Annual Report of the Scientific Network on BSE‐TSE 2022
Published: 10 November 2022
Approved: 27 October 2022
EFSA Journal 2022;19(11):EN-7656
On request from: EFSA Question Number: EFSA‐Q‐2021‐00660
Contact: biohaw@efsa.europa.eu
Abstract
Establishing a system of Networks of organisations operating in the fields within EFSA's mission is among the tasks of EFSA, according to Regulation (EC) No 178/2002, in order to facilitate a scientific cooperation framework by the coordination of activities, the exchange of information, the development and implementation of joint projects, the exchange of expertise and best practices. The EFSA Scientific Network on bovine spongiform encephalopathies and other transmissible spongiform encephalopathies (BSE‐TSE) was established in 2006 and held its 17th annual meeting on 13‐14 October 2022, as a hybrid meeting. The meeting served as an opportunity to exchange scientific information on BSE‐TSE related issues among EU Member States, countries from the European Free Trade Association, EU candidate countries, EFSA, the European Commission (EC) and ad hoc participants [in the 2022 meeting, represented by the World Organisation for Animal Health (WOAH)]. The topics discussed included: update on the situation and surveillance of CWD in North America and Norway, the molecular characterization of emergent CWD strains in Europe, the impact of feed ban on L‐BSE, the history experimental models using non‐human primates assessing transmissibility, pathogenesis and resistance of prions, a online real‐time pulse survey on the network and TSE in Europe, recent and ongoing activities on TSE of EFSA, WOAH and EC, as well as the preliminary results of the 2021 EU TSE summary report.
© European Food Safety Authority
2.2.2 Assessment of zoonotic potential of TSEs: the contribution of in vivo experimental models
Emmanuel Comoy, Veterinary researcher and member of CEA (FR) presented an overview of major inoculation experiments of prion diseases on primates and provided additional details on this experimental model that has shed light on transmissibility, pathogenesis and resistance of prions. Additionally, he has recently published a review paper on the topic: Comoy et al. (2022)6 “Non-human primates in prion diseases” of which he presented the main conclusions of this paper, highlighting the achievement of the experimental studies in non-human primates in the history of TSE. He showed how the experimental models using non-human primates complement the use of transgenic humanised mice, and have biochemical and physiological relevance, while providing the opportunity to better challenge different exposure routes akin to natural conditions. The long life expectancy (30 years), the gyrencephalic brain, similar physiology as humans and comparable size are advantages of this type of models. The successful transmission of multiple human and animal TSE to different species of non-human primates like macaque, lemur, salmiri, etc., is a proof of the validity of these models to demonstrate the zoonotic potential of animal TSE.
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2.3.3EU TSE annual report 2020: preliminary results
Giuseppe Ru, Director of the EURL on TSE, presented the main findings that will be included in the EU annual report on TSE for 2021.
The report presents the results of surveillance of TSE in different animal species and is expected to be published by the end of November 2022 by EFSA.
It includes data from 27 Member States (MS), 8 non-European Reporting Countries (Bosnia and Herzegovina, Iceland, Montenegro, North Macedonia, Norway, Serbia, Switzerland and Turkey), and the United Kingdom (as non-MS from 1 February 2020).
In total, 1,021,252 cattle were tested by EU27 and XI (-9%, compared with 2020 when data from the United Kingdom were not restricted to Northern Ireland), and 66,121 cattle by eight non-EU reporting countries, with two cases of H-BSE in France and Spain, and four L-BSE in France (2), Germany and Spain.
In total, 311,174 sheep and 118,457 goats were tested in the EU27 and XI (-6.4% and -1.8%, respectively, compared to 2020 when data from the whole United Kingdom were considered).
In sheep, 551 cases of scrapie were reported by 17 MS and XI: 448 classical scrapie (CS) by six MS [80 index cases (IC) with genotypes of susceptible groups in 97% of the cases], 103 atypical scrapie (AS) (96 IC) by 13 MS and XI.
In the other non-EU reporting countries 27,594 sheep were tested with 55 CS and 1 AS in Iceland and 8 AS in Norway.
Ovine random genotyping was reported by nine MS and genotypes of susceptible groups accounted for 7.9%.
In goats, 224 cases of scrapie were reported by six EU MS: 219 CS (30 IC) by six MS, and five AS (5 IC) by three MS.
In total, 5,854 cervids were tested for chronic wasting disease by eight MS; all resulted negative.
Norway tested 21,670 cervids with two moose and one red deer positive.
In total, 149 animals from four other species tested negative in Finland and Turkey
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2.3.4Update on the activities of WOAH in the TSE field
Aurelio Cabezas, Disease Status Officer of the Status Department of the WOAH, updated the Network on the TSE-related activities ongoing at WOAH since the previous Network meeting.
During 2021 there have been many activities in WOAH:
a) the official recognition of BSE risk status: France recognized as having a negligible BSE risk and Russia as having a controlled BSE risk. 53 members and 3 zones have negligible risk, 4 countries and 2 zones have controlled risk;
b) and revision of the BSE standards: the work continued with the latest revision by the Code Commission in September 2022 and possible submission of the new chapters in the Terrestrial Animal Health Code to the General Assembly on Delegates in May 2023 for approval;
c) development of BSE surveillance guidelines: under peer review at the moment; d) impact of revised BSE standards on member or zones currently having a BSE risk status: this work has finalized with the conclusion that the revised BSE standards won’t have any Annual report of the Scientific Network on BSE-TSE 2022 impact on the maintenance of status of the Members and zones currently having a BSE risk status.
An assessment of atypical BSE against the listing criteria in the Terrestrial Animal Health Code was conducted and the results can be consulted in the reports of the September 2022 meetings of the Scientific and Code Commissions soon to be available at the WOAH website.
Finally, following a meeting in September 2021, WOAH decided not to progress on the delisting of scrapie.
The Code Commission will start the revision of the Chapter 14.8 of the Terrestrial Animal Health Code soon.
EFSA Network on BSE-TSE Minutes of the 17th meeting Held on 13-14 October 2022
EFSA
European Food Safety Authority
BIOLOGICAL HAZARDS AND ANIMAL HEALTH AND WELFARE
Network on BSE-TSE Minutes of the 17th meeting Held on 13-14 October 2022
Meeting room: SEAT M07/M08 and web-meeting (Agreed on 22 October 2022)
Participants
• Network Participants of Member States (including EFTA Countries and IPA countries):
Country Name
Austria Hermann Schildorfer Belgium Severine Matthijs Croatia Tihana Miškić, Karmen Branović Čakanić Cyprus Georgia Aparin Czechia Pavel Vodrážka, Klara Jelinkova Denmark Anne Sofie Hammer Estonia Luisa Leinberg Finland Suvi Joutsen France Thomas Maignien Germany Christine Fast Hungary Tamas Lancsak Ireland Justin Byrne, John McConville Italy Romolo Nonno, Giuseppe Ru, Elena Maria Bozzetta Latvia Edvins Olsevskis Lithuania Viktoras Maskaliovas Malta Sergio Fiore, Steve Farrugia The Netherlands Linda Kox Norway Michael Tranulis Poland Miroslaw Pavel Polak Portugal Sofia Quintas Romania Theodora Chesnoiu Vasile Slovakia Martin Mojzis Slovenia Polona Juntes Spain Maria Esther Prieto Caballero Sweden Maria Nöremark Norway Michael A. Tranulis
European Food Safety Authority Via Carlo Magno 1A – 43126 Parma, Italy Tel. +39 0521 036 111 │ www.efsa.europa.eu
Switzerland Torsten Seuberlich Albania Lisa Aldin Bosnia and Herzegovina Slobodan Dojcinovic Montenegro Rados Mkovic, Nikola Pejovic North Macedonia Aleksandar Jovanchev Serbia Sanja Aleksic Kovacevic Kosovo Saranda Ametaj Turkey Anil Demeli
EFSA: BIOHAW Unit: Ernesto Liebana, Kateryna Chuzhakina, Ancuta Cezara Simon, Angel Ortiz Pelaez, Pietro Stella, Frank Verdonck.
