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Pathogenic prion protein is degraded by a manganese oxide mineral found in soils

Posted Jan 15 2009 4:52pm
Short Communication

Pathogenic prion protein is degraded by a manganese oxide mineral found in soils Fabio Russo1,,, Christopher J. Johnson2,,, Chad J. Johnson2, Debbie McKenzie2, Judd M. Aiken2 and Joel A. Pedersen1

1 Department of Soil Science and Molecular and Environmental Toxicology Center, University of Wisconsin, Madison, WI, USA 2 Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI, USA

Correspondence Joel A. Pedersen

Prions, the aetiological agents of transmissible spongiform encephalopathies, exhibit extreme resistance to degradation. Soil can retain prion infectivity in the environment for years. Reactive soil components may, however, contribute to the inactivation of prions in soil. Members of the birnessite family of manganese oxides (MnO2) rank among the strongest natural oxidants in soils. Here, we report the abiotic degradation of pathogenic prion protein (PrPTSE) by a synthetic analogue of naturally occurring birnessite minerals. Aqueous MnO2 suspensions degraded the PrPTSE as evidenced by decreased immunoreactivity and diminished ability to seed protein misfolding cyclic amplification reactions. Birnessite-mediated PrPTSE degradation increased as a solution's pH decreased, consistent with the pH-dependence of the redox potential of MnO2. Exposure to 5.6 mg MnO2 ml–1 (PrPTSE : MnO2=1 : 110) decreased PrPTSE levels by 4 orders of magnitude. Manganese oxides may contribute to prion degradation in soil environments rich in these minerals.

These authors contributed equally to this work.

Present address: Dipartimento di Scienze del Suolo della Pianta dell'Ambiente e delle Produzioni Animali, Università Federico II, Portici (NA), Italy.

Present address: US Geological Survey, Biological Resources Division, National Wildlife Health Center, Madison, WI, USA.

Three supplementary figures are available with the online version of this paper.

Common soil mineral degrades the nearly indestructible prion Published: Thursday, January 15, 2009 - 08:42 in Health & Medicine Learn more about: brain ailments chemical disinfectants contaminated soil journal of general virology mad cow disease mineral In the rogues' gallery of microscopic infectious agents, the prion is the toughest hombre in town. Warped pathogens that lack both DNA and RNA, prions are believed to cause such fatal brain ailments as chronic wasting disease (CWD) in deer and moose, mad cow disease in cattle, scrapie in sheep and Creutzfeldt-Jakob disease in humans. In addition to being perhaps the weirdest infectious agent know to science, the prion is also the most durable. It resists almost every method of destruction from fire and ionizing radiation to chemical disinfectants and autoclaving, which reduce prion infectivity but fail to completely eliminate it.

Now, however, a team of Wisconsin researchers has found that a common soil mineral, an oxidized from of manganese known as birnessite, can penetrate the prion's armor and degrade the protein.

The new finding, which was reported earlier this month (Jan. 2) in the Journal of General Virology, is important because it may yield ways to decontaminate soil and other environments where prions reside.

"Prions are resistant to many of the conventional means of inactivating pathogens," says Joel Pedersen, a University of Wisconsin-Madison environmental chemist and the senior author of the new study. For example, autoclaving, a standard method for sterilization in the laboratory, will reduce the concentration of prions in solution, but fails to eliminate them altogether, as it does for virtually all other types of pathogens.

Because prions infect both wild and domesticated animals, the agent can contaminate barnyards and other areas where infected livestock are kept, as well as persist in natural environments where deer, elk and other animals can become infected by contact with contaminated soil.

Other studies have shown that prions can survive in the soil for at least three years, and that soil is a plausible route of transmission for some animals, Pedersen says. "We know that environmental contamination occurs in deer and sheep at least," he notes.

Prion reservoirs in the soil, Pedersen explains, are likely critical links in the chain of infection because the agent does not appear to depend on vectors — intermediate organisms like mosquitoes or ticks — to spread from animal to animal.

That the birnessite family of minerals possessed the capacity to degrade prions was a surprise, Pedersen says. Manganese oxides like birnessite are commonly used in such things as batteries and are among the most potent oxidants occurring naturally in soils, capable of chemically transforming a substance by adding oxygen atoms and stripping away electrons. The mineral is most abundant in soils that are seasonally waterlogged or poorly drained.

"A variety of manganese oxide minerals exist and one of the most common is birnessite. They are common in the sense that you find them in many soils, but in low concentrations," says Pedersen. "They are among the strongest oxidants in soil."

