritt K, Woods TO, Busick DN.  2005. Effects on instruments
of the World Health Organization-recommended protocols for
decontamination after possible exposure to transmissible spongiform
encephalopathy-contaminated tissue.  J. Biomed Mater Res Part B; Appl
Biomater 72B: 186-190.

Burger D, Hartsough GR.  1965.  .Encephalopathy of mink. II.
Experimental and natural transmission.  J Infect Dis. Oct;115(4):393-9.

Calzolai, L., Lysek, D. A., Pérez, D. A., Güntert, P., and Wüthrich,
K. 2005.  Prion protein NMR structures of chickens, turtles, and frogs.
Proc. Nat. Acad. Sci.  102:651-655.

Carrell, RW.  Prion dormancy and disease.  2004. Science 306
(5702):1692-1693.

Castilla J, Saά P, Hetz C, and Soto C.  2005. In vitro generation of
infectious scrapie prions.  Cell 121: 195-206.

Castilla J, Morales R, Saά P, Barria M, Gambetti P, and Soto C.  2008.
Cell-free propagation of prion strains.  The EMBO Journal 27:2557-2566.

Chesebro B, Trifilo M, Race R, Meade-White K, Teng C, LaCasse R, Raymond
L, Favara C, Baron G, Priola S, Caughey B, Masliah E, and Oldstone M.
2005. Anchorless prion protein results in infectious amyloid disease
without clinical scrapie.  Science 308: 1435-1439.

Colby DW, Zhang Q, Wang S, Groth D, Legname G, Riesner D, and Prusiner
SB.  2007.  Prion detection by an amyloid seeding assay.  PNAS
104:20914-20919.

Deleault NR, Harris BT, Rees JR, and Supattapone S. 2007.  Formation of
native prions from minimal components in vitro.  PNAS 104:9741-9746.

Enserink M.  2005.  After the crisis: more questions about prions.
Science 310: 1756-1758.

Gajdusek DC.  1977.  Unconventional viruses and the origin and
disappearance of kuru.  Science. Sep 2;197(4307):943-960.

Goldberg AL.  2007.  On prions, proteasomes, and mad cows.  N. Engl. J.
Med. 357:1150-1152.

Haase B, Doherr MG, Seuberlich T, Drögenmüller C, Dolf G, Nicken P,
Schiebel K, Ziegler U, Groschup MH, Zurbiggen, A, and Leeb T. 2007.
PRNP promoter polymorphisms are associated with BSE susceptibility in
Swiss and German cattle.  BMC Genetics, 16 April, 8:15.

Harris, D., A., Falls, D., L., Johnson, F. A., Fischbach, G. D. 1991.  A
prion-like protein from chicken brain copurifies with an
acetylcholine-inducing activity.  Proc. Natl. Acad. Sci.  88:7664-7668.

Hartsough GR, Burger D.  Encephalopathy of mink. 1965.  I.
Epizootiologic and clinical observations. J Infect Dis.
Oct;115(4):387-92.

Heaton, M.P., et al. 2008. Prevalence of the prion protein gene E211K
variant in U.S. cattle. BMC Veterinary Research, 4:25.

Jackson WS, Borkowski AW, Faas H, Steele AD, King OD, Watson N, Jasanoff
A, and Lindquist S. (2009).  Spontaneous generation of prion infectivity
in fatal familial insomnia knockin mice. Cell 63: 438-450.

Jeffrey M, González L, Espenes A, Press CMcL, Martin S, Chaplin M,
Davis L, Landsverk T, MacAldowie C, Eaton S, McGovern, G.  2006.
Transportation of prion protein across the intestinal mucosa of
scrapie-susceptible and scrapie-resistant sheep.  J. Pathol. 209:4-14.

Jonietz E. 2007.  Prion infectivity pinpointed.  Technology Review. May
09, 2007.

Lau AL, Yam AY, Michelitsch MMD, Wang X, Gao C, Goodson R J,. Shimizu R,
Timoteo G, Hall H, Medina-Selby A, Coit D, McCoin C, Pehlps B, Wu P, Hu
C, Chien D, and Peretz D. 2007.  Characterization of prion protein
(PrP)-derived peptides that discriminate full-length PrPSc from PrPC.
PNAS 104:11551-11556.

Legname G, Baskakov IV, Nguyen OAB, Riesner D, Cohen FE, DeArmond SJ,
Prusiner SB.  Synthetic mammalian prions.  2004. Science 305; 673-676.