European Commission (EC): Fabien Schneegans (DG SANTE G5- Food Hygiene, Feed and Fraud)
Others: WOAH: Aurelio Cabezas Murillo
External speakers: Emmanuel Comoy, Sylvie Benestad, Mike Miller, Atle Mysterud, Takateru Daikai
Day 1, 13 October 2022
1. Welcome and apologies for absence The Chair welcomed the participants from 24 EU Member States, Norway, Switzerland, Albania, Bosnia and Herzegovina, Kosovo, Montenegro, North Macedonia, Serbia and Turkey.
Apologies were received from representative of Greece.
2. Adoption of agenda
The agenda was adopted without changes.
3. Agreement of the minutes of the 16th meeting of the Network on BSE/TSE held on 18- 19 October 2021, Parma1 .
The minutes were agreed by written procedure on 5 November 2021 and published on the ESA website on 22 November 2021.
4. Chronic wasting disease (CWD): update
Three presentations provided updates of the situation of CWD in North America and Norway, and an overview into European CWD situation was addressed.
4.1 CWD in North America: update. Should we get worried in Europe Michael W Miller (external speaker), wildlife veterinarian and CWD epidemiologist, presented an update of the situation of CWD in North America, lessons learnt from the long experience of handling
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the disease in North America and potential management strategies adopted, and highlighting the parallelism of the situations in North America and Europe, as well as final recommendations.
4.2 Characterization of CWD strains in Europe2
Sylvie Benestad, veterinary pathologist from the Norwegian Veterinary Institute of Norway, presented the latest evidence on the characterization of CWD strains in Europe, not only of Norway but also of Sweden and Finland. She showed some of the latest results, in particular, the emerging evidence of the presence of multiple strains of the TSE agent circulating in Europe at the moment. In particular the transmission study in bank voles of Norwegian CWD, showing that CWD prion strains affecting Norwegian cervids are distinct from those found in North America2 .
4.3 CWD in Norway: update. What’s next. Future prospects
Atle Mysterud, Professor of Ecology at University of Oslo, after providing an overview of past strategies on depopulation of reindeer in one of the populations of wild reindeer in Norway affected by CWD, presented ongoing integrated wildlife disease management and surveillance of semi-domestic reindeer population in Norway.
4.4 Q&A
To presentation 4.2 there were questions about the interpretation of the possible evolution of moose strains into more contagious reindeer-like strains through passage in animal models, and about the presence of these strains, some of them contagious, evolving already in the field for a long time. Dr Benestad answered it was difficult to transfer those results of experimental studies to natural conditions and that the surveillance efforts were very small before 2016 to speculate about how long CWD strains could have been evolving in Norway.
To presentation 4.3 there were questions about the impact of age variability in the presentation of the disease and about the reintroduction of reindeer in the culled population of Nordfjella. Prof Mysterud replied that the agent can be adapted in the host hence the age of the population needs to be taken into consideration. It has been decided to wait 5 years for the restocking, taking it from the practice in the North America. Dr Miller informed there were no clear basis to justify the 5-year clearance before restocking.
5. Zoonotic potential: species barrier
Two presentations were provided on different aspects of the same theme: the zoonotic potential of TSE. Two approaches were presented, mathematical modelling and experimental animal models.
5.1 L-BSE: verification of spontaneity. Impact of feed ban on L-BSE Takateru Daikai, Assistant Director from Animal health division, Ministry of Agriculture, Forestry and Fisheries (MAFF) Japan, presented the main outcomes of the paper by Daikai and Yamamoto, 2022. “Epidemiological verification of the mechanism of occurrence of atypical L-type bovine spongiform encephalopathy” 3 . regarding the effectiveness of the feed ban in reducing the cases of C-BSE and LBSE, using data from nine European countries to assess whether L-BSE could be transmitted orally to new birth cohorts. In addition, recent results from the surveillance programme were presented.
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5.2 Assessment of zoonotic potential of TSEs: the contribution of in-vivo experimental models
Emmanuel Comoy, veterinary researcher and member of CEA (Alternative Energies and Atomic Energy Commission) and Université Paris-Saclay, presented an overview of major inoculation experiments of prion diseases on primates and provided additional details on these experimental models that has shed light on transmissibility, pathogenesis and resistance of prions. This review has been recently published: Comoy et al. (2022)4 “Non-human primates in prion diseases”. He presented the main conclusions of this paper, highlighting the achievement of the experimental studies in non-human primates in the history of TSE.
Day 2, 14 October 2022
6. Welcome and apologies for absence The Chair welcomed the participants to the second day of the Network meeting.
7. Pulse Survey: EFSA Network on BSE/TSE and TSE in Europe Angel Ortiz (EFSA) prepared a pulse survey for Network participants regarding the scientific network format, attendance by members and observers, awareness of experts and public in Europe about the risk of TSE for humans. The main outcomes of the survey highlighted that:
The majority of the members attend the meeting to catch up with scientific developments in the field, to meet colleagues from other countries and network and to listen to the updates by the EC, WOAH and EFSA.
Over 90% of the participants want to keep the two sessions in the annual meeting, and about half of them consider sufficient and adequate just attend the annual meeting and not to engage in other activities of the network, whereas approx. 30% recognised this was not sufficient.
Regarding the follow up and use of MS Teams, half of the participants regularly follows the posts and updates while the other half is not active on MS Teams platform. Among the main reasons for not proposing topics in the annual meeting it was stated that 44% (n= 14/32) of the survey participants prefer the topics proposed by EFSA; some participants explained that research on TSE is not performed in their MS, while in other countries BSE cases are not recurrent.
The EFSA Scientific network on BSE/TSE is identified with the words knowledge, interaction, scientific, science, exchange and useful
Half of the attendees considered that although TSE diseases in Europe are in decline, many aspects are still not well understood, and we should strengthen the research and risk assessment efforts, and 40% considers that despite being in decline, we should continue with the current level of research and risk assessment efforts.
Approximately 55% of the attendees believe that currently there is very little level of awareness of the public in Europe about the risk of TSE for humans, and approx. 25% believes that younger generations do not know anything about TSE and older generations have forgotten or do not perceive risks anymore.
Most of the participants consider that CWD is not a problem that should only worry Scandinavian countries
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5 In decreasing order, the animal TSEs causing the most worry (in both classical and atypical forms) and should be prioritised in terms of research and risk assessment are BSE, scrapie and CWD (45%), followed by CWD, BSE and scrapie (20%), and BSE, CWD and scrapie (12.9%).
