Resistance of Bovine Spongiform Encephalopathy (BSE) Prions to Inactivation

Resistance of Bovine Spongiform Encephalopathy (BSE) Prions to Inactivation

Kurt Giles1,2, David V. Glidden3, Robyn Beckwith1, Rose Seoanes1, David Peretz1,2¤, Stephen J. DeArmond1,4, Stanley B. Prusiner1,2,5*

1 Institute for Neurodegenerative Diseases, University of California San Francisco, San Francisco, California, United States of America, 2 Department of Neurology, University of California San Francisco, San Francisco, California, United States of America, 3 Department of Epidemiology and Biostatistics, University of California San Francisco, San Francisco, California, United States of America, 4 Department of Pathology, University of California San Francisco, San Francisco, California, United States of America, 5 Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California, United States of America

Abstract Distinct prion strains often exhibit different incubation periods and patterns of neuropathological lesions. Strain characteristics are generally retained upon intraspecies transmission, but may change on transmission to another species. We investigated the inactivation of two related prions strains: BSE prions from cattle and mouse-passaged BSE prions, termed 301V. Inactivation was manipulated by exposure to sodium dodecyl sulfate (SDS), variations in pH, and different temperatures. Infectivity was measured using transgenic mouse lines that are highly susceptible to either BSE or 301V prions. Bioassays demonstrated that BSE prions are up to 1,000-fold more resistant to inactivation than 301V prions while Western immunoblotting showed that short acidic SDS treatments reduced protease-resistant PrPSc from BSE prions and 301V prions at similar rates. Our findings argue that despite being derived from BSE prions, mouse 301V prions are not necessarily a reliable model for cattle BSE prions. Extending these comparisons to human sporadic Creutzfeldt-Jakob disease and hamster Sc237 prions, we found that BSE prions were 10- and 106-fold more resistant to inactivation, respectively. Our studies contend that any prion inactivation procedures must be validated by bioassay against the prion strain for which they are intended to be used.


http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1000206


Tuesday, November 11, 2008

Transmission of atypical bovine prions to mice transgenic for human prion protein

DOI: 10.3201/eid1412.080941

http://bse-atypical.blogspot.com/2008/11/transmission-of-atypical-bovine-prions.html


MARSH

http://www.bseinquiry.gov.uk/files/mb/m09/tab05.pdf


In Confidence - Perceptions of unconventional slow virus diseasesof animals in the USA - APRIL-MAY 1989 - G A H Wells


http://www.bseinquiry.gov.uk/files/mb/m11b/tab01.pdf


Wednesday, August 20, 2008

Bovine Spongiform Encephalopathy Mad Cow Disease typical and atypical strains, was there a cover-up ?


http://bse-atypical.blogspot.com/2008/08/bovine-spongiform-encephalopathy-mad.html


TME hyper/drowsy, INTER-SPECIES TRANSMISSION CWD and strain properties

Date: October 22, 2007 at 12:48 pm PSTCompletely Edited VersionPRION ROUNDTABLETME hyper/drowsy, INTER-SPECIES TRANSMISSION CWD and strain propertiespage 19 of 62. ...tss

Dr. Detwiler: How would you explain that biochemically?

snip...see full text ;

http://transmissible-mink-encephalopathy.blogspot.com/2007/12/phenotypic-similarity-of-transmissible.html


Phenotypic Similarity of Transmissible Mink Encephalopathy in Cattle and L-type Bovine Spongiform Encephalopathy in a Mouse Model

Thierry Baron,* Anna Bencsik,* Anne-Gaëlle Biacabe,* Eric Morignat,* andRichard A. Bessen?*Agence Française de Sécurité Sanitaire des Aliments-Lyon, Lyon, France; and?Montana State University, Bozeman, Montana, USA

Abstract

Transmissible mink encepholapathy (TME) is a foodborne transmissiblespongiform encephalopathy (TSE) of ranch-raised mink; infection with aruminant TSE has been proposed as the cause, but the precise origin of TMEis unknown. To compare the phenotypes of each TSE, bovine-passaged TMEisolate and 3 distinct natural bovine spongiform encephalopathy (BSE) agents(typical BSE, H-type BSE, and L-type BSE) were inoculated into an ovinetransgenic mouse line (TgOvPrP4). Transgenic mice were susceptible toinfection with bovine-passaged TME, typical BSE, and L-type BSE but not toH-type BSE. Based on survival periods, brain lesions profiles,disease-associated prion protein brain distribution, and biochemicalproperties of protease-resistant prion protein, typical BSE had a distintphenotype in ovine transgenic mice compared to L-type BSE and bovine TME.The similar phenotypic properties of L-type BSE and bovine TME in TgOvPrP4mice suggest that L-type BSE is a much more likely candidate for the originof TME than is typical BSE.snip...ConclusionThese studies provide experimental evidence that the Stetsonville TME agentis distinct from typical BSE but has phenotypic similarities to L-type BSEin TgOvPrP4 mice. Our conclusion is that L-type BSE is a more likelycandidate for a bovine source of TME infection than typical BSE. In thescenario that a ruminant TSE is the source for TME infection in mink, thiswould be a second example of transmission of a TSE from ruminants tonon-ruminants under natural conditions or farming practices in addition totransmission of typical BSE to humans, domestic cats, and exotic zoo animals(37). The potential importance of this finding is relevant to L-type BSE,which based on experimental transmission into humanized PrP transgenic miceand macaques, suggests that L-type BSE is more pathogenic for humans thantypical BSE (24,38).