Lundmark K, Westermark GT, Nyström S, Murphy CL, Solomon A, and
Westermark P.  2002.  Transmissibility of systemic amyloidosis by a
prion-like mechanism.  PNAS 99: 6979-6984..

Mallucci G, Dickinson A, Linehan J, Klohn PC, Brandner S, and Collinge
J. 2003.  Depleting neuronal PrP in prion infection prevents disease and
reverses spongiosis.  Science 302: 871-874.

Manuelidis L.  2007. A 25 nm virion is the likely cause of transmissible
spongiform encephalopathies.  J. Cell Biochem. 100:897-915.

Manuelidis L, Yu Z-X, Barquero N, and Mullins B.  2007.  Cells infected
witih scrapie and Creutzfeldt_Jacob disease agents produce intracellular
25-nm virus-like particles.  PNAS 104:1965-1970.

McKenzie D, Bartz J, Mirwald J, Olander D, Marsh R, and Aiken J.  1998.
Reversibility of scrapie inactivation is enhanced by copper.  J. Biol.
Chem. 273:25545-25547.

Mead S, Stumpf MPH, Whitfield J, Beck JA, Poulter M, Campbell T, Uphill
JB, Goldstein D, Alpers M., Fisher EMC, and Collinge J.  2003. Balancing
selection at the prion protein gene consistent with prehistoric kurulike
epidemics.  Science 300: 64-643.

Jaton AL, Vigouret J-M, Paganetti P, Walsh DM, Mathews PM, Ghiso J,
Staufenbeil M, Walker LC, and Jucker M.  Exogenous induction of cerebral
β-amyloidogenesis is governed by agent and host.  2006. Science
313:1781-1784.

Moore, RC, Mastrangelo P, Bouzamondo E, Heinrich C, Legname G, prusiner
SB, Hood L, Westaway D, DeArmond SJ, and Tremblay P.  2001.
Doppel-induced cerebellar degeneration in transgenic mice.  PNAS 98:
15288-15293.

Müller H, Stitz L, Wille H, Prusiner SB, and Riesner D.  Influence of
water, fat, and glycerol on the mechanism of thermal prion inactivation.
 2007. J. Biol. Chem. 282(49):35855-35867.  Epub 9-18-07.

Oesch B, Westaway D., Walchli M., McKinley M.P, Kent SBH., Aebersold R.,
Barry RA, Tempst P, Teplow DB, Hood LE, Prusiner SB, and Weissmann C.
1985. A cellular gene encodes scrapie Prp 27-30 protein.  Cell 40:
735-746.

Palmer MS, Dryden AJ, Hughes JT, and Collinge J.  1991.  Homozygous
prion protein genotype predisposes to sporadic Creutzeldt-Jakob disease.
Nature 352: 340-342.

Piccardo P, Manson, JC, King D, Ghetti B, and Barron RM.  2007.
Accumulation of prion protein in the brain that is not associated with
transmissible disease.  PNAS 104:4712-4717.

Premzl M, Gready  JE, Jermiin LS, Simonic T, Graves JAM.  2004.
Evolution of vertebrate genes related to prion and Shadoo proteins –
clues from comparative genomic analysis.  Mol. Biol. Evol.
21 :2210-2231.

Premzl, M, Sangiorgio L, Strumbo B, Graves JAM, Simonic T, Gready J.
2003.  Gene 314:89-102.

Prusiner SB.  Novel proteinaceous infectious particles cause scrapie.
1982. Science 216:136-144..

Prusiner SB, Gajdusekk C, and Aplers MP.  1982. Kuru with incubation
periods exceeding two decades.  Ann. Neurol. 12:1-9.

Prusiner SB, Groth DF, Bolton DC, Kent SB, and Hood LE.  Prusiner, S.B.
1984. Purification and structural studies of a major scrapie  prion
protein.  Cell 38:127-134.

Sakaguchi S. 2005. Prion protein, prion protein-like protein and
neurodegeneration, in Brown D.R. Neurodegeneration and Prion Disease,
Springer, New York, pp 167-193.

Scientific American.  2007.  Scientists closer to unfolding the
mysteries of prion formation in mad cow disease.  Scientific American,
May 09.

Simoneau S, Rezaei H, Salès N, Kaiser-Schulz G, Lefebvre-Roque M, Vidal
C, Fournier J-G, Comte, J, Wopfner F, Grosclaude J, Schätzl H, and
Lasmézas CI. 2007.  In vitro and in vivo neurotoxicity of prion protein
oligomers.  PLoS Pathogens 3(8)e125.

Soto, C.  2006.  Prions.  The new biology of proteins.  CRC Press, FL.