8. General session
8.1 EFSA activities on TSE 2021-2022
Angel Ortiz (EFSA) updated the Network on the TSE-related risk assessment activities that took place in EFSA since the 2021 Annual Network meeting. In particular, he presented the main findings of the scientific opinion regarding the use of a multi-step catalytic co-processing hydro-treatment for the production of renewable fuels using Category 3 animal fat and used cooking oil. The scientific opinion was adopted on 28 September 2022 and will be published by the end of October 2022. Angel provided also updates on the ongoing scientific opinion on the monitoring of CWD to be submitted by March 2023 as well on the PAP pig opinion to be submitted by June 2023. Finally, future prospects regarding Czechia that submitted on 12 May 2022 to the EC the application to be recognized as a Member State with negligible risk of classical scrapie were discussed.
8.2 EU TSE annual report 2021: preliminary results
Giuseppe Ru, Director of the EURL on TSE and contractor for the production of the EFSA’s EU summary report on TSE, presented the preliminary findings that will be included in the EU annual report on TSE for 2021. The report presents the results of surveillance of TSE in different animal species and is expected to be published by the end of November 2022 by EFSA. It includes data from 27 Member States (MS), 8 non-European Reporting Countries (Bosnia and Herzegovina, Iceland, Montenegro, North Macedonia, Norway, Serbia, Switzerland and Turkey), and the United Kingdom (as non-MS from 1 February 2020).
8.3 Update on the activities of the World Organisation for Animal Health (WOAH) in the TSE field
Aurelio Cabezas, Disease Status Officer of the Status Department of the WOAH, updated the Network on the TSE-related activities ongoing in WOAH since the previous Network meeting. During 2021 there have been many activities in WOAH linked to the revision of the BSE chapters in the Terrestrial Animal Health Code, which will be presented to the General Assembly for approval in May 2023.
8.4 Update on the regulatory activities of the EC in the TSE field
Fabien Schneegans, legislative officer of G5 – Food Hygiene, Feed and Fraud, Directorate-General for Health and Food Safety, European Commission, presented the activities of the EC since the last Network meeting. Two horizontal topics were presented: 1) Feed ban; 2) Trade-related issues. Three disease-specific topics were also presented: 1) BSE; 2) scrapie; 3) CWD.
8.5 Q&A
The chair asked participants for further comments and questions but there were none.
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8.6 Round-the-table discussion on the topics discussed in the EFSA Scientific Network on BSE-TSE and country updates
Open floor for all participants (not by country):
FRANCE informed of the forthcoming publication of a risk assessment of the use of fat tissue for feed due to cross-contamination with specified risk materials (SRM) when splitting the carcass. Some aspects concerning the use of gelatine and ruminant collagen for feed have also be included in this opinion.
IRELAND suggested the use of email as the main channel for communication because Microsoft Teams is not a supported platform in their institution. SWEDEN concurred with this proposal. The chair explain that it is EFSA policy to use Microsoft Teams for the scientific networks but, exceptionally, important communications could be also channelled by email.
EFSA showed the summary of the answers to the pulse survey and asked for feedback. The members supported the continuation of the annual meeting of the EFSA network and consider the meeting a unique opportunity since there are not meetings anymore in the area of TSE. The general consensus was to hold the meetings physically in Parma or somewhere else, but if needed, doing the meeting in Parma every second year would be a minimum
SLOVENIA asked WOAH if and when the name of the OIE Manual will be amended, because of the requirements related to QA system and accreditation according to ISO170025, where all changes of relevant references should be followed and corrected. Auditors are very thorough with that kind of changes. WOAH representative said that that will not be soon, it can take several months. That information is important for laboratories, due to audits and modification of the SOPs, etc.
Croatia asked WOAH if the BSE active surveillance will be eliminated in the future, following the new provisions of the terrestrial manual. The representative of WOAH replied that, in the revised provision, it will go from active to passive surveillance plus high risk animals, and that the point system will disappear. The representative of the EC expressed the opinion that in the EU active surveillance should stay in place, and that may eventually ask EFSA opinion on this matter, but eventually EU would align with WOAH provisions.
9. Any Other Business
The draft minutes will be circulated soon following the requirement to publish the network minutes within 14 working days. At least one Network meeting will be planned for 2023. Additional meetings/activities will be organised depending on the need and topics for discussion identified by the Network.
Angel Ortiz (EFSA) invited the Network participants to the upcoming info session for potential applicants on alternative methods to produce animal-by-products under the EU work frame legislation, which will be held on Thursday 8 December 20225 .
A discussion followed regarding the format and participation of European and International experts. Most of the Network participants endorsed the current hybrid format and the participation of EU and WOAH representatives. In general, it was proposed to implement the interaction between participants and the representative from The Netherlands committed to bring forward more topics for the interactive sessions for the next annual meeting, such as the risk of relaxing the feed ban. Information about the EFSA panel self-tasks and grant & procurements were also proposed to be addressed next year.
No other businesses were discussed.
5 The date has now been changed to 1 December 2022
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The Chair thanked all participants for their attendance and closed the meeting.
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This information is critical, and should continue to be collected.
The TSE prion is spreading across the USA in Cervid as in CWD TSE Prion.
The mad cow surveillance, feed ban, testing, and SRM removal there from, has been, and still is, a terrible failure.
WE know that the USA Food and Drug Administration's BSE Feed Regulation (21 CFR 589.2000) of August 1997 was/is a colossal failure, and proven to be so year after year, decade after decade, and this was just admitted by the FDA et al (see below FDA Reports on VFD Compliance Sept. 2019 report).
God, all these decades you hear from all the warning letters on SRM that were released to the public for consumption, that even if they did eat a SRM, the BSE Feed Regulation (21 CFR 589.2000) of August 1997 would save that tissue from that animal from having a TSE Prion, was nothing but lies. what about those children all across the USA that were fed the most high risk cattle for mad cow disease, i.e. dead stock downer cows via the USDA School lunch program, who will watch those kids for the next 50 years for cjd tse prion aka mad cow disease, let alone all the folks consuming SRMs that have been exposed to mad cow type disease in different livestock species, due to the fact the USA colossal failure of the BSE Feed Regulation (21 CFR 589.2000) of August 1997. it's all documented below, see for yourself; SUNDAY, SEPTEMBER 1, 2019 FDA Reports on VFD Compliance
Tuesday, September 10, 2019
FSIS [Docket No. FSIS–2019–0021] Notice of Request To Renew an Approved Information Collection: Specified Risk Materials Singeltary Submission
Sunday, January 10, 2021
APHIS Concurrence With OIE Risk Designation for Bovine Spongiform Encephalopathy [Docket No. APHIS-2018-0087] Singeltary Submission June 17, 2019
APHIS Concurrence With OIE Risk Designation for Bovine Spongiform Encephalopathy [Docket No. APHIS-2018-0087] Singeltary Submission
Greetings APHIS et al,
I would kindly like to comment on APHIS Concurrence With OIE Risk Designation for Bovine Spongiform Encephalopathy [Docket No. APHIS-2018-0087], and my comments are as follows, with the latest peer review and transmission studies as references of evidence.
