Prions: Novel Pathogens of Environmental Concern?

On March 13, 2006, the U.S. Department of Agriculture announced the third case of bovine spongiform encephalopathy (BSE or “mad cow” disease) in the United States, the eighth confirmed case in North America. A ninth case was announced a month later by Canadian officials. While North America is not expected to experience a BSE outbreak of the magnitude as that in the United Kingdom (180,927 cases as of March 1, 2006, ⟨www.defra.gov.uk⟩), the causal link between BSE and variant Creutzfeldt-Jakob disease (vCJD) in humans has focused government and industry attention on preventing human exposure to the disease agent. The geographic range of a related disease, chronic wasting disease (CWD) of North American cervids (members of the deer family), has expanded rapidly in the last six years. Once believed to be confined to parts of northeastern Colorado and southeastern Wyoming, CWD is now present in captive and wild deer populations in fourteen states and two Canadian provinces ⟨www.nwhc.usgs.gov/disease-information/chronic-wasting-disease/north-america-CWD-map.jsp⟩. Management responses in several states and provinces have involved significant reductions in wild cervid populations and depopulation of game farms. There is no evidence that CWD is transmissible to humans, however, recent research has demonstrated the experimental transmission of CWD to squirrel monkeys (Marsh et al. 2005) and that CWD agent is present in deer muscle (Angers et al. 2006). These findings may intensify concern by scientists, wildlife managers, and the general public about potential human exposure to CWD agent.

BSE, CWD, CJD, and scrapie (a natural infection of sheep and goats) belong to a family of infectious, neurodegenerative diseases known as transmissible spongiform encephalopathies (TSEs) or prion diseases. TSEs are characterized by long incubation periods, spongiform degeneration of the brain, accumulation of ${\mathrm{PrP}}^{\mathrm{Sc}}$ [a misfolded form of a normally benign cell-surface prion protein $\left({\mathrm{PrP}}^{\mathrm{C}}\right)$ ], loss of coordination and, in humans, personality changes and memory loss (Prusiner 1998). These diseases have long preclinical phases, and once clinical symptoms manifest, they progress inexorably to death. There is no specific immune response and no cure. The etiological agents in TSEs, termed “prions” (derived from proteinaceous and infectious, Prusiner 1982), are apparently devoid of nucleic acid and are remarkably difficult to inactivate. The abnormally folded form of the prion protein, ${\mathrm{PrP}}^{\mathrm{Sc}}$ , is thought to constitute the infectious agent, mainly because it co-purifies with prion infectivity. The amount of TSE agent in a sample or tissue is often described in terms of median infectious units $\left({\mathrm{IU}}_{50}\right)$ , which are determined by bioassay. One ${\mathrm{IU}}_{50}$ represents the amount of infectious agent required to infect half the test animals in a given study. The brain of a clinically affected cow contains $\sim {10}^6$ ${\mathrm{IU}}_{50}{\mathrm{g}}^{-1}$ when assayed by intracerebral inoculation in cattle (Scientific Steering Committee 2002). Other tissues can contain lower, but significant amounts of infectivity. The amount of infectious agent accumulating in the brains of infected deer and elk is unknown.

Prions are extraordinarily resistant to most treatments that inactivate conventional pathogens. Inactivation methods shown to be ineffective include exposure to UV and ionizing radiation, treatment with proteases and many chemical disinfectants including alkylating agents (e.g., formalin, ethylene oxide), organic solvents, ${\mathrm{ClO}}_2$ , and ${\mathrm{H}}_2{\mathrm{O}}_2$ (Taylor 2000). Prion infectivity is not eliminated by boiling, standard autoclaving conditions, or incineration at temperatures below $600°\mathrm{C}$ (Taylor 2000; Brown et al. 2004).

An environmental reservoir of prion infectivity appears to contribute to the natural transmission of CWD (Miller et al. 1998; Miller et al. 2004) and, possibly, scrapie (Pálsson 1979; Andréoletti et al. 2000). The presence of decomposed infected carcasses or residual excreta from infected animals on the landscape was sufficient to transmit CWD to healthy mule deer (Miller et al. 2004). CWD agent can enter soil when carcasses of infected animals decompose and, presumably, through shedding in feces and saliva (Williams and Miller 2002). Urinary shedding by individuals with concurrent kidney infections (Seeger et al. 2005) may represent an additional route of prion entry into soils. A number of observations support the hypothesis that soil serves as an environmental reservoir of prion infectivity: (1) herbivores, including deer, ingest soil both deliberately at salt and mineral licks, and incidentally during grazing and grooming (Beyer et al. 1994; Atwood and Weeks 2003); (2) the early involvement of gut-associated lymph tissues in CWD infection suggests an oral route of exposure; (3) the presence of prions in gut-associated lymph tissues also suggests alimentary shedding to the environment; (4) prions can persist in soils for at least three years (Brown and Gajdusek 1991); (5) vertical migration of prions is expected to be limited in fine textured soils (Brown and Gajdusek 1991; Johnson et al. 2006), keeping CWD agent near the surface of some soils; and (6) despite remarkably strong binding to particular soil minerals, prions exhibit undiminished, and perhaps enhanced, infectivity via the intracerebral route of exposure (Johnson et al. 2006). The existence of such a dispersed and heterogeneous environmental reservoir for infectivity complicates efforts to limit the spread of TSEs in free-ranging populations.

