Environ. Sci. Technol. 2007, 41, 811-817
Fate of Prions in Soil: Detergent Extraction of PrP from Soils C I N D Y M . C O O K E , * ,†,| J O A N N E R O D G E R , ‡,⊥ A L L I S T E R S M I T H , ‡ KAREN FERNIE,‡ GEORGE SHAW,§ AND ROBERT A. SOMERVILLE‡ Centre for Environmental Policy, Imperial College London, Silwood Park, Buckhurst Road, Ascot, Berkshire, SL5 7PY, U.K., Institute for Animal Health, Neuropathogenesis Unit, West Mains Road, Edinburgh, EH9 3JF, U.K., and Division of Agricultural and Environmental Sciences, University Park, University of Nottingham, Nottingham, NG7 2RD, U.K.
The transmissible spongiform encephalopathies (TSEs) are caused by infectious agents whose structures have not been fully characterized but include abnormal forms of the host protein PrP, designated PrPSc, which are deposited in infected tissues. The transmission routes of scrapie and chronic wasting disease (CWD) seem to include environmental spread in their epidemiology, yet the fate of TSE agents in the environment is poorly understood. There are concerns that, for example, buried carcasses may remain a potential reservoir of infectivity for many years. Experimental determination of the environmental fate requires methods for assessing binding/elution of TSE infectivity, or its surrogate marker PrPSc, to and from materials with which it might interact. We report a method using Sarkosyl for the extraction of murine PrPSc, and its application to soils containing recombinant ovine PrP (recPrP). Elution properties suggest that PrP binds strongly to one or more soil components. Elution from a clay soil also required proteinase K digestion, suggesting that in the clay soil binding occurs via the N-terminal of PrP to a component that is absent from the sandy soils tested.
Introduction Transmissible spongiform encephalopathies (TSEs) (prion diseases) are a group of infectious, neurodegenerative disorders that affect both humans and animals. Scrapie in sheep, the archetype of TSE diseases, has been recognized for over 250 years, yet remarkably little is known about its origin or transmission routes. TSE diseases were brought to our attention with the outbreak of bovine spongiform encephalopathy (BSE) in the United Kingdom in the 1980s (1). Lower level epizootics have subsequently been reported in many other European countries and several cases were identified in North America (2). Chronic wasting * Corresponding author phone: (+44) 118 3787910; fax: (+44) 0118 3786666; e-mail:
[email protected]. † Imperial College London. ‡ Institute for Animal Health. § University of Nottingham. | Present address: Department of Soil Science, School of Human and Environmental Sciences, University of Reading, Whiteknights, Reading RG6 6DW, U.K. ⊥ Present address: NHS Grampian, Foresterhill House Annex, Foresterhill, Aberdeen, AB25 2ZB, U.K. 10.1021/es0618189 CCC: $37.00 Published on Web 12/13/2006
2007 American Chemical Society
disease (CWD) in deer was first recognized in North America in 1967 (3) and the number and distribution of cases is increasing (2). The transmission routes of scrapie are poorly understood but may include environmental pathways, and there is also now substantial evidence that CWD is spread via the environment (4-6). By contrast, the primary infection pathway for BSE has been via dietary exposure to industrial animal feed (7) and there is little evidence of an environmental component in its spread, although there are continuing concerns that, for example, buried carcasses may remain reservoirs of infectivity for many years (8). There are many unanswered questions relating to sources of TSE infection and the fate and behavior of TSE infectivity in the natural environment following the death and decomposition of an infected animal. The causal agent of TSE diseases has not been fully characterized but is thought to include the host protein PrP in its structure. Abnormal forms of PrP, designated PrPSc, with which the causal agents of TSEs are closely associated, are deposited in infected brain and other organs. TSE infectivity is highly resistant to inactivation with chemicals, heat, and proteases (9), raising questions about long-term survival and hence their persistence in the environment. Infected tissues may be introduced into the environment by on-farm carcass burial, mass burial by landfill, TSE infectivity in effluents from rendering plants, and in ash following incineration of carcasses (10). Another area of concern involves materials that are applied to the land (contaminated organic fertilizers, manures, sewage sludge, and composts) (11) and substances left in situ (excrement, placentae, and excreted body fluids of scrapie-infected sheep and CWDinfected cervids) (12). The presence of TSE infectivity in soil may indicate a potential infection pathway via soil ingestion, or via biological vectors such as ectoparasites or necrophagous insects (13-15). However, it has been difficult to quantify the entry of TSE infectivity or its surrogate, PrPSc, into the soil environment, or to provide a suitable quantitative risk assessment model to validate statements regarding the relative importance of various entry routes, since extraction and detection methods that can be easily applied to soils or other environmental matrices had not been developed until recently (16-18). Because of the close association between TSE infectivity and PrPSc it is sometimes possible to use the protein as a surrogate marker for TSE infectivity, although it is important to determine, ultimately, that TSE infectivity properties mirror those of the surrogate. In order to investigate the fate and behavior of TSE infectivity in the environment a method is required for PrPSc extraction from environmental matrices. Studies by Ogunseitan with soil proteins provided a useful starting point for method development (16, 19). However, while numerous methods, extractants, and conditions have been employed for the soil extraction of proteins (20-24), many of these methods are likely to denature protein and inactivate TSE infectivity, and thus were found not to be applicable to PrPSc. Recent research has suggested that soils can differ dramatically in their capacity to adsorb PrPSc due to differences in surface area and mineral composition (18). In one report, while nearly all the PrPSc was found to bind to clay particle surfaces, much remained in solution in a sand mixture (18). This suggests that PrP fate and behavior will depend on specific soil conditions. Differences in extractability should be expected. PrPSc interactions are complex because the protein molecule consists of both hydrophobic VOL. 41, NO. 3, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
811
TABLE 1. Physicochemical Characteristics of the Three Soils soil characteristic pH (H2O) particle sizeb % clay (
