Evaluation of the application for new alternative biodiesel production process for rendered fat including Category 1 animal by-products (BDI-RepCat® process, AT)
Published: 15 April 2021
Adopted: 10 March 2021
EFSA Journal 2021;19(4):6511
Keywords: animal by-product, ABP, biodiesel, prion, TSE, methanol, tallow, category 1
On request from: Austrian Competent Authority
Question Number: EFSA-Q-2020-00450
Biological Hazards Contact:
A new alternative method for the production of biodiesel from rendered fat, including animal by-product (ABP) Category 1 tallow, was evaluated. The method consists of a conversion phase, based on esterification and transesterification in a single step (at temperature ≥ 200°C, pressure ≥ 70 bar with a retention time ≥ 15 min), using MgO as a catalyst and in the presence of methanol (10–15%), followed by vacuum distillation (at ≥ 150°C, ≤ 10 mbar) of the end-product, biodiesel and the co-product, glycerine. Prions (PrPSc), which are abnormal isoforms of the prion protein, were considered by the applicant to be the most resistant hazard. In accordance with previous EFSA Opinions and current expert evaluation, a reduction in prion infectivity, or detectable PrPSc, of at least 6 log10 should be achieved for the process to be considered equivalent to the processing method laid down in the Regulation (EU) No 142/2011. Published data from an experimental replication of the conversion step of the biodiesel production process under consideration were provided, which showed an at least 6 log10 reduction in detectable PrPSc, by Western blot, in tallow that had been spiked with murine and human prion strains. In addition, it was demonstrated that the presence of methanol does not affect the recovery or detection of PrPSc from a biodiesel substrate. Based on scientific literature, the vacuum distillation step has been shown to be capable of achieving an additional 3 log10 reduction in PrPSc. Therefore, the proposed alternative method is considered to be at least equivalent to the processing method laid down in the legislation for the production of biodiesel from raw materials including Category 1 ABP.
© European Food Safety Authority, 2021
On 10 June 2020, the European Food Safety Authority (EFSA) received from the Austrian Competent Authority (Federal Ministry of Social Affairs, Health, Care and Consumer Protection) an application (mandate and technical dossier: EFSA-Q-2020-00450) under Regulation (EC) No 1069/2009 referring to the evaluation of an alternative method for the production of biodiesel from rendered fat, including animal by-product (ABP) Category 1 tallow, submitted by BDI-BioEnergy International GmbH(hereinafter referred to as the applicant).
The proposed new method will be applied using feedstock treated according to method 1 (pressure sterilisation) and after removal of insoluble impurities in excess of 0.15% w/w. The process, called RepCat®, consists of a conversion phase of esterification and transesterification in a single step (at temperature ≥ 200 °C, pressure ≥ 70 bar with a retention time ≥ 15 min), using MgO as a catalyst and in the presence of methanol (10–15%), followed by vacuum distillation (at ≥ 150°C, ≤ 10 mbar) of the end-product, biodiesel.
The material to be treated is rendered fat, including ABP Category 1 tallow. Under Article 20 of Regulation (EC) No 1069/2009, EFSA is required to assess whether the method submitted ensures that any risks to public or animal health are reduced to a degree that is at least equivalent to that achieved by the processing method laid down in the legislation for the same category of ABP. Prions (PrPSc), which are the abnormal isoforms of the prion protein, were considered to be the most resistant hazard by the applicant and the BIOHAZ Panel agreed with the approach of focusing on the capability of the alternative method to reduce prion infectivity or detectable PrPSc. In accordance with previous EFSA Opinions and current expert evaluation, a reduction in prion infectivity, or detectable PrPSc, of at least 6 log10 should be achieved for the process to be considered at least equivalent to the processing method laid down in Regulation (EU) No 142/2011.
The applicant provided data in the form of published studies in which it was shown that methanol does not affect the recovery or detection of prions from a biodiesel substrate, the RepCat®esterification and transesterification process can achieve a reduction in prions of at least 6 log10, as detected by Western blot, and vacuum distillation can provide a further reduction of 3 log10.
The application includes sufficient information on the HACCP plan and the risks of interdependent processes and those associated wit h the intended end use of biodiesel. The measures proposed in the dossier to deal with these risks are compliant with the relevant legislation.
The application and supporting references have been thoroughly reviewed and the data provided support the conclusion that the RepCat®process can achieve at least a 6 log10 reduction of the hazard. The process can therefore be considered at least equivalent to the processing method laid down in the legislation for the production of biodiesel from raw materials including Category 1 AB P
•The method under assessment (RepCat®process) for the production of biodiesel from rendered animal fats, including animal by-product (ABP) Category 1 tallow, consists of esterification and transesterification at ≥ 200°C, ≥ 70 bar, ≥ 15 min in a single step, using MgOas a catalyst and in the presence of methanol (10–15%), followed by vacuum distillation (at≥ 150°C, ≤ 10 mbar) of the end-product biodiesel.
•Since the starting material includes Category 1 ABP tallow, the applicant considered that, of any biological hazards that may be present, prions would be the most resistant. The BIOHAZ Panel agrees with the approach used by the applicant of focusing on prions.
•Previous EFSA Opinions established that a reduction in prion infectivity, or detectable PrPSc of at least 6 log10 should be achieved to consider a method at least equivalent, for the relevant category of ABP (i.e. Category 1), to the processing method laid down in the legislation.
•The applicant provided evidence in the form of published studies in which it was shown that methanol does not affect the recovery or detection of prions from a biodiesel substrate. It was also shown that the RepCat®esterification and transesterification process can achieve a reduction in sCJD(MM1) prions of at least 6 log10, as estimated through Western blot, from the complete loss of recoverable PrPSc, and that vacuum distillation can provide a further reduction of 3 log10.
•The application includes sufficient information on the HACCP plan and about the risks of interdependent processes and those associated with the intended end use of biodiesel. The measures proposed in the dossier to deal with these risks are compliant with the relevant legislation.
•The alternative method is capable of achieving at least the 6 log10 reduction in detectable PrPSc that enables it to be considered at least equivalent to the processing method laid down in the legislation for the production of biodiesel from raw materials including category 1 ABP
Evaluation biodiesel Cat. 1 ABP (BDI-RepCat®, AT)
Appendix A – Methodological limitations of prion-related hazard reduction assessments Inactivation studies based on experimentally derived laboratory prion strains, and using WB to assess the presence of detectable PrPSc following treatment, are increasingly being considered suboptimal as predictors of the resistance of natural TSE agents when they are subjected to any particular inactivation protocol, due to the inherently limited analytical sensitivity and biological relevance of WB for the detection of prion infectivity (Marın-Moreno et al., 2019; EFSA BIOHAZ Panel,2020a,b). However, similar methods have been used in previous inactivation studies and appear in official guidelines (EMA, 2019 ). The latter acknowledges that most studies aimed at following the partition/removal of PrPSc and/or infectivity during plasma fractionation processes also use ‘rodent-adapted TSE agent (263K hamster strain) brain homogenate and microsomal brain fractions as a spike and rely on direct [PrPSc] immunodetection tools (WB or conformation-dependent immunoassay) to demonstrate a drop in the TSE agent content in processed fractions and on bioassay infectivity measurements to confirm the results’.
Historically, EFSA opinions have used the terms ‘infectivity’ and ‘the presence of detectable abnormal form of PrP (PrPSc) as a proxy for infectivity ’ interchangeably, but data now support the view that they cannot be considered biologically interchangeable. The presence of PrPSc in tissues is generally indicative of the presence of TSE infectivity, but there is not an absolute correlation between the amount of detectable PrP and the amount of detectable infectivity. It has been demonstrated that high titres of TSE infectivity can be present in brain tissue from animals that show clinical and vacuolar signs of TSE disease but contain low or undetectable levels of PrPSc (Barron et al., 2007). Also, in many cases, during the incubation phase of the disease, infectivity (as detected by the rodent bioassay) can be demonstrated before detectable accumulation of PrPSc (as determined using existing in vitro detection methods). In addition, the structure of the polymer aggregates of different strains results in different levels of infectivity as shown for fast hamster prions (Riesner et al., 1996) and confirmed more recently by Tixador et al. (2010), in what has been described as the decoupling between infectivity measured by bioassay and the amount of PK-resistant PrPSc estimated by WB, with respect to the size of particles.
While a few well-characterised rodent-adapted strains were widely used for decontamination/inactivation studies at the start of the bovine spongiform encephalopathy (BSE) epidemic, it has subsequently been shown that individual strains can respond very differently to different physico-chemical processes, and extrapolation from one strain to another should be carried out with extreme caution, unless there are robust parallel studies that allow direct comparisons (EFSA BIOHAZ Panel,2020a). This also applies to prions derived from naturally occurring TSE. Thus, it cannot be assumed that even prions from the same host species, e.g. classical BSE (C-BSE), or atypical BSE (L-BSE and H-BSE), will respond similarly to such processes. In fact, the unique comparative study that has been undertaken for these strains and their response to Method 1 sterilisation indicated substantial differences in outcome (Chapman et al., 2020).