Chronic wasting disease outbreaks in captive cervid facilities (e.g., game farms, research facilities) result in higher rates of CWD prevalence than observed in wild populations and require decontamination of land inhabited by infected animals. No effective means of decontaminating prion-infected land has been reported. Miller et al. (1998) describe an attempt to decontaminate paddocks that had contained CWD-infected elk. Soil in the enclosures was treated with $1000\mathrm{ppm}$ aqueous $\mathrm{Ca}(\mathrm{ClO}{)}_2$ (65% available chlorine), plowed to $\sim 0.3\mathrm{m}$ , and then treated again with hypochlorite. Structures within the paddocks were either treated twice with $\mathrm{Ca}(\mathrm{ClO}{)}_2$ or replaced. After lying fallow for $>12$ months, these paddocks were repopulated with elk. These elk contracted CWD after $\ge 3$ years.

Efforts to control TSE transmission involve depopulation of herds associated with infected animals. For example, hundreds of cattle were slaughtered, and 2000 tons of potentially infected beef products were discarded when the first U.S. BSE case was discovered in 2003 (Matthews 2005). In Wisconsin and other states, CWD management efforts include herd reduction and postmortem screening to limit intraspecies transmission. Large volumes of infected waste are generated by these responses, creating a need for safe, effective disposal options for carcasses and other materials. Potentially viable disposal methods include landfilling, incineration, and alkaline hydrolysis. Rendering may have some utility when combined with other disposal techniques, since rendering processes significantly decrease the volume of material requiring ultimate disposal.

Landfilling represents an economically attractive disposal option. The risks associated with landfilling TSE-contaminated wastes are, however, not known because only limited data are available on prion fate and transport in porous materials (e.g., solid waste, soils). The extreme recalcitrance of prions implies that they may persist for many years within a landfill. Brown and Gajdusek (1991) demonstrated that prions remain infectious in soil environments for $\ge 3$ years. After three-year in situ incubation of prion-spiked soil samples, water washes of the soil still contained ${10}^{5.6}$ – ${10}^{6.4}$ ${\mathrm{IU}}_{50}$ . Johnson et al. (2006) demonstrated that ${\mathrm{PrP}}^{\mathrm{Sc}}$ incubated with soil particles is strongly bound and is not extractable by water, suggesting that soil may harbor more TSE infectivity than previously recognized. The strong binding of ${\mathrm{PrP}}^{\mathrm{Sc}}$ to smectitic clay minerals suggests limited mobility in fine-textured soils (Johnson et al. 2006). Our research group is actively investigating the mobility of prions in landfill environments.

Prion degradation by landfill microorganisms has not yet been investigated. In addition to being resistant to chemical inactivation, ${\mathrm{PrP}}^{\mathrm{Sc}}$ is characteristically resistant to enzymatic degradation. Very few microorganisms have been isolated from any source that are capable of degrading ${\mathrm{PrP}}^{\mathrm{Sc}}$ (Müller-Hellwig et al. 2006).

Under appropriate conditions, incineration can effectively eliminate prion infectivity. Using a laboratory incineration apparatus, Brown et al. (2004) demonstrated that prion infectivity in scrapie-infected hamster brain macerates ( $>{10}^9$ ${\mathrm{IU}}_{50}{\mathrm{g}}^{-1}$ ) was abolished by 15-min exposure to temperatures approaching $1000°\mathrm{C}$ under both normal and air-starved conditions. A small amount of infectivity survived burning at $600°\mathrm{C}$ in normal air. Air emissions from the incineration apparatus were not infectious under the conditions examined. The ability to achieve similar and reliable results in full-scale thermal units would depend on operating conditions and facility design. We are not aware of any studies addressing such important parameters, and no data currently exist on the destruction of prions in full-scale facilities.

Hot alkali processes can inactivate prions. Although both autoclaving and 2-h exposure to $2\mathrm{N}$ $\mathrm{NaOH}$ do not cause total inactivation, these processes do result in substantial reductions in the amount of infectious agent in a sample (Taylor 2000). However, combination of these treatments results in complete inactivation. Alkaline hydrolysis tissue digesters are currently used for disposal of CWD-infected carcasses and are considered the preferred method for disposal of BSE-contaminated carcasses (USDA 2003). While the efficacy of these tissue digesters for inactivating TSE agents has not been demonstrated experimentally, the elevated temperature and pressure and extended digestion times may be adequate to inactivate prions.