http://www.cdc.gov/eid/content/13/12/1887.htm?s_cid=eid1887_e


http://transmissible-mink-encephalopathy.blogspot.com/2007/12/phenotypic-similarity-of-transmissible.html


A New Prionopathy OR more of the same old BSe and sporadic CJD

http://creutzfeldt-jakob-disease.blogspot.com/2008/08/new-prionopathy-or-more-of-same-old-bse.html


HUMAN and ANIMAL TSE Classifications i.e. mad cow disease and the UKBSEnvCJD only theory JUNE 2008

snip...Tissue infectivity and strain typing of the many variants of the human and animal TSEs are paramount in all variants of all TSE. There must be a proper classification that will differentiate between all these human TSE in order to do this. With the CDI and other more sensitive testing coming about, I only hope that my proposal will some day be taken seriously.

...snip...

http://cjdmadcowbaseoct2007.blogspot.com/2008/06/human-and-animal-tse-classifications-ie.html

ONE HUNDRED AND FIRST MEETING OF THE SPONGIFORM ENCEPHALOPATHY ADVISORY COMMITTEEOct 23, 2008 at 9:00 AM


http://seac992007.blogspot.com/2008/10/one-hundred-and-first-meeting-of_23.html

http://flounder068.vox.com/library/post/one-hundred-and-first-meeting-of-the-spongiform-encephalopathy-advisory-committee.html


***The pathology features of Nor98 in the cerebellum of the affected sheep showed similarities with those of sporadic Creutzfeldt-Jakob disease in humans.

http://www.prion2007.com/pdf/Prion%20Book%20of%20Abstracts.pdf


Here we report that both Nor98 and discordant cases, including three sheep homozygous for the resistant PrPARR allele (A136R154R171), efficiently transmitted the disease to transgenic mice expressing ovine PrP, and that they shared unique biological and biochemical features upon propagation in mice. These observations support the view that a truly infectious TSE agent, unrecognized until recently, infects sheep and goat flocks and may have important implications in terms of scrapie control and public health.Edited by Stanley B. Prusiner, University of California, San Francisco, CA, and approved September 12, 2005 (received for review March 21, 2005)

http://www.pnas.org/cgi/content/abstract/0502296102v1


Tuesday, June 3, 2008 SCRAPIE USA UPDATE JUNE 2008 NOR-98 REPORTED PA

http://nor-98.blogspot.com/2008/06/scrapie-usa-update-june-2008-nor-98.html


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

Abstract

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

Introduction

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;

http://www.pnas.org/cgi/content/full/97/7/3418

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).

http://europa.eu.int/comm/food/fs/sc/ssc/out58_en.pdf

PAUL BROWN SCRAPIE SOIL TEST

http://www.bseinquiry.gov.uk/files/sc/seac07/tab03.pdf

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]

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8006664&dopt=Abstract

LANCET

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].

http://www.thelancet.com/newlancet/sub/issues/vol351no9110/body.commentary1146.html

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. ...

http://www.bseinquiry.gov.uk/files/yb/1989/04/03006001.pdf

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

http://www.bse.org.uk/files/ws/s019b.pdf

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:cathrin.bruederle@gmail.com

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.

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2493038

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.

P04.61

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.

snip...end....NEUROPRION 2007...TSS

Public release date: 11-Aug-2008

Contact: Dr. Björn Seidel mhtml:%7B33B38F65-8D2E-434D-8F9B-8BDCD77D3066%7Dmid://00000089/!x-usc:mailto:bjoern.seidel@ime.fraunhofer.de 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.

http://www.eurekalert.org/pub_releases/2008-08/f-rp081108.php#

Disposal of meat and bone meal (MBM) derived from specified risk material (SRM) and over thirty month scheme carcasses by landfill The Committee was asked to consider a quantitative risk assessment of the disposal of meat and bone meal derived from specified risk material and over thirty month scheme carcasses by landfill, prepared in response to a request from the Committee at its June 1999 meeting.