The BIOHAZ Panel has performed evaluations of several applications involving the use of Category1 material for the production of fuels, and other oleochemical processes (EFSA BIOHAZ Panel, 2010b,2011, 2015, 2017, 2020b). The evaluation published in 2015 included an extensive description of the opinions published prior to that. In the more recent opinions, it was noted that there was a limitation of data in the scientific literature that hampered the assessment of the required infectivity reduction in the proposed processes in some of the cases (EFSA BIOHAZ Panel, 2015, 2017, 2020b).
In 2020, an alternative method for production of biodiesel from processed fats from Category 1, 2 and 3 animal by-products was assessed (EFSA BIOHAZ Panel, 2020b). Experimental data from studies commissioned to quantify the required reduction in detectable PrPSc in material spiked with a TSE strain and measured by WB were presented. The proposed method was considered to be at least equivalent to the methods previously approved to produce biodiesel from all categories of animal by-products, although it was subject to the methodological caveats described above.
New in vitro methods are now being developed that may offer improved alternatives to bioassays. However, until the relevant guidance documents are updated with regard to the need to use situation-specific strains, and comparatively evaluated quantification methods, the BIOHAZ Panel considers that assessments using proxy strains such as rodent-adapted prion strains and WB detection methods remain a necessarily acceptable approach. Despite the methodological caveats that may lead to an under-/overestimation of the actual level of hazard reduction, they still enable comparison of new biodiesel methodologies relative to pre-existing approved methods that have been assessed by similar means.
Appendix B – Extract from 2017 EFSA BIOHAZ Panel Scientific Opinion Extract verbatim from the EFSA BIOHAZ Panel ‘Scientific Opinion on the evaluation of the Application for new alternative biodiesel production process for rendered fat of Cat 1 (BDI-RepCatprocess, AT)’ (EFSA BIOHAZ Panel, 2017):
‘3.3 Full description of the material to be treated
Category 1 ABP tallow, pretreated according to Method 1, is used as feedstock with a maximum permitted level of insoluble impurities of 0.15%.
3.4 Hazard identification Although the Applicant did not present the complete hazard identification, taking into consideration the type of process, TSEs must be considered the most relevant hazards. This application is specifically aimed at using Category 1 animal fat, a high-risk material due to the potential presence of TSE agents. The method proposed by the Applicant is suitable for all kinds of animal fats as a feedstock. Besides TSE agents, Category 1 material can contain other biological hazards (including some highly heat-resistant bacterial spores and viruses). However, given the high resistance to destruction, and, in particular, the high thermostability of the infectious agents causing TSEs (Somerville et al., 2009), it is assumed that if the alternative method ensures the inactivation of the TSE agent, then all microorganisms, including spore-forming bacteria and thermo resistant viruses, will be completely inactivated. Therefore, the focus will be on the risk reduction in relation to TSE agents.
3.5.3 Assessment of the level of risk reduction (distillation)[...] The distillation phase of the RepCat process is equivalent to the distillation step of the approved biodiesel production process and similar to the one described by Mittelbach et al. (2007). A 3 log10 reduction factor in PrP27– 30 was obtained by these authors, and therefore, a similar level of PrP27–30 reduction could be expected for the distillation phase of the proposed process.’
SCIENTIFIC OPINION ADOPTED: 19 October 2017 doi: 10.2903/j.efsa.2017.5053
Evaluation of the Application for new alternative biodiesel production process for rendered fat of Cat 1 (BDI-RepCat process, AT)
EFSA Panel on Biological Hazards (BIOHAZ), Antonia Ricci, Ana Allende, Declan Bolton, Marianne Chemaly, Robert Davies, Rosina Girones, Lieve Herman, Konstantinos Koutsoumanis, Roland Lindqvist, Birgit Nørrung, Lucy Robertson, Giuseppe Ru, Moez Sanaa, Marion Simmons, Panagiotis Skandamis, Emma Snary, Niko Speybroeck, Benno Ter Kuile, John Threlfall, Helene Wahlstrom, Avelino Alvarez Ordo € nez, ~ John Griffin, John Spiropoulos, Emmanuel Vanopdenbosch, Sandra Correia and Pablo Salvador Fernandez Esc amez
A new alternative method for the production of biodiesel from rendered fat of all categories of animal by-products was assessed. The process was compared to the approved biodiesel production process described in Chapter IV Section 2 D of Annex IV of Commission Regulation (EU) 142/2011. Tallow derived from Category 1 material is treated according to Method 1 from the same Regulation (133°C, 20 min, 3 bar) and subsequently mixed with 15% methanol, heated to reaction temperature (220°C) in several heat exchangers and transferred into the continuous conversion reactor by means of a high pressure pump (80 bar) for 30 min. In the conversion phase, there is an exposure to methanol in the absence of alkaline or acidic conditions. The impact of this procedure on the thermostability of transmissible spongiform encephalopathy (TSE) has not been assessed in the literature. After the reaction, the biodiesel/glycerol mixture is distilled under vacuum at a minimum temperature of 150°C and a maximum pressure of 10 mbar, which is equivalent to the distillation step in the approved biodiesel production process, for which a 3 log10 reduction factor in PrP27–30 was obtained. Therefore, a similar level of TSE infectivity reduction could be expected for that phase of the method. A previous EFSA Opinion established that a reduction of 6 log10 in TSE infectivity should be achieved by any proposed alternative method in order to be equivalent to the approved processing method. This level of reduction has not been shown with experimental trials run under conditions equivalent to the ones described for the RepCat process. It was not possible to conclude whether or not the level of TSE infectivity reduction in the RepCat process is at least of 6 log10. Therefore, it was also not possible to conclude about the equivalence with the approved biodiesel production process.
© 2017 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority. Keywords: animal by-product, category 1, TSE, risk reduction, biodiesel, rendered fat, conversion Requestor: European Commission Question number: EFSA-Q-2017-00136 Correspondence: firstname.lastname@example.org
Title: Prion infected Meat-and-Bone Meal is still infectious after biodiesel production
Author item Bruederle, Cathrin item Hnasko, Robert item KRAEMER, THOMAS item Garcia, Rafael item Haas, Michael item Marmer, William item Carter, John Submitted to: PLoS ONE Publication Type: Peer Reviewed Journal Publication Acceptance Date: 7/24/2008 Publication Date: 8/13/2008 Citation: Bruederle, C.E., Hnasko, R.M., Kraemer, T., Garcia, R.A., Haas, M.J., Marmer, W.N., Carter, J.M. 2008. Prion infected Meat-and-Bone Meal is still Infectious After Biodiesel Production. PLoS One 3(8): e2969. doi:10.1371/journal.pone.0002969. Interpretive Summary: The problem addressed by this research is safe disposal of slaughterhouse by-products. Some parts of cattle and sheep have low commercial value as human food. Much of this material has traditionally been processed into Meat-and-Bone Meal (MBM), a highly nutritious animal feed supplement. But MBM was linked to the spread of Bovine Spongiform Encephalopathy (BSE), and the BSE epidemic in the UK and EU was probably ended by banning MBM from feed. This has created a problem for MBM as an agricultural commodity. MBM is high in fat, and rising prices for crude oil have recently created a favorable market shift for biodiesel, which can be produced from this fat through a simple but harsh chemical reaction. Biodiesel is usually manufactured from relatively pure fat or oil, but we found the reaction worked equally well on MBM. We then used a hamster scrapie model of BSE, with two different methods to test whether the biodiesel reaction also destroyed infectivity in the MBM. When we used antibody-based detection (Western blot) we were unable to detect the proteinase-resistant protein marker for disease in the biodiesel or solid residue. This result agrees with published reports implying the method produces safe biodiesel. When we used a bioassay (intracranial inoculation) we found that the biodiesel method destroyed all infectivity in the biodiesel and 99.9999% of the infectious material in the solid residue. This suggests the biodiesel fraction is safe for use as fuel. However, this level of decontamination/disinfection is unfortunately not sufficient to allow use of the solid residue as a feed supplement for cows and sheep. We think minor changes to the biodiesel recipe, such as increased time and temperature, could make it strong enough to completely decontaminate MBM.
Technical Abstract: The epidemic of bovine spongiform encephalopathy (BSE) has led to world-wide drop in the market for beef by-products, such as Meat-and-Bone Meal (MBM), a fat-containing 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 vacuolization. Re-inoculation of these brains into a new cohort of hamsters led to onset of clinical scrapie symptoms within 75 days, suggesting that the intrinsic properties of the prion protein were not changed during the biodiesel process. Results from our study demonstrate biochemical detection by Western Blot is insufficient to conclude an absence of TSE infectivity. Furthermore the biodiesel reaction can not be considered a viable prion decontamination method despite increased survival time of hamsters and reduced infectivity greater than 6 log orders in the solid MBM residue.
Prion Infected Meat-and-Bone Meal Is Still Infectious after Biodiesel Production
Cathrin E. Bruederle1 *, Robert M. Hnasko1 , Thomas Kraemer2 , Rafael A. Garcia3 , Michael J. Haas3 , William N. Marmer3 , John Mark Carter1
1 USDA-ARS WRRC, Foodborne Contaminants Research Unit, Albany, California, United States of America, 2 Forensic Toxicology, Institute of Legal Medicine, Saarland University, Homburg/Saar, Germany, 3 USDA-ARS ERRC, Fats, Oils and Animal Coproducts Research Unit, Wyndmoor, Pennsylvania, United States of America
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.