THE OIE/USDA BSE Minimal Risk Region MRR is nothing more than free pass to import and export the Transmissible Spongiform Encephalopathy TSE Prion disease. December 2003, when the USDA et al lost it's supposedly 'GOLD CARD' ie BSE FREE STATUS (that was based on nothing more than not looking and not finding BSE), once the USA lost it's gold card BSE Free status, the USDA OIE et al worked hard and fast to change the BSE Geographical Risk Statuses i.e. the BSE GBR's, and replaced it with the BSE MRR policy, the legal tool to trade mad cow type disease TSE Prion Globally. The USA is doing just what the UK did, when they shipped mad cow disease around the world, except with the BSE MRR policy, it's now legal.
Also, the whole concept of the BSE MRR policy is based on a false pretense, that atypical BSE is not transmissible, and that only typical c-BSE is transmissible via feed. This notion that atypical BSE TSE Prion is an old age cow disease that is not infectious is absolutely false, there is NO science to show this, and on the contrary, we now know that atypical BSE will transmit by ORAL ROUTES, but even much more concerning now, recent science has shown that Chronic Wasting Disease CWD TSE Prion in deer and elk which is rampant with no stopping is sight in the USA, and Scrapie TSE Prion in sheep and goat, will transmit to PIGS by oral routes, this is our worst nightmare, showing even more risk factors for the USA FDA PART 589 TSE PRION FEED ban.
The FDA PART 589 TSE PRION FEED ban has failed terribly bad, and is still failing, since August 1997. there is tonnage and tonnage of banned potential mad cow feed that went into commerce, and still is, with one decade, 10 YEARS, post August 1997 FDA PART 589 TSE PRION FEED ban, 2007, with 10,000,000 POUNDS, with REASON, Products manufactured from bulk feed containing blood meal that was cross contaminated with prohibited meat and bone meal and the labeling did not bear cautionary BSE statement. you can see all these feed ban warning letters and tonnage of mad cow feed in commerce, year after year, that is not accessible on the internet anymore like it use to be, you can see history of the FDA failure August 1997 FDA PART 589 TSE PRION FEED ban here, but remember this, we have a new outbreak of TSE Prion disease in a new livestock species, the camel, and this too is very worrisome.
WITH the OIE and the USDA et al weakening the global TSE prion surveillance, by not classifying the atypical Scrapie as TSE Prion disease, and the notion that they want to do the same thing with typical scrapie and atypical BSE, it's just not scientific.
WE MUST abolish the BSE MRR policy, go back to the BSE GBR risk assessments by country, and enhance them to include all strains of TSE Prion disease in all species. With Chronic Wasting CWD TSE Prion disease spreading in Europe, now including, Norway, Finland, Sweden, also in Korea, Canada and the USA, and the TSE Prion in Camels, the fact the the USA is feeding potentially CWD, Scrapie, BSE, typical and atypical, to other animals, and shipping both this feed and or live animals or even grains around the globe, potentially exposed or infected with the TSE Prion. this APHIS Concurrence With OIE Risk Designation for Bovine Spongiform Encephalopathy [Docket No. APHIS-2018-0087], under it's present definition, does NOT show the true risk of the TSE Prion in any country. as i said, it's nothing more than a legal tool to trade the TSE Prion around the globe, nothing but ink on paper.
AS long as the BSE MRR policy stays in effect, TSE Prion disease will continued to be bought and sold as food for both humans and animals around the globe, and the future ramifications from friendly fire there from, i.e. iatrogenic exposure and transmission there from from all of the above, should not be underestimated. ...
Control of Chronic Wasting Disease OMB Control Number: 0579-0189 APHIS-2021-0004 Singeltary Submission
Docket No. APHIS-2018-0011 Chronic Wasting Disease Herd Certification
RE-Inactivation of porcine endogenous retrovirus in pigs using CPISPR-Cas9
seems that the USA feed ban for ruminant protein is still a serious problem, so there seems to still be a risk factor for pigs and Transmissible Spongiform Encephalopathy TSE prion disease. now with the updated science showing that pigs are susceptible to the Chronic Wasting Disease TSE Prion ORALLY, and cwd running rampant in the USA, any use of porcine organs should be tested for the CWD TSE Prion...
Research Project: TRANSMISSION, DIFFERENTIATION, AND PATHOBIOLOGY OF TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES
Location: Virus and Prion Research
Title: Disease-associated prion protein detected in lymphoid tissues from pigs challenged with the agent of chronic wasting disease
Author item Moore, Sarah item Kunkle, Robert item Kondru, Naveen item Manne, Sireesha item Smith, Jodi item Kanthasamy, Anumantha item West Greenlee, M item Greenlee, Justin
Submitted to: Prion Publication Type: Abstract Only Publication Acceptance Date: 3/15/2017
Publication Date: N/A Citation: N/A Interpretive Summary:
Technical Abstract: Aims: Chronic wasting disease (CWD) is a naturally-occurring, fatal neurodegenerative disease of cervids. We previously demonstrated that disease-associated prion protein (PrPSc) can be detected in the brain and retina from pigs challenged intracranially or orally with the CWD agent. In that study, neurological signs consistent with prion disease were observed only in one pig: an intracranially challenged pig that was euthanized at 64 months post-challenge. The purpose of this study was to use an antigen-capture immunoassay (EIA) and real-time quaking-induced conversion (QuIC) to determine whether PrPSc is present in lymphoid tissues from pigs challenged with the CWD agent.
Methods: At two months of age, crossbred pigs were challenged by the intracranial route (n=20), oral route (n=19), or were left unchallenged (n=9). At approximately 6 months of age, the time at which commercial pigs reach market weight, half of the pigs in each group were culled (<6 month challenge groups). The remaining pigs (>6 month challenge groups) were allowed to incubate for up to 73 months post challenge (mpc). The retropharyngeal lymph node (RPLN) was screened for the presence of PrPSc by EIA and immunohistochemistry (IHC). The RPLN, palatine tonsil, and mesenteric lymph node (MLN) from 6-7 pigs per challenge group were also tested using EIA and QuIC.
Results: PrPSc was not detected by EIA and IHC in any RPLNs. All tonsils and MLNs were negative by IHC, though the MLN from one pig in the oral <6 month group was positive by EIA. PrPSc was detected by QuIC in at least one of the lymphoid tissues examined in 5/6 pigs in the intracranial <6 months group, 6/7 intracranial >6 months group, 5/6 pigs in the oral <6 months group, and 4/6 oral >6 months group. Overall, the MLN was positive in 14/19 (74%) of samples examined, the RPLN in 8/18 (44%), and the tonsil in 10/25 (40%). Conclusions:
This study demonstrates that PrPSc accumulates in lymphoid tissues from pigs challenged intracranially or orally with the CWD agent, and can be detected as early as 4 months after challenge.
CWD-infected pigs rarely develop clinical disease and if they do, they do so after a long incubation period. This raises the possibility that CWD-infected pigs could shed prions into their environment long before they develop clinical disease.
Furthermore, lymphoid tissues from CWD-infected pigs could present a potential source of CWD infectivity in the animal and human food chains.