The Committee was asked whether, in the light of the results of the risk assessment, it held to its earlier published (June 1999) view that landfill was an acceptable outlet for MBM of any origin, although it retained a preference for incineration. The Committee reiterated that it had a strong preference for incineration as the favoured route for the disposal of MBM and were uneasy about the use of landfill for the disposal of this material. If there were cases where incineration was not practical the Committee felt it would be preferable for any material going to landfill to be pressure-cooked first or possibly stored above ground prior to incineration.

http://www.seac.gov.uk/summaries/summ_0700.htm

Disposal of BSE suspect carcases It is the Department's policy to dispose of BSE suspects by incineration wherever feasible. No BSE suspect carcases have been landfilled since 1991.

http://www.defra.gov.uk/animalh/bse/publichealth/notification.html#disp

OPINION ON

THE USE OF BURIAL FOR DEALING WITH ANIMAL

CARCASSES AND OTHER ANIMAL MATERIALS THAT

MIGHT CONTAIN BSE/TSE

ADOPTED BY THE

SCIENTIFIC STEERING COMMITTEE

MEETING OF 16-17 JANUARY 2003

The details of the SSC’s evaluation are provided in the attached report. The SSC

concludes as follows:

(1) The term “burial” includes a diversity of disposal conditions. Although burial is

widely used for disposal of waste the degradation process essential for BSE/TSE

infectivity reduction is very difficult to control. The extent to which such an

infectivity reduction can occur as a consequence of burial is poorly characterised.

It would appear to be a slow process in various circumstances.

(2) A number of concerns have been identified including potential for groundwater

contamination, dispersal/transmission by birds/animals/insects, accidental

uncovering by man.

(3) In the absence of any new data the SSC confirms its previous opinion that animal

material which could possibly be contaminated with BSE/TSEs, burial poses a

risk except under highly controlled conditions (e.g., controlled landfill).

SNIP...

4. CONCLUSION

In the absence of new evidence the opinion of the SSC “Opinion on Fallen Stock”

(SSC 25th June 1999) must be endorsed strongly that land burial of all animals and

material derived from them for which there is a possibility that they could

incorporate BSE/TSEs poses a significant risk. Only in exceptional circumstances

where there could be a considerable delay in implementing a safe means of disposal

should burial of such materials be considered. Guidelines should be made available

to aid on burial site selection.

4 PAGES;

http://europa.eu.int/comm/food/fs/sc/ssc/out309_en.pdf

During the 2001 outbreak of FMD in the UK, the

Department of Health prepared a rapid qualitative

assessment of the potential risks to human health

associated with various methods of carcass disposal

(UK Department of Health, 2001c). The most

relevant hazards to human health resulting from

burial were identified as bacteria pathogenic to

humans, water-borne protozoa, and BSE. The main

potential route identified was contaminated water

supplies, and the report generally concluded that an

engineered licensed landfill would always be

preferable to unlined burial. In general terms, the

findings of the qualitative assessment relative to

biological agents are summarized in Table 13.

TABLE 13. Potential health hazards and associated pathways of exposure resulting from landfill or burial of

animal carcasses (adapted from UK Department of Health, 2001c).

PLEASE SEE TABLE AT;

http://www.k-state.edu/projects/fss/research/books/carcassdispfiles/PDF%...les/CH%201%20-%20Burial.pdf

PART 2

Rendering and fixed-facility incineration were

preferred, but the necessary resources were not

immediately available and UK officials soon learned

that the capacity would only cover a portion of the

disposal needs. Disposal in commercial landfills was

seen as the next best environmental solution, but

legal, commercial, and local community problems

limited landfill use. With these limitations in mind,

pyre burning was the actual initial method used but

was subsequently discontinued following increasing

public, scientific, and political concerns. Mass burial

and on-farm burial were last on the preferred

method list due to the complicating matter of bovine

spongiform encephalopathy (BSE) and the risk posed

to groundwater (Hickman & Hughes, 2002).

http://www.k-state.edu/projects/fss/research/books/carcassdispfiles/PDF%...les/Introduction%20to%20Part%202%20-%20Cross-Cutting%20&%20Policy%20Issues.pdf

Carcase disposal:

A Major Problem of the

2001 FMD Outbreak

Gordon Hickman and Neil Hughes, Disposal Cell,

FMD Joint Co-ordination Centre, Page Street

snip...

http://www.defra.gov.uk/animalh/svj/fmd/pages27-40.pdf

3. Prof. A. Robertson gave a brief account of BSE. The US approach was to accord it a _very low profile indeed_. Dr. A Thiermann showed the picture in the ''Independent'' with cattle being incinerated and thought this was a fanatical incident to be _avoided_ in the US _at all costs_...

snip...

http://www.bseinquiry.gov.uk/files/mb/m11b/tab01.pdf

Some unofficial information from a source on the inside looking out -

Confidential!!!!

As early as 1992-3 there had been long studies conducted on small pastures containing scrapie infected sheep at the sheep research station associated with the Neuropathogenesis Unit in Edinburgh, Scotland. Whether these are documented...I don't know. But personal recounts both heard and recorded in a daily journal indicate that leaving the pastures free and replacing the topsoil completely at least 2 feet of thickness each year for SEVEN years....and then when very clean (proven scrapie free) sheep were placed on these small pastures.... the new sheep also broke out with scrapie and passed it to offspring. I am not sure that TSE contaminated ground could ever be free of the agent!! A very frightening revelation!!! ...

----------

More here:

http://www.bseinquiry.gov.uk/files/ws/s018.pdf

INCINERATION TEMPS

Requirements include:

a. after burning to the range of 800 to 1000*C to eliminate smell;

well heck, this is just typical public relations fear factor control. do you actually think they would spend the extra costs for fuel, for such extreme heat, just to eliminate smell, when they spread manure all over your veg's. i think not. what they really meant were any _TSE agents_.

b. Gas scrubbing to eliminate smoke -- though steam may be omitted;

c. Stacks to be fitted with grit arreaters;

snip...

1.2 Visual Imact

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

full text;

http://www.bseinquiry.gov.uk/files/yb/1989/04/03006001.pdf

http://europa.eu.int/comm/food/fs/sc/ssc/out311_en.pdf


Oral Transmissibility of Prion Disease Is Enhanced by Binding to Soil Particles

Christopher J. Johnson1,2, Joel A. Pedersen3, Rick J. Chappell4, Debbie McKenzie2, Judd M. Aiken1,2*

1 Program in Cellular and Molecular Biology, University of Wisconsin-Madison, Madison, Wisconsin, United States of America, 2 Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin, United States of America, 3 Department of Soil Science and Molecular and Environmental Toxicology Center, University of Wisconsin-Madison, Madison, Wisconsin, United States of America, 4 Biostatistics and Medical Informatics, University of Wisconsin Medical School, Madison, Wisconsin, United States of America

Soil may serve as an environmental reservoir for prion infectivity and contribute to the horizontal transmission of prion diseases (transmissible spongiform encephalopathies [TSEs]) of sheep, deer, and elk. 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 prions adsorbed to the common soil mineral montmorillonite (Mte) retain infectivity following intracerebral inoculation. Here, we assess the oral infectivity of Mte- and soil-bound prions. We establish that prions bound to Mte are orally bioavailable, and that, unexpectedly, binding to Mte significantly enhances disease penetrance and reduces the incubation period relative to unbound agent. Cox proportional hazards modeling 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 organic carbon-rich soil was equivalent to that of unbound agent. Enhanced transmissibility of soil-bound prions may explain the environmental spread of some TSEs despite the presumably low levels shed into the environment. Association of prions with inorganic microparticles represents a novel means by which their oral transmission is enhanced relative to unbound agent.

snip...


Discussion


These experiments address the critical question of whether soil particle­bound prions are infectious by an environmentally relevant exposure route, namely, oral ingestion. Oral infectivity of soil particle­bound prions is a conditio sine qua non for soil to serve as an environmental reservoir for TSE agent. The maintenance of infectivity and enhanced transmissibility when TSE agent is bound to the common soil mineral Mte is remarkable given the avidity of the PrPTSE­Mte interaction [22]. One might expect the avid interaction of PrPTSE with Mte to result in the mineral serving as a sink, rather than a reservoir, for TSE infectivity. Our results demonstrate this may not be the case. Furthermore, sorption of prions to complex whole soils did not diminish bioavailability, and in two of three cases promoted disease transmission by the oral route of exposure. While extrapolation of these results to environmental conditions must be made with care, prion sorption to soil particles clearly has the potential to increase disease transmission via the oral route and contribute to the maintenance of TSE epizootics.

Two of three tested soils potentiated oral prion disease transmission. The reason for increased oral transmissibility associated with some, but not all, of the soils remains to be elucidated. One possibility is that components responsible for enhancing oral transmissibility were present at higher levels in the Elliot and Bluestem soils than in the Dodge soil. The major difference between the Dodge soil and the other two soils was the extremely high natural organic matter content of the former (34%, [22]). The Dodge and Elliot soils contained similar levels of mixed-layer illite/smectite, although the contribution of smectite layers was higher in the Dodge soil (14%­16%, [22]). The organic matter present in the Dodge soil may have obstructed access of PrPTSE to sorption sites on smectite (or other mineral) surfaces.

The mechanism by which Mte or other soil components enhances the oral transmissibility of particle-bound prions remains to be clarified. Aluminosilicate minerals such as Mte do not provoke inflammation of the intestinal lining [39]. Although such an effect is conceivable for whole soils, soil ingestion is common in ruminants and other mammals [25]. Prion binding to Mte or other soil components may partially protect PrPTSE from denaturation or proteolysis in the digestive tract [22,40] allowing more disease agent to be taken up from the gut than would otherwise be the case. Adsorption of PrPTSE to soil or soil minerals may alter the aggregation state of the protein, shifting the size distribution toward more infectious prion protein particles, thereby increasing the specific titer (i.e., infectious units per mass of protein) [41]. In the intestine, PrPTSE complexed with soil particles may be more readily sampled, endocytosed (e.g., at Peyer's patches), or persorbed than unbound prions. Aluminosilicate (as well as titanium dioxide, starch, and silica) microparticles, similar in size to the Mte used in our experiments, readily undergo endocytotic and persorptive uptake in the small intestine [42­44]. Enhanced translocation of the infectious agent from the gut lumen into the body may be responsible for the observed increase in transmission efficiency.

Survival analysis indicated that when bound to Mte, prions from both BH and purified PrPTSE preparations were more orally infectious than unbound agent. Mte addition influenced the effective titer of infected BH to a lesser extent than purified PrPTSE. Several nonmutually exclusive factors may explain this result: (1) other macromolecules present in BH (e.g., lipids, nucleic acids, other proteins) compete with PrPTSE for Mte binding sites; (2) prion protein is more aggregated in the purified PrPTSE preparation than in BH [45], and sorption to Mte reduces PrPTSE aggregate size, increasing specific titer [41]; and (3) sorption of macromolecules present in BH to Mte influences mineral particle uptake in the gut by altering surface charge or size, whereas the approximately 1,000-fold lower total protein concentration in purified PrPTSE preparations did not produce this effect.

We previously showed that other inorganic microparticles (kaolinite and silicon dioxide) also bind PrPTSE [22]. All three types of microparticles are widely used food additives and are typically listed as bentonite (Mte), kaolin (kaolinite), and silica (silicon dioxide). Microparticles are increasingly included in Western diets. Dietary microparticles are typically inert and considered safe for consumption by themselves, do not cause inflammatory responses or other pathologies, even with chronic consumption, and are often sampled in the gut and transferred from the intestinal lumen to lymphoid tissue [39,46,47]. Our data suggest that the binding of PrPTSE to dietary microparticles has the potential to enhance oral prion disease transmission and warrants further investigation.

In conclusion, our results provide compelling support for the hypothesis that soil serves as a biologically relevant reservoir of TSE infectivity. Our data are intriguing in light of reports that naïve animals can contract TSEs following exposure to presumably low doses of agent in the environment [5,7­9]. We find that Mte enhances the likelihood of TSE manifestation in cases that would otherwise remain subclinical (Figure 3B and 3C), and that prions bound to soil are orally infectious (Figure 5). Our results demonstrate that adsorption of TSE agent to inorganic microparticles and certain soils alter transmission efficiency via the oral route of exposure.

snip...full text is here:

http://pathogens.plosjournals.org/perlserv/?request=get-document&doi=10.1371/journal.ppat.0030093

http://pathogens.plosjournals.org/perlserv/?request=get-pdf&file=10.1371_journal.ppat.0030093-L.pdf

http://pathogens.plosjournals.org/perlserv/?request=get-pdf&file=10.1371_journal.ppat.0030093-S.pdf


Greetings,

Considering all the above, and the fact that there are over 20 documented strains of the so called typical scrapie, with new and emerging atypical Scrapie i.e. the NOR-98, and not to forget BSE transmission to sheep and goat. CWD in deer and elk, and just how many strains do we have there? cBSE (typical), hBSE, both documented in North America, and the fact several outbreaks of TME in the USA, no l-BSE ? i question no l-BSE in the USA, due to the fact of the total bungling of the USDA's so called enhanced BSE surveillance program, and then the sudden dramatic decrease in the USDA BSE testing right after being forced to come clean on the negative atypical BSE case in Texas due to the Fong effect, and the Alabama atypical BSE case. i would bet my mothers life on the l-BSE being circulating among the other TSE strains in the USA, just undetected for all the obvious reasons. but how many other strains? now mind you, all of the above strains of TSE transmitting to primates, and mind you, the _typical_ scrapie transmitting to the primate by THERE NON-FORCED ORAL CONSUMPTION. oh yes, but don't forget, typical scrapie has never been documented to transmit to humans. r i g h t ! with the new and emerging human TSEs, some showing up right here in the USA, why is it that the human TSE (all age groups) why are they not all reportable in the USA in every state and Internationally? This must be done. the ramifications from proven transmission studies via the medical and surgical arena's are real, they have been real for some time. Blood now a real issue, now a threat. We must act now, let science take it's course, and put the politics and the industry aside, the conflicts of interest are just to great. these studies take too long due to the incubation period, for partisan, industry, political issues to stand in the way. people are dying. this old study, and some newer ones, always bring it home for me. there is much more to this story than the infamous UKBSEnvCJD hamburger eating adolescents only story. ...TSS




J Neurol Neurosurg Psychiatry. 1994 June; 57(6): 757–758. PMCID: PMC1072988

Copyright notice

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

C J Gibbs, Jr, D M Asher, A Kobrine, H L Amyx, M P Sulima, and D C Gajdusek Laboratory of Central Nervous System Studies, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892


Abstract

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.

FULL TEXT ;

http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1072988&blobtype=pdf



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: cathrin.bruederle@gmail.com

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.




Abstract

The epidemic of bovine spongiform encephalopathy (BSE) has led to a world-wide drop in the market for beef by-products, such as Meat-and-Bone Meal (MBM), a fat-containing but mainly proteinaceaous product traditionally used as an animal feed supplement. While normal rendering is insufficient, the production of biodiesel from MBM has been suggested to destroy infectivity from transmissible spongiform encephalopathies (TSEs). In addition to producing fuel, this method simultaneously generates a nutritious solid residue. In our study we produced biodiesel from MBM under defined conditions using a modified form of alkaline methanolysis. We evaluated the presence of prion in the three resulting phases of the biodiesel reaction (Biodiesel, Glycerol and Solid Residue) in vitro and in vivo. Analysis of the reaction products from 263K scrapie infected MBM led to no detectable immunoreactivity by Western Blot. Importantly, and in contrast to the biochemical results the solid MBM residue from the reaction retained infectivity when tested in an animal bioassay. Histochemical analysis of hamster brains inoculated with the solid residue showed typical spongiform degeneration and vacuolation. Re-inoculation of these brains into a new cohort of hamsters led to onset of clinical scrapie symptoms within 75 days, suggesting that the specific infectivity of the prion protein was not changed during the biodiesel process. The biodiesel reaction cannot be considered a viable prion decontamination method for MBM, although we observed increased survival time of hamsters and reduced infectivity greater than 6 log orders in the solid MBM residue. Furthermore, results from our study compare for the first time prion detection by Western Blot versus an infectivity bioassay for analysis of biodiesel reaction products. We could show that biochemical analysis alone is insufficient for detection of prion infectivity after a biodiesel process.

snip...


DiscussionDecontamination of pathogenic prions has turned out to be a challenging endeavor. Prions are known to be unusually resistant to common decontamination methods. BSE is believed to be a result of insufficient decontamination and rendering methods of ruminant coproducts that were used as animal feed. Although this led to a devastating feed-borne epidemic among cattle, a major concern here is the overwhelming evidence for the zoonotic transmission of bovine prions to humans [20]. Total elimination of TSEs requires methods that completely destroy any potential prion infectivity in a large scale format. Production of biodiesel from bovine fat and brain tissue has been proposed to be a useful tool for decontamination of prions resulting in safe biodiesel [21]. In our study we evaluated an inexpensive large scale method (in situ transesterification) for production of biodiesel for TSE decontamination potential. Furthermore we investigated potential infectivity present not only in the biodiesel but also in the two other phases developed from the process, a solid MBM residue and glycerol. The solid MBM residue is of particular interest for its potential as a nutritious feed additive for ruminants such as cattle. In our hands, under optimal conditions for transesterification, the solid MBM residue retained 7% of the initial triglyceride and 90% of the initial protein content [17]

The alkaline methanolysis method efficiently produced biodiesel from MBM spiked with hamster brain and the method eliminated PrPsc detection in all products as determined by Western blot. Our biochemical results are comparable to previous studies, at least with regards to the biodiesel and glycerol phase [15]. Biodiesel and glycerol products had no detectable infectivity in our long term animal assay (survival>200d). In contrast to the biodiesel and glycerol phase, we show that the remaining solid MBM residue that had been spiked with scrapie brain retained infectivity in our sensitive bioassay. All animals inoculated with the infected solid MBM residue developed scrapie. However, increased survival time suggests the reaction did reduce infectivity in solid MBM residue from 10-3 ID50 to 10-9 ID50 (a partial decontamination of ~6 logs), based on a standard hamster survival curve that we established in our laboratory according to previous reported results [18]. The broad distribution of time-to-death for these animals is likely due to uneven distribution of infectious material in the inoculum, as the residue produced a relatively coarse suspension in the syringe. We suggest that, in addition to disinfection by the alkaline methanolysis reaction, we observe significant partitioning of infectivity, from the liquid phases into the solid residue. Another possible explanation for increased survival of animals inoculated with the solid MBM residue could be a high binding affinity of the prion protein to MBM and thus a sustained release from MBM in the brain. A phenomenon like this was described previously for prion binding to soil minerals [22]. In our study, when spiked into MBM, PrPsc was only detectable by Western Blot after boiling of sample in detergent. On the other hand we could show that control animals that received infected MBM not subjected to the reaction (MBM sc) developed disease in a time frame comparable to a standard scrapie brain homogenate.

Our results clearly show that Western Blot detection alone is insufficient to conclude on the absence of infectious prion, particularly when assessing a grossly heterogeneous sample such as MBM. This study illustrates that lack of prion detection in vitro does not necessarily exclude infectivity as determined by bioassay.

Furthermore the residual scrapie infectivity detected in the solid MBM residue probably limits the use of ruminant MBM as a feed additive to only non-ruminants, such as fish and fowl, as they are not susceptible to TSEs. Relatively minor variations of this reaction (e.g., more heat and/or alkali) may prove fully effective for complete destruction of infectivity in the solid MBM residue, but must be cost-effective if suspect MBM is to be considered as a ruminant feed additive.


http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2493038



RESULTS ON HUMAN SCLERA

SEAC considered preliminary results provided by the HPA and National CJD Surveillance Unit from tests on eye tissue (sclera) from a vCJD case. The results suggest the presence of infectivity and, in contrast with previous testing of samples from other vCJD cases, abnormal prion protein in this tissue. However, as the sclera is very difficult to remove from surrounding eye tissues, which are themselves known to carry vCJD infectivity, the findings may have arisen as a result of contamination at autopsy. Nevertheless, even if the data are reliable, they indicate that there may only be a relatively low level of infectivity present in sclera. As preliminary unpublished data were considered, this issue was discussed in a reserved business session in accordance with the SEAC Code of Practice.5 Gambetti et al. (2008) A novel human disease with abnormal prion protein sensitive to protease. Ann. Neurol. 63, 697-708. 5 © SEAC 2008


SEE FULL TEXT ;


http://www.seac.gov.uk/papers/101-summary.pdfhttp://www.mad-cow.org/dec99_news.html#bbbhttp://creutzfeldt-jakob-disease.blogspot.com/2008/01/cjd-hgh-body-snatchers.html


5.5 There is now convincing evidence of human to human transmission of vCJD via blood transfusion with 3 clinical cases of the disease and one of sub-clinical infection believed to have been transmitted via this route. However, in humans little is known about the level, distribution and temporal development of infectivity in blood. Estimates of prevalence of asymptomatic infection in the UK population remain uncertain, as does the susceptibility of recipients to infection. 5.6 To assess the cost-effectiveness of future measures to reduce the risk of vCJD by blood components 8 scenarios relating to prevalence, susceptibility and infectivity were modelled: a prevalence of 1:20,000 (LOW) and1:4000 (HIGH), infectivity of 0.1 ID/ml (LOW) and 30 ID/ml (HIGH), and susceptibility of recipients to development of clinical disease of 10% (LOW) and 100% (HIGH). It was noted that the high susceptibility scenario is not consistent with the observed number of clinical cases. It was noted that SEAC reviewed data available to date from The National Anonymised Tonsil Archive (NATA) Study at their meeting on 25th April 2008 and has not revised its estimate of prevalence of sub-clinical infection as a result.


http://www.advisorybodies.doh.gov.uk/acsbto/2nd_meeting_minutes_290408.pdf


these minutes are not available yet ;1st Public Meeting of SaBTO (Advisory Committee on the Safety of Blood, Tissues and Organs)Tuesday 21st October 2008, 2pm-4pm


http://www.advisorybodies.doh.gov.uk/acsbto/Public_Meeting-21_October_2008.htm


vCJD case study highlights blood transfusion risk Friday 8th December 2006


http://www.mrc.ac.uk/consumption/groups/public/documents/content/mrc003431.pdf


Public release date: 29-Aug-2008Contact: Tara Womersley tara.womersley@ed.ac.uk 44-131-650-9836 University of Edinburgh

Study confirms vCJD could be transmitted by blood transfusion

A 9-year study in sheep has added to the evidence that vCJD can be transmitted through blood transfusion in humans The findings underline the importance of precautions against vCJD transmission, such as the Government decision in 2004 to ban blood donations from anyone who had received a blood transfusion since 1980.The study published in Blood, the journal of the American Society of Hematology, looked at BSE transmission between sheep through infected blood with the aim of quantifying how vCJD - the human form of BSE - could be spread through transfusions.Researchers (Fiona Houston, Nora Hunter and colleagues) at the Neuropathogenesis Unit at the Institute of Animal Health, which is now part of The Roslin Institute, University of Edinburgh, found that the likelihood of BSE being transmitted between sheep through transfusion of infected sheep blood was 36 per cent, with rates of 43 per cent found for scrapie.Fiona Houston, now at the University of Glasgow, who led the research, said: "It is apparent that the stage of disease incubation in infected donors played a large role in the likelihood of transmission. The longer that BSE or scrapie had been carried by donors, the greater the likelihood of the disease being transmitted with transfusions of infected blood."While cases of vCJD are tailing off there are concerns that up to 4,000 people could be carrying the disease in the UK, which could then be transmitted through infected blood causing further infections.Scientists are working to develop a test for vCJD that can be used before symptoms develop and a filter is also being trialled to remove prions - infective proteins - from donated blood.Dr Houston said: "The study shows that, for sheep infected with BSE or scrapie, transmission rates via blood transfusion can be high, particularly when donors are in the later stages of infection. This suggests that blood transfusion represents an efficient route of transmission for these diseases and justifies the current control measures put in place to safeguard human blood supplies."While it may not correlate directly to what happens in the human population, due to factors such as species differences in genetic susceptibility to disease, it provides greater insight into the role of how vCJD may be carried through infected blood. By understanding how vCJD can be transmitted through blood transfusions, we can ensure the most effective control measures to minimise human to human infection."BSE is one of a group of rare neurodegenerative disorders called transmissible spongiform encephalopathies (TSEs), which include scrapie and vCJD. Of 22 sheep that received BSE infected blood, eight showed evidence of infection. Nine out of 21 sheep receiving scrapie-infected blood developed the disease.To date 167 cases of vCJD have been recorded in the United Kingdom, of which three patients are thought to have received vCJD through infected blood.### Tara Womersley, Press and PR office, University of Edinburgh, Tel 0131 650 9836 or 07791 355 804 Email: Tara.womersley@ed.ac.ukThe University of Edinburgh is a charitable body, registered in Scotland, with registration number SC005336.


http://www.eurekalert.org/pub_releases/2008-08/uoe-scv082908.php


Tuesday, November 11, 2008

SaBTO Summary of 1st Public Meeting - variant CJD and blood Tuesday 21st October 2008, 2pm-4pm

http://vcjdblood.blogspot.com/2008/11/sabto-summary-of-1st-public-meeting.html


Thursday, October 23, 2008
Creutzfeldt-Jakob Disease Associated with Cadaveric Dura Mater Grafts - Japan, 1979-2008 : UPDATE

http://creutzfeldt-jakob-disease.blogspot.com/2008/10/creutzfeldt-jakob-disease-associated.html


SEAC TSE DENTAL


7. SEAC considered that the experiments appear well designed and the conclusions justified and reliable, while recognising that the research is incomplete and confirmatory experiments have yet to be completed. It is recommended that the research be completed, submitted for peer-review and widely disseminated as soon as possible so others can consider the implications. Nevertheless, these preliminary data increase the possibility that some oral tissues of humans infected with vCJD may potentially become infective during the preclinical stage of the disease. In addition, they increase the possibility that infection could potentially be transmitted not only via accidental abrasion of the lingual tonsil or endodontic procedures but a variety of routine dental procedures. Implications for transmission risks

8. The new findings help refine assumptions made about the level of infectivity of dental pulp and the stage of incubation period when it becomes infective in the risk assessment of vCJD transmission from the reuse of endodontic files and reamers10. For example, if one patient in 10 000 were to be carrying infection (equivalent to about 6 000 people across the UK – the best current estimate11), the data suggest that in the worst case scenario envisaged in the risk assessment, re-use of endodontic files and reamers might lead to up to 150 new infections per annum. It is not known how many of those infected would go on to develop clinical vCJD. In addition, transmission via the re-use of endodontic files and reamers could be sufficiently efficient to cause a self-sustaining vCJD epidemic arising via this route.



http://www.seac.gov.uk/statements/state-vcjd-dentrstry.htm

http://www.seac.gov.uk/papers/paper100-2.pdf


TSS