1

Scientific I nformation Concerning the Issue of Whether a Prion Is a “Pest” under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) ( 2 / 17/ 10 ) I. I ntroduction and Background T his paper reviews currently available scientific information pert aining to the issue of whether a prion (“ pr oteinaceous in fectious particles”) should be considered to be a “pest” as defined b y 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 20 09), 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 potentiall y relevant to prions include :  FIFRA Section 2(t): A pest is “... (2) an y 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 an y 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: o Prions are unquestionably injurious to human and animal health. o Prions share man y characteristics of other deleterious microorganisms. o Prions share enough characteristics of “other micro - organism” or “form of life” to fall within the scope of FIFRA section 2(t)(2). T hey originate in living organisms, replicate within host organisms, and can be transmitted to other hosts. o If prion products were not regulated under FIFRA, EPA could not provide assurance that such produc ts would be effective or safe. o 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.2

II. The Science of Prions A. 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 prop erties 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 b y the catastrophic outbreak of mad cow disease, or bovine spongiform encephalitis (BSE), originating in Essex C ounty , 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 ) l ed 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 b y unconventional viruses, virus - like agents, or slow vir uses because the time interval between exposure and clinical disease was years or decades ( Gajdusek, 1977; Prusiner et al. , 1982). While a number o f scientists have suggested that an unknown virus or co - factor i s the causative agent of TSEs ( Manuelidis, 2 007; Manuelidis et al. 2007), no such virus or co - factor has been isolated afte r 40 ye ars 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 moi e ties) infectious agent was suggested b y Griffith ( 1967 ) and Brown et al. ( 1990a) . This protein - only hy pothesis is now generally accepted by strong weight of evidence , especially Prusiner’s hy pothesis of a novel proteinaceous infectious agent

– the prion (Prusiner et al. 1982; Bolton et al. 1982) . In particular, experimental transmission of TSE diseases b y prion protein s ynthesized or propagated in cell free s ys tems (Legname et al. 2004; Castilla et al. 2005 ; Deleault et al., 2007 ) is a ccepted by man y as definitive proof of the prion hy pothesis. Though prions are now generally accepted as the agent responsible for TSE disease s, the precise structural properties of the prion protein responsible for infectivity

and the c hain of events that produce neural degeneration and fatal TSEs ( e . g . , Barron et al. 2007) , and what

– if an y - cofactors might be featured in the mechanisms of disease , remain unknown . As a result of the novelty of the prion hy

pothesis and controversy

surrounding it as the agent for infectious diseases, the prion protein and its isoforms have been studied with unusual intensity ; yet the phy siological 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 ye ars. As detailed below (B. Key Characteristics), the normal isoform of the prion protein (PrP) is now known to b e encoded b y 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 conform ation s (Soto, 2006) by unknown events, the resultant protein aggregates by

passes the normal proteosomal degradation pathway . This process ty picall y produces the non - amyloid or am yl oid deposits featured in the TSE diseases, and3

acquire s the property of TSE infectivity . The clin ical 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 i n 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 precisel y the structural determinants of infect ivity . In humans, genetic variation is common at the PRNP gene codon for amino acid 129 , resulting in high frequencies of amino acid substitution s (129M , 129 V) in the translated prion protein. H owever, inheritance of identical codons from both parents results in prion protein homozygosity (129 MM or 129 VV ) , which produce s a cellular pool of identical prion proteins that are predisposed to misfolding and aggregation through homologous interaction . In contrast, inheritance of two different codons result s in prion protein heterozygosity (129 M/V ) , which produces a cellular pool of poly morphic protein s that resist misfolding (Mead et al. 2003). In addition to mutations in the PRNP protein coding region , g enetic mutations in the PRNP promoter region are tho ught 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 poly peptides recruit and convert normal PrP poly peptides to adopt the misfolded infectious conformation ; however, a low level of prion - like transmission of altered protein may occur in other amyloid plaque dise ases (Aguzzi, 2009) , and “prinoid” behavior may

be a feature of other protein aggregating neuropathologies . For example, experimental inflammatory am yl oidosis 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 sy nthesized and eventuall y degraded through normal metabolic processes, whereas misfolding o f 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, consequentl y, for the slow accumulation of amyloid deposits or brain plaques , which are often observed in some TSEs . Furth ermore, 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 transm issible encephalopathies ( e.g., kuru, scrapie, BSE ) feature a novel disease mechanism. Inthe prion hy pothesis of disease, it is thought that prion protein poly pepti d es in the misfolded , aggregation prone isoform convert poly peptides 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 necessaril y refers to an unknown, slow virus, but alternativel y to describe this novel process of recruitme nt 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) . H owever, the potential for long, unresolved dormancy of prion diseases in humans is a matter of great public4

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 extraordi narily difficult to decontaminate (Brown et al. 1990 , 2000 and 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, ro ughl y 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 th e presence of nucleic acids (Prusiner et al. 1984). Through the work of more than two decades, it is now believed that the infectious iso form of the prion protein is a misfolded isoform of a normal cell membrane protein, and various terminologies have app eared as numerous researchers have joined the investigations. The normal cellular isoform of the protein is ty pically designated PrP C , and the infectious isoform in scrapie is PrP Sc . Because a variety of isoforms produc e different transmissible spongifor

m encephalopathies, PrP d has been introduced recently to connote an y disease is o form (Brown, 2005) . Also, PrP res has been used to connote a PrP d poly peptide fragment that features the resistance to proteinase K digestion (Brown, 2005) . Other less common usages also appear in the literature. More recently , PrP TSE has been used genericall y for the infectious conformation of the transcripts of any prion strain, which lends simplicity to communicating about the homologous prion proteins of different human an d 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 St atus of Dr. Stanley Prusiner’s (1982) protein only hypothesis Amyloid poly

merization of recombinant prion protein produced in bacteria has been shown to accompan y the acquisition of prion infectivity (L egname et al. 2004 ; Colby et al. 2007 ) that is chara cteristic of the TSE s. F ibrils consisting of recMoPrP(89 - 230) were inoculated intracerebrall y into transgenic (Tg) mice expressing MoPrP(89 - 231). B etween 380 and 660 day s 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 hy pothesis, the amino acid sequence of the human prion protein , for example, has been extensively probed and two 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 principle of recruitment of PrP C and conversion to infectious PrP Sc (Lau et al. 2007) . By using guanidi ne hydrochloride as a denaturant and copper binding, apparent reductions in prion infectivity

have been shown to be partl y reversible , which supports the concept that the property of infectivity reside s with the fibrillar , protease resistant conformation (McKenzie et al. 1998).5

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 b y protein misfolding cyclic amplification in a cell free s ys tem (Castilla et al. 2005 ; 2008 ). Hamsters inoculated with PrP res produced in vitro developed scrapie disease and died after about 170 day s. Significant recent work by L indquist (e . g ., Tessier and L indquist 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 “prion o genic” infectivity . The publication of this work has been widel y acclaimed in a series of news featur es that accompanied publication of Tessier and Lindquist (2007), which is regarded b y man y as a breakthrough paper that proves the prion hy pothesis (Surewicz 2007; Jonietz 2007; Scientific American 2007).

3. Normal Pr ion P rotein T he infectious prion protein is any one of several isoforms of an otherwise normal cell membrane protein (Stahl et al. 1990), encoded b y a highl y 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 P rnp gene has been conserved across species from yeast to primates (Westaway and Prusiner 1986) and may share an ance stral 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 neuro protective 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 p ri on gene family

also includes Prnd which encodes Doppel, a protein with striking sequence similarity to PrP C

. Doppel does not possess the conversion and recruitment properties of the prion protein but is neurotoxic, causing cerebellar degeneration when overexpressed in the brain (Why

te et al. 2003; Moore et al. 2001). Across the vertebrates, amino acid sequence homology

between the PrP prot eins 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 PrP C (Harris et al. 1991; Calzolai et al. 2005 ). Another view is that the similarity of DNA sequence s from ye ast to vertebrate prion protein genes is an evolutionary convergence in the yeast and an example of non - Mendelian inheritance of phenotypes benefiting ye ast adaptations to diverse environments (Alberti et al. 2009). Based on the limited understan ding 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 ye ast to vertebrates, or the interpretation of convergent evolution in the yeast to conserve phenoty pic 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 PrP C 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 PrP C and stability of the PrP Sc infectious conformation .6

D isruption of normal PrP C function in vivo by binding with monoclonal antibodies produces rapid cell death in neurons (S o lforosi et al. 2004), which is consistent with indications that PrP C functions include neuroprotection and that disruption of these norm al functions produces neuronal cell death. Doppel - induced disease can be rescued by coexpression of wild - ty

pe PrP C , attributing antioxidant properties to the cellular prion protein ( Wong et al. 2001). Similar to the aggregates of neurotoxic proteins foun d in other neurodegenerative diseases (review Taylor et al. 2002), the neurotoxic PrP isoforms are produced b y misfolding and aggregation of  - rich isomers and accumulation of plaques in neurons. Homozygous PRNP gene codons for prion protein 129 methionin e ( 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 s ys tem ( CNS ) tissues (Mead et al. 2003). Hetero zygosity at codon 129 confers some resistance to prion disease, probabl y b y 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 large ly been studied in the brain. While most abund antly expressed in the brain, PrP C has been detected in the liver, kidney , heart and skeletal muscle, among other tissues (Bosque et al. 2002). N ew evidence indicates the normal isoform of the protein has imp ortant functions in non - neural tissues. Zhang et al. (2006) show that the prion protein is a biomarker for hematopo i etic 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 hy pothesis is h ow misfolding of an oth erwise normal cellular protein lead s 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 th e biochemistry of the prion proteins and related poly peptide 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 ( Jackso n et al. 2009) support s the core tenets of the prion hy pothesis 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 association s between the accumulation of prion protein aggregates and neuronal cell death , and the weight of these cumulative studies hint s at the major features of the neurotoxicity pathway and infectivity

of prions . R emoval of misfolded and damaged proteins is a normal part of cellular metabolism; however, misfolded prion protein accumulates in cellular aggregates. These aggregates are7

thought to result from failure of the ubiquitin - proteasome pathway to adequately

degrade misfolded protein, which is a feature prion disease s s hare 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 d evelopment of prion disease may require the presence of normal PrP expression and the continued conversion of PrP C to PrP Sc (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 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 Sc shows that grafted tissues accumulate PrP Sc 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 infection arrests disease progression, even though extraneuronal PrP Sc 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 PrP Sc several weeks before PRNP inactivation. Within days after the PRNP gene was inactivated, the mice became depleted of PrP C and remained alive and disease fre e a ye ar later. Inoculated control mice lacking the transgenic downregulation of PRNP all died.

 PrP C protein is normally anchored to the neuronal cell membrane b y a gl ycos ylphosphatidylinositol ( GPI) linkage. Disabling the GPI linkage in transgenic mice does not prevent the accumulation of PrP Sc plaques in inoculated mice, but does lower the prion titer and alters the development of scrapie disease (Chesebro et al. 20 0 5). That the relationships between prion aggregatio n, plaque accumulation, infectivity and spongiform degeneration remain incompletel y 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 c ircumstances plaque accumulation can occur in the absence of infectivity and degeneration (Piccardo et al. 2007). While the mechanisms of prion neurotoxicity and infectivity

will likel y 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. C. TSE Diseases Prion diseases are neurodegenerative diseases also referred to as transmissible spongiform encephalopathies ( TSE s ) due to the large vacuo les (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 widely8

known TSE naturally

occurring in she ep ; 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 spo ngiform encephalopathy ( BSE -- mad cow disease) (Wells et al. 1987), feline spongiform encephalopathy (FSE) (W yatt et al. 1991), and transmissible mink encephalopathy (TME) ( Hartsough and Burger, 1965; Burger and Hartsough, 1965; Marsh 1992). In humans, p rion based diseases are generall y thought to include Creutzfeldt - Jakob disease (CJD), Gerstmann - Strä ussler - 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 b y a rapid dementia occurring in individuals betwe en 45 and 75 years of age. Death can result as earl y as 2 - 3 months from onset (Collinge 2001). Other sy mptoms include fatigue, insomnia, depression, weight loss, headaches, ataxia, blindness and random pain sensations . On the other hand, variant CJD (vCJ D ) 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). Creutzfeldt - Jacob disease - human Kuru – human

Bovine spongi form encephalopathy -

(cow) Scrapie - (sheep)9

Figure 1. Pa thology 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 encephalopath y  Transmissible mink encephalopath y  Chronic wasting disease of mule deer , elk , and moose  Feline spongiform encephalopath y  Encephalopathies in zoo animals*

 Gerstmann - Strä

ussler Scheinker disease (GSS)

 Variant CJD * ankole, Arabian ory

nx, bison, cheetah, eland, gemsbok, greater kudu, nya la, ocelot, puma, Scimitar horned ory

x, and tiger (Jeffrey

and Wells 1988) . D. History of TSE D is eases 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 . P rion - 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 19 5 7 in Papua New Guinea among native tribes known to practice ritualistic cannibalism (Zigas and Gadjusek 1957). The disease was called ”k uru ” and was closely

related to Cruetzfeldt Jaco b Disease (CJD) although the prion hy pothesis 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 earl y 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). T he first major outbreak of transmissible encephalopathies having catastrophic public consequences linked to prions was not diagnosed until 1985 when m ad c ow 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 e ach we ek, and in 1995 , at the height of the epidemic, there were approximately10 Distribution of CWD ( http://www.aphis.usda.gov/ anim al_health/animal_diseases/cwd/downlo ads/distribution_cwd.pdf ) 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; S pecified B ovine O ffals ban 1990; M eat and B one M eal ban 1996) , the epidemic waned and feed security was assured 1996 (Brown et al. 2001; Univ. of Wiscons in 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, Ital y, Jap an, 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 vCJ D cases has declin ed , from a high of 28 in 2000 to 1 in 200 8 (http://www.cjd.ed.ac.uk/cjdq56.pdf ). 1 The 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 protei n in ruminant feed (diagnosis final in June 2005) (http://www.cfsan.fda.gov/~comm/bs efaq.html). The third and most recent case of BSE was discovered in Alabama (http://www.aphis.usda.gov/newsroom/hot_issues/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, respectivel y) 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 likel y to have originated in Saudi Arabia where the patient was born, raised and resided until 2005. None of the patients had m edical h i stories to which transmission could be attributed (http://www.cdc.gov/ ncidod/ dvrd/vcjd/factsheet_nvcjd.htm). However, there is no epidemiological evidence to suggest that any of these vCJD patients ac quired the disease in the United States. They are presumed to have been infected in the United Kingdom or Saudi Arabia, respectivel y. Chronic Wasting Disease (CWD) was first recognized as a clinical "wasting" s yndrome in 1967 in captive mule deer in a wildlife research facility in Colorado. Itwas identified as a TSE in 1978. In1981, 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 W yoming. Since nationwide wildlife surveillance w as instituted in 1997, CWD has been identified in wild deer and elk in several additional states: Il l inois, 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 endemic11

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 earl y 18th century . Affected sheep were observed rub bing or “scraping” their coat against a tree or a fence post as if they itche d . The disease is generall y fatal after a long incubation period. It is estimated that scrapie costs the U.S. sheep industry $20 million per year in dire ct 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 earl y 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 c ases in domestic cats dropped off once measures were put into place to prevent materials potentially contaminated with BSE prions from entering the food chain . N o new cases have been reported since 1999. No cases in U . S . cats have ever been reported (Cor nell 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 (Mar s h and Bessen, 1993), possibly associated with a transmission of a rare TSE of cattle to mink (L iberski et al., 2009). E. Transmission and Potential Routes o f Exposure TSE s 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, intrali ngual, 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 generall y thought to explain the natural pathogenesis of scrapie in sheep, the spread of kuru, and the human acquisition of vCJD from BSE - contaminated beef; howeve r, 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 infec ted sheep and goats (Pattison et al. 1972) although scrapie - contaminated feces have also been proposed (Weissmann et al. 2002b) . B oth scenarios represent dire c t horizontal transmission. Some studies suggest that indirect, horizontal transmission occurs v ia the oral route , such as from contaminated environments (Pal s son 1979) or by prionuria ( Shanked12

et al., 2001; Li gios 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 earl y 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 e t al. 2007) and may serve as a reserv oir of disease . P urified PrP Sc protein has been shown to adhere to soil minerals and remain infectious (Johnson et al. 2006 ; Ma et al. 2007 ), and this s oil binding may actually enhance infectivity

(Johnson et al. 2007) , possibly by a mechanism comparable to the recognized durability and seemingl y enhanced infectivity of prions bound to surgical steel instrument surface s ( Zobeley et al. 1999; Flechig et al. 2001; Peretz et al. 2006 ). It is also perceived that, as in th e 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, m eat and bone meal we re 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 th is 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 an y additional aty pical BSE cases in the U.S. with K211 would be vanishi n gl y 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 th e 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 likel y to result from a somatic mutation, the spontaneous conversion of PrP C to PrP Sc , or reduced cl earance of low levels of P rP Sc that may normally be present (Peretz et al. 2001). However, CJD can be transmitted by exposure to contaminated surgical instruments or electroencephalogram e lectrodes, or exposure to infectious brain, pituitary , or ocular tissue or associated products via organ transplantation or other medical procedures (Blattler 2002).13

Both kuru and vCJD are caused b y 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 orophary ngeal 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 transfu sion have also been reported (one case manifested clinicall y and the second was identified at autopsy following death from an unrelated cause) (Llewely

n et al. 2004, Peden et al. 2004) , and at the present time it is likely these numbers will continue to ri se 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, b ecause so little is understood about prions, they will remain an important public health issue for the foreseeable future , particularl y since it is not clear if measures taken so far will adequatel y prevent future occurrences. In addition, scientists do n ot 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) . Fi nall y, the epidemic of CWD in deer and elk populations in the Western USA continues to spread and has certainly entered states e ast of the Mississippi, but the implications of this disease for humans remains unknown. Rapid advances in recombinant and cell free te chnologies 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 especiall y 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 b y using contaminated bone meal to feed other cattle. The resultant intense surveillance now shows low leve l 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 TSE s 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 co ntaminated with prions. T hese 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 proced ures include administrative and engineering controls, special work practices, personal hygiene measures, personal protective clothing and decontamination methods.14

The Centers for Disease Control and Prevention (CDC) has published two documents that provi de guidance for health - care workers pertaining to environmental infection control generally and to “special pathogens” (including prions) specificall y. Guidelines for H andwashing and H ospital E nvironmental C

ontrol ( Garner and Favero, 1986; Bol yard et al. 1998: CDC/HI CPAC 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 C ontrol in Health - Care Facilities (CDC 2003) provides extensive, detailed guidance on all aspects of infection control in health - care facilities and specificall y addresses Creutzfeldt - Jakob Disease (CJD) in patient - care areas. This guidance notes that a li mited number of cases result from direct exposure to prion - containing material (usuall y central nervous s ys tem tissue or pituitary hormones) acquired as a result of health care (i.e., iatrogenic cases). S ix 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 ac tivities ), 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 o perating 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 be en contaminated with central nervous s ys tem tissue or cerebral spinal fluid (see decontamination methods described in next section). In 2000 , the World Health Organization issued its guidance document, WH O Infection Control Guidelines for Transmissible S pongiform Encephalopathies (WHO 2000 ), which focuses specificall y on prion diseases. In this document, WHO designate d central nervous sy stem tissues and fluids as “high - infectivity ,” which should be managed more conservativel y than other disease s. Key

re commendations include the following :

 Covering p atient 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 viso r, 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 h igh - 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.  L ow - and high - infectivity instruments are to be stored separatel y between and during uses.  Bulk fluids are to be decontaminated or absorbed into sawdust, then incinerated.15

 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

work ers after occupational exposures. Safety recommendations are provided also for the transport of contaminated specimens b y 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 b y TSE agents in animal tissues and waste materials (AAVLD 2004). The AAVLD recommends:

 Using standard BSL2 safety precautions such as restricted access to laboratories, prot ective clothing and facial protection, special care with sharp instruments, and avoidance of aerosols.  Using numerous specific protective measures for necrops y 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. G. Decontaminatio n 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 espe ciall y 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 decontaminat e environmental surfaces and instruments in veterinary

facilities, research laborat ories, 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 d econtamination methods for other biological pathogens, including bacterial spores. In particular, prions bind tightly to stainless steel , resist elution by exhaustive washing, and remain highl y infectious after formaldeh yde 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 limited16

academic research, and a number of issues bring into question whether these methods can demonstrate a disinfectant’s ability to totally inactivate prion s on inanimate surfaces and objects. 1. The recommended prion - inactivation methods have not been tested using a validated effi cacy 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. 2. The risk of transmissible i nfectivity persists at prion titers far below the limits of detection using anal yt ical 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 sur

viving prion infectivity

in hamsters was 1000 fold more sensitive than visual detection b y Western 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 Sy rian hamster (Marsh and Kimberlin 1975) and mouse (Chandler 1961) have a long history of utility in prion research, the life span of th ese 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 o f 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 re sidual prion infectivity

at very low titers, as might be expected with cleaning procedures that have been largely , but not entirely , effective. Ideall y, 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 inf ectivity by using sy nthetic 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 way s to solve the lifespan problem, but these approaches create new uncertainties b y using animals already predisposed to overexpression of host or foreign prion strains. 4. In addition to the common use of two species of test animals -- hamster and mouse -- man y different strains of prions are emplo yed in decontamin ation research. T hese experimental variables might account for the discrepant results for Environ LpH obtained by Ernst and Race (1993) and Race and Ray mond (2004), using Sy

rian hamsters as the test model, with those of Jackson et al. (2005), using mice. 5. There are important barriers to the transmission of prion diseases between animal species and human s , and the degree of transmission of prion diseases from animals to humans17

varies greatly . For example , experimental studies using human and elk prion gen es 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 BS E epidemic in the UK (1986 - 2000) demonstrates that BSE can cross the species barrier and become in fectious to humans as vCJD (Brown 2001). Since there are no s ys tematic studies of comparative infectivity

of prion strains across different animal species and humans, it is not clear how precisel y one can extrapolate human risk from efficacy studies perfo rmed in various animal species. Specificall y, some of the strains used in efficacy studies may pose little or no risk to humans. Conversel y , 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. 6. Similarly , c ompariso ns 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 examp le, 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 compariso n 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. Accordingl y , although a number of academic studies have demonstrated that certain chemical and/or ph ys ical decontamination methods provide some assurance of reduction of prion infectivity , current test methods cannot demonstrate that a disinfectant can total ly inactivat e prions or TSE agents on inanimate surfaces and o bjects. III. The Scienc e of Prions As It Relates to the Definition s 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.” Th is section address es the exte nt to which the available scientific information on prions summarized in Section II relate s to the issue of whether prions fit within the definition of “pest” specified in FIFRA Section s 2(t) or 25(c)(1). A. Definition s of “Microorganism ” The defini tion 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.” B ased on the18

available information described in Section II., prions clearl y are deleterio us to humans and animals . E xposure 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, bacte ria or other microorganism .... ” Prions are neither viruses nor bacteria, as discussed in the preceding sections, so if prions are to be considered p e sts under FIFRA they

would have to be “other micro - organisms . ” Below, two approaches have been taken to th e 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. 1 The Oxford D ictionary of Biology defines “microorganism” as “ An y organism that can be observed onl y with the aid of a microscope. Microorganisms include bacteria, viruses, protists (including certain algae), and fungi. ” “ O rganism” is further defined by the Oxford Di ctionary as “ An individual living sy stem, 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 b y which organisms ge nerate others of the same kind  Growth—the normal process of increase in size of an organism b y accretion of tissue similar to that originally

present  Maintenance—the process of holding a stable , steady state over a long peri od Therefore, whether prions fall within th e above definition of “microorganism” as it is generall y used b y biologists and the medical community arguabl y depends on whether they exhibit reproduction, growth and maintenance. However, this interpretati on 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 discusse d 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 L oeffler developed a list of general guidelines that, if met, would indicate that a microorganism would likely cause disease (i.e., be a pathogen).

1 This definition and those that f ollow 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

.19

Koch later published these characteristics as four “postulates” ( Koch 189 3 ) . While a number of infecti ous 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 or ganisms suffering from the disease, but should not be found in healthy animals. (2) T he microorganism must be isolated from a diseased organism and grown in pure 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. R egarding 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 equall y cl ear that prions can be detected in CNS, blood and perhaps in other tissues in sub - clini cal cases of disease (section II. E., for example) . T his is also the case for other diseases having as ymptomatic manifestations or asy mptomatic carriers , which Koch himself observed when he discovered asy mptomatic carriers of cholera and ty

phoid fever . Regarding the 2 nd postulate, prions can be isolated from diseased CNS (and other tissues). Prions can be grown i n 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 PrP C with prion isofo r ms, such as in the cell - free protein misfolding cyclic amplificat ion s ys tems 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 3 rd and 4 th postulates are satisfied b y th e numerous demonstrations that prions enriched from diseased animals or sy nthesized de novo in cell - free s ys tems, are both infectious and produce disease (see II.A.2, above) . Thus, the mechanisms of prion disease and infectivity generall y conform with Koc h’s postulates for the microbial basis of disease. B. Definition s 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 kn own 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:

“ The aggregate of vital phenomena; a certain peculiar stimulated condition of organized matter; that obscure principle whereb y organized beings are peculiarl y endowed with certain powers and functions not associated with inorganic matter. Generally , living things share, in varying degrees, the following characteristics: organ ization, irritability , movement, growth, reproduction, and adaptation.”20

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 generall y 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 M icroorganism s 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 way s to view the issue of whether prions may be classified as eithe r alive or a microorganism . One can focus on the biochemical properties of prions as a ty pe of protein molecule and argue that prions are neither alive nor a microorganism . A lternativel y, 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) P rions are not microorganism s or alive : From the perspective of protein chemistry , the research studies summ ariz ed 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, carboh ydrate s 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 transc ribed b y 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 convert s other normal prion protein molecules to adopt the same misfolded conformation through recruitment and conversion , which i s analogous to the way cry stals grow . Thus, a misfolded prion protein cannot reproduce itself; it can only induce a copy

of its shape in oth er prion prote ins . A ll other infectious agents

—algae, fungi, bacteria, viruses and viroids

— produce infectious diseases b y infecting and propagating their numbers with in the host species, which is accomplished through genetic reproduction and the propagati on 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 l imited to that of misfolded prion protein converting other, existi ng, homologous protein molecules to adopt by copying the template of the misfolded conformation. There is n either sy nthesis of new prion protein21

no r of new genetic material . From this pers pective, 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, p rions 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 geneticall y adapt to their environment or pass along their characteristics because they contain no DNA . Finally , individual prion particle s cannot modify themse lves to make themselves “more fit for existence” although prions can adapt or evolve non - geneticall y, as described in the next section . Applying the definitions of “microorganism” and “life” narrowl y, therefore, one could argue that prions are neither mic roorganisms n or alive. 2) P rions are microorganisms and /or alive : By focus ing on the properties of prions that make them infectious to humans and animals , and distinct from conventional protein toxin s , one can argue that prions are microorganism s and/or are alive . T he term s “microorganism” and “life” have previousl y been expanded to accommodate newl y 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 microo rganisms even though viruses are not considered to be alive, and do not have all of the characteristics of a micro organism as listed above. Viruses do not grow, have no metabolism, and cannot reproduce b y 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. 2 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.

2 Microbiology textb ooks also routinely devote significant space to viruses; see, for example: Textbook of Diagnostic Microbiology, Connie Mahon, George Manuselis, Donald Lehman (3d ed., Elsevier 2006); Microbiology: Principles and Explorations, Jacquelyn G. Black (6th ed. Wi ley 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 Scien ces 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 Medical Microbiology: An Introduction to In fectious Diseases, Kenneth J. Ryan, C. George Ray, John C. Sherris (McGraw Hill 2003) .22

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 (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. P rions have been characterized as non - Mendelian elements of phenotypic inheritance (Alberti et al. 2009) because they are capable of transmitting structurall y and functionally distinct states of the protein molecule. Recognizing that prions are the agents of TSEs , Miller (1999) classifies prions a s “ Subviral Agents , ” and B ü chen - Osmond (2003) list s 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 . Regardi ng 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, dy sfunctional prion proteins (additional prions) over a long period of time, leading to neurological disease and death. Unlike conventional chemical toxicants , prions success full y replicate themselves , increase in number, and spread from one host organism to another , much as pathogenic viruses and bacteria. P rions move within an organism, move to other organisms, and move through the environment , and these movements are close ly 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 man y organisms universall y regarded as living . Finally , prions can adapt or evol ve (non - genetically) in two way s: (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 point s , prions could be considered to have some of the characteristics of “life.” T he FIFRA SAP ha s also weighed in on th e 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 co ntended on scientific basis that prions lack the significant features ty pical of microorganisms. (USEPA 2009) S everal 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.23  R eplication of prions has been ampl y 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 onl y occur if prions can replicate themselves.

 As demonstrated b y the isolation and careful characterization of multiple mouse and hamster - adapted sheep scrapie strains, prions can adapt to new sp ecies.

 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.

 P rions most definitely exhibit a stable steady

state over a long period of tim e (maintenance). Established stocks of TSE infectivity can be maintained for decades with no change in either their titer or their biological characteristics.

 P rions 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 b y 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” broadl y, 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 defini tions that prions are not microorganism s or alive , and ( 2) a broader interpretation of current definitions that prions are a unique typ e of microorganism and/or are alive . In either case, prions are infectious and do produce disease, and in doing so, the y generall y 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 fo rmulating a regulatory position on whether prions should be considered to be a “pest” under FIFRA.24

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