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.
Citation: Bruederle CE, Hnasko RM, Kraemer T, Garcia RA, Haas MJ, et al. (2008) Prion Infected Meat-and-Bone Meal Is Still Infectious after Biodiesel
Production. PLoS ONE 3(8): e2969. doi:10.1371/journal.pone.0002969
Editor: Neil Mabbott, University of Edinburgh, United Kingdom
Received April 21, 2008; Accepted July 24, 2008; Published August 13, 2008
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.
Funding: CRIS 5325-32000-007-00D and CRIS 5325-32000-008-00D
Competing Interests: The authors have declared that no competing interests exist.
TOP STUDY ***> Published data from an experimental replication of the conversion step of the biodiesel production process under consideration were provided, which showed an at least 6 log10 reduction in detectable PrPSc, by Western blot, in tallow that had been spiked with murine and human prion strains.
BOTTOM STUDY***> 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.
Biodiesel from Specified Risk Material Tallow: An Appraisal of TSE Risks and their Reduction
This document presents a systematic survey of current knowledge about the risk to human and animal health posed by the processing of tissues from animals potentially infected with transmissible spongiform encephalopathy (TSE, or ‘prion disease’) into biodiesel. It is organised into an introductory background section on TSE, followed by chapters treating the sequential stages of biodiesel production. The principal conclusions are:
Animal tissue sources. The choice of geographic origin, based on published scientific evaluations of the risk of TSE to be present in a given country, can largely reduce or even eliminate the entry of contaminated tissue into the biodiesel feedstock. Further safeguards can be provided by selection of animal species not susceptible to TSE, and of tissues without any detectable infectivity even in susceptible species. None of these measures, however, would be applied to the biodiesel projects under consideration, which have the specific aim of using animals and tissues (including specified risk material, or SRM) considered to have sufficient potential risk to be unacceptable for use in food, feed, fertilisers, or pharmaceuticals (including biologicals and medical devices) and therefore are designated for other approved uses, or destruction. Tissue rendering to produce tallow. Experiments to test the survival of TSE infectivity in the products of rendering failed to detect any infectivity in the crude tallow fraction, even following processing methods that allowed survival of infectivity in the co-produced meat and bone meal fraction. It is therefore extremely unlikely that pure tallow originating from diseased animals would be infectious; however, lower grade tallow might contain infectious impurities. A requirement that tallow derived from SRM for use in biodiesel contain no detectable protein would reduce this possibility.
Transesterification of tallow to biodiesel. Several steps in the manufacturing process have at least the potential to inactivate TSE infectivity, for example, exposure to strong acids or bases and, depending on the process, exposure to high temperature or purification by chromatography or precipitation. However, the two-phase aqueouslipidic nature of transesterification introduces an element of uncertainty about the effect of these procedures on infectivity.
Biodiesel use. Combustion temperatures, even at millisecond exposures, might partially inactivate contaminated biodiesel. But experimental data have so far been limited to saline tissue suspensions of infected tissues and several minute exposure times in static rather than dynamic combustion processes. A substantial body of epidemiological and laboratory evidence indicates that TSE is not transmitted by aerosol inhalation.
Overall conclusion. Biodiesel produced from animals infected with TSE poses a negligible risk to animal and public health. This conclusion extends even to the use of SRM as a source of tallow, based on experimental evidence showing that rendered tallow from infected animal tissues does not transmit disease to inoculated susceptible animals. Although infectivity reductions during biodiesel manufacturing steps should therefore be redundant, any such reduction would still be desirable as an added measure of safety. At present, the potential for infectivity reduction through biodiesel manufacturing and combustion can only be estimated from analogy to methods known to inactivate infectivity in saline suspensions of infected tissue. Studies of the actual biodiesel process, using experimentally contaminated input tallow, are recommended as the only means by which a scientifically-based conclusion can be made about the capacity of these processes to reduce or eliminate TSE infectivity.
6.1.4 Use - Biodiesel Combustion The effect of combustion in a diesel engine at more than 600 ºC could have some TSE inactivating capacity; however, the very short dwell time at high temperature may be insufficient for complete inactivation to occur if the fuel were to be contaminated by a TSE agent. Although all evidence to date indicates that TSE is not transmissible by inhalation, the potential risk of contaminated fuel aerosols cannot be entirely dismissed, particularly in closed environments.
Aerosols Transmit Prions to Immunocompetent and Immunodeficient Mice
Johannes Haybaeck1.¤a , Mathias Heikenwalder1.¤b , Britta Klevenz2., Petra Schwarz1 , Ilan Margalith1 , Claire Bridel1 , Kirsten Mertz1,3, Elizabeta Zirdum2 , Benjamin Petsch2 , Thomas J. Fuchs4 , Lothar Stitz2 *, Adriano Aguzzi1 * 1 Department of Pathology, Institute of Neuropathology, University Hospital Zurich, Zurich, Switzerland, 2 Institute of Immunology, Friedrich-Loeffler-Institut, Tu¨bingen, Germany, 3 Department of Pathology, Clinical Pathology, University Hospital Zurich, Zurich, Switzerland, 4 Department of Computer Science, Machine Learning Laboratory, ETH Zurich, Zurich, Switzerland
Prions, the agents causing transmissible spongiform encephalopathies, colonize the brain of hosts after oral, parenteral, intralingual, or even transdermal uptake. However, prions are not generally considered to be airborne. Here we report that inbred and crossbred wild-type mice, as well as tga20 transgenic mice overexpressing PrPC , efficiently develop scrapie upon exposure to aerosolized prions. NSE-PrP transgenic mice, which express PrPC selectively in neurons, were also susceptible to airborne prions. Aerogenic infection occurred also in mice lacking B- and T-lymphocytes, NK-cells, follicular dendritic cells or complement components. Brains of diseased mice contained PrPSc and transmitted scrapie when inoculated into further mice. We conclude that aerogenic exposure to prions is very efficacious and can lead to direct invasion of neural pathways without an obligatory replicative phase in lymphoid organs. This previously unappreciated risk for airborne prion transmission may warrant re-thinking on prion biosafety guidelines in research and diagnostic laboratories.
In summary, our results establish aerosols as a surprisingly efficient modality of prion transmission. This novel pathway of prion transmission is not only conceptually relevant for the field of prion research, but also highlights a hitherto unappreciated risk factor for laboratory personnel and personnel of the meat processing industry. In the light of these findings, it may be appropriate to revise current prion-related biosafety guidelines and health standards in diagnostic and scientific laboratories being potentially confronted with prion infected materials. While we did not investigate whether production of prion aerosols in nature suffices to cause horizontal prion transmission, the finding of prions in biological fluids such as saliva, urine and blood suggests that it may be worth testing this possibility in future studies.
Prion transmission via aerosols
Prion aerosols were produced by a nebulizing device with brain homogenates at concentrations of 0.1–20% (henceforth always indicating weight/volume percentages) derived from terminally scrapie-sick or healthy mice, and immitted into an inhalation chamber. As per the manufacturer's specifications, aerosolized particles had a maximal diameter of <10 µm, and approximately 60% were <2.5 µm .
Groups of mice overexpressing PrPC (tga20; n = 4–7) were exposed to prion aerosols derived from infectious or healthy brain homogenates (henceforth IBH and HBH) at various concentrations (0.1, 2.5, 5, 10 and 20%) for 10 min (Fig. 1A, Table 1). All tga20 mice exposed to aerosols derived from IBH (concentration: ≥2.5%) succumbed to scrapie with an attack rate of 100%. The incubation time negatively correlated with the IBH concentration (2.5%: n = 4, 165±54 dpi; 5%: n = 4, 131±7 dpi; 10%: n = 5, 161±27 dpi; 20%: n = 6, 133±8 dpi; p = 0.062, standard linear regression on standard ANOVA; Fig. 1A and F, Table 1, Table S1A).
Figure 1. Prion transmission through aerosols.
(A) tga20 mice were exposed to aerosols generated from 0.1%, 2.5%, 5%, 10% or 20% prion-infected mouse brain homogenates (IBH) for 10 min. (B) Groups of tga20 and (C) CD1 mice were exposed for 1, 5 or 10 min to aerosols generated from a 20% IBH. Experiments were performed twice (different colors). (D) C57BL/6, (E) 129SvxC57BL/6, and Prnpo/o mice were exposed for 10 min to aerosols generated from 20% IBH. Kaplan-Meier curves describe the percentage of survival after particular time points post exposure to prion aerosols (y-axis represents percentage of living mice; x-axis demonstrates survival time in days post inoculation). Different colors and symbols describe the various experimental groups. (F) Jittered scatter plot of survival time against concentration of prion aerosols generated out of IBH with added linear regression fit (p = 0.0622). (G) Jittered scatter plot of survival time against exposure time for tga20 mice with added linear regression fit. The negative correlation between survival time and exposure time is significant (p<0.001***). (H) Consecutive paraffin sections of the right hippocampus of Prnpo/o, tga20, CD1 and C57BL/6 mice stained with HE (for spongiosis, gliosis, neuronal cell loss), SAF84 (PrPSc deposits), GFAP (astrogliosis) and Iba-1 (microglia). All animals had been exposed to aerosols generated from 20% IBH for 10 min. Scale bars: 100µm.