CONFIDENTIAL
EXPERIMENTAL PORCINE SPONGIFORM ENCEPHALOPATHY
While this clearly is a cause for concern we should not jump to the conclusion that this means that pigs will necessarily be infected by bone and meat meal fed by the oral route as is the case with cattle. ...
we cannot rule out the possibility that unrecognised subclinical spongiform encephalopathy could be present in British pigs though there is no evidence for this: only with parenteral/implantable pharmaceuticals/devices is the theoretical risk to humans of sufficient concern to consider any action.
Our records show that while some use is made of porcine materials in medicinal products, the only products which would appear to be in a hypothetically ''higher risk'' area are the adrenocorticotrophic hormone for which the source material comes from outside the United Kingdom, namely America China Sweden France and Germany. The products are manufactured by Ferring and Armour. A further product, ''Zenoderm Corium implant'' manufactured by Ethicon, makes use of porcine skin - which is not considered to be a ''high risk'' tissue, but one of its uses is described in the data sheet as ''in dural replacement''. This product is sourced from the United Kingdom.....
snip...see much more here ;
OIE Bulletin
Camel prion disease: a possible emerging disease in dromedary camel populations?
The identification of a new prion disease in dromedary camels in Algeria and Tunisia, called camel prion disease (CPD), extends the spectrum of animal species naturally susceptible to prion diseases and opens up new research areas for investigation.
Camel prion disease was identified in 2018 in adult camels showing clinical signs at the ante mortem inspection at slaughterhouses in the region of Ouargla (Algeria), and in 2019 in the region of Tataouine (Tunisia). It adds to the group of existing animal prion diseases, including scrapie in sheep and goats, chronic wasting disease (CWD) in cervids and BSE (mainly in bovines). The detection of a new prion disease in the dromedary population requires attention and investigation needs to be carried out to assess the risks of this disease to animal and public health. As of today, very limited epidemiological information is available to assess the prevalence, geographical distribution and dynamic of the transmission of the disease.
Based on the clinical signs suggesting prion disease, CPD seems to have occurred in 3.1% of the dromedaries brought to the abattoir in Ouargla. Pathognomonic neurodegeneration and disease specific prion protein (PrPSc) were detected in brain tissue from three symptomatic animals (source:
In May 2019, the OIE received a report from Tunisia on a single case of a 12-year-old slaughtered dromedary camel showing neurological signs confirmed as CPD by the Istituto Superiore di Sanità (ISS) based in Italy.
©B. Babelhadj/University Kasdi Merbah, Algeria
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Is camel prion disease transmissible in natural conditions?
The involvement of lymphoid tissue in prion replication, observed both in the Algeria and Tunisia cases, is suggestive of a peripheral pathogenesis, which is thought to be a prerequisite for prion shedding into the environment. As with other animal prion diseases, such as scrapie and CWD, in which lymphoid tissues are extensively involved and horizontal transmission occurs efficiently under natural conditions, the detection of prion proteins in lymph nodes is suggestive of the infectious nature of CPD and concurs to hypothesise the potential impact of CPD on animal health. No evidence is currently available with which to argue for the relevance of CPD for human health. However, no absolute species barrier exists in prion diseases and minimising the exposure of humans to prion-infected animal products is an essential aspect of public health protection. As for the relationship between CPD and other animal prion diseases, preliminary analyses suggest that CPD prions have a different molecular signature from scrapie and BSE.
Actions on the follow up of CPD
Since the first description of CPD, the OIE promoted discussions on the impact of this new disease through the OIE Scientific Commission for Animal Diseases (Scientific Commission). The Scientific Commission consulted two OIE ad hoc Groups, one on BSE risk status evaluation of Members and the other on camelids. It analysed the information available from the Algeria and Tunisia cases to evaluate if CPD should be considered an ‘emerging disease’ based on the criteria listed in the Terrestrial Animal Health Code1 .
The OIE Scientific Commission noted that limited surveillance data were available on the prevalence of CPD and that the evidence was not sufficient to measure, at that time, the impact of the disease on animal or public health. Therefore, it was concluded that, with the current knowledge, CPD did not currently meet the criteria to be considered an emerging disease. Nonetheless, it was emphasised that CPD should be considered as a new disease not to be overlooked and called for the collection of further scientific evidence through research and surveillance in the affected countries and in countries with dromedary camel populations to measure the impact of the disease. As new scientific evidence becomes available, the OIE Scientific Commission will reassess whether this disease should be considered as an emerging disease.
The worldwide camel population is ~35 million head (FAO, 2019), 88% of which is found in Africa. The camel farming system is evolving rapidly, and these animals represent vital sources of meat, milk and transportation for millions of people living in the most arid regions of the world. This makes it necessary to assess the risk for animal and human health and to develop evidence-based policies to control and limit the spread of the disease in animals, and to minimise human exposure. As a first step, the awareness of Veterinary Services about CPD and its diagnostic capacity needs to be improved in all countries where dromedaries are part of the domestic livestock.
At the regional level, CPD was first discussed in the 18th Joint Permanent Committee of the Mediterranean Animal Health Network (REMESA) held in Cairo, Egypt, in June 2019 where an expert 1 a new occurrence in an animal of a disease, infection or infestation, causing a significant impact on animal or public health resulting from a) a change of a known pathogenic agent or its spread to a new geographic area or species, or b) a previously unrecognised pathogenic agent or disease diagnosed for the first time www.oiebulletin.com
3
from ISS, Italy, shared the knowledge available on the new disease with the 15 REMESA Member Countries. The discussion highlighted the need to strengthen surveillance systems in order to collect epidemiological data to inform the risk assessments. The results of these risk assessments will support the implementation of evidence-based policies to manage the risks in both animals and humans.
CPD was recently discussed atthe 15thConference of the OIE Regional Commission for the Middle East in November. During this conference, the CAMENET (Camel Middle East Network) launched a wide ranging proposal for training, coordinated surveillance and research on CPD. In addition, the ERFAN (Enhancing Research for Africa Network), a platform aimed at enhancing scientific cooperation between Africa and Italy, during its 2nd ERFAN meeting for North Africa, presented a project on CPD with the objective of increasing CPD coordinated surveillance in North Africa.
The OIE, through its Reference Laboratories for prion diseases, and by involving the above scientific initiatives, is keeping a close watch on the evolution of the disease to gather scientific evidence and to allow a proper and more thorough assessment of the risk associated with this novel disease.
◼ December 2019
Tuesday, April 27, 2021
Working Document on Camel Prion Disease (CPrD) 14/09/2020
Very low oral exposure to prions of brain or saliva origin can transmit chronic wasting disease
Nathaniel D. Denkers ,Clare E. Hoover ,Kristen A. Davenport,Davin M. Henderson,Erin E. McNulty,Amy V. Nalls,Candace K. Mathiason,Edward A. Hoover
Published: August 20, 2020
We report that oral exposure to as little as 300 nanograms (ng) of CWD-positive brain or to saliva containing seeding activity equivalent to 300 ng of CWD-positive brain, were sufficient to transmit CWD disease. This was true whether the inoculum was administered as a single bolus or divided as three weekly 100 ng exposures. However, when the 300 ng total dose was apportioned as 10, 30 ng doses delivered over 12 weeks, no infection occurred. While low-dose exposures to prions of brain or saliva origin prolonged the time from inoculation to first detection of infection, once infection was established, we observed no differences in disease pathogenesis. These studies suggest that the CWD minimum infectious dose approximates 100 to 300 ng CWD-positive brain (or saliva equivalent), and that CWD infection appears to conform more with a threshold than a cumulative dose dynamic.