The new study, which was led by Fabio Russo of the University of Naples and Christopher J. Johnson of UW-Madison, was conducted on prions in solution in the laboratory. The group's working hypothesis, according to Pedersen, is that the mineral oxidizes the prion, a chemical process that can be seen in things like iron oxidizing to form rust or how cut pears and apples turn brown when exposed to oxygen.

The next step, Pedersen says, is to mix the mineral with contaminated soil to see if it has the same effect. If it does, birnessite may become a useful tool for cleaning up contaminated farmyards and other places where the prion may be concentrated in the soil.

"I expect that its efficacy would be somewhat diminished in soil," says Pedersen. "It's something we'll explore."

Source: University of Wisconsin-Madison

Chronic Wasting Disease UW finding could be 'first step' in fight against chronic wasting disease prion By Mark Johnson of the Journal Sentinel

Posted: Jan. 14, 2009

Scientists at the University of Wisconsin-Madison might have found a chink in one of biology's most fearsome infectious agents: the prion, believed responsible for the family of brain-wasting diseases that includes Creutzfeldt-Jakob in humans and chronic wasting disease in deer.

Previous studies have showed that the mutant proteins called prions have the insidious ability to survive in soil and retain their ability to infect animals.

But Wisconsin researchers reported in the Journal of General Virology that an oxidized form of manganese called birnessite degraded prions in laboratory test tubes.

"It looks pretty good," said Patrick Bosque, a prion researcher and associate professor of neurology at Denver Health Medical Center. "They really do show that this does accelerate the degradation of this (prion) protein."

Chronic wasting disease was first detected in Wisconsin's deer herd in 2001, prompting considerable worry in a state where deer hunting holds a treasured place.

Other prion diseases are mad cow disease and its human variant and scrapie in sheep.

Prions can survive on metal surgical instruments. An autoclave, which uses steam and pressure to kill bacteria in minutes, requires a much longer period to eliminate prions.

Bosque cautioned that researchers still must test birnessite on animals to see whether it will protect them from contaminated soil. This is important because animals ingest soil - deliberately to supplement their mineral nutrition, but also by accident while feeding and grooming.

Very resilient Once they get inside soil, prions have proved a resilient foe.

Bosque said a case has been documented in which a field where sheep were infected with scrapie was kept empty for several years and then repopulated with sheep from a country that did not have scrapie.

Despite the precautions, the new sheep were infected with the disease.

"It's a first step," said Joel Pedersen, an environmental chemist at UW and one of the authors of the study. "It's promising, but we have much more to do."

Pedersen said researchers conducted their study on hamsters infected with a prion disease. Scientists isolated prions from the infected hamsters, put them in solution and introduced birnessite, a soft, dark brown or black mineral oxide. Scientists then monitored the decline of the prions.

In one scenario, Pedersen said, birnessite dramatically reduced the number of prions.

He said the scientists are preparing to follow up this work on two fronts. They will add prions to soil, mix in birnessite and then measure the prions to see whether the material is effective in decontaminating soil.

This, Pedersen stressed, is a technically challenging experiment.

Researchers also will expose prions to birnessite and then inject them into hamsters to learn whether treated prions will infect the animals.

The experiments in the new paper did not involve the deer illness, chronic wasting disease, but rather a different prion disease.

Pedersen said later experiments will focus on chronic wasting disease.

Sunday, November 16, 2008 Resistance of Bovine Spongiform Encephalopathy (BSE) Prions to Inactivation


Published online before print March 14, 2000, 10.1073/pnas.050566797; Proc. Natl. Acad. Sci. USA, Vol. 97, Issue 7, 3418-3421, March 28, 2000

Medical Sciences New studies on the heat resistance of hamster-adapted scrapie agent: Threshold survival after ashing at 600°C suggests an inorganic template of replication

Paul Brown*,, Edward H. Rau, Bruce K. Johnson*, Alfred E. Bacote*, Clarence J. Gibbs Jr.*, and D. Carleton Gajdusek§

* Laboratory of Central Nervous System Studies, National Institute of Neurological Disorders and Stroke, and Environmental Protection Branch, Division of Safety, Office of Research Services, National Institutes of Health, Bethesda, MD 20892; and § Institut Alfred Fessard, Centre National de la Recherche Scientifique, 91198 Gif sur Yvette, France

Contributed by D. Carleton Gajdusek, December 22, 1999


One-gram samples from a pool of crude brain tissue from hamsters infected with the 263K strain of hamster-adapted scrapie agent were placed in covered quartz-glass crucibles and exposed for either 5 or 15 min to dry heat at temperatures ranging from 150°C to 1,000°C. Residual infectivity in the treated samples was assayed by the intracerebral inoculation of dilution series into healthy weanling hamsters, which were observed for 10 months; disease transmissions were verified by Western blot testing for proteinase-resistant protein in brains from clinically positive hamsters. Unheated control tissue contained 9.9 log10LD50/g tissue; after exposure to 150°C, titers equaled or exceeded 6 log10LD50/g, and after exposure to 300°C, titers equaled or exceeded 4 log10LD50/g. Exposure to 600°C completely ashed the brain samples, which, when reconstituted with saline to their original weights, transmitted disease to 5 of 35 inoculated hamsters. No transmissions occurred after exposure to 1,000°C. These results suggest that an inorganic molecular template with a decomposition point near 600°C is capable of nucleating the biological replication of the scrapie agent.

transmissible spongiform encephalopathy scrapie prion medical waste incineration


The infectious agents responsible for transmissible spongiform encephalopathy (TSE) are notoriously resistant to most physical and chemical methods used for inactivating pathogens, including heat. It has long been recognized, for example, that boiling is ineffective and that higher temperatures are most efficient when combined with steam under pressure (i.e., autoclaving). As a means of decontamination, dry heat is used only at the extremely high temperatures achieved during incineration, usually in excess of 600°C. It has been assumed, without proof, that incineration totally inactivates the agents of TSE, whether of human or animal origin. It also has been assumed that the replication of these agents is a strictly biological process (1), although the notion of a "virus" nucleant of an inorganic molecular cast of the infectious -pleated peptide also has been advanced (2). In this paper, we address these issues by means of dry heat inactivation studies.

full text;

infectivity surviving ashing to 600*C is (in my opinion) degradable but infective. based on Bown & Gajdusek, (1991), landfill and burial may be assumed to have a reduction factor of 98% (i.e. a factor of 50) over 3 years. CJD-infected brain-tissue remained infectious after storing at room-temperature for 22 months (Tateishi et al, 1988). Scrapie agent is known to remain viable after at least 30 months of desiccation (Wilson et al, 1950). and pastures that had been grazed by scrapie-infected sheep still appeared to be contaminated with scrapie agent three years after they were last occupied by sheep (Palsson, 1979).


1: J Neurol Neurosurg Psychiatry 1994 Jun;57(6):757-8

Transmission of Creutzfeldt-Jakob disease to a chimpanzee by electrodes contaminated during neurosurgery.

Gibbs CJ Jr, Asher DM, Kobrine A, Amyx HL, Sulima MP, Gajdusek DC.

Laboratory of Central Nervous System Studies, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892.

Stereotactic multicontact electrodes used to probe the cerebral cortex of a middle aged woman with progressive dementia were previously implicated in the accidental transmission of Creutzfeldt-Jakob disease (CJD) to two younger patients. The diagnoses of CJD have been confirmed for all three cases. More than two years after their last use in humans, after three cleanings and repeated sterilisation in ethanol and formaldehyde vapour, the electrodes were implanted in the cortex of a chimpanzee. Eighteen months later the animal became ill with CJD. This finding serves to re-emphasise the potential danger posed by reuse of instruments contaminated with the agents of spongiform encephalopathies, even after scrupulous attempts to clean them.

PMID: 8006664 [PubMed - indexed for MEDLINE]


Volume 351, Number 9110 18 April 1998 [Previous] [Next]

BSE: the final resting place

How to dispose of dangerous waste is a question that has vexed the human race for hundreds of years. The answer has usually been to get it out of sight--burn it or bury it. In Periclean Athens, victims of the plague were incinerated in funeral pyres; in 14th century Venice, a law stipulated that Black Death corpses should be buried to a minimum depth of 5 feet; and now, as the 20th century draws to a close, we are challenged by everything from industrial mercury to the smouldering reactors of decommissioned atomic submarines.

The Irish Department of Agriculture will convene an expert panel on April 27-29 to discuss the disposal of tissues from animals with bovine spongiform encephalopathy (BSE). Proper disposal of tissues from infected cattle has implications for both human and animal safety. Safety for human beings is an issue because there is now unassailable if still indirect evidence that BSE causes infections in man in the form of "new variant" Creutzfeld-Jakob disease (nvCJD).1-3 Safety for animals is also an issue because BSE-affected cattle could possibly transmit disease to species other than cattle, including sheep, the species that was almost surely the unwitting source of the BSE epidemic.

The first matter to consider is the distribution of infectivity in the bodies of infected animals. The brain (and more generally, the central nervous system) is the primary target in all transmissible spongiform encephalopathies (TSE), and it contains by far the highest concentration of the infectious agent. In naturally occuring disease, infectivity may reach levels of up to about one million lethal doses per gram of brain tissue, whether the disease be kuru, CJD, scrapie, or BSE. The infectious agent in BSE-infected cattle has so far been found only in brain, spinal cord, cervical and thoracic dorsal root ganglia, trigeminal ganglia, distal ileum, and bone marrow.4 However, the much more widespread distribution of low levels of infectivity in human beings with kuru or CJD, and in sheep and goats with scrapie, suggests that caution is advisable in prematurely dismissing as harmless other tissues of BSE-infected cattle.

A second consideration relates to the routes by which TSE infection can occur. Decades of accumulated data, both natural and experimental, have shown clearly that the most efficient method of infection is by direct penetration of the central nervous system; penetration of peripheral sites is less likely to transmit disease. Infection can also occur by the oral route, and the ingestion of as little as 1 g of BSE brain tissue can transmit disease to other cattle.5 Infection by the respiratory route does not occur (an important consideration with respect to incineration), and venereal infection either does not occur or is too rare to be detected.

How can tissue infectivity be destroyed before disposal? The agents that cause TSE have been known almost since their discovery to have awesome resistance to methods that quickly and easily inactivate most other pathogens. Irradiation, chemicals, and heat are the three commonest inactivating techniques. Irradiation has proved entirely ineffective, and only a handful of a long catalogue of chemicals have produced more than modest reduction in infectivity. The most active of these are concentrated solutions of sodium hypochlorite (bleach) or sodium hydroxide (lye). As for heat, even though the agent shares with most other pathogens the feature of being more effectively damaged by wet heat than by dry heat, boiling has little effect, and steam heat under pressure (autoclaving) at temperatures of 121ºC is not always sterilising. To date, the most effective heat kill requires exposure of infectious material to steam heat at 134ºC for 1 h in a porous-load autoclave.6 Exposure to dry heat even at temperatures of up to 360ºC for 1 h may leave a small amount of residual infectivity.7 The standard method of incineration, ***heating to about 1000ºC for at least several seconds, has been assumed to achieve total sterilisation, but needs experimental verification in the light of suggestions that rendered tissue waste might find some useful purpose as a source of heating fuel.

Thus, TSE agents are very resistant to virtually every imaginable method of inactivation, and those methods found to be most effective may, in one test or another, fail to sterilise. It seems that even when most infectious particles succumb to an inactivating process, there may remain a small subpopulation of particles that exhibit an extraordinary capacity to withstand inactivation, and that, with appropriate testing, will be found to retain the ability to transmit disease. Also, almost all available inactivation data have come from research studies done under carefully controlled laboratory conditions, and it is always difficult to translate these conditions to the world of commerce. Even when the data are applied in the commercial process, the repetitive nature of the process requires vigilance in quality control and inspection to ensure adherence to its regulations.

The final issue that must be addressed is the "lifespan" of the infectious agent after disposal if it has been only incompletely inactivated beforehand. Given the extraordinary resistance of the agent to decontamination measures, the epidemiological and experimental evidence indicating that TSE agents may endure in nature for a long time should come as no surprise. The first real clue to this possibility came from the Icelandic observation that healthy sheep contracted scrapie when they grazed on pastures that had lain unused for 3 years after having been grazed by scrapie-infected sheep.8

Support for this observation was obtained from an experiment in which scrapie-infected brain material was mixed with soil, placed in a container, and then allowed to "weather" in a semi-interred state for 3 years.9 A small amount of residual infectivity was detected in the contaminated soil, and most of the infectivity remained in the topmost layers of soil, where the tissue had originally been placed--in other words, there had been no significant leaching of infectivity to deeper soil layers.

It is therefore plausible for surface or subsurface disposal of TSE-contaminated tissue or carcasses to result in long-lasting soil infectivity. Uncovered landfills are a favourite feeding site for seagulls, which could disperse the infectivity.10 Other animals might do likewise, and if the landfill site were later used for herbivore grazing, or tilled as arable land, the potential for disease transmission might remain. A further question concerns the risk of contamination of the surrounding water table, or even surface waste-water channels, by effluents and discarded solid waste from treatment plants.

A reasonable conclusion from existing data is that there is a potential for human infection to result from environmental contamination by BSE-infected tissue residues. The potential cannot be quantified because of the huge number of uncertainties and assumptions that attend each stage of the disposal process.

On the positive side, spongiform encephalopathy can be said to be not easily transmissible. Although the level of infectivity to which creatures are exposed is not known, it is probably very low, since sheep that die from scrapie, cattle that die from BSE, and human beings who die from nvCJD represent only a small proportion of their respective exposed populations.

Whatever risk exists is therefore extremely small, but not zero, hence all practical steps that might reduce the risk to the smallest acceptable level must be considered. What is practical and what is acceptable are concepts that will be hammered out on the anvil of politics: scientific input, such as it is, already waits in the forge. A fairly obvious recommendation, based on the science, would be that all material that is actually or potentially contaminated by BSE, whether whole carcasses, rendered solids, or waste effluents, should be exposed to lye and thoroughly incinerated under strictly inspected conditions. Another is that the residue is buried in landfills to a depth that would minimise any subsequent animal or human exposure, in areas that would not intersect with any potable water-table source. Certainly, it has been, and will continue to be, necessary in many instances to accept less than the ideal.

Paul Brown

Laboratory of Central Nervous System Studies, National Institute of Neurological Disorders and Stroke, Bethesda, MD 20892, USA

1 Will RG, Ironside JW, Zeidler M, et al. A new variant of Creutzfeldt-Jakob disease in the UK. Lancet 1996; 347: 921-25 [PubMed].

2 Bruce M, Will RG, Ironside JW, et al. Transmissions to mice indicate that 'new variant' CJD is caused by the BSE agent. Nature 1997: 389: 498-501.

3 Collinge J, Sidle KCL, Heads J, Ironside J, Hill AF. Molecular analysis of prion strain variation and the aetiology of 'new variant' CJD. Nature 1996; 383: 685-90 [PubMed].

4 Wells GAH, Hawkins SAC, Green RB, et al. Preliminary observations on the pathogenesis of experimental bovine spongiform encephalopathy (BSE): an update. Vet Rec 1998; 142: 103-06 [PubMed].

5 Collee JG, Bradley R. BSE: a decade on--part 2. Lancet 1997; 349: 715-21 [PubMed].

6 Taylor DM. Exposure to, and inactivation of, the unconventional agents that cause transmissible degenerative encephalopathies. In: Baker HF, Ridley RM, eds. Methods in molecular medicine: prion diseases. Totawa NJ: Humana Press, 1996: 105-18.

7 Brown P, Liberski PP, Wolff A, Gajdusek DC. Resistance of scrapie infectivity to steam autoclaving after formaldehyde fixation and limited survival after ashing at 360°C: practical and theoretical implications, J Infect Dis 1990; 161: 467-72 [PubMed].

8 Palsson PA. Rida (scrapie) in Iceland and its epidemiology. In: Prusiner SB, Hadlow WJ, eds. Slow transmissible diseases of the nervous system, vol I. New York: Academic Press, 1979: 357-66.

9 Brown P, Gajdusek DC. Survival of scrapie virus after 3 years' interment. Lancet 1991; 337; 269-70.

10 Scrimgoeur EM, Brown P, Monaghan P. Disposal of rendered specified offal. Vet Rec 1996; 139: 219-20 [PubMed].

1.2 Visual Impact

It is considered that the requirement for any carcass incinerator design would be to ensure that the operations relating to the reception, storage and decapitation of diseased carcasses must not be publicly visible and that any part of a carcass could not be removed or interfered with by animals or birds. ...

88. Natural decay: Infectivity persists for a long time in the environment. A study by Palsson in 1979 showed how scrapie was contracted by healthy sheep, after they had grazed on land which had previously been grazed by scrapie-infected sheep, even though the land had lain fallow for three years before the healthy sheep were introduced. Brown also quoted an early experiment of his own (1991), where he had buried scrapie-infected hamster brain and found that he could still detect substantial infectivity three years later near where the material had been placed. 89. Potential environmental routes of infection: Brown discusses the various possible scenarios, including surface or subsurface deposits of TSE-contaminated material, which would lead to a build-up of long-lasting infectivity. Birds feeding on animal remains (such as gulls visiting landfill sites) could disperse infectivity. Other animals could become vectors if they later grazed on contaminated land. "A further question concerns the risk of contamination of the surrounding water table or even surface water channels, by effluents and discarded solid wastes from treatment plants. A reasonable conclusion is that there is a potential for human infection to result from environmental contamination by BSE-infected tissue residues. The potential cannot be quantified because of the huge numbers of uncertainties and assumptions that attend each stage of the disposal process". These comments, from a long established authority on TSEs, closely echo my own statements which were based on a recent examination of all the evidence. 90. Susceptibility: It is likely that transmissibility of the disease to humans in vivo is probably low, because sheep that die from scrapie and cattle that die from BSE are probably a small fraction of the exposed population. However, no definitive data are available. 91. Recommendations for disposal procedures: Brown recommends that material which is actually or potentially contaminated by BSE should be: 1) exposed to caustic soda; 2) thoroughly incinerated under carefully inspected conditions; and 3) that any residue should be buried in landfill, to a depth which would minimise any subsequent animal or human exposure, in areas that would not intersect with any potable water-table source. 92. This review and recommendations from Brown have particular importance. Brown is one of the world's foremost authorities on TSEs and is a senior researcher in the US National Institutes of Health (NIH). It is notable that such a respected authority is forthright in acknowledging the existence of potential risks, and in identifying the appropriate measures necessary to safeguard public health. Paper by SM Cousens, L Linsell, PG Smith, Dr M Chandrakumar, JW Wilesmith, RSG Knight, M Zeidler, G Stewart, RG Will, "Geographical distribution of variant CJD in the UK (excluding Northern Ireland)". Lancet 353:18-21, 2 nd January 1999 93. The above paper {Appendix 41 (02/01/99)} (J/L/353/18) examined the possibility that patients with vCJD (variant CJD) might live closer to rendering factories than would be expected by chance. All 26 cases of vCJD in the UK with onset up to 31 st August 1998 were studied. The incubation period of vCJD is not known but by analogy with other human TSEs could lie within the range 5-25 years. If vCJD had arisen by exposure to rendering products, such exposure might plausibly have occurred 8-10 years before the onset of symptoms. The authors were able to obtain the addresses of all rendering plants in the UK which were in production in 1988. For each case of vCJD, the distance from the place of residence on 1st January 1998 to the nearest rendering plant was calculated................SNIP...END

PLoS ONE. 2008; 3(8): e2969. Published online 2008 August 13. doi: 10.1371/journal.pone.0002969. PMCID: PMC2493038

Copyright This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.

Prion Infected Meat-and-Bone Meal Is Still Infectious after Biodiesel Production

Cathrin E. Bruederle,1* Robert M. Hnasko,1 Thomas Kraemer,2 Rafael A. Garcia,3 Michael J. Haas,3 William N. Marmer,3 and John Mark Carter1 1USDA-ARS WRRC, Foodborne Contaminants Research Unit, Albany, California, United States of America 2Forensic Toxicology, Institute of Legal Medicine, Saarland University, Homburg/Saar, Germany

3USDA-ARS ERRC, Fats, Oils and Animal Coproducts Research Unit, Wyndmoor, Pennsylvania, United States of America Neil Mabbott, Editor

University of Edinburgh, United Kingdom * E-mail: mhtml:%7B33B38F65-8D2E-434D-8F9B-8BDCD77D3066%7Dmid://00000089/!x-usc:mailto:cat

Conceived and designed the experiments: CEB RMH WNM JMC. Performed the experiments: CEB RMH TK. Analyzed the data: CEB TK JMC. Contributed reagents/materials/analysis tools: CEB RMH TK RAG MJH JMC. Wrote the paper: CEB. Received April 21, 2008; Accepted July 24, 2008.

P04.08 Environmental Persistence of TSEs - Extraction of PrPSc from Soil Smith, A; Fernie, Karen; Somerville, R Neuropathogenesis Unit, UK Background: There are concerns about the potential spread of transmissible spongiform encephalopathies (TSEs) by environmental routes following, for example, burial of infected carcasses or the disposal of waste water. The extent to which TSE infectivity survives or is disseminated within soil and soil water is unclear as is the likelihood of ensuing infection. Aim: As part of this environmental project, soil samples are being collected from lysimeters containing either infected bovine heads or boluses of infectivity. The aim of this experiment is to devise a method for the extraction of PrPSc from soil and examines the interaction between soil and its components and TSE infectivity. Methods: Samples from two soil types (clay and sandy loam) were spiked with known amounts of TSE infected brain homogenate and subjected to various extraction methods including combinations of freeze/thaw, boiling, sonication and mixing with various solvents and detergents. Any recovery was determined on western blot using PrPSc as a surrogate marker for the presence of TSE infectivity. Results: These experiments have shown that PrPSc binds strongly to both sandy and clay soil, and to pure sand (quartz). Elution from quartz and the soils was only achieved in the presence of the detergent sarkosyl, and in the case of clay soil, satisfactory elution was only achieved if PrPSc was digested with proteinase K. This finding suggests that components in clay soil may bind differently to PrP than those of sandy soil, and that the N-terminal domain of PrP is involved in this binding. Conclusion: These results form the basis of a method for the extraction of PrPSc from soil and will be used to assay samples from a large scale lysimeter experiment. Samples testing positive for the presence of PrPSc will be selected for bioassay in mice. Results to date suggest that TSE infectivity may bind strongly to soil components and could therefore persist in the environment for long periods of time.


Survival of PrPSc during Simulated Wastewater Treatment Processes

Pedersen, J1; Hinckley, G1; McMahon, K2; McKenzie, D3; Aiken, JM3 1University of Wisconsin, Soil Science/Civil and Environmental Engineering, USA; 2University of Wisconsin, Civil and Environmental Engineering, USA; 3University of Wisconsin, Comparative Biosciences, USA

Concern has been expressed that prions could enter wastewater treatment systems through sewer and/or septic systems (e.g., necropsy laboratories, rural meat processors, private game dressing) or through leachate from landfills that have received TSE-contaminated material. Prions are highly resistant to degradation and many disinfection procedures raising concern that they could survive conventional wastewater treatment. Here, we report the results of experiments examining the partitioning and survival of PrPSc during simulated wastewater treatment processes including activated and mesophilic anaerobic sludge digestion. We establish that PrPSc can be efficiently extracted from activated and anaerobic digester sludges with 1% sodium dodecyl sulfate, 10% sodium undecyl sulfate, and 1% sodium N-lauryl sarcosinate. Activated sludge digestion does not result in significant degradation of PrPSc. The protein partitions strongly to the activated sludge solids and is expected to enter biosolids treatment processes. A large fraction of PrPSc survived simulated mesophilic anaerobic sludge digestion. Our results suggest that if prions were to enter municipal waste water treatment systems, most of the agent would partition to activated sludge solids, survive mesophilic anaerobic digestion, and be present in treated biosolids. Land application of biosolids containing prions could represent a route for their unintentional introduction into the environment. Our results argue for excluding inputs of prions to municipal wastewater treatment facilities that would result in unacceptable risk of prion disease transmission via contaminated biosolids.

P04.71 Oral Transmission of Prion Disease Is Enhanced by Binding to Soil Particles Johnson, C; Pedersen, J; Chappell, R; McKenzie, D; Aiken, J University of Wisconsin - Madison, USA A long-unanswered question in prion biology is how certain transmissible spongiform encephalopathies (TSEs), such as sheep scrapie and cervid chronic wasting disease, spread from animal to animal. Anecdotal evidence and controlled field experiments have suggested the presence of an environmental TSE reservoir. We, and others, have speculated that soil may harbor TSE agent in the environment and allow its transfer to naïve hosts. TSE infectivity can persist in soil for years, and we previously demonstrated that the disease-associated form of the prion protein binds to soil particles and that prions adsorbed to the common soil mineral montmorillonite (Mte) retain infectivity following intracerebral inoculation. We assessed the oral infectivity of Mte- and soil-bound prions and found that prions bound to Mte are orally bioavailable and that, unexpectedly, binding to Mte significantly enhances disease penetrance and reduces incubation period relative to unbound agent. Cox proportional hazards modelling revealed that across the doses of TSE agent tested, Mte increased the effective infectious titer by a factor of 680 relative to unbound agent. Oral exposure to Mte-associated prions led to TSE development in experimental animals even at doses too low to produce clinical symptoms in the absence of the mineral. We tested the oral infectivity of prions bound to three whole soils differing in texture, mineralogy and organic carbon content, and found soil-bound prions to be orally infectious. Two of the three soils increased oral transmission of disease, and the infectivity of agent bound to the third soil was equivalent to that of unbound agent. Enhanced infectivity of soilbound prions may explain the environmental transmission of some TSEs despite the presumably low levels shed into the environment.

P04.104 Survival of Prion Proteins in Environmental Matrices Maluquer de Motes, C1; Torres, JM2; Pumarola, M3; Girones, R1 1University of Barcelona, Spain; 2Centro de Investigacion en Sanidad Animal, Spain; 3Autonomous University of Barcelona, Spain Several publications have suggested the environment as a possible route of transmission, especially for sheep scrapie and cervid Chronic Wasting Disease (CWD). The role of the environment as a reservoir for these disorders is difficult to prove and faces a considerable lack of information. In this work, different methodologies have been developed to evaluate the survival and inactivation of TSE agents in environmental matrices. Different slaughterhouse and urban sewage samples were spiked with diverse strains of either scrapie or BSE agents and kept under controlled conditions for extended periods of time. Aliquots of every experiment were sequentially collected and concentrated according to a methodology specifically selected for each type of matrix. Sensitivity of the methods developed was estimated among 2-10 ƒÊg of infected tissue. PrPres was finally detected by western blot. Films were then transformed into digital pictures, signal intensities were quantified and regression models were computed. According to the results obtained, scrapie agent showed higher stability than BSE in all the environments studied. However, no significant differences were observed among mouse-passaged scrapie strains and sheep scrapie. The regression models provided t90 and t99 values (times of incubation necessaries for 90% and 99% reduction of PrPres levels). In urban sewage, i.e., t99 was estimated as about 50 and 22 days for scrapie and BSE respectively. In general, the effect of the matrix was clearly observed in all the experiments, showing up to a 6-8 fold higher reduction of PrPres levels in comparison to PBS controls. As some of the inocula were titrated in terms of infectious doses, we approximated the decay of PrPres levels to the reduction of infectivity for both agents. In slaughterhouse wastewater, i.e., two-log reduction was observed for both agents after 30-35 days of incubation. Data on infectivity will be confirmed by a series of bioassay experiments.

P04.125 Environmental Persistence of TSE Infectivity: Field Studies Fernie, K; Smith, A; Somerville, R Neuropathogenesis Unit, Roslin Institute, UK Background: There is concern about the consequences of contamination of the environment with TSE infectivity. Infectivity may enter the environment by various routes, persist in the ground and spread from the original source to contaminate an extended area and groundwater. Aims: We are studying this problem by addressing the following questions: 1. Does infectivity with some containment (e.g. in a carcass) survive in the carcass over time; 2. Does infectivity without containment survive, and is it disseminated into the surrounding soil and water? 3. Do the environmental conditions, e.g. soil type and pH, affect the survival and/or transport of infectivity through soil? Methods: To address these questions, we are performing two field experiments (with appropriate containment) each using two soil types. Air temperature, rainfall, soil temperature and moisture content are being monitored. In one experiment a series of 10 bovine heads have been spiked with the BSE derived TSE strain 301V and buried in the two soils, contained within individual lysimeters, for exhumation and analysis at yearly intervals. Rainwater flowing through and collected as groundwater is also being analysed. In the second experiment a bolus of infected brain is buried at the centre of two 3 meter diameter lysimeters and soil samples taken from them at regular intervals. Water flow-through is also analysed. Results: To date, the first two bovine heads have been exhumed and the surrounding soil sampled. Both of the exhumed heads were apparently largely decomposed but on examination of the brain cavity were found to contain significant amounts of brain tissue. These have been sampled and are presently being analysed. The soil samples taken from around the heads and five sets of core samples taken from the soil surrounding the buried brain in the two large lysimeters are presently being analysed for PrPSc, the abnormal protein associated with the TSEs and for infectivity. Water samples have also been collected for analyses. Discussion: We will use the acquired data to build a predictive model of TSE behaviour in the environment which will inform future risk assessments.

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Public release date: 11-Aug-2008

Contact: Dr. Björn Seidel mhtml:%7B33B38F65-8D2E-434D-8F9B-8BDCD77D3066%7Dmid://00000089/!x-usc:mailto:bjo 49-297-230-2330 Fraunhofer-Gesellschaft

Resistant prions

A flock of sheep at pasture – a seemingly idyllic scene. But appearances can be deceptive: If the animals are suffering from scrapie, entire flocks may perish. Scrapie is an infectious disease in which prions destroy the animal's brain, rather like BSE. The brain becomes porous, the sheep lose their orientation, they suffer from strong itching sensations and scrape off their fleece. Eventually, the infected animals die.

It is difficult to contain the disease – all too often, scrapie will break out again on the same farm several months or years after it has apparently been eradicated. Are the prions transmitted not only by direct contact, but also by the environment – perhaps by the pastures? How long do prions that get into the pasture via the saliva and excrements of the sick animals, persist in the ground?

Together with fellow-scientists from the Robert Koch Institute in Berlin and the Friedrich Loeffler Institute (Federal Research Institute for Animal Health) on the island of Riems, research scientists from the Fraunhofer Institute for Molecular Biology and Applied Ecology IME in Schmallenberg investigated these questions on behalf of the German Ministry for Environment, Nature Conservation and Nuclear Safety BMU. "We mixed soil samples with scrapie pathogens to find out how long the pathogens would survive," says Dr. Björn Seidel, who headed the investigations at IME. "Even after 29 months, in other words more than two years, we were still able to detect prions in the soil." But are these prions still infectious? "The soil actually seems to increase the infectiousness of the pathogens. The incubation period – the time it takes for the disease to break out – is exceedingly short even after the prions have persisted in the soil for 29 months. All of the animals that were given contaminated soil became sick within a very short time.

These results indicate that fresh incidences of scrapie among sheep are due to contaminated pastures," says Seidel in summary. The results of the study reveal that sheep may even become infected from the surface water, though the risk of infection is much lower in this case. There is no danger to humans, however: scrapie pathogens seem unable to affect them.

Another cause for concern is chronic wasting disease (CWD). Like BSE and scrapie, this is caused by prions, but it mainly affects deer. The numbers of infected animals in North America are rising steeply. How long do BSE and CWD prions survive in the ground? "To find this out, we urgently need to carry out further tests. The appropriate research applications have already been submitted," says Seidel. #

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Monday, September 1, 2008


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