Little is known regarding the fate of prions in wastewater treatment (WWT) systems. Gale and Stanfield (2001) conducted a risk assessment for BSE agent in sewage sludge to evaluate the potential for transmission of BSE to cattle and vCJD to humans via application of biosolids to agricultural fields. They concluded that exposure of cattle to BSE agent in biosolids was not adequate to explain the U.K. epizootic and that the risk to humans was acceptably low (Gale and Stanfield 2001); however, the authors noted that their risk assessment was hampered by lack of data on prion inactivation by sorption to sewage sludge and degradation by sludge microorganisms. The lower incidence of known BSE cases in North America suggests the risk of TSE transmissionvia biosolids would be substantially lower than in theUnited Kingdom.

The concentration of prions entering WWT systems is not known. Risk assessments are based on best estimates of the amount of TSE agent entering sewage during the slaughtering and processing of BSE-infected cows in the United Kingdom (Gale and Stanfield 2001). There are, however, other potential routes of TSE agents to enter WWT systems; these include agricultural digesters (particularly those used for disposal of “downer” cows), some septic systems (e.g., private game dressing, rural meat processors, necropsy laboratories for CWD surveillance), and systems receiving leachate from landfills that have received TSE-contaminated materials. The degree to which conventional WWT processes inactivate TSE agents is unknown and is currently being investigated by our research group.

Reports of urinary excretion of ${\mathrm{PrP}}^{\mathrm{Sc}}$ by CJD patients and TSE-infected animals (Shaked et al. 2001) has led to concern that CJD agent may enter WWT plants and be subsequently applied to agricultural fields in treated biosolids. These reports of urinary shedding have not been substantiated by others (Head et al. 2005) and appear to have been artifactual (Furukawa et al. 2004). Urinary prion excretion at levels typically $\le 0.5$ intracerebral ${\mathrm{IU}}_{50}{\mathrm{mL}}_{\mathrm{urine}}^{-1}$ has, however, been demonstrated in scrapie-infected mice that have concurrent kidney infection (Seeger et al. 2005). If CJD patients are assumed to excrete prions at the same level as was found by Seeger et al. (2005), back-of-the-envelope calculations show that the amount of infectivity in biosolids-amended soils would be exceedingly small. For example, ingestion on the order of $\mathrm{60,000}\mathrm{kg}$ of biosolids-amended soil would be necessary to acquire a single oral ${\mathrm{IU}}_{50}$ , if a single CJD patient with a kidney infection contributed prions to a 80 MGD wastewater treatment plant. The incidence of sporadic CJD is approximately one in ${10}^6$ (Prusiner 1998), so if urinary excretion were to occur, the number of individuals potentially contributing CJD prions to a given wastewater treatment plant would be quite small.

An improved understanding of prion fate in natural and engineered environments is needed before the potential risks posed by various disposal options can be addressed. Significant challenges in studies of prions in the environment include the lack of sufficiently sensitive and quantitative detection methods and difficulties in extracting TSE agents from environmental matrices. Current detection strategies are based on immunochemical methods (usually immunoblotting and enzyme linked immunosorbent assays), which suffer from a limited linear response range and high detection limits. The unique properties of ${\mathrm{PrP}}^{\mathrm{Sc}}$ make the use of noninfectious surrogates problematic. For example, recombinant, $\beta$ -sheet-enriched $\mathrm{PrP}$ exhibits properties significantly different from the infectious disease agent (Rezaei et al. 2005).

The persistence and transport of prions in soil matrices warrant further research because soils may contribute to the horizontal transmission of CWD and sheep scrapie. Better understanding of prion fate in landfills is needed, given the ongoing need to dispose of large quantities of TSE-infected carcasses and other material. In addition, the survival of these agents during biological treatment processes, alkaline hydrolysis in tissue digesters, and full-scale thermal destruction should be studied. The role played by environmental contamination in interspecies transmission also merits investigation. Societal concern over these devastating diseases requires that environmental engineers and scientists generate the data needed to conduct quantitative risk assessments for TSE agents and to carefully communicate their findings to government agencies and the public.

The authors gratefully acknowledge financial support from the National Science Foundation (NSF) through CAREER grant BSE-0547484 (JAP), and the U.S. Environmental Protection Agency (USEPA) through grants 4C-R070-NAEX (CHB and JAP) and 4C-R156-NAEX (KDM, CHB and JAP). This editorial does not necessarily represent the views of NSF or USEPA. Endorsement by NSF or USEPA is not implied and should not be assumed.