Stahl N, Borchelt DR and Prusiner SB.  1990. Differential release of
cellular and scrapie prion protein form cellular membranes of
phosphatidylinositol specific phospholipase. Biochemistry 29: 5405-5412.

St Rose SG, Hunter, N, Matthews L, Foster JD, Chase-Topping ME, Kruuk,
LEB, Shaw D, Rhind SM, Will RG, and Woolhouse MEJ.  2006.  Comparative
evidence for a link between Peyer’s patch development and
susceptibility to transmissible spongiform encephalopathies.  BMC
Infect. Dis.  11 Jan, 6:5.

z S, Sάnchez-Alvarez M., Sugama S, DeGiorgio LA, Volpe BT, Wiseman E,
Abalos G, Masliah E, Gilden D, Oldstone MB, Conti B, and Williamson RA.
2004. Cross-linking cellular prion protein triggers neuronal apoptosis
in vivo.  Science 303: 1514-1516.

Surewicz WK. 2007.  Discriminating taste of prions.  Nature 447:541-542.

Taylor JP, Hardy J, and Fischbeck KH. 2002. Toxic proteins in
neurodegenerative disease.  Science 296: 1991-1995.

Tessier PM and Lindquist S. 2007.  Prion recognition elements govern
nucleation, strain specificity and species barriers.  Nature
447:556-561.

Watts JC, Drisaldi B, Ng V, Yang J, Strome B, Horne P, Sy M-S, Yoong L,
Young R, Mastrangelo P, Bergeron C, Fraser PE, Carlson GA, Mount HTJ,
Schmitt-Ulms G, and Westaway D.  2007.  The CNS protein Shadoo has
PrPC-like protective properties and displays reduced levels in prion
infections.  The EMBO Journal 26:4038-4050.

Webb S, Lekishvili T, Loeschner C, Sellarajah S, Prelli F, Wisniewski T,
Gilbert IH, and Brown DR.  2007.  Mechanistic insights into the cure of
prion disease by novel antiprion compounds.  J. Virol. 81:10729-10741.

Wells GA, Scott AC, Johnson CT, Gunning RF, Hancock RD, Jeffrey M,
Dawson M, Bradley R.  1987.  A novel progressive spongiform
encephalopathy in cattle.  Vet Rec. Oct 31;121(18):419-420.

Westaway, D. and Prusiner, S. B. 1986.  Conservation of the cellular
gene encoding scrapie prion protein.  Nuc. Acid Res. 14:2035-2044.

WHO Infection Control Guidellines for Transmissible Spongiform
Encephalopathies.  WHO/CDS/CSR/APH/2000.3.

Whyte SM, Sylvester ID, Martin SR, Gill AC, Wopfner F, Schatzl HM,
Dodson GG, and Bayley PM.  2003.  Stability and conformational
properties of doppel, a prion-like protein, and its single-disulphide
mutant.  Biochem J 373:  485-494.

  HYPERLINK
"http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22W
ong%20BS%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_Result
sPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus"  Wong BS ,
HYPERLINK
"http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22L
iu%20T%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsP
anel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus"  Liu T ,   HYPERLINK
"http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22P
aisley%20D%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_Resu
ltsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus"  Paisley D ,
HYPERLINK
"http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22L
i%20R%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPa
nel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus"  Li R ,   HYPERLINK
"http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22P
an%20T%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsP
anel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus"  Pan T ,   HYPERLINK
"http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22C
hen%20SG%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_Result
sPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus"  Chen SG ,
HYPERLINK
"http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22P
erry%20G%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_Result
sPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus"  Perry G ,
HYPERLINK
"http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22P
etersen%20RB%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_Re
sultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus"  Petersen RB ,
HYPERLINK
"http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22S
mith%20MA%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_Resul
tsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus"  Smith MA ,
HYPERLINK
"http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22M
elton%20DW%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_Resu
ltsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus"  Melton DW ,
HYPERLINK
"http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22G
ambetti%20P%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_Res
ultsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus"  Gambetti P ,
HYPERLINK
"http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22B
rown%20DR%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_Resul
tsPanel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus"  Brown DR , and
HYPERLINK
"http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22S
y%20MS%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsP
anel.Pubmed_DiscoveryPanel.Pubmed_RVAbstractPlus"  Sy MS .  2001.
Induction of HO-1 and NOS in doppel-expressing mice devoid of PrP:
implications for doppel function.  Mol Cell Neurosci 17:768-775.

Zhang CC, Steele AD, Lindquist S, and Lodish HF.  2006. Prion protein is
expressed on long-term repopulating hematopoetic stem cells and is
important to their self-renewal.  PNAS 103:2184-2189.

References to Section II.C.

Brown DR. 2005. Chapter 1. Introduction. IN: Neurodegeneration and Prion
Disease. Editor DR Brown. Springer+Business Media, Inc., NY. pp 1-11.

Collinge J. 2001. Prion diseases of humans and animals: Their causes and
molecular basis. Annu.Rev. Neurosci. 24:519-550.

Jeffrey M and Wells GA. 1988. Spongiform encephalopathies in a nyala
(Tragelaphus angasi). Vet Pathol. 25:398-399.

Marsh RF. 1992. Transmissible mink encephalopathy. See Collinge, 2001.

Wells GA, Hawkins SA, Johnson CT, Gunning RF, Hancock RD, Jeffrey M,
Dawson M, and Bradley, R., 1987. A novel progressive spongiform
encephalopathy in cattle. Vet Rec. 31:419-420.

  HYPERLINK
"http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&itoo
l=PubMed_Abstract&term=%22Wickner+RB%22%5BAuthor%5D" \o "Click to search
for citations by this author."  Wickner RB ,   HYPERLINK
"http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&itoo
l=PubMed_Abstract&term=%22Edskes+HK%22%5BAuthor%5D" \o "Click to search
for citations by this author."  Edskes HK ,   HYPERLINK
"http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&itoo
l=PubMed_Abstract&term=%22Roberts+BT%22%5BAuthor%5D" \o "Click to search
for citations by this author."  Roberts BT ,   HYPERLINK
"http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&itoo
l=PubMed_Abstract&term=%22Baxa+U%22%5BAuthor%5D" \o "Click to search for
citations by this author."  Baxa U ,   HYPERLINK
"http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&itoo
l=PubMed_Abstract&term=%22Pierce+MM%22%5BAuthor%5D" \o "Click to search
for citations by this author."  Pierce MM ,   HYPERLINK
"http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&itoo
l=PubMed_Abstract&term=%22Ross+ED%22%5BAuthor%5D" \o "Click to search
for citations by this author."  Ross ED ,   HYPERLINK
"http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&itoo
l=PubMed_Abstract&term=%22Brachmann+A%22%5BAuthor%5D" \o "Click to
search for citations by this author."  Brachmann A . 2004. Prions:
proteins as genes and infectious entities. Genes Dev. 18: 470-485.

Williams ES and Young S. 1980. Chronic wasting disease of captive mule
deer: a spongiform encephalopathy. J. Wildl. Dis. 16:89-98.

Wyatt JM, Pearson GR, Smerdon TN, Gruffydd-Jones TJ, Wells GAH, and
Wilesmith JW. 1991. Naturally occurring scrapie-like spongiform
encephalopathy in five domestic cats. Vet. Rec. 129:233-236.

References to Section II.D.

Alpers MP. 1987. Epridemiology and clinical aspects of kuru. (see
Collinge, 2001)

Cornell Feline Health Center. 2004. Feline Spongiform Encephalopathy.
HYPERLINK "http://www.vet.cornell.edu/fhc/resources/madcow.htm"
http://www.vet.cornell.edu/fhc/resources/madcow.htm . Last update 7
July, 2004.

Fischer A and Fischer JL. 1961.  Culture and Epidemiology: A theoretical
investigation of kuru.  J. Health and Human Behavior 2:16-25.

Gajdusek DC.  1977.  Unconventional viruses and the origin and
disappearance of kuru. Science 197:943-960.

Miller MW, Williams ES, McCarty CW, Spraker TR, Kreeger TJ, Larsen CT,
and Thorne ET. 2000. Epizootiology of chronic wasting disease in
free-ranging cervids in Colorado and Wyoming. Journal of
Wildlife-Diseases 36[4], 676-690.   HYPERLINK
"http://whyfiles.org/193prion/2.html"
http://whyfiles.org/193prion/2.html

U.S. Department of Agriculture. Federal Register notice, March 17, 2000,
Volume 65, Number 53.    HYPERLINK
"http://www.who.int/mediacentre/factsheets/fs113/en/"
www.who.int/mediacentre/factsheets/fs113/en/

Williams, ES., Miller, MW., and Thorne, ET. 2002. Chronic Wasting
Disease: Implications and Challenges for Wildlife Managers. Transactions
of the 67th North American Wildlife and Natural Resources Conference 67,
87-103.

Zigas V. and Gajdusek DC.  1957.  Kuru: clinical study of a new syndrome
resembling paralysis agitans in natives of the Eastern Highlands of
Australian New Guinea. Med. J. Aust. 44(21):745-54.

References to Section II.E.

Andreoletti O, Lacroux C, Chabert A, Monnereau L, Tabouret G, Lantier F,
Berthon P, Eychenne F, Lafond-Benestad F, Elsen JM, Schelcher F. 2002.
PrP(Sc) accumulation in  placentas of ewes exposed to natural scrapie:
Influence of foetal PrP genotype and effect on ewe-to-lamb transmission.
J Gen Virol 83:2607-16.

Belay ED, Maddox RA, Williams ES, Miller MW, Gambetti P, Schonberger LB.
2004. Chronic wasting disease and potential transmission to humans.
Emerg Infect Dis10:977-84.   HYPERLINK "http://www.cdc.gov/eid"
www.cdc.gov/eid .

Blattler T. 2002.  Implications of prion diseases for neurosurgery.
Neurosurg Rev 25:195-203.

Brown, P and Gajdusek DC. 1991.  Survival of scrapie virus after 3
years' interment.  Lancet 337:269-270.

Bruce ME, Will RG, Ironside JW, McConnell I, Drummond D, Suttie A,
McCardle L, Chree A, Hope J, Birkett C, Cousens S, Fraser H, Bostock CJ.
1997. Transmissions to mice indicate that “new variant” CJD is
caused by the BSE agent. Nature 389:498-501.

Chesebro B. 2003.  Introduction to the transmissible spongiform
encephalopathies or prion diseases. Br Med Bull 66:1-20.

Collinge J, Sidle KCL, Meads J, Ironside J, Hill AF. 1996.  Molecular
analysis of prion strain variation and the aetiology of “new
variant” CJD. Nature 383:685-90.

Dong J, Bloom MD, Goncharov V, Chattopadhyay M, Milhauser GL, Lynn, DG,
Scheibel T, Lindquist S.  Probing the role of PRP repeats in
conformational conversion and amyloid assembly of chimeric yeast prions.
 2007.  J. Biol. Chem.  24 Sept. [E-publication ahead of print].

Fflechsig E, Hegyi I, Schwarz P, Collinge J and Weissman C.  2001.
Transmission of scrapie by steel-surface-bound  prions.  Mol. Med.
7:679-684.

Ingrosso L, Pisani F, Picchiari M.  1999.  Transmission of the 263K
scrapie strain by the dental route. J Gen Vir 80:3043-7.

Johnson CJ, Phillips KE, Schramm PT, McKenzie D, Aiken JM, and Pedesen,
J . 2006.  Prions adhere to soil minerals and remain infectious.  PLoS
Pathogens 2: 296-302.

Johnson CJ, Pedersen, JA, Chappell RJ, McKenzie D, and Aiken JM.  2007.
Oral transmissibility of prion disease is enchanced by binding to soil
particles.  PLoS Pathogens Jul 6;3(7):e93 [E-pub ahead of print].

Karpuj, MB, Giles K, Gelibter-Niv S, Scot MR, Lingappa VR, Szoka FC,
Peretz D, Denetclaw W, and Prusiner SB.  2007.  Phosphorothionate
oligonucleotides reduce PrPSc levels and prion infectivity cultured
cells.  J. Mol. Med.  13:190-198.

Ligios C, Cancedda GM, Margalith I, Santucciu C, Madau L, Maestrale C,
Basagni M, Saba M, and Heikenwalder M.  2007.  Intraepithelial and
interstitial deposition of pathological prion protein in kidneys of
scrapie-affected sheep.  PLoS ONE Sept 12;2(9):e859.

Leninger AL. 1970.  Biochemistry. The Molecular Basis of Cell Structure
and Function, Worth Publisher's Inc., New York.

Liberski PP, Sikorska B, Guiory D, and Bessen RA. 2009.  Transmissible
mink encephalopathy – review of the etiology.  Folia Neuropathol.
47:195-204.

Llewelyn CA, Hewitt RE, Knoght RSG, Amar K, Cousens S, MacKenzie J, Will
RG. 2004. Possible transmission of variant Creutzfeldt-Jakob disease by
blood transfusion. Lancet 363:417-421.

Ma, X.; Benson, C. H., McKenzie, D., Aiken, J. M., Pedersen, J. A.
2007.  Adsorption of Pathogenic Prion Protein to Quartz Sand, Environ.
Sci. Technol. 41(7); 2324-2330.

Marsh RF and Bessen RA. 1993. Epidemiological and experimental studies
of transmissible mink encephalopathy.  Dev. Biol. Scand. 80:111-118.

Mathiason CK, Powers JG, Dahmes SJ, Osborn DA, Miller KV, Warren RJ,
Mason GL, Hays SA, Hayes-Klug  J, Seellig DM, Wild MA, Wolfe LL, Spraker
TR, Miller MW, Sigurdson CJ, Telling GC, Hoover EA.  2006. Infectious
Prions in the Saliva and Blood of Deer with Chronic Wasting Disease.
Science 13:133-136.

Miller MW and Williams ES. 2003.  Prion disease: horizontal prion
transmission in mule deer. Nature 425:35-6.

Miller MW, Williams ES, Hobbs NT, Wolfe LL. 2004.  Environmental sources
of prion transmission in mule deer. Emerging Infectious Diseases
10:1003-6.   HYPERLINK "http://www.cdc.gov/eid"  www.cdc.gov/eid .

Müller H, Stitz L, Wille H, Prusiner SB, and Riesner D.  Insluence of
water, fat, and glycerol on the mechanism of thermal prion inactivation.
 2007.  J. Biol. Chem. [18 Sept. E-pub ahead of print].

Palsson PA.  1979.  Rida (scrapie) in Iceland and its epidemiology. In:
Slow Transmissible Diseases of the Nervous System (Eds. Prusiner SB,
Hadlow WJ). Academic Press, New York, NY, pp 357-366.

Pattison IH, Hoare MN, Jebbett JN, Watson WA.  1972.  Spread of scrapie
to sheep and goats by oral dosing with foetal membranes from
scrapie-affected sheep. Vet Rec 90:465-468.

Peden AH, Head MW, Ritchie DL, Bell JE, Ironside JW.  2004.  Autopsy
detection of preclinical vCJD transmission following blood transfusion
from a PRNP codon 129 heterozygote. Lancet 364:527-529.

Peretz D, Supattapone S, Giles K, Vergara J, Freyman Y, Lessard P, Safar
JG, Glidden DV, McCulloch C, Nguyen H-OB, Scott M, DeArmond SJ, and
Prusiner SB.  2006.  Inactivation of prions by sodium dodecyl sulfate.
J. Virol. 80:322-331.

Peretz D, Williamson RA, Kaneko K, Vergara J, Leclerc E, Schmitt-Ulms G,
Mehlhorn IR, Legname G, Wormald MR, Rudd PM, Dwek RA, Burton DR,
Prusiner SB.  2001.  Antibodies inhibit prion propagation and clear cell
culture of prion infectivity. Nature 412:739-43.

Prusiner SB, Scott MR, DeArmond SJ (2004) Transmission and Replication
of Prions.  In: Prion Biology and Diseases, Second Edition (Ed. Prusiner
SB).  Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York,
NY, pp187-242.

Scherbel C, Pichner R, Groschup MH, Mueller-Hellwig S, Scherer S,
Dietrich R, Maertlbauer E, and Gareis M.  2007.  Infectivity of scrapie
prion protein (PrPSc ) following in vitro digestion with bovine
gastrointestinal microbiota.  Zoonoses and Public Health  54:185-190.

Seidel B, Thomzig A, Buschmann A, Groschup MH, Peters R, Beekes M, and
Terytze K.  2007.  Scrapie agent (strain 263K) can transmit disease via
the oral route after persistence in soil over years.  PLoS ONE
2(5):e435.

Shaked GM, Shaked Y, Kariv-Inbal Z, Halimi M, Avaraham, I, and Gabizon
R. 2001. A protease-resistant prion protein isoform is present in urine
of animals and humans affected with prion diseases.  J. Biol. Chem.
276:31479-31482.

Solomon A, Richey T, Murphy CL, Weiss DT, Wall JS, Westermark GT, and
Westermark P, 2007.  Amyloidogenic potential of foie gras. PNAS
104:10998-11001.

Supattapone S, Nguyen H-OB, Cohen FE, Prusiner SB, and Scott MR.   1999.
Elimination of prions by branched polyamines and implications for
therapeutics.  PNAS 96:14529-14534.

Supattapone S, Wille H, Uyechi L, Safar J, Tremblay P, Szoka FC, Cohen
FC, Prusiner SB, and Scott MR.  2001. Branched polyamins cure
prion-infected neuroblastoma cell. J. Virol. 75:3453-3461.

Tessier PM and Lindquist S.  Prion recognition elements govern
nucleation, strain specificity and species baarriers.  2007.  Nature
447:556-561.

Wells GAH and Wilesmith JW.  2004.  Bovine spongiform encephalopathy and
related diseases.  In: Prion Biology and Diseases, Second Edition (Ed.
Prusiner SB).  Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
New York, NY, pp 595-628.

Weissmann C, Enari M, Klohn PC, Rossi P, Flechsig E.  2002a.
Transmission of prions. J Infect Dis 186 Suppl 2:S157-65.

Weissmann C, Enari M, Klohn PC, Rossi P, Flechsig E.  2002b.
Transmission of prions. PNAS 99 Suppl 4:16378-83.

Wilesmith JW, Ryan JBM, Atkinson JM. 1991. Bovine spongiform
encephalopathy: Epidemiologic studies on the origin. Vet Rec
128:199-203.

Will RG, Cousens SN, Farrington CP, Smith PG, Knight RSG, Ironside JW.
1999.  Deaths from variant Creutzfeldt-Jakob disease. Lancet 353:979.

Will RG, Ironside, JW, Zeidler M, Cousens SN, Estibeiro K, Alperovitch
A, Poser S, Pocchiari M, Hofman A, Smith PG.  1996. A new variant of
Creutzfeldt-Jakob disease in the UK. Lancet 347:921-5.

Zobeley E, Flechsig E, Cozzio A, Enari M, Weissman C.  1999.
Infectivity of scrapie prions bound to stainless steel surface.
Molecular Medicine 5:240-243.

References to Section II.F.

Garner JS and favero MS. 1986.  CDC guideline for handwashing and
hospital environmental control.  Infect. Control.  7:231-243.

ference to III.B.

References to Section II.G.

AAVLD.  2004.  Best management practices for handling suspect biosafety
level 2 animal transmissible spongiform encephalopathy (TSE) diagnostic
samples (scrapie, chronic wasting disease, and transmissible mink
encephalopathy)  in animal health laboratories.

Alper T, Haig, DA and Carke MC.  1966.  The exceptionally small size of
the scrapie agent.  Biochem. Biophys. Res. Commun.  22:278-284.

Alper T, Cramp WA, Haig, DA and Carke MC.  1967. Does the agent of
scrapie replicate without nucleic acid?  Nature 214:764-766.

Bernoullli C, Siegfried J, Baumgartner G, Regli F, Rabinowicz T,
Gajdusek DC, et al. 1977.  Danger of accidental person-to-person
transmission of Creutzfeldt-Jacob disease by surgery.  Lancet 1:278-479.

Brown P. 2001.  Bovine spongiform encephalopathy and variant
Creutzfeldt-Jacob disease.  BMJ 322:841-844.

Brown P, Rau EH, Lemieux P, Johnson BK, and Gajdusek DC.  2004.
Infectivity Studies of both ash and air emissions fro simulated
incineration of scrapie-contaminated tissues.  Environ. Sci. Technol.
38:6155-6160.

Brown SA, Merritt K, Woods TO, and Busick DN. 2005.  Effects on
instruments of the World Health Organization-recommended protocols for
decontamination after possible exposure to transmissible spongiform
encephalopathy-contaminated tissue.  J. Biomed. Mater. Res. Part B:
Appl. Biomater. 72B:186-190.

Canadian Food Inspection Agency; Science Branch; Biohazard Containment
and Safety Unit.  2005.  Containment standards for laboratories, animal
facilities, and post mortem room handling prion disease agents.

CDC; 1999.  Office of Health and Safety; BMBL Section VII.  Agent
Summary Statements Section VII-D: Prions.

Chandler RL. 1961.  Encephalopathy in mice produced by incolulation with
scrapie brain.  Lancet i: 1378-1379.

Ernst Dr and Race RE.  1993.  Comparative analysis of scrapie
inactivation methods.  J. Virol. Methods 41:193-202.

Giles K, Peretz D, Glidden DV, and Prusiner SB. 2006.  Poster SA-05.
Prion 2006: strategies advances and trends towards protection of
society.  Turin, Italy, 3-6 October.

Giles et al., 2008, in press PLoS Pathogens.

Gordon, WS.  1946. Advances in veterinary research.  Vet. Res.
58:516-520.

Gregori L, Maring JA, MacAuley C, Dunston D, Rentsch M, Kempf C, and
Rohwer RG.  2003.  Partitioning of TSE infectivity during ethanol
fractionation of human plasma.  Biologicals 32:1-10.

Jackson GS, McKintosh E, Flechsig E, Prodromidou K, Hirsch P, Linhan J,
Brandner S, Clarke AR, Weissmann C, and Collinge J.  2005.  An
enzyme-detergent method for effective prion decontamination of surgical
steel.  J. Gen. Virol. 86:869-878.

Kong Q, Huang S, Zou W, Vanegas D, Wang M, Wu D, Yuan J, Zheng M, Bai H,
Deng H, Chen K, Jenny AL, O’Rourke K, Belay E, Schonberger LB,
Peterseon RB, Sy MS, Chen SG, and Gambetti P. 2005.  Chronic wasting
disease of elk: transmissibility to humans examined by transgenic mouse
models.  J. Neurosci.  25:7944-7949.

Legname G, Baskakov IV, Nguyen H-O B, Riesner D, Cohen FE, DeArmond SJ,
and Prusiner SB.  2004.  Synthetic mammalian prions.  Science
305:673-676.

Marsh RF, Kimberlin RH.  1975. Comparison of scrapie and transmissible
mink encephalopathy in hamsters.  II. Clinical signs, pathology, and
pathogenesis.  J. Infect. Dis.  131: 104-110.

McDonnell G, Fichet G, Antloga K, Kaiser H, Bernardo M, Dehen C, Duval
C, Comoy E, and Deslys JP.  2005.  Cleaning investigations to reduce the
risk of pron contamination on manufacturing surfaces and materials.
Euro. J. Parenteral Pharm. Sci. 10:67-72.

Michigan State University.  2005.  Recommended Biosafety Practices for
Handling Prions and Prion-Infected Tissues.

Peretz D, Supattapone S, Giles K, Vergara J, Freyman Y, Lessared P,
Safar JG, Glidden DV, McCullooch C, Nguyen O-H B, Scott M, DeArmond SJ,
and Prusiner SB.  2006.  Inactivation of prions by sodium dodecyl
sulfate.  J. Virol.  80:322-331.

Prusiner SB, Groth DF, McKinley MP, Cochran SP, Bowman KA, and Kasper
KC.  1981.  Thiocyanate and hydroxyl ions inactivate the scrapie agent.
Proc. Natl. Acad. Scie. 78:4606-4610.

Race RE and Raymond GJ.  2004. Inactivation of transmissible spongiform
encephalopathy (prion) agents  by Environ LpH.  J. Virol. 78:2164-2165.

Solassol J, Arlotto A, and Lehman S.  2004.  Detection of prion after
decontamination procedures: comparative study of standard Western blot,
filter retention and scrapie-cell assay.  J. Hosp. INf.  57:156-161.

Solassol J, Pastore M, Crozet c, Perrier V, and Lehmann S.  2006.  A
novel copper-hydrogen peroxide formulation for prion decontamination.
J. Infect. Dis.  194:865-869.

Taylor DM. 1991.  Inactivatio of BSe agent.  Develop. Biol. Standard.
75:97-102.

Yamamoto M, Horiuchi M, Ishiguro N, Shingawa M, Matsuo T, and Kaneko K.
2001.  Glycidol degrades scrapie mouse prion protein.  J. Vet. Med. Sci.
 63:983-990.

WHO. 2000.  WHO Infection and control guidelines for transmissible
spongiform encephalopathies.

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Infectivity of scrapie prions bound to a stainless steel surface.
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References to Section III.

Alberti S, Halfmann R, King O, Kapila A, and Lindquist S. (2009).
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Bűchen-Osmond C. (2003) Taxonomy and Classification of Viruses.  In
Manual of Clinical Microbiology, 2:1217-1226, ASM Press, Washington, DC.

Koch R (1893). "Über den augenblicklichen Stand der bakteriologischen
Choleradiagnose" (in German). Zeitschrift für Hygiene und
Infectionskrankheiten. 14: 319–333.

Jacomo V, Kelly P, Raoult D (2002). "Natural history of Bartonella
infections (an exception to Koch's postulate)". Clin Diagn Lab Immunol 9
(1): 8–18.   HYPERLINK
"http://en.wikipedia.org/wiki/Digital_object_identifier" \o "Digital
object identifier"  doi :  HYPERLINK
"http://dx.doi.org/10.1128%2FCDLI.9.1.8-18.2002"
10.1128/CDLI.9.1.8-18.2002

Miller MJ.  1999.  Viral Taxonomy.  Clinical Infectious Diseases
29:731-733.

 This definition and those that follow were chosen from among several
available to EPA.  Those discussed herein were selected based on their
inclusion in authoritative sources, their completeness, and their
utility in addressing the scientific arguments considered.

&fectious Diseases, Kenneth J. Ryan, C. George Ray, John C. Sherris
(McGraw-Hill 2003).

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Distribution of CWD (http://www.aphis.usda.gov/
animal_health/animal_diseases/cwd/downloads/distribution_cwd.pdf)