Citation: Haybaeck J, Heikenwalder M, Klevenz B, Schwarz P, Margalith I, et al. (2011) Aerosols Transmit Prions to Immunocompetent and Immunodeficient Mice. PLoS Pathog 7(1): e1001257. doi:10.1371/journal.ppat.1001257 Editor: David Westaway, University of Alberta, Canada Received March 22, 2010; Accepted December 13, 2010; Published January 13, 2011 Copyright: 2011 Haybaeck et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by EU grants ANTEPRION and PRIORITY (LS), and the TSE-Forschungsprogramm des Landes Baden-Wuerttemberg, Germany (LS). This work was also supported by grants from the UK Department of Environment, Food and Rural Affairs (AA), the EU grants LUPAS and PRIORITY (AA), the Novartis Research Foundation (AA), and an Advanced Grant of the European Research Council to AA. MH was supported by the Foundation for Research at the Medical Faculty, the Prof. Dr. Max-Cloetta foundation and the Bonizzi-Theler Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: email@example.com (AA); firstname.lastname@example.org (LS) ¤a Current address: Institute of Pathology, Medical University Graz, Graz, Austria ¤b Current address: Institute of Virology, Technical University Mu¨nchen/Helmholtz Zentrum Mu¨nchen, Munich, Germany . These authors contributed equally to this work
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]
New studies on the heat resistance of hamster-adapted scrapie agent: Threshold survival after ashing at 600°C suggests an inorganic template of replication
Prion Infected Meat-and-Bone Meal Is Still Infectious after Biodiesel Production
Detection of protease-resistant cervid prion protein in water from a CWD-endemic area
A Quantitative Assessment of the Amount of Prion Diverted to Category 1 Materials and Wastewater During Processing
Rapid assessment of bovine spongiform encephalopathy prion inactivation by heat treatment in yellow grease produced in the industrial manufacturing process of meat and bone meals
Survival and Limited Spread of TSE Infectivity after Burial
Survival and Limited Spread of TSE Infectivity after Burial
Karen Fernie, Allister Smith and Robert A. Somerville The Roslin Institute and R(D)SVS; University of Edinburgh; Roslin, Scotland UK
Scrapie and chronic wasting disease probably spread via environmental routes, and there are also concerns about BSE infection remaining in the environment after carcass burial or waste 3disposal. In two demonstration experiments we are determining survival and migration of TSE infectivity when buried for up to five years, as an uncontained point source or within bovine heads. Firstly boluses of TSE infected mouse brain were buried in lysimeters containing either sandy or clay soil. Migration from the boluses is being assessed from soil cores taken over time. With the exception of a very small amount of infectivity found 25 cm from the bolus in sandy soil after 12 months, no other infectivity has been detected up to three years. Secondly, ten bovine heads were spiked with TSE infected mouse brain and buried in the two soil types. Pairs of heads have been exhumed annually and assessed for infectivity within and around them. After one year and after two years, infectivity was detected in most intracranial samples and in some of the soil samples taken from immediately surrounding the heads. The infectivity assays for the samples in and around the heads exhumed at years three and four are underway. These data show that TSE infectivity can survive burial for long periods but migrates slowly. Risk assessments should take into account the likely long survival rate when infected material has been buried.
The authors gratefully acknowledge funding from DEFRA.
PRION CONFERENCE 2010 ABSTRACT REFERENCE
2018 - 2019
***> This is very likely to have parallels with control efforts for CWD in cervids.
Rapid recontamination of a farm building occurs after attempted prion removal
Kevin Christopher Gough, BSc (Hons), PhD1, Claire Alison Baker, BSc (Hons)2, Steve Hawkins, MIBiol3, Hugh Simmons, BVSc, MRCVS, MBA, MA3, Timm Konold, DrMedVet, PhD, MRCVS3 and Ben Charles Maddison, BSc (Hons), PhD2
The transmissible spongiform encephalopathy scrapie of sheep/goats and chronic wasting disease of cervids are associated with environmental reservoirs of infectivity.
Preventing environmental prions acting as a source of infectivity to healthy animals is of major concern to farms that have had outbreaks of scrapie and also to the health management of wild and farmed cervids.
Here, an efficient scrapie decontamination protocol was applied to a farm with high levels of environmental contamination with the scrapie agent.
Post-decontamination, no prion material was detected within samples taken from the farm buildings as determined using a sensitive in vitro replication assay (sPMCA).
A bioassay consisting of 25 newborn lambs of highly susceptible prion protein genotype VRQ/VRQ introduced into this decontaminated barn was carried out in addition to sampling and analysis of dust samples that were collected during the bioassay.
Twenty-four of the animals examined by immunohistochemical analysis of lymphatic tissues were scrapie-positive during the bioassay, samples of dust collected within the barn were positive by month 3.
The data illustrates the difficulty in decontaminating farm buildings from scrapie, and demonstrates the likely contribution of farm dust to the recontamination of these environments to levels that are capable of causing disease.
As in the authors' previous study,12 the decontamination of this sheep barn was not effective at removing scrapie infectivity, and despite the extra measures brought into this study (more effective chemical treatment and removal of sources of dust) the overall rates of disease transmission mirror previous results on this farm. With such apparently effective decontamination (assuming that at least some sPMCA seeding ability is coincident with infectivity), how was infectivity able to persist within the environment and where does infectivity reside? Dust samples were collected in both the bioassay barn and also a barn subject to the same decontamination regime within the same farm (but remaining unoccupied). Within both of these barns dust had accumulated for three months that was able to seed sPMCA, indicating the accumulation of scrapie-containing material that was independent of the presence of sheep that may have been incubating and possibly shedding low amounts of infectivity.
This study clearly demonstrates the difficulty in removing scrapie infectivity from the farm environment. Practical and effective prion decontamination methods are still urgently required for decontamination of scrapie infectivity from farms that have had cases of scrapie and this is particularly relevant for scrapiepositive goatherds, which currently have limited genetic resistance to scrapie within commercial breeds.24 This is very likely to have parallels with control efforts for CWD in cervids.
Acknowledgements The authors thank the APHA farm staff, Tony Duarte, Olly Roberts and Margaret Newlands for preparation of the sheep pens and animal husbandry during the study. The authors also thank the APHA pathology team for RAMALT and postmortem examination.
Funding This study was funded by DEFRA within project SE1865.
Competing interests None declared.
Saturday, January 5, 2019
Rapid recontamination of a farm building occurs after attempted prion removal
The effectiveness of on-farm decontamination methods for scrapie - SE1865
Scrapie infectivity persists on farms where infected animals have been removed1. Recently we have demonstrated that it is possible to detect environmental scrapie contamination biochemically using serial Protein Misfolding Cyclic Amplification (sPMCA)2, allowing the monitoring of scrapie infectivity on farm premises. Ongoing Defra study SE1863 has compared pen decontamination regimes on a scrapie-infected farm by both sheep bioassay and sPMCA. For bioassay, scrapie-free genetically susceptible lambs were introduced into pens decontaminated using distinct methodologies, all pens contained scrapie-positive lambs within 1 year. Remarkably this included lambs housed within a pen which had been jet washed/chloros treated, followed by regalvanisation/ replacement of all metalwork and painting of all other surfaces.
We have recently demonstrated using sPMCA, that material collected on swabs from vertical surfaces at heights inaccessible to sheep within a barn on the same scrapie affected farm contained scrapie prions (unpublished observations). We hypothesise that scrapie prions are most likely to have been deposited in these areas by bioaerosol movement. We propose that this bioaerosol movement contributes to scrapie transmission within the barn, and could account for the sheep that became positive within the pen containing re-galvanised/new metalwork and repainted surfaces (project SE1863). It is proposed that a thorough decontamination that would minimise prion-contaminated dust, both within the building and its immediate vicinity, is likely to increase the effectiveness of current methods for decontaminating farm buildings following outbreaks of scrapie. The proposed study builds on our previous data and will thoroughly investigate the potential for farm building scrapie-contamination via the bioaerosol route, a previously unrecognised route for dissemination of scrapie infectivity. This route could lead to the direct infection of healthy animals and/or indirect transmission of disease via contamination of surfaces within animal pens. The proposed study would analyse material collected using air samplers set up within “scrapie-infected” barns and their immediate vicinity, to confirm that prion containing material can be airborne within a scrapie infected farm environment. The study would incorporate a biochemical assessment of different surface decontamination methods, in order to demonstrate the best methodology and then the analysis of air and surface samples after a complete building decontamination to remove sources of dust and surface bound prions from both the building and its immediate vicinity. Analysis of such surface and air samples collected before and after treatment would measure the reduction in levels of infectivity. It is envisaged that the biochemical demonstration of airborne prions and the effective reduction in such prion dissemination would lead to a sheep bioassay experiment that would be conducted after a full farm decontamination. This would fully assess the effectiveness of an optimised scrapie decontamination strategy.
This study will contribute directly to Defra policy on best practice for on-farm decontamination after outbreaks of scrapie; a situation particularly relevant to decontamination after scrapie cases on goat farms where no genetic resistance to scrapie has currently been identified, and where complete decontamination is essential in order to stop recurrence of scrapie after restocking.
• Determine the presence and relative levels of airborne prions on a scrapie infected farm.
• Evaluate different pen surface decontamination procedures.
• Determine the presence of any airborne prions in a barn after a full decontamination.
• Further assess the efficacy of the decontamination procedure investigated in phase 2 by sheep bioassay.
Time-Scale and Cost
Contractor / Funded Organisations
A D A S UK Ltd (ADAS)
Keywords Animals Fields of Study Animal Health
The Effectiveness of on-Farm Decontamination Methods for Scrapie
Start date 2012
End date 2016
Objective Phase 1
Determine the presence and relative levels of airborne prions on a scrapie infected farm. Evaluate different pen surface decontamination procedures.
Determine the presence of any airborne prions in a barn after a full decontamination.
Further assess the efficacy of the decontamination procedure investigated in phase 2 by sheep bioassay.
Scrapie infectivity persists on farms where infected animals have been removed1. Recently we have demonstrated that it is possible to detect environmental scrapie contamination biochemically using serial Protein Misfolding Cyclic Amplification (sPMCA)2, allowing the monitoring of scrapie infectivity on farm premises. Ongoing Defra study SE1863 has compared pen decontamination regimes on a scrapie-infected farm by both sheep bioassay and sPMCA. For bioassay, scrapie-free genetically susceptible lambs were introduced into pens decontaminated using distinct methodologies, all pens contained scrapie-positive lambs within 1 year. Remarkably this included lambs housed within a pen which had been jet washed/chloros treated, followed by regalvanisation/replacement of all metalwork and painting of all other surfaces.
We have recently demonstrated using sPMCA, that material collected on swabs from vertical surfaces at heights inaccessible to sheep within a barn on the same scrapie affected farm contained scrapie prions (unpublished observations). We hypothesise that scrapie prions are most likely to have been deposited in these areas by bioaerosol movement. We propose that this bioaerosol movement contributes to scrapie transmission within the barn, and could account for the sheep that became positive within the pen containing re-galvanised/new metalwork and repainted surfaces (project SE1863). It is proposed that a thorough decontamination that would minimise prion-contaminated dust, both within the building and its immediate vicinity, is likely to increase the effectiveness of current methods for decontaminating farm buildings following outbreaks of scrapie. The proposed study builds on our previous data and will thoroughly investigate the potential for farm building scrapie contamination via the bioaerosol route, a previously unrecognised route for dissemination of scrapie infectivity. This route could lead to the direct infection of healthy animals and/or indirect transmission of disease via contamination of surfaces within animal pens. The proposed study would analyse material collected using air samplers set up within “scrapie-infected” barns and their immediate vicinity, to confirm that prion containing material can be airborne within a scrapie infected farm environment. The study would incorporate a biochemical assessment of different surface decontamination methods, in order to demonstrate the best methodology and then the analysis of air and surface samples after a complete building decontamination to remove sources of dust and surface bound prions from both the building and its immediate vicinity. Analysis of such surface and air samples collected before and after treatment would measure the reduction in levels of infectivity. It is envisaged that the biochemical demonstration of airborne prions and the effective reduction in such prion dissemination would lead to a sheep bioassay experiment that would be conducted after a full farm decontamination. This would fully assess the effectiveness of an optimised scrapie decontamination strategy.
This study will contribute directly to Defra policy on best practice for on-farm decontamination after outbreaks of scrapie; a situation particularly relevant to decontamination after scrapie cases on goat farms where no genetic resistance to scrapie has currently been identified, and where complete decontamination is essential in order to stop recurrence of scrapie after restocking.
Department for Environment, Food and Rural Affairs
View this project
Policy and Planning
Circulation of prions within dust on a scrapie affected farm
Kevin C Gough1 , Claire A Baker2 , Hugh A Simmons3 , Steve A Hawkins3 and Ben C Maddison2*
Prion diseases are fatal neurological disorders that affect humans and animals. Scrapie of sheep/goats and Chronic Wasting Disease (CWD) of deer/elk are contagious prion diseases where environmental reservoirs have a direct link to the transmission of disease. Using protein misfolding cyclic amplification we demonstrate that scrapie PrPSc can be detected within circulating dusts that are present on a farm that is naturally contaminated with sheep scrapie. The presence of infectious scrapie within airborne dusts may represent a possible route of infection and illustrates the difficulties that may be associated with the effective decontamination of such scrapie affected premises.
Discussion We present biochemical data illustrating the airborne movement of scrapie containing material within a contaminated farm environment. We were able to detect scrapie PrPSc within extracts from dusts collected over a 70 day period, in the absence of any sheep activity. We were also able to detect scrapie PrPSc within dusts collected within pasture at 30 m but not at 60 m distance away from the scrapie contaminated buildings, suggesting that the chance of contamination of pasture by scrapie contaminated dusts decreases with distance from contaminated farm buildings. PrPSc amplification by sPMCA has been shown to correlate with infectivity and amplified products have been shown to be infectious [14,15]. These experiments illustrate the potential for low dose scrapie infectivity to be present within such samples. We estimate low ng levels of scrapie positive brain equivalent were deposited per m2 over 70 days, in a barn previously occupied by sheep affected with scrapie. This movement of dusts and the accumulation of low levels of scrapie infectivity within this environment may in part explain previous observations where despite stringent pen decontamination regimens healthy lambs still became scrapie infected after apparent exposure from their environment alone . The presence of sPMCA seeding activity and by inference, infectious prions within dusts, and their potential for airborne dissemination is highly novel and may have implications for the spread of scrapie within infected premises. The low level circulation and accumulation of scrapie prion containing dust material within the farm environment will likely impede the efficient decontamination of such scrapie contaminated buildings unless all possible reservoirs of dust are removed. Scrapie containing dusts could possibly infect animals during feeding and drinking, and respiratory and conjunctival routes may also be involved. It has been demonstrated that scrapie can be efficiently transmitted via the nasal route in sheep , as is also the case for CWD in both murine models and in white tailed deer [18-20].
The sources of dust borne prions are unknown but it seems reasonable to assume that faecal, urine, skin, parturient material and saliva-derived prions may contribute to this mobile environmental reservoir of infectivity. This work highlights a possible transmission route for scrapie within the farm environment, and this is likely to be paralleled in CWD which shows strong similarities with scrapie in terms of prion dissemination and disease transmission. The data indicate that the presence of scrapie prions in dust is likely to make the control of these diseases a considerable challenge.
Research Project: TRANSMISSION, DIFFERENTIATION, AND PATHOBIOLOGY OF TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES Location: Virus and Prion Research
Title: Scrapie transmits to white-tailed deer by the oral route and has a molecular profile similar to chronic wasting disease
item Greenlee, Justin item Moore, S - Orise Fellow item Smith, Jodi - Iowa State University item Kunkle, Robert item West Greenlee, M - Iowa State University Submitted to: American College of Veterinary Pathologists Meeting Publication Type: Abstract Only Publication Acceptance Date: 8/12/2015 Publication Date: N/A Citation: N/A
Technical Abstract: The purpose of this work was to determine susceptibility of white-tailed deer (WTD) to the agent of sheep scrapie and to compare the resultant PrPSc to that of the original inoculum and chronic wasting disease (CWD). We inoculated WTD by a natural route of exposure (concurrent oral and intranasal (IN); n=5) with a US scrapie isolate. All scrapie-inoculated deer had evidence of PrPSc accumulation. PrPSc was detected in lymphoid tissues at preclinical time points, and deer necropsied after 28 months post-inoculation had clinical signs, spongiform encephalopathy, and widespread distribution of PrPSc in neural and lymphoid tissues. Western blotting (WB) revealed PrPSc with 2 distinct molecular profiles. WB on cerebral cortex had a profile similar to the original scrapie inoculum, whereas WB of brainstem, cerebellum, or lymph nodes revealed PrPSc with a higher profile resembling CWD. Homogenates with the 2 distinct profiles from WTD with clinical scrapie were further passaged to mice expressing cervid prion protein and intranasally to sheep and WTD. In cervidized mice, the two inocula have distinct incubation times. Sheep inoculated intranasally with WTD derived scrapie developed disease, but only after inoculation with the inoculum that had a scrapie-like profile. The WTD study is ongoing, but deer in both inoculation groups are positive for PrPSc by rectal mucosal biopsy. In summary, this work demonstrates that WTD are susceptible to the agent of scrapie, two distinct molecular profiles of PrPSc are present in the tissues of affected deer, and inoculum of either profile readily passes to deer.
THURSDAY, FEBRUARY 28, 2019
BSE infectivity survives burial for five years with only limited spread
***> CONGRESSIONAL ABSTRACTS PRION CONFERENCE 2018
P69 Experimental transmission of CWD from white-tailed deer to co-housed reindeer
Mitchell G (1), Walther I (1), Staskevicius A (1), Soutyrine A (1), Balachandran A (1)
(1) National & OIE Reference Laboratory for Scrapie and CWD, Canadian Food Inspection Agency, Ottawa, Ontario, Canada.
Chronic wasting disease (CWD) continues to be detected in wild and farmed cervid populations of North America, affecting predominantly white-tailed deer, mule deer and elk. Extensive herds of wild caribou exist in northern regions of Canada, although surveillance has not detected the presence of CWD in this population. Oral experimental transmission has demonstrated that reindeer, a species closely related to caribou, are susceptible to CWD. Recently, CWD was detected for the first time in Europe, in wild Norwegian reindeer, advancing the possibility that caribou in North America could also become infected. Given the potential overlap in habitat between wild CWD-infected cervids and wild caribou herds in Canada, we sought to investigate the horizontal transmissibility of CWD from white-tailed deer to reindeer.
Two white-tailed deer were orally inoculated with a brain homogenate prepared from a farmed Canadian white-tailed deer previously diagnosed with CWD. Two reindeer, with no history of exposure to CWD, were housed in the same enclosure as the white-tailed deer, 3.5 months after the deer were orally inoculated. The white-tailed deer developed clinical signs consistent with CWD beginning at 15.2 and 21 months post-inoculation (mpi), and were euthanized at 18.7 and 23.1 mpi, respectively. Confirmatory testing by immunohistochemistry (IHC) and western blot demonstrated widespread aggregates of pathological prion protein (PrPCWD) in the central nervous system and lymphoid tissues of both inoculated white-tailed deer. Both reindeer were subjected to recto-anal mucosal associated lymphoid tissue (RAMALT) biopsy at 20 months post-exposure (mpe) to the white-tailed deer. The biopsy from one reindeer contained PrPCWD confirmed by IHC. This reindeer displayed only subtle clinical evidence of disease prior to a rapid decline in condition requiring euthanasia at 22.5 mpe. Analysis of tissues from this reindeer by IHC revealed widespread PrPCWD deposition, predominantly in central nervous system and lymphoreticular tissues. Western blot molecular profiles were similar between both orally inoculated white-tailed deer and the CWD positive reindeer. Despite sharing the same enclosure, the other reindeer was RAMALT negative at 20 mpe, and PrPCWD was not detected in brainstem and lymphoid tissues following necropsy at 35 mpe. Sequencing of the prion protein gene from both reindeer revealed differences at several codons, which may have influenced susceptibility to infection.
Natural transmission of CWD occurs relatively efficiently amongst cervids, supporting the expanding geographic distribution of disease and the potential for transmission to previously naive populations. The efficient horizontal transmission of CWD from white-tailed deer to reindeer observed here highlights the potential for reindeer to become infected if exposed to other cervids or environments infected with CWD.
SOURCE REFERENCE 2018 PRION CONFERENCE ABSTRACT
Research Project: TRANSMISSION, DIFFERENTIATION, AND PATHOBIOLOGY OF TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES Location: Virus and Prion Research
Title: Horizontal transmission of chronic wasting disease in reindeer
item MOORE, SARAH - ORISE FELLOW item KUNKLE, ROBERT item WEST GREENLEE, MARY - IOWA STATE UNIVERSITY item Nicholson, Eric item RICHT, JUERGEN item HAMIR, AMIRALI item WATERS, WADE item Greenlee, Justin
Submitted to: Emerging Infectious Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/29/2016
Publication Date: 12/1/2016
Citation: Moore, S., Kunkle, R., Greenlee, M., Nicholson, E., Richt, J., Hamir, A., Waters, W., Greenlee, J. 2016. Horizontal transmission of chronic wasting disease in reindeer. Emerging Infectious Diseases. 22(12):2142-2145. doi:10.3201/eid2212.160635.
Interpretive Summary: Chronic wasting disease (CWD) is a fatal neurodegenerative disease that occurs in farmed and wild cervids (deer and elk) of North America and was recently diagnosed in a single free-ranging reindeer (Rangifer tarandus tarandus) in Norway. CWD is a transmissible spongiform encephalopathy (TSE) that is caused by infectious proteins called prions that are resistant to various methods of decontamination and environmental degradation. Little is known about the susceptibility of or potential for transmission amongst reindeer. In this experiment, we tested the susceptibility of reindeer to CWD from various sources (elk, mule deer, or white-tailed deer) after intracranial inoculation and tested the potential for infected reindeer to transmit to non-inoculated animals by co-housing or housing in adjacent pens. Reindeer were susceptible to CWD from elk, mule deer, or white-tailed deer sources after experimental inoculation. Most importantly, non-inoculated reindeer that were co-housed with infected reindeer or housed in pens adjacent to infected reindeer but without the potential for nose-to-nose contact also developed evidence of CWD infection. This is a major new finding that may have a great impact on the recently diagnosed case of CWD in the only remaining free-ranging reindeer population in Europe as our findings imply that horizontal transmission to other reindeer within that herd has already occurred. Further, this information will help regulatory and wildlife officials developing plans to reduce or eliminate CWD and cervid farmers that want to ensure that their herd remains CWD-free, but were previously unsure of the potential for reindeer to transmit CWD.
Technical Abstract: Chronic wasting disease (CWD) is a naturally-occurring, fatal prion disease of cervids. Reindeer (Rangifer tarandus tarandus) are susceptible to CWD following oral challenge, and CWD was recently reported in a free-ranging reindeer of Norway. Potential contact between CWD-affected cervids and Rangifer species that are free-ranging or co-housed on farms presents a potential risk of CWD transmission. The aims of this study were to 1) investigate the transmission of CWD from white-tailed deer (Odocoileus virginianus; CWDwtd), mule deer (Odocoileus hemionus; CWDmd), or elk (Cervus elaphus nelsoni; CWDelk) to reindeer via the intracranial route, and 2) to assess for direct and indirect horizontal transmission to non-inoculated sentinels. Three groups of 5 reindeer fawns were challenged intracranially with CWDwtd, CWDmd, or CWDelk. Two years after challenge of inoculated reindeer, non-inoculated negative control reindeer were introduced into the same pen as the CWDwtd inoculated reindeer (direct contact; n=4) or into a pen adjacent to the CWDmd inoculated reindeer (indirect contact; n=2). Experimentally inoculated reindeer were allowed to develop clinical disease. At death/euthanasia a complete necropsy examination was performed, including immunohistochemical testing of tissues for disease-associated CWD prion protein (PrPcwd). Intracranially challenged reindeer developed clinical disease from 21 months post-inoculation (months PI). PrPcwd was detected in 5 out of 6 sentinel reindeer although only 2 out of 6 developed clinical disease during the study period (< 57 months PI). We have shown that reindeer are susceptible to CWD from various cervid sources and can transmit CWD to naïve reindeer both directly and indirectly.
***> Infectious agent of sheep scrapie may persist in the environment for at least 16 years
***> Nine of these recurrences occurred 14–21 years after culling, apparently as the result of environmental contamination, but outside entry could not always be absolutely excluded.
JOURNAL OF GENERAL VIROLOGY Volume 87, Issue 12
Infectious agent of sheep scrapie may persist in the environment for at least 16 years Free
Gudmundur Georgsson1, Sigurdur Sigurdarson2, Paul Brown3
First Published: 01 December 2006 https://doi.org/10.1099/vir.0.82011-0 ABSTRACT In 1978, a rigorous programme was implemented to stop the spread of, and subsequently eradicate, sheep scrapie in Iceland. Affected flocks were culled, premises were disinfected and, after 2–3 years, restocked with lambs from scrapie-free areas. Between 1978 and 2004, scrapie recurred on 33 farms. Nine of these recurrences occurred 14–21 years after culling, apparently as the result of environmental contamination, but outside entry could not always be absolutely excluded. Of special interest was one farm with a small, completely self-contained flock where scrapie recurred 18 years after culling, 2 years after some lambs had been housed in an old sheep-house that had never been disinfected. Epidemiological investigation established with near certitude that the disease had not been introduced from the outside and it is concluded that the agent may have persisted in the old sheep-house for at least 16 years.
Gudmundur Georgsson,1 Sigurdur Sigurdarson2 and Paul Brown3
Gudmundur Georgsson email@example.com
1 Institute for Experimental Pathology, University of Iceland, Keldur v/vesturlandsveg, IS-112 Reykjavı´k, Iceland
2 Laboratory of the Chief Veterinary Officer, Keldur, Iceland
3 Bethesda, Maryland, USA
Received 7 March 2006 Accepted 6 August 2006
In 1978, a rigorous programme was implemented to stop the spread of, and subsequently eradicate, sheep scrapie in Iceland. Affected flocks were culled, premises were disinfected and, after 2–3 years, restocked with lambs from scrapie-free areas. Between 1978 and 2004, scrapie recurred on 33 farms. Nine of these recurrences occurred 14–21 years after culling, apparently as the result of environmental contamination, but outside entry could not always be absolutely excluded. Of special interest was one farm with a small, completely self-contained flock where scrapie recurred 18 years after culling, 2 years after some lambs had been housed in an old sheephouse that had never been disinfected. Epidemiological investigation established with near certitude that the disease had not been introduced from the outside and it is concluded that the agent may have persisted in the old sheep-house for at least 16 years.
TITLE: PATHOLOGICAL FEATURES OF CHRONIC WASTING DISEASE IN REINDEER AND DEMONSTRATION OF HORIZONTAL TRANSMISSION
*** DECEMBER 2016 CDC EMERGING INFECTIOUS DISEASE JOURNAL CWD HORIZONTAL TRANSMISSION
Back around 2000, 2001, or so, I was corresponding with officials abroad during the bse inquiry, passing info back and forth, and some officials from here inside USDA aphis FSIS et al. In fact helped me get into the USA 50 state emergency BSE conference call way back. That one was a doozy. But I always remember what “deep throat” I never knew who they were, but I never forgot;
Some unofficial information from a source on the inside looking out -
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!!!
---end personal email---end...tss
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).
Dr. Paul Brown Scrapie Soil Test BSE Inquiry Document
Using in vitro Prion replication for high sensitive detection of prions and prionlike proteins and for understanding mechanisms of transmission.
Claudio Soto Mitchell Center for Alzheimer's diseases and related Brain disorders, Department of Neurology, University of Texas Medical School at Houston.
Prion and prion-like proteins are misfolded protein aggregates with the ability to selfpropagate to spread disease between cells, organs and in some cases across individuals. I n T r a n s m i s s i b l e s p o n g i f o r m encephalopathies (TSEs), prions are mostly composed by a misfolded form of the prion protein (PrPSc), which propagates by transmitting its misfolding to the normal prion protein (PrPC). The availability of a procedure to replicate prions in the laboratory may be important to study the mechanism of prion and prion-like spreading and to develop high sensitive detection of small quantities of misfolded proteins in biological fluids, tissues and environmental samples. Protein Misfolding Cyclic Amplification (PMCA) is a simple, fast and efficient methodology to mimic prion replication in the test tube. PMCA is a platform technology that may enable amplification of any prion-like misfolded protein aggregating through a seeding/nucleation process. In TSEs, PMCA is able to detect the equivalent of one single molecule of infectious PrPSc and propagate prions that maintain high infectivity, strain properties and species specificity. Using PMCA we have been able to detect PrPSc in blood and urine of experimentally infected animals and humans affected by vCJD with high sensitivity and specificity. Recently, we have expanded the principles of PMCA to amplify amyloid-beta (Aβ) and alphasynuclein (α-syn) aggregates implicated in Alzheimer's and Parkinson's diseases, respectively. Experiments are ongoing to study the utility of this technology to detect Aβ and α-syn aggregates in samples of CSF and blood from patients affected by these diseases.
***>>> Recently, we have been using PMCA to study the role of environmental prion contamination on the horizontal spreading of TSEs. These experiments have focused on the study of the interaction of prions with plants and environmentally relevant surfaces. Our results show that plants (both leaves and roots) bind tightly to prions present in brain extracts and excreta (urine and feces) and retain even small quantities of PrPSc for long periods of time. Strikingly, ingestion of prioncontaminated leaves and roots produced disease with a 100% attack rate and an incubation period not substantially longer than feeding animals directly with scrapie brain homogenate. Furthermore, plants can uptake prions from contaminated soil and transport them to different parts of the plant tissue (stem and leaves). Similarly, prions bind tightly to a variety of environmentally relevant surfaces, including stones, wood, metals, plastic, glass, cement, etc. Prion contaminated surfaces efficiently transmit prion disease when these materials were directly injected into the brain of animals and strikingly when the contaminated surfaces were just placed in the animal cage. These findings demonstrate that environmental materials can efficiently bind infectious prions and act as carriers of infectivity, suggesting that they may play an important role in the horizontal transmission of the disease.
Since its invention 13 years ago, PMCA has helped to answer fundamental questions of prion propagation and has broad applications in research areas including the food industry, blood bank safety and human and veterinary disease diagnosis.
source reference Prion Conference 2015 abstract book
Grass Plants Bind, Retain, Uptake, and Transport Infectious Prions
Sandra Pritzkow,1 Rodrigo Morales,1 Fabio Moda,1,3 Uffaf Khan,1 Glenn C. Telling,2 Edward Hoover,2 and Claudio Soto1, * 1Mitchell Center for Alzheimer’s Disease and Related Brain Disorders, Department of Neurology, University of Texas Medical School at Houston, 6431 Fannin Street, Houston, TX 77030, USA
2Prion Research Center, Department of Microbiology, Immunology, and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, USA
3Present address: IRCCS Foundation Carlo Besta Neurological Institute, 20133 Milan, Italy *Correspondence: firstname.lastname@example.org http://dx.doi.org/10.1016/j.celrep.2015.04.036
Prions are the protein-based infectious agents responsible for prion diseases. Environmental prion contamination has been implicated in disease transmission. Here, we analyzed the binding and retention of infectious prion protein (PrPSc) to plants. Small quantities of PrPSc contained in diluted brain homogenate or in excretory materials (urine and feces) can bind to wheat grass roots and leaves. Wild-type hamsters were efficiently infected by ingestion of prion-contaminated plants. The prion-plant interaction occurs with prions from diverse origins, including chronic wasting disease. Furthermore, leaves contaminated by spraying with a prion-containing preparation retained PrPSc for several weeks in the living plant. Finally, plants can uptake prions from contaminated soil and transport them to aerial parts of the plant (stem and leaves). These findings demonstrate that plants can efficiently bind infectious prions and act as carriers of infectivity, suggesting a possible role of environmental prion contamination in the horizontal transmission of the disease.
This study shows that plants can efficiently bind prions contained in brain extracts from diverse prion infected animals, including CWD-affected cervids. PrPSc attached to leaves and roots from wheat grass plants remains capable of seeding prion replication in vitro. Surprisingly, the small quantity of PrPSc naturally excreted in urine and feces from sick hamster or cervids was enough to efficiently contaminate plant tissue. Indeed, our results suggest that the majority of excreted PrPSc is efficiently captured by plants’ leaves and roots. Moreover, leaves can be contaminated by spraying them with a prion-containing extract, and PrPSc remains detectable in living plants for as long as the study was performed (several weeks). Remarkably, prion contaminated plants transmit prion disease to animals upon ingestion, producing a 100% attack rate and incubation periods not substantially longer than direct oral administration of sick brain homogenates.
Finally, an unexpected but exciting result was that plants were able to uptake prions from contaminated soil and transport them to aerial parts of the plant tissue. Although it may seem farfetched that plants can uptake proteins from the soil and transport it to the parts above the ground, there are already published reports of this phenomenon (McLaren et al., 1960; Jensen and McLaren, 1960;Paungfoo-Lonhienne et al., 2008). The high resistance of prions to degradation and their ability to efficiently cross biological barriers may play a role in this process. The mechanism by which plants bind, retain, uptake, and transport prions is unknown. We are currently studying the way in which prions interact with plants using purified, radioactively labeled PrPSc to determine specificity of the interaction, association constant, reversibility, saturation, movement, etc.
Epidemiological studies have shown numerous instances of scrapie or CWD recurrence upon reintroduction of animals on pastures previously exposed to prion-infected animals. Indeed, reappearance of scrapie has been documented following fallow periods of up to 16 years (Georgsson et al., 2006), and pastures were shown to retain infectious CWD prions for at least 2 years after exposure (Miller et al., 2004). It is likely that the environmentally mediated transmission of prion diseases depends upon the interaction of prions with diverse elements, including soil, water, environmental surfaces, various invertebrate animals, and plants.
However, since plants are such an important component of the environment and also a major source of food for many animal species, including humans, our results may have far-reaching implications for animal and human health. Currently, the perception of the riskfor animal-to-human prion transmission has beenmostly limited to consumption or exposure to contaminated meat; our results indicate that plants might also be an important vector of transmission that needs to be considered in risk assessment.
RIGINAL RESEARCH ARTICLE
Front. Vet. Sci., 14 September 2015 | https://doi.org/10.3389/fvets.2015.00032
Objects in contact with classical scrapie sheep act as a reservoir for scrapie transmission
imageTimm Konold1*, imageStephen A. C. Hawkins2, imageLisa C. Thurston3, imageBen C. Maddison4, imageKevin C. Gough5, imageAnthony Duarte1 and imageHugh A. Simmons1
1Animal Sciences Unit, Animal and Plant Health Agency Weybridge, Addlestone, UK
2Pathology Department, Animal and Plant Health Agency Weybridge, Addlestone, UK
3Surveillance and Laboratory Services, Animal and Plant Health Agency Penrith, Penrith, UK
4ADAS UK, School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington, UK
5School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington, UK
Classical scrapie is an environmentally transmissible prion disease of sheep and goats. Prions can persist and remain potentially infectious in the environment for many years and thus pose a risk of infecting animals after re-stocking. In vitro studies using serial protein misfolding cyclic amplification (sPMCA) have suggested that objects on a scrapie-affected sheep farm could contribute to disease transmission. This in vivo study aimed to determine the role of field furniture (water troughs, feeding troughs, fencing, and other objects that sheep may rub against) used by a scrapie-infected sheep flock as a vector for disease transmission to scrapie-free lambs with the prion protein genotype VRQ/VRQ, which is associated with high susceptibility to classical scrapie. When the field furniture was placed in clean accommodation, sheep became infected when exposed to either a water trough (four out of five) or to objects used for rubbing (four out of seven). This field furniture had been used by the scrapie-infected flock 8 weeks earlier and had previously been shown to harbor scrapie prions by sPMCA. Sheep also became infected (20 out of 23) through exposure to contaminated field furniture placed within pasture not used by scrapie-infected sheep for 40 months, even though swabs from this furniture tested negative by PMCA. This infection rate decreased (1 out of 12) on the same paddock after replacement with clean field furniture. Twelve grazing sheep exposed to field furniture not in contact with scrapie-infected sheep for 18 months remained scrapie free. The findings of this study highlight the role of field furniture used by scrapie-infected sheep to act as a reservoir for disease re-introduction although infectivity declines considerably if the field furniture has not been in contact with scrapie-infected sheep for several months. PMCA may not be as sensitive as VRQ/VRQ sheep to test for environmental contamination.
Classical scrapie is an environmentally transmissible disease because it has been reported in naïve, supposedly previously unexposed sheep placed in pastures formerly occupied by scrapie-infected sheep (4, 19, 20).
Although the vector for disease transmission is not known, soil is likely to be an important reservoir for prions (2) where – based on studies in rodents – prions can adhere to minerals as a biologically active form (21) and remain infectious for more than 2 years (22).
Similarly, chronic wasting disease (CWD) has re-occurred in mule deer housed in paddocks used by infected deer 2 years earlier, which was assumed to be through foraging and soil consumption (23).
Our study suggested that the risk of acquiring scrapie infection was greater through exposure to contaminated wooden, plastic, and metal surfaces via water or food troughs, fencing, and hurdles than through grazing.
Drinking from a water trough used by the scrapie flock was sufficient to cause infection in sheep in a clean building.
Exposure to fences and other objects used for rubbing also led to infection, which supported the hypothesis that skin may be a vector for disease transmission (9).
The risk of these objects to cause infection was further demonstrated when 87% of 23 sheep presented with PrPSc in lymphoid tissue after grazing on one of the paddocks, which contained metal hurdles, a metal lamb creep and a water trough in contact with the scrapie flock up to 8 weeks earlier, whereas no infection had been demonstrated previously in sheep grazing on this paddock, when equipped with new fencing and field furniture.
When the contaminated furniture and fencing were removed, the infection rate dropped significantly to 8% of 12 sheep, with soil of the paddock as the most likely source of infection caused by shedding of prions from the scrapie-infected sheep in this paddock up to a week earlier.
This study also indicated that the level of contamination of field furniture sufficient to cause infection was dependent on two factors: stage of incubation period and time of last use by scrapie-infected sheep.
Drinking from a water trough that had been used by scrapie sheep in the predominantly pre-clinical phase did not appear to cause infection, whereas infection was shown in sheep drinking from the water trough used by scrapie sheep in the later stage of the disease.
It is possible that contamination occurred through shedding of prions in saliva, which may have contaminated the surface of the water trough and subsequently the water when it was refilled.
Contamination appeared to be sufficient to cause infection only if the trough was in contact with sheep that included clinical cases.
Indeed, there is an increased risk of bodily fluid infectivity with disease progression in scrapie (24) and CWD (25) based on PrPSc detection by sPMCA.
Although ultraviolet light and heat under natural conditions do not inactivate prions (26), furniture in contact with the scrapie flock, which was assumed to be sufficiently contaminated to cause infection, did not act as vector for disease if not used for 18 months, which suggest that the weathering process alone was sufficient to inactivate prions.
PrPSc detection by sPMCA is increasingly used as a surrogate for infectivity measurements by bioassay in sheep or mice.
In this reported study, however, the levels of PrPSc present in the environment were below the limit of detection of the sPMCA method, yet were still sufficient to cause infection of in-contact animals.
In the present study, the outdoor objects were removed from the infected flock 8 weeks prior to sampling and were positive by sPMCA at very low levels (2 out of 37 reactions).
As this sPMCA assay also yielded 2 positive reactions out of 139 in samples from the scrapie-free farm, the sPMCA assay could not detect PrPSc on any of the objects above the background of the assay.
False positive reactions with sPMCA at a low frequency associated with de novo formation of infectious prions have been reported (27, 28).
This is in contrast to our previous study where we demonstrated that outdoor objects that had been in contact with the scrapie-infected flock up to 20 days prior to sampling harbored PrPSc that was detectable by sPMCA analysis [4 out of 15 reactions (12)] and was significantly more positive by the assay compared to analogous samples from the scrapie-free farm.
This discrepancy could be due to the use of a different sPMCA substrate between the studies that may alter the efficiency of amplification of the environmental PrPSc.
In addition, the present study had a longer timeframe between the objects being in contact with the infected flock and sampling, which may affect the levels of extractable PrPSc.
Alternatively, there may be potentially patchy contamination of this furniture with PrPSc, which may have been missed by swabbing.
The failure of sPMCA to detect CWD-associated PrP in saliva from clinically affected deer despite confirmation of infectivity in saliva-inoculated transgenic mice was associated with as yet unidentified inhibitors in saliva (29), and it is possible that the sensitivity of sPMCA is affected by other substances in the tested material.
In addition, sampling of amplifiable PrPSc and subsequent detection by sPMCA may be more difficult from furniture exposed to weather, which is supported by the observation that PrPSc was detected by sPMCA more frequently in indoor than outdoor furniture (12).
A recent experimental study has demonstrated that repeated cycles of drying and wetting of prion-contaminated soil, equivalent to what is expected under natural weathering conditions, could reduce PMCA amplification efficiency and extend the incubation period in hamsters inoculated with soil samples (30).
This seems to apply also to this study even though the reduction in infectivity was more dramatic in the sPMCA assays than in the sheep model.
Sheep were not kept until clinical end-point, which would have enabled us to compare incubation periods, but the lack of infection in sheep exposed to furniture that had not been in contact with scrapie sheep for a longer time period supports the hypothesis that prion degradation and subsequent loss of infectivity occurs even under natural conditions.
In conclusion, the results in the current study indicate that removal of furniture that had been in contact with scrapie-infected animals should be recommended, particularly since cleaning and decontamination may not effectively remove scrapie infectivity (31), even though infectivity declines considerably if the pasture and the field furniture have not been in contact with scrapie-infected sheep for several months. As sPMCA failed to detect PrPSc in furniture that was subjected to weathering, even though exposure led to infection in sheep, this method may not always be reliable in predicting the risk of scrapie infection through environmental contamination.
These results suggest that the VRQ/VRQ sheep model may be more sensitive than sPMCA for the detection of environmentally associated scrapie, and suggest that extremely low levels of scrapie contamination are able to cause infection in susceptible sheep genotypes.
Keywords: classical scrapie, prion, transmissible spongiform encephalopathy, sheep, field furniture, reservoir, serial protein misfolding cyclic amplification
Chemical Inactivation of Prions Is Altered by Binding to the Soil Mineral Montmorillonite
Clarissa J. Booth, Stuart Siegfried Lichtenberg, Richard J. Chappell, and Joel A. Pedersen* Cite this: ACS Infect. Dis. 2021, XXXX, XXX, XXX-XXX Publication Date:March 31, 2021 https://doi.org/10.1021/acsinfecdis.0c00860 © 2021 American Chemical Society
Environmental routes of transmission contribute to the spread of the prion diseases chronic wasting disease of deer and elk and scrapie of sheep and goats. Prions can persist in soils and other environmental matrices and remain infectious for years. Prions bind avidly to the common soil mineral montmorillonite, and such binding can dramatically increase oral disease transmission. Decontamination of soil in captive facilities and natural habitats requires inactivation agents that are effective when prions are bound to soil microparticles. Here, we investigate the inactivation of free and montmorillonite-bound prions with sodium hydroxide, acidic pH, Environ LpH, and sodium hypochlorite. Immunoblotting and bioassays confirm that sodium hydroxide and sodium hypochlorite are effective for prion deactivation, although montmorillonite appears to reduce the efficacy of hypochlorite. Acidic conditions slightly reduce prion infectivity, and the acidic phenolic disinfectant Environ LpH produces slight reductions in infectivity and immunoreactivity. The extent to which the association with montmorillonite protects prions from chemical inactivation appears influenced by the effect of chemical agents on the clay structure and surface pH. When clay morphology remains relatively unaltered, as when exposed to hypochlorite, montmorillonite-bound prions appear to be protected from inactivation. In contrast, when the clay structure is substantially transformed, as when exposed to high concentrations of sodium hydroxide, the attachment to montmorillonite does not slow degradation. A reduction in surface pH appears to cause slight disruptions in clay structure, which enhances degradation under these conditions. We expect our findings will aid the development of remediation approaches for successful decontamination of prion-contaminated sites.
Terry S. Singeltary Sr.