WE know now, and we knew decades ago, that 5.5 grams of suspect feed in TEXAS was enough to kill 100 cows.
look at the table and you'll see that as little as 1 mg (or 0.001 gm) caused 7% (1 of 14) of the cows to come down with BSE;
Risk of oral infection with bovine spongiform encephalopathy agent in primates
Corinne Ida Lasmézas, Emmanuel Comoy, Stephen Hawkins, Christian Herzog, Franck Mouthon, Timm Konold, Frédéric Auvré, Evelyne Correia, Nathalie Lescoutra-Etchegaray, Nicole Salès, Gerald Wells, Paul Brown, Jean-Philippe Deslys
Summary The uncertain extent of human exposure to bovine spongiform encephalopathy (BSE)--which can lead to variant Creutzfeldt-Jakob disease (vCJD)--is compounded by incomplete knowledge about the efficiency of oral infection and the magnitude of any bovine-to-human biological barrier to transmission. We therefore investigated oral transmission of BSE to non-human primates. We gave two macaques a 5 g oral dose of brain homogenate from a BSE-infected cow. One macaque developed vCJD-like neurological disease 60 months after exposure, whereas the other remained free of disease at 76 months. On the basis of these findings and data from other studies, we made a preliminary estimate of the food exposure risk for man, which provides additional assurance that existing public health measures can prevent transmission of BSE to man.
snip...
BSE bovine brain inoculum
100 g 10 g 5 g 1 g 100 mg 10 mg 1 mg 0·1 mg 0·01 mg
Primate (oral route)* 1/2 (50%)
Cattle (oral route)* 10/10 (100%) 7/9 (78%) 7/10 (70%) 3/15 (20%) 1/15 (7%) 1/15 (7%)
RIII mice (ic ip route)* 17/18 (94%) 15/17 (88%) 1/14 (7%)
PrPres biochemical detection
The comparison is made on the basis of calibration of the bovine inoculum used in our study with primates against a bovine brain inoculum with a similar PrPres concentration that was inoculated into mice and cattle.8 *Data are number of animals positive/number of animals surviving at the time of clinical onset of disease in the first positive animal (%). The accuracy of bioassays is generally judged to be about plus or minus 1 log. ic ip=intracerebral and intraperitoneal.
Table 1: Comparison of transmission rates in primates and cattle infected orally with similar BSE brain inocula
Published online January 27, 2005
It is clear that the designing scientists must
also have shared Mr Bradley’s surprise at the results because all the dose
levels right down to 1 gram triggered infection.
6. It also appears to me that Mr Bradley’s answer (that it would take less than say 100
grams) was probably given with the benefit of hindsight; particularly if one
considers that later in the same answer Mr Bradley expresses his surprise that it
could take as little of 1 gram of brain to cause BSE by the oral route within the
same species. This information did not become available until the "attack rate"
experiment had been completed in 1995/96. This was a titration experiment
designed to ascertain the infective dose. A range of dosages was used to ensure
that the actual result was within both a lower and an upper limit within the study
and the designing scientists would not have expected all the dose levels to trigger
infection. The dose ranges chosen by the most informed scientists at that time
ranged from 1 gram to three times one hundred grams. It is clear that the designing
scientists must have also shared Mr Bradley’s surprise at the results because all the
dose levels right down to 1 gram triggered infection.
***> cattle, pigs, sheep, cwd, tse, prion, oh my!
***> In contrast, cattle are highly susceptible to white-tailed deer CWD and mule deer CWD in experimental conditions but no natural CWD infections in cattle have been reported (Sigurdson, 2008; Hamir et al., 2006).
Sheep and cattle may be exposed to CWD via common grazing areas with affected deer but so far, appear to be poorly susceptible to mule deer CWD (Sigurdson, 2008). In contrast, cattle are highly susceptible to white-tailed deer CWD and mule deer CWD in experimental conditions but no natural CWD infections in cattle have been reported (Sigurdson, 2008; Hamir et al., 2006). It is not known how susceptible humans are to CWD but given that the prion can be present in muscle, it is likely that humans have been exposed to the agent via consumption of venison (Sigurdson, 2008). Initial experimental research suggests that human susceptibility to CWD is low and there may be a robust species barrier for CWD transmission to humans (Sigurdson, 2008), however the risk appetite for a public health threat may still find this level unacceptable.
DEFRA
Friday, December 14, 2012
DEFRA U.K. What is the risk of Chronic Wasting Disease CWD being introduced into Great Britain? A Qualitative Risk Assessment October 2012
snip.....
In the USA, under the Food and Drug Administration's BSE Feed Regulation (21 CFR 589.2000) most material (exceptions include milk, tallow, and gelatin) from deer and elk is prohibited for use in feed for ruminant animals. With regards to feed for non-ruminant animals, under FDA law, CWD positive deer may not be used for any animal feed or feed ingredients. For elk and deer considered at high risk for CWD, the FDA recommends that these animals do not enter the animal feed system. However, this recommendation is guidance and not a requirement by law. Animals considered at high risk for CWD include:
1) animals from areas declared to be endemic for CWD and/or to be CWD eradication zones and
2) deer and elk that at some time during the 60-month period prior to slaughter were in a captive herd that contained a CWD-positive animal.
Therefore, in the USA, materials from cervids other than CWD positive animals may be used in animal feed and feed ingredients for non-ruminants.
The amount of animal PAP that is of deer and/or elk origin imported from the USA to GB can not be determined, however, as it is not specified in TRACES.
It may constitute a small percentage of the 8412 kilos of non-fish origin processed animal proteins that were imported from US into GB in 2011.
Overall, therefore, it is considered there is a __greater than negligible risk___ that (nonruminant) animal feed and pet food containing deer and/or elk protein is imported into GB.
There is uncertainty associated with this estimate given the lack of data on the amount of deer and/or elk protein possibly being imported in these products.
snip.....
36% in 2007 (Almberg et al., 2011). In such areas, population declines of deer of up to 30 to 50% have been observed (Almberg et al., 2011). In areas of Colorado, the prevalence can be as high as 30% (EFSA, 2011). The clinical signs of CWD in affected adults are weight loss and behavioural changes that can span weeks or months (Williams, 2005). In addition, signs might include excessive salivation, behavioural alterations including a fixed stare and changes in interaction with other animals in the herd, and an altered stance (Williams, 2005). These signs are indistinguishable from cervids experimentally infected with bovine spongiform encephalopathy (BSE). Given this, if CWD was to be introduced into countries with BSE such as GB, for example, infected deer populations would need to be tested to differentiate if they were infected with CWD or BSE to minimise the risk of BSE entering the human food-chain via affected venison. snip..... The rate of transmission of CWD has been reported to be as high as 30% and can approach 100% among captive animals in endemic areas (Safar et al., 2008).
snip.....
In summary, in endemic areas, there is a medium probability that the soil and surrounding environment is contaminated with CWD prions and in a bioavailable form. In rural areas where CWD has not been reported and deer are present, there is a greater than negligible risk the soil is contaminated with CWD prion. snip..... In summary, given the volume of tourists, hunters and servicemen moving between GB and North America, the probability of at least one person travelling to/from a CWD affected area and, in doing so, contaminating their clothing, footwear and/or equipment prior to arriving in GB is greater than negligible... For deer hunters, specifically, the risk is likely to be greater given the increased contact with deer and their environment. However, there is significant uncertainty associated with these estimates.
snip.....
Therefore, it is considered that farmed and park deer may have a higher probability of exposure to CWD transferred to the environment than wild deer given the restricted habitat range and higher frequency of contact with tourists and returning GB residents.
snip.....
-------- Original Message --------
Subject: re-BSE prions propagate as either variant CJD-like or sporadic CJD
Date: Thu, 28 Nov 2002 10:23:43 -0000
From: "Asante, Emmanuel A" e.asante@ic.ac.uk
To: "'flounder@wt.net'" flounder@wt.net
Dear Terry,
I have been asked by Professor Collinge to respond to your request. I am a Senior Scientist in the MRC Prion Unit and the lead author on the paper. I have attached a pdf copy of the paper for your attention.
Thank you for your interest in the paper.
In respect of your first question, the simple answer is, ***yes. As you will find in the paper, we have managed to associate the alternate phenotype to type 2 PrPSc, the commonest sporadic CJD. It is too early to be able to claim any further sub-classification in respect of Heidenhain variant CJD or Vicky Rimmer's version. It will take further studies, which are on-going, to establish if there are sub-types to our initial finding which we are now reporting. The main point of the paper is that, as well as leading to the expected new variant CJD phenotype, BSE transmission to the 129-methionine genotype can lead to an alternate phenotype which is indistinguishable from type 2 PrPSc.
I hope reading the paper will enlighten you more on the subject. If I can be of any further assistance please to not hesitate to ask. Best wishes.
Emmanuel Asante
<<Asante et al 2002.pdf>>
____________________________________
Dr. Emmanuel A Asante MRC Prion Unit & Neurogenetics Dept. Imperial College School of Medicine (St. Mary's) Norfolk Place, LONDON W2 1PG Tel: +44 (0)20 7594 3794 Fax: +44 (0)20 7706 3272 email: e.asante@ic.ac.uk (until 9/12/02) New e-mail: e.asante@prion.ucl.ac.uk (active from now)
____________________________________
''This study demonstrates that the H-type BSE agent is transmissible by the oronasal route. Cattle with the EK211 genotype are oronasally susceptible to small doses of the H-BSE agent from either EK211 or EE211 (wild type) donors. Wild-type EE211 cattle remained asymptomatic for the duration of the experiment with this small dose (0.1g) of inoculum. These results reinforce the need for ongoing surveillance for classical and atypical BSE to minimize the risk of potentially infectious tissues entering the animal or human food chains.''
***Moreover, sporadic disease has never been observed in breeding colonies or primate research laboratories, most notably among hundreds of animals over several decades of study at the National Institutes of Health25, and in nearly twenty older animals continuously housed in our own facility.***
Even if the prevailing view is that sporadic CJD is due to the spontaneous formation of CJD prions, it remains possible that its apparent sporadic nature may, at least in part, result from our limited capacity to identify an environmental origin.
O.05: Transmission of prions to primates after extended silent incubation periods: Implications for BSE and scrapie risk assessment in human populations
Emmanuel Comoy, Jacqueline Mikol, Valerie Durand, Sophie Luccantoni, Evelyne Correia, Nathalie Lescoutra, Capucine Dehen, and Jean-Philippe Deslys Atomic Energy Commission; Fontenay-aux-Roses, France
Prion diseases (PD) are the unique neurodegenerative proteinopathies reputed to be transmissible under field conditions since decades. The transmission of Bovine Spongiform Encephalopathy (BSE) to humans evidenced that an animal PD might be zoonotic under appropriate conditions. Contrarily, in the absence of obvious (epidemiological or experimental) elements supporting a transmission or genetic predispositions, PD, like the other proteinopathies, are reputed to occur spontaneously (atpical animal prion strains, sporadic CJD summing 80% of human prion cases).
Non-human primate models provided the first evidences supporting the transmissibiity of human prion strains and the zoonotic potential of BSE. Among them, cynomolgus macaques brought major information for BSE risk assessment for human health (Chen, 2014), according to their phylogenetic proximity to humans and extended lifetime. We used this model to assess the zoonotic potential of other animal PD from bovine, ovine and cervid origins even after very long silent incubation periods.
*** We recently observed the direct transmission of a natural classical scrapie isolate to macaque after a 10-year silent incubation period,
***with features similar to some reported for human cases of sporadic CJD, albeit requiring fourfold long incubation than BSE. Scrapie, as recently evoked in humanized mice (Cassard, 2014),
***is the third potentially zoonotic PD (with BSE and L-type BSE),
***thus questioning the origin of human sporadic cases.
We will present an updated panorama of our different transmission studies and discuss the implications of such extended incubation periods on risk assessment of animal PD for human health.
===============
***thus questioning the origin of human sporadic cases***
===============
***our findings suggest that possible transmission risk of H-type BSE to sheep and human. Bioassay will be required to determine whether the PMCA products are infectious to these animals.
==============
PRION 2015 CONFERENCE
***Transmission data also revealed that several scrapie prions propagate in HuPrP-Tg mice with efficiency comparable to that of cattle BSE. While the efficiency of transmission at primary passage was low, subsequent passages resulted in a highly virulent prion disease in both Met129 and Val129 mice.
***Transmission of the different scrapie isolates in these mice leads to the emergence of prion strain phenotypes that showed similar characteristics to those displayed by MM1 or VV2 sCJD prion.
***These results demonstrate that scrapie prions have a zoonotic potential and raise new questions about the possible link between animal and human prions.
PRION 2016 TOKYO
Saturday, April 23, 2016
SCRAPIE WS-01: Prion diseases in animals and zoonotic potential 2016
Prion. 10:S15-S21. 2016 ISSN: 1933-6896 printl 1933-690X online
Taylor & Francis
Prion 2016 Animal Prion Disease Workshop Abstracts
WS-01: Prion diseases in animals and zoonotic potential
Transmission of the different scrapie isolates in these mice leads to the emergence of prion strain phenotypes that showed similar characteristics to those displayed by MM1 or VV2 sCJD prion.
These results demonstrate that scrapie prions have a zoonotic potential and raise new questions about the possible link between animal and human prions.
Title: Transmission of scrapie prions to primate after an extended silent incubation period)
*** In complement to the recent demonstration that humanized mice are susceptible to scrapie, we report here the first observation of direct transmission of a natural classical scrapie isolate to a macaque after a 10-year incubation period. Neuropathologic examination revealed all of the features of a prion disease: spongiform change, neuronal loss, and accumulation of PrPres throughout the CNS.
*** This observation strengthens the questioning of the harmlessness of scrapie to humans, at a time when protective measures for human and animal health are being dismantled and reduced as c-BSE is considered controlled and being eradicated.
*** Our results underscore the importance of precautionary and protective measures and the necessity for long-term experimental transmission studies to assess the zoonotic potential of other animal prion strains.
PRION CONFERENCE 2022 ABSTRACTS CWD TSE PrP ZOONOSIS
Transmission of prion infectivity from CWD-infected macaque tissues to rodent models demonstrates the zoonotic potential of chronic wasting disease.
Samia Hannaouia, Ginny Chenga, Wiebke Wemheuerb, Walter J. Schulz-Schaefferb, Sabine Gilcha, and Hermann M. Schätzla aDepartment of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine & Hotchkiss Brain Institute; University of Calgary, Calgary, Canada; bInstitute of Neuropathology, Medical Faculty, Saarland University, Homburg/Saar, Germany
Aims: Chronic wasting disease (CWD) is a prion disease of cervids. Its rapid geographic expansion, shedding of infectivity and persistence in the environment for many years are of concern for humans. Here, we provide the first evidence by transmission experiments to different transgenic mouse models and bank voles that Cynomolgus macaques inoculated via different routes with CWD-positive cervid tissues harbor infectious prions that elicit clinical disease in rodents.
Material and Methods: We used tissue materials from macaques inoculated with CWD to inoculate transgenic mice overexpressing cervid PrPCfollowed by transmission into bank voles. We used RT-QuIC, immunoblot and PET blot analysis to assess brains, spinal cords, and tissues of the gastrointestinal tract (GIT) for the presence of prions.
Results: Our results show that of the macaque materials that induced clinical disease in transgenic mice,73% were from the CNS (46% spinal cord and 27% brain), and 27% were from the spleen, although attack rates were low around 20%. Clinical mice did not display PK-resistant PrPSc(PrPres) in immunoblot, but showed low-levels of prion seeding activity. Transmission into bank voles from clinical transgenic mice led to a 100% attack rate with typical PrPressignature in immunoblot, which was different from that of voles inoculated directly with CWD or scrapie prions. High-level prion seeding activity in brain and spinal cord and PrPresdeposition in the brain were present. Remarkably, we also found prion seeding activity in GIT tissues of inoculated voles. Second passage in bank voles led to a 100% attack rate in voles inoculated with brain, spinal cord and small intestine material from first round animals, with PrPresin immunoblot, prion seeding activity, and PrPresdeposition in the brain. Shortened survival times indicate adaptation in the new host. This also shows that prions detected in GIT tissues are infectious and transmissible. Transmission of brain material from sick voles back to cervidized mice revealed transmission in these mice with a 100% attack rate, and interestingly, with different biochemical signature and distribution in the brain.
Conclusions: Our findings demonstrate that macaques, considered the best model for the zoonotic potential of prions, were infected upon CWD challenge, including oral one. The disease manifested as atypical in macaques and transgenic mice, but with infectivity present at all times, as unveiled in the bank vole model with an unusual tissue tropism.
Funded by: The National Institutes of Health, USA, and the Alberta Prion Research Institute/Alberta Innovates Canada. Grant number: 1R01NS121016-01; 201,600,023
Acknowledgement: We thank Umberto Agrimi, Istituto Superiore di Sanità, Rome, Italy, and Michael Beekes, Robert-Koch Institute Berlin, Germany, for providing the bank vole model. We thank the University of Calgary animal facility staff and Dr. Stephanie Anderson for animal care.
Transmission of Cervid Prions to Humanized Mice Demonstrates the Zoonotic Potential of CWD
Samia Hannaouia, Irina Zemlyankinaa, Sheng Chun Changa, Maria Immaculata Arifina, Vincent Béringueb, Debbie McKenziec, Hermann M. Schatzla, and Sabine Gilcha
aDepartment of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine; Hotchkiss Brain Institute; University of Calgary, Calgary, Canada; bUniversité Paris-Saclay, INRAE, UVSQ, VIM, Jouy-en-Josas, France; cDepartment of Biological Sciences, Center for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Canada
Aims: Chronic wasting disease (CWD), a prion disease of cervids, spreads efficiently among wild and farmed animals. Potential transmission to humans of CWD is a growing concern due to its increasing prevalence. Here, we aimed to determine the zoonotic potential of CWD using a mouse model for human prion diseases.
Material and Methods: Transgenic mice overexpressing human PrPChomozygous for methionine at codon 129 (tg650) were inoculated intracerebrally with brain homogenates of white-tailed deer infected with Wisc-1/CWD1 or 116AG CWD strains. Mice were monitored for clinical signs and were euthanized at terminal disease. Brains were tested by RT-QuIC, western blot upon PK digestion, and immunohistochemistry; fecal homogenates were analyzed by RT-QuIC. Brain/spinal cord and fecal homogenates of CWD-inoculated tg650 mice were inoculated into tg650 mice or bank voles. Brain homogenates of bank voles inoculated with fecal homogenates of CWD-infected tg650 mice were used for second passage in bank voles.
Results: Here, we provide the strongest evidence supporting the zoonotic potential of CWD prions, and their possible phenotype in humans. Inoculation of mice expressing human PrPCwith deer CWD isolates (strains Wisc-1 and 116AG) resulted in atypical clinical manifestations in > 75% of the mice, with myoclonus as leading clinical sign. Most of tg650 brain homogenates were positive for seeding activity in RT-QuIC. Clinical disease and presentation was transmissible to tg650 mice and bank voles. Intriguingly, protease-resistant PrP in the brain of tg650 mice resembled that found in a familial human prion disease and was transmissible upon passage. Abnormal PrP aggregates upon infection with Wisc-1 were detectable in thalamus, hypothalamus, and midbrain/pons regions.
Unprecedented in human prion disease, feces of CWD-inoculated tg650 mice harbored prion seeding activity and infectious prions, as shown by inoculation of bank voles and tg650 with fecal homogenates.
Conclusions: This is the first evidence that CWD can infect humans and cause disease with a distinctive clinical presentation, signature, and tropism, which might be transmissible between humans while current diagnostic assays might fail to detect it. These findings have major implications for public health and CWD-management.
Funded by: We are grateful for financial support from the Natural Sciences and Engineering Research Council of Canada, the National Institutes of Health, Genome Canada, and the Alberta Prion Research Institute. SG is supported by the Canada Research Chairs program.
Acknowledgement: We thank Dr. Trent Bollinger, WCVM, University of Saskatchewan, Saskatoon, Canada, for providing brain tissue from the WTD-116AG isolate, Dr. Stéphane Haïk, ICM, Paris, France, for providing brain tissue from vCJD and sCJD cases, and Dr. Umberto Agrimi, Istituto Superiore di Sanità, Italy, for the bank vole model. We thank animal facility staff for animal care, Dr. Stephanie Anderson for veterinary oversight, and Yo-Ching Cheng for preparing recombinant PrP substrates. Thank you to Dr. Stephanie Booth and Jennifer Myskiw, Public Health Agency of Canada, Canada.
The chronic wasting disease agent from white-tailed deer is infectious to humanized mice after passage through raccoons
Eric Cassmanna, Xu Qib, Qingzhong Kongb, and Justin Greenleea
aNational Animal Disease Center, Agricultural Research Service, US Department of Agriculture, Ames, IA, USA bDepartments of Pathology, Neurology, National Center for Regenerative Medicine, and National Prion Disease Pathology Surveillance Center, Case Western Reserