Scientific Information Concerning the Issu e of Whether Prions Are a “Pest” under the Federal Insecticide, Fungic ide, and Rodenticide Act (FIFRA) (12/17/08 Draft)

I. Introduction and Background

This paper reviews currently available scien tific information pertai ning to the issue of whether a prion (“ pr oteinaceous in fectious particles”) should be c onsidered to be a “pest” as defined by the Federal Insecticide, Fungicide, an d Rodenticide Act (FIFRA). The scientific

information in this review has been considered by the EPA work group that developed the Notice of Proposed Rulemaking that proposes to es tablish a prion as a pest under FIFRA.

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

• FIFRA Section 2(t): A pest is “... (2) any other formof terrestrial or aquatic plant or animal life or virus, bacteria or other micro-orga nism...which the Administrator declares to be a pest under se ction 25(c)(1).”

• FIFRA Section 25(c)(1): “The Administrator, after not ice 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 Prev ention, Pesticides, and Toxic Substances (EPA/OPPTS) decided that a pri on should be considered to be a “pest” under FIFRA and that products intended to inactivate prions (i.e., “pri on products”) should be regulated under FIFRA (USEPA, 2004). The Agency’s rationale may be summarized as the following key points:

o Prions are unquestionably injurious to human and animal health.

o Prions share many characteristics of other deleterious microorganisms.

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

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

o It appears that the inte nt of Congress in the 1972 FIFRA was that EPA should regulate products used agains t deleterious forms of life share the characteristics of viruses and other microorganisms

.

In order to provide sound, sc ientific input into the issue of whether a prion may be considered to be a “pest” under FIFRA, EPA has again examined the relevant, available

scientific evidence relevant to the question. This document princi pally addresses the scientific evidence. Legal and policy considerations that factor into the Agency’s decision to issue a NPRM will be described in more depth in the Preamble to the NPRM.

1II. The Science of Prions

A. Description

The leading theory on the cau sative 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 distincti on between molecular properties of the novel, proteinaceous infectious particles that cause scra pie disease and molecular properties of particles, such as viruses, plasmids, a nd viroids that feature a core of nucleic acid. Fueled by the catastrophic outbreak of mad cow disease, or bovine spongiform en cephalitis (BSE), originating in Essex County, England (1986), re search to characterize the pr ecise nature of prions and resolve how they cause the diseases known as TSEs 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 th e time interval between exposure and clinical disease was years or decades (Prusi ner et al. 1982). While a number of scientists have not ruled out an unknown virus as the agen t of the TSEs (Manuelidis, 2007; Manuelidis et al. 2007), no virus producing a TSE disease has been isolated after 40 years of searching. Instead, the proteinonly hypothesis for the origin of the TSEs (Alper et al. 1966; 1967; Griffith, 1967; Brown et al. 1990a) is now generally accepted by overwhelming we ight of evidence, especially Prusiner’s hypothesis of a novel proteinaceous infectious agen t – the prion (Prusiner et al. 1982; Bolton et al. 1982). In particular, experimental transmi ssion of TSE diseases by purified prion protein synthesized or propagated in cel l free systems (Legname et al . 2004; Castilla et al. 2005) is accepted by many as definitive proof of the pri on hypothesis. Though prions are now generally accepted as the agent responsible for the TSEs, th e precise structural pr operties of the prion protein responsible for infectivity and the chain of events that produce neural degeneration and fatal TSEs remain unknown (Barron et al. 2007). As a result of the novelty of the prion hypothesis and controversy surroundi ng 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). Hi ghlights of prion research are pr esented in section II of this

paper.

Numerous breakthroughs have been made in th e 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 propert y, so that when the normal α -helix rich conformation is converted to a misfolded, β -sheet rich conformation, the resu ltant protein by-passes the normal proteosomal degradation pathway, produces a TSE whose clinical features vary somewhat depending on the species (eg., cow, sheep, human, et c), and acquires the prope rty of infectivity. Experimental intervention to restore proteo somal degradation of the misfolded protein conformation “cured” prion infect ivity in cultured cells (Webb et al. 2007). The property of infectivity appears to reside in the β -sheet-rich conformation (Mülle r et al. 2007), which is the focal point of numerous ongoing investigations to define precisely the struct ural determinants of infectivity. In humans, polymorphism at PRNP gene codon 129 (129M/V) is common;

2however, homozygous genotypes at 129 predispo se PrP transcripts to misfolding and heterozygous transcripts resist misfolding (Mead et al. 2003). Genetic polymorphism in the PRNP promoter may also influence susceptibility to TSEs, as in the case of BSE (Haase et al. 2007). What distinguishes prion diseases from other neurodegenerative diseases featuring neurotoxic accumulations of prot ein plaques (such as Alzheimer’s, Parkinson’s, and ALS) is that misfolded PrP transcripts recru it and convert normal PrP transc ripts to adopt the misfolded conformation; however, a low le vel of recruitment and convers ion may be common to other amyloid plaque diseases. For example, experi mental 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 administ ered extracts from human Alzhemier’s brain (Meyer-Luehmann et al. 2006). These misfolded β sheet proteins produce aggregate particles (known as plaques in histology), which are linked to cell death in neurons, and produce a number of fatal spongiform encephalopathies in humans, livestock and game animals (review Brown, 2005).

In summary, normal cellular protein is s ynthesized and eventually degraded through normal metabolic processes, whereas misfolding of the protein produces a shape change that accounts for the resistance of prion protein particles to proteosomal degradation and, consequently, for the slow accumulation of brain pla ques. 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 clea r that the transmissible encephalopathies (kuru, scrapie, mad cow, etc), in fact , feature a novel disease mechanism. In the prion hypothesis of disease, it is thought that pri on protein polypeptides in the mi sfolded, laque-forming isoform convert polypeptides in the norma l 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 refers to an unknown, slow virus, but ra ther to this novel process of recruitment and conversion. Furthermore, as will be detailed below, Legname et al. (2004) show evidence that normal PrP can spontaneously convert to the misfolded isoform.

While the outbreak of mad cow diseases in the UK appears to have effectively ended in cows (review Brown, 2001), the lo ng, unresolved dormancy of prion diseases is a matter of great public concern (Prusiner et al . 1982; Carrell, 2004; Enserink, 2005) , with the risk that prion disease might reappear in human populations lo ng after the consumption of food contaminated with diseased CNS tissues. Also, surgical tool s contaminated with prions are extraordinarily difficult to decontaminate (Brown et al. 1990; 2000; 2005; WHO 1999).

B. 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 partic ular, enrichment of the protein correlated with th e 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 th e work of more than two decades, it is now

3believed that the infectious isoform of the pri on 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 isof orm of the protein is ty pically designated PrP C

, the infectious isoform in scrapie is PrP

P Sc . Because a variety of isoforms produce different transmissible spongiform encephalopathies, PrP d has been introduced recently to connote any disease isoform. Also, PrP res has been used to connote a purified protein or recombinant polypeptide fragment that features the resistance to proteinase K digestion that is characteristic of TSEs. Other less common usages also appe ar in the literature. More recently, PrP TSE has been used generically for the inf ectious 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 pape r, “prion” refers to a misfolded isoform of any prion protein; “prion protei n” can refer to both the normal and misfolded isoforms.

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

Amyloid polymerization of recombinant pri on protein produced in bacteria has been shown to accompany the acquisition of prion inf ectivity (Legname et al. 2004; Colby et al. 2007)

that is characteristic of the TSEs. Between 380 and 660 days after inoculation of normal mice with these transgenic fibrils, al l mice developed neuropathology, a nd their brain tissue, in turn,

was infectious to other mice. The amino acid sequence of the human prion protein has been extensively probed and tw o cationic domains (PrP 19-30 and PrP 100-111) have been shown to be capable of homologous binding between PrP C and PrP Sc , which would account for the

recruitment of PrP C and conversion to infectious PrP Sc (Lau et al. 2007). By using guanidine hydrochloride as a denaturant a nd copper binding, apparent reductio ns in prion infectivity have been shown to be partly reversible, which suppor ts the concept that the property of infectivity resides with the fibrillar, protease resistant conforma tion (McKenzie et al. 1998).

Large quantities of infectious PrP res can be produced in vitro by seeding the normal PrP C

isoform with small amounts of PrP res , followed by protein misfolding cyclic amplification in a cell free system (Castilla et al. 2005; 2008). Hamsters inoculated with PrP res produced in vitro developed scrapie disease and died after about 170 days.

3. Normal Prion Protein

It is now known that the infectious prion pr otein 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 ( review Sakaguchi, 2005) located on chromosome 20 in humans and on 2 in mice (Oesch et al. 1985; Basler et al. 1986). Th e Prnp gene appears to be conserved across species from yeast to pr imates (Westaway and Prusiner, 1986) and may share an ancestral origin by di vergence 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 PrP C . 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 homology to PrP C . Doppel does not possess the conversi on and recruitment properties of the prion protein but is neurotoxic, causing cerebellar degeneration when overexpressed in the brain

4(Whyte et al. 2003; Moore et al. 2001). Across the vertebrates, amino acid sequence homology between the PrP proteins of frog, turtle, chicke n, and mammal is conserved at a rate of about 30%, which is sufficient to pr eserve the same molecular architecture of mammalian PrP C (Harris et al. 1991; Calzol ai et al. 2005).

While attention has been focused on the dis ease producing isoforms, it appears that the

normal isoform of PrP C is a copper binding metalloprotei n (Brown DR, 1997), and it may function as an antioxidant. As noted above (M cKenzie et al. 1998), copper binding status may also be an important cof actor in conversion of PrP C and stability of the PrP

P Sc infectious conformation. Disruption of normal PrP C function in vivo by binding with monoclonal antibodies produces rapid cell death in neurons (Solforosi et al. 2004), which is consistent with

indications that PrP C functions include neuroprotection a nd that disruption of these normal functions produces neuronal cell death. Doppel-i nduced disease can be rescued by coexpression of wild-type PrP C , attributing antioxidant pr operties to the cellular pr ion 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 isofor ms are produced by misfolding and aggregation of β -rich isomers and accumulation of plaque s in neurons. Homozygous codons for methionine (Met, M) or valine (Val,V) at codon 129 in the human PRNP gene predispose PrP to recruitment and misfolding, so that in kuru, for ex ample, homozygotes have an earlier onset of disease following cannibalism of contaminated CNS tissues (Mead et al . 2003). Heterozygosity at codon 129 confers some resistance to pr ion 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, spongiform encephalopathies, the PrP has la rgely been studied in the brain. While most abundantly expressed in the brain, PrP

P C has been detected in the live r, 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-neur al tissues. Zhang et al . (2006) show that the prion protein is a biomarker for hematopoetic st em cells where it is normally expressed on the

cell surface. When bone marrow is depleted of prion protein expr essing stem cells, the capacity to regenerate is also lost. This evidence indi cates normal prion protein expression is required for bone marrow stem cells to regenerate.

4. Neurotoxic mechanism

Numerous breakthroughs have been made in th e past several years re garding associations between the accumulation of prion protein aggregat es and neuronal cell death. These aggregates are thought to result from failure of the ubiqu itin-proteasome pathway to adequately degrade misfolded protein, which is a feature of pri on disease share with other neurodegenerative diseases, such as ALS, Parkinson’s disease, a nd Alzheimer’s disease (G oldberg, 2007). In the case of the TSE diseases, the misfolded prion protein conformation does not appear to be intrinsically neurotoxic. Rather, it appears that the scrapie prion kills neurons by disrupting the function of the normal cell surface protein, and development of prion disease requires the presence of normal PrP expression. Uncoupli ng normal PrP from the cell surface also uncouples

5the presence of PrP Sc from the progression of disease. Key steps in resolving the mechanisms of the prion diseases include:

• Grafting transgenic tissue overexpressing PrP into embryonic forebrain of PrP deficient mice (Prnp 0/0 ) and inoculating with PrP

P Sc shows that grafted tissues accumulate PrP Sc

P and exhibit scrapie histology, whereas Prnp 0/0 tissue remain unaffected (Brandner et al. 1996). In other words, the β -sheet rich prion isoform is not intrinsically neurotoxic.

• Depletion of PrP C in neurons during established in fection arrests disease progression, even though extraneuronal PrP Sc continues to accumulate (Mallucci et al. 2003). Transgenic mice programmed to disable the PR NP gene in neurons at 12 weeks of age

were inoculated with PrP

P Sc several weeks before PRNP in activation. Within days after the PRNP gene was inactivated, the mice became depleted of PrP C and remained alive and disease free a year later. Inoculat ed control mice lacking the transgenic downregulation of PRNP all died.

• PrP C protein is normally anchored to the neuronal cell membrane by a glycosylphosphatidylinositol (G TI) linkage. Disabling the GTI linkage in transgenic mice does not prevent the accumulation of PrP Sc plaques in inoculated mice, but does prevent the development of scrapi e disease (Chesebro et al. 2005).

Relationships between prion aggregation, plaque accumulation, infectivity, and spongiform degeneration are in completely understood, as show n by seemingly inconsistent evidence that the formation of oligomers (or sma ll PrP aggregates) may be an intermediate step in neuronal degeneration (Simon eau et al. 2007), although in some circumstances plaque

accumulation can occur in the absence of infect ivity and degeneration (Piccardo et al. 2007).

C. TSE Diseases

Prion diseases are neurodegenerative dis eases also referred to as transmissible spongiform encephalopathies (TSE) due to the large vacuoles (Fi gure 1) that are seen upon post

mortem examination of the cortex and cerebe llum from affected individuals (Collinge, 2001).

These diseases are seen in both humans and anim als (Table 1). Scrapie is the most widely known TSE naturally occurring in sh eep; it was first recognized so me 200 years ago in Europe, and now present worldwide (Collinge, 2001). More recently, other type s of TSE have been recognized such as chronic wasting disease in deer, moose, and elk (Williams and Young, 1980), bovine spongiform encephalopathy (mad cow disease) (Wells et al. 1987), feline encephalopathy (Wyatt et al. 1991), and transmissible mink encephalopathy (Marsh, 1992).

In humans, prion based diseases are ge nerally thought to include Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler syndrome (GSS), fatal familial insomnia (FFI) and kuru. They are further categorized into three etiologi cal categories: sporadic, inherited or acquired (Collinge, 2001, Wickner et al. 2004, Brown, 2005). The incidence of sporadic CJD worldwide is about one per million (Collinge, 2001). 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 in clude fatigue, insomnia, depression, weight loss,

6headaches, ataxia, blindness and random pain sensations. 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).

Most prion disease in humans is acquired. 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 populati on. Because of the different roles adult men and women and children played in Fore co mmunities, kuru was prev alent among women and children until the practice of cannibalism ended in the 1950s (Fischer and Fischer, 1961; Alpers, 1987 as cited in Collinge, 2001).

Creutzfeldt-Jacob disease - human 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): sporadic, iatrogenic,

familial

 Kuru

 Sporadic and familial

insomnia

 Acquired variant CJD Spongiform

 Scrapie – sheep

 Bovine spongiform encephalopathy

 Transmissible mink encephalopathy

 Chronic wasting disease of

mule deer & elk

7(vCJD)  Feline spongiform encephalopathy

 Encephalopathies in zoo

animals*

Dominantly Inherited

 Gerstmann-StrausslerScheinker Syndrome (GSS)

Acquired by ingestion

 Variant CJD

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

D. History of TSE Diseases

Though sporadic CJD occurs spontaneously in th e human population at the rate of one per million people per year, prion-related disease epid emics tend to result from acquired TSEs. The first prion-related epidemic disease was docum ented 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 Cruetzfe ldt Jacob Disease (CJD). However, the first major outbreak of transmissible encephalopath ies was not diagnosed until 1985 when mad cow disease (i.e., bovine spongiform encephalopathy; BSE) first appear ed in Great Britain. By 1990, 14,324 cases of BSE were confirme d 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 146,000 cases of BSE. Other European countries also reported cases of BSE during this time including Switzerland (200 cases), Ireland (120 cases), and Portugal (30 cases). As the practi ce of including animal parts in cattle feed was stopped, the epidemic waned (Brown et al., 2001; Univ. of Wisconsin, 2004). However, since the introduction of monitoring pr ograms to detect BSE in d ead and slaughtered cattle, 12

countries have found their firs t cases of BSE: Austria, C zech Republic, Finland, Germany, Greece, Israel, Italy, Japan, Poland, Slovakia, Sloveni a, and Spain. Small numbers of cases have also been reported in Canada, 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 varian t CJD 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 Br itain. Since then, th e annual number of new vCJD cases has declined, from a high of 28 in 2000 to 1 in 2007 (http://www.cjd.ed.ac.uk/cjdq56.pdf). 1 The first case of mad cow 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 November 2004 in a cow of unknown origin and born before the U.S. ban on the use of mammalian protein in rumina nt 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

8( http://www.aphis.usda.gov/newsroom/hot_i ssues/bse/downloads/EPI_Final5-2-06.pdf ; Heaton et al., 2008).

To date, three cases of vCJD have been reported in the Unites States. The first and second patients (dates of onset 2001 and 2005, re spectively) 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 di agnosis, 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 tran smission could be attributed (http://www.cdc.gov/ncidod/dvrd/ vcjd/factsheet_nvcjd.htm).

Distribution of CWD (www.aphis.usda.gov) Chronic Wasting Disease (CWD) was first r ecognized as a clinical "wasting" syndrome in 1967 in captive mule deer in a wildlife research facility in Colorado. It was identified as a Transmissible Spongiform Encephalopathy (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 and elk in northeastern Colorado and southeastern Wy oming. Since nationwide

wildlife surveillance was inst ituted in 1997, CWD has been

identified in wild deer and el k 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 in fected deer populations although it is not known how the disease might beco me epidemic (Miller et al. 2000). There is no evidence of human cases of pr ion disease linked with CWD.

Scrapie, a neurological diseas e 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 or a fence post as if it itched. The disease is genera lly fatal after a long incubation period. It is estimated that scrapie costs the U.S. sheep indus try $20 million per year in direct losses (USDA, 2000). There are no known human cases associated with scrapie inf ected sheep. Scrapie

infected offal in cattle feed ma y have been the source of BSE.

Approximately 100 cases of feline spongiform encephalopathy (FSE) have been reported in Great Britain and Europe dur ing the BSE epidemic in the ear ly to mid 1990's (Cornell Feline Health Center, 2004). British cats were belie ved to have gotten the disease by eating BSEcontaminated commercial cat food and butcher scra ps. The number of new cases in domestic cats dropped off once measures were put into plac e 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 (C ornell Feline Health Cent er, 2006). Britain also reported cases in captive exotic cats including nine cases in cheetahs (three were diagnosed

9abroad but originated in Britain), three in pumas, three in ocelots, two in tigers and two in lions (Cornell Feline Health Center, 2004).

E. Transmission and Potential Routes of Exposure

TSEs can be transmitted naturally and expe rimentally. The most efficient route of experimental transmission is intracerebral injec tion of infectious brain tissue (Weissman et al. 2002a). Intraocular, intraspinal, intravenous, in tramuscular, intraperitoneal, or subcutaneous injections, scarification, or oral administration have also been shown to be effective methods of transmission in the laboratory se tting. Experimental transmissi on via the dental route has also been reported (Ingrosso et al. 1999 ). Consumption of contaminated food is generally thought to explain the natural pathogenesi s of scrapie in sheep, the sp read of kuru, and the human acquisition of vCJD from BSE-contaminated beef; however, the pr ecise details about the fate of infectious prions in association with other f ood 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; Sc herbet 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. Th e 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 studi es suggest that indirect, hori zontal transmission occurs via the oral route, such as from contaminated envi ronments (Palson, 1979) or by prionuria (Ligios et al. 2007). Vertical transmission (m other to offspring) of scrapi e may be possible, but probably not significant. It is thought th at a more likely route of transm ission involves an early horizontal, post-natal event (Andreole tti et al. 2002).

Transmission of CWD among deer and elk is not completely understood. Both direct and

indirect horizontal transmissi ons are known to occur via contact with infected animals and

environments contaminated with excreta or deco mposed carcasses of infect ed animals (Miller et al. 2004). In this regard, prion contaminated mate rial in soil remains infectious for years (Brown and Gajdusek 1991; Seidel et al. 2007) and may serve as a reservoi r of disease. Purified PrP Sc
protein has been shown to adhere to soil minera ls and remain infectious (Johnson et al. 2006; Ma et al. 2007), and this soil bindi ng may actually enhance infectivity (Johnson et al. 2007), possibly by a mechanism comparable to the recognized dura bility and seemingly enhanced infectivity of prions bound to surgical steel instrument su rfaces (Zobeley et al. 1999; Flechig et al. 2001; Peretz et al. 2006). It is also recognized that, as with BS E, maternal transmission of CWD likely occurs, but at low levels (M iller and Williams, 2003; Wells and Wilesmith, 2004). A recent study confirms transmission of CWD in deer via blood and saliva, ra ising 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 case s 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 epidem ic transmitted by the feeding of BSE prioncontaminated meat and bone meal to cattle (W ilesmith et al. 1991). Meat and bone meal are

10prepared from the offal of cattle, sheep, pigs, and chicken and fed to cattle as a high protein nutritional supplement. For the U.S., a recent study by Heaton et al. (2008) surveyed over 6,000 cattle, including five commercial beef processing plants, and determined that BSE cases with the

K211 allele that occurred in Alabama would be unlikely. Similarly, in cats, feline spongiform encephalopathy (FSE) has been linked with c onsumption of commercial cat food and butcher scraps contaminated with BSE, and transmissibl e mink encephalopathy (TME ) is associated with the feeding of tissue from scra pie-infected sheep or BSE-inf ected cattle (Chesebro, 2003).

Human TSEs are associated with a variety of transmission routes. Most cases of CJD are classified as sporadic and ar e likely to result from a so matic mutation, the spontaneous

conversion of PrP C to PrP Sc , or reduced clearance of low levels of PrP Sc 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 tr ansplantation or other medical procedures (Blattler, 2002).

Both kuru and vCJD are caused by ingestion of contaminated material . Kuru, the TSE of New Guinea natives, is associated with consumption of infected human tissue during cannibalistic rituals. Exposu re is also believed to have occurred through abrasions of oropharyngeal or conjunctival mucous membra nes, as well as via open wounds on the hands (Gajdusek, 1977). Variant CJD (vCJD) is associ ated 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 transm ission of vCJD via blood transfusion have also been reported (one case was manifested clinically and the se cond was identified at autopsy following death from an unrelated cause) (Llewe lyn et al. 2004, Pe den et al. 2004).

Prions have presented a major public health problem in the UK with the emergence of mad cow disease. Through a major effort in th e 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 fu ture. Certain prion diseases are infectious to humans, including BSE or mad cow disease, but it is not clear if the measures taken so far will adequately prevent future occurrences. In addition, scientists do not completely understand several aspects of prion dis eases, or whether effective tr eatments might be found through understanding of the misfolding an d infectivity of the fibrillar structure (Karpuj et al. 2007; Supattapone et al. 1999, 2001). Finall y, the epidemic of chronic wast ing disease in deer and elk populations in the Western USA continues to spre ad, but the implications of this disease for

humans remains unknown.

Rapid advances in recombinant and cell free technologies are attack ing 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 misfoldi ng diseases such as Alzheimer’s and similar amyloidoses are not? In addition to known heredi tary prion diseases, it is an especially worrisome health problem th at normal prion protein can also misfold under conditions that have not been defined to produc e spontaneous TSE disease. For example, the UK commission report concluded that the cat astrophic epidemic of mad cow (1986 – 2000) began with the spontaneous emergence of an abnormal prion, followed by using contaminated

11bone 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.

F. Existing Occupational Safety Procedures

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

The Centers for Disease Control and Preven tion (CDC) has published two documents that provide guidance for health-care workers pertaini ng to environmental infe ction control generally and to “special pathogens” (inc luding prions) specifically. Guidelines for Handwashing and Hospital Environmental Control (Bolyard et al. 1998: CDC/ HICPAC 2003) encompasses general infection control, with emphasis on th e handling of air, water, and environmental surfaces; environmental sampling; and disposal of regulated and unregulated medical waste. Guidelines for Environmental Infecti on 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 pr ion-containing material (usually central nervous system ti ssue or pituitary hormones) acquire d as a result of health care (i.e., iatrogenic cases). Six doc umented iatrogenic cases were a ssociated with neurosurgical

instruments and devices that intr oduced residual contamination dire ctly 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 activ ities), 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 w ith known or suspected CJD and implement prionspecific infection-control measures for the ope rating 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 flui d (see decontamination methods described in next section).

In 2000, the World Health Organiza tion issued its guidance document, WHO Infection Control Guidelines for Transmissible Spongifiorm Encephalopathies (WHO, 2000), which focuses specifically on prion diseases. In this document, the WHO designated central nervous

12system tissues and fluids as “high-infectivity,” which should be managed more conservatively than others. Key recommenda tions include the following:

 Covering patient treatment surf aces 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 liqui d-repellent aprons over plastic gowns).

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

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

 Reusable instruments are to be kept mois t until cleaning and decontamination; this measure is needed to prevent dryi ng 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 inci nerated, as are the dis posable materials.

 All incineration is to be conducte d in hazardous-waste facilities.

The WHO document provides similar specifi c guidelines for mortuary and laboratory workers who may contact contaminated material s, 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 ag ents in animal tissues and waste materials (AAVLD, 2004). The AAVLD recommends:

 Using standard BSL2 safety precautions such as restricted acce ss to laboratories, protective clothing, and facial protection, special care with sharp instruments, and

avoidance of aerosols.

 Numerous specific protective measures for necr opsy laboratories, hi stology laboratories, and laboratories handling fresh tissues from TSE suspect animals, such as extensive protective clothing, screened drains to tr ap tissue fragments, use of appropriate disinfectants to inactivate TSE agents on su rfaces and non-disposable items, and disposal

of contaminated tissu es by certain methods.

G. Decontamination Methods

A number of decontamination methods have be en 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 s purred 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

13veterinary facilities, research laboratories, health care settings, a nd other settings in which prioncontamination is a possibility .

The infectious conformation of prion prot eins is extraordinarily durable, and the measures necessary to destroy such prion proteins are extrem e compared to decontamination methods for other biological pat hogens, including bacter ial spores. In particular, prions bind tightly to stainless steel and are most difficult to inactivate on this surface (Zobeley et al. 1999)

Some of the currently recommended decontam ination 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 fo r 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 pr ocedures are found in several advi sory reports, such as those of the CDC (1999), WHO (2000), AAVLD (2004), Cana dian Food Inspection Agency (2005) and MSU (2005). However, these recommended de contamination methods are based on limited academic research, and a number of issues br ing into question whether these methods can demonstrate a disinfectant’s ability to totally inactivate prions on inanim ate surfaces and objects.

1. The recommended prion-inactivation methods ha ve 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 forma lly 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.

2. The risk of transmissible infectivity persists at prion titers far below the limits of detection using analytical methods, such as gel electrophoresis a nd 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 Wester n blot assay. Consequently, adequate confirmation in an animal model is required to confirm effective elimination of all prion infectivity.

3. 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 hist ory of utility in pr ion research, the life span of these laboratory animal s is relatively short, whereas the time interval from prion exposure to the development of clinical diseas e 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 c linical signs during the short lifespan of the

14animal model might be mistaken as evidence of successful elimina tion of infectivity. Attempts to accelerate the progression of infectivity by using synthetic mammalian prions (Legname et al. 2004), as well as Pr P over-expressing cell lin es (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.

4. In addition to the common use of different sp ecies of test animals, hamster and mouse, many different strains of prions are empl oyed 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 Jack son et al. (2005), using mice.

5. There are important barriers to the transmissi on 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 m ouse indicate that elk chr onic wasting disease may not transmit readily to humans (Kong et al. 2005). On the other hand, the mad cow epidemic in the UK (1986-2000) demonstrates that BSE re adily crosses the species barrier and is 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. Converse ly, 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 pr ocedures may have consequences for risks to humans that vary from inconsequential to very significant, depe nding on the strain of prion being inactivated. For example, sheep scrapie is clinically unknown in humans and so the risks to humans resulting from impe rfect decontamination of scrapie would be expected to be negligible compared to th e risks from imperfect inactivation of BSE prions on surfaces to which humans are exposed.

6. 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 eith er 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. Prelimin ary additional studies i ndicate that the cow BSE prion may be even more resistant to inact ivation 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 wh en tested with more durable human and cow prions.

15Accordingly, 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 demonstr ate that a disinfectant can totally inactivate prions or TSE agents on inanimate surfaces and objects.

III. The Science of Prions As It Relates to the FIFRA Definition of “Pest”

The Introduction to this paper described two s ections 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 de finition of “pest” specified in FI FRA Sections 2(t) or 25(c)(1).

A. How Do Prions Relate to Co mmon 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 delete rious to man or the environment.” Based on the available information described in Section II., prions clearly are dele terious to humans and animals. Exposure may lead to infectious acqui sition 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 pr ions are to be considered pests under FIFRA they would have to be “other micro-organisms”.

Although there is no single, definitive definition of “microorganism”, the definition in the Oxford Dictionary of Biology (Fourth Edition, 2000) is representative. 1 The Oxford Dictionary of Biology defines “microorganism” as “Any organi sm that can be observed only with the aid of a microscope. Microorganisms include bacteria, viruses, protists (incl uding certain algae), and fungi.” “Organism” is further defined by the Oxford Dictinonary as “An individual living system, such as an animal, plant, or microorga nism, that is capable of reproduction, growth, and

maintenance.” These latter terms are define d by Dorland’s Medical Dictionary as follows:

• Reproduction— the sexual or asexual process by whic h 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 this definition of “mic roorganism” as it is generally used by biologists and the medical commun ity would depend on whether they exhibit reproduction, growth and maintenance. The resear ch studies summarized in Chapter II indicate

1 This definition and those that fo llow were chosen from among several available to EPA. Those

discussed herein were selected based on thei r inclusion in authorit ative sources, their completeness, and their utility in addre ssing the scientific arguments considered

.

16that a prion protein (whether normal or misfolded) does not have these characteristics. The normal prion protein is a cellular protein that is transcribed by a gene ubiquitous in vertebrate species. It cannot reproduce, grow or perform self-maintenan ce as microorganisms do. When a prion protein becomes misfolded, it converts normal prion proteins to adopt the same misfolded conformation through recruitment and conversio n. Thus, a misfolded prion protein cannot reproduce itself; it can only induce other prion prot eins to take on a misfolded shape. All other infectious agents—algae, fungi, bacteria, viru ses and viroids—produce infectious diseases by infecting and propagating their numbers in th e host species, which is accomplished through genetic reproduction and the propagation of new ge nerations from genes comprised of DNA or

RNA. Unlike these other infecti ous agents, the aggregated partic les of a prion protein are devoid of any genetic element and have no mechanis m for genetic reproducti on. The property of infectivity is limited to that of misfolded pr ion protein converting other, existing, homologous protein to adopt the same misfolded conformati on. There is neither synthesis of new prion protein nor of genetic material. Finally, recognition that prions are distinct from th e entities that biologists and the medical community generally re cognize as microorganisms lies at the heart of Nobel prize for the discovery that prion particles are (a) comprise d solely of prot ein and (b) are the causative agent of a novel infectious process (Prusi ner, Nobel Prize, 1997).

Although prions do not fit the criteria biologist s generally use to define microorganisms, the term “microorganism” is sometimes used more broadly. For example, the Oxford Dictionary of Biology’s definition for “microorganism” cite d above includes viruses as microorganisms, even though they do not have all of the characteristics of an “organism” as listed above. Viruses cannot reproduce by themselves, but do replicat e themselves by injecting their DNA into host cells and taking over the host cell’s reproductive machinery. Nevertheless, they are often grouped with bacteria and other entities that m eet the entire microorganism criteria described

above. 2 In the absence of a more general term that expressly includes viruses and other infectious agents that do not meet all the criteri a of microorganisms, this broader use of the term “microorganism” is common among professionals in the biological, medical and public health sciences, at least in casual speech.

B. Are Prions Alive?

As mentioned above, 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:

2 Microbiology textbooks al so routinely devote signif icant space to viruses; see, for example: Textbook of Diagnostic Microbiology, Connie Mahon, George Ma nuselis, Donald Lehman (3d ed., Elsevier 2006); Microbiology: Principles and Explorations, Jacquelyn G. Black (6th ed. Wiley 2005); Bailey & Scott's Diagnostic Microbiology, Betty A. Forbes, Alice S. Weissfeld, Daniel F. Sahm (Elsevier Health Sciences 2007); Medical Microbiology, Patrick R. Murray, Ken S. Rosenthal, Michael A. Pfaller, Ken S. Rosenthal (Elsevier Health Sciences 2005); Koneman’s Color Atlas and Textbook of Diagnostic Microbiology, Elmer W. Koneman, Stephen D. Allen, Gail L. Woods, William M. Janda, Paul C. Schreckenberger (Lippincott Williams & Wilkins 2005); Sherris MedicalMicrobiology: An Introduction to Infectious Diseases, Kenneth J. Ryan, C. George Ray, John C. Sherris (McGrawHill 2003).

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

This definition adds four more characteristic s to the characteristics beyond those included in the definition of “microorganism” and “organis m” and addressed in the preceding section (i.e., reproduction, growth, metabolism). These additional characteristics of 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.

Of the six properties of living things id entified in the Dorland definition, the only property of living things that pri ons share is organization. Prions are made of protein, which has four levels of organi zation (Leninger, 1970):

• Primary structure is the sequence of amino aci ds that comprise one polypeptide chain.

• Secondary structure describes the polypept ide chain's repetitive domains of alpha (helical) and beta (pleated sheets) structures.

• Tertiary structure describes the full, 3- dimensional folding conformation of the polypeptide, which is determined by the content of alpha and beta structures, as well as by disulfide cross-linkages.

• Quaternary describes the orga nization of individual polypeptid e chains to form a larger, more complex molecule comprised of indivi dual subunits, such as first described in hemoglobin. [Note: prions are single polypeptide chains, so they do not have this level of organization.]

Although prions feature the above kinds of “organization,” they do not differ in this respect from any other biological proteins. In asmuch as other biologi cal proteins are not generally considered to be forms of life, these kinds of “organization” do not appear indicative of forms of life. With respect to “irritability” and “movement,” prions are not capable of reacting to stimuli or initiating movement on their own. Fina lly, with respect to “ad aptation,” prions have

no known mechanism for either adapting to their envir onment or passing along their

characteristics via DNA because th ey contain no nucleic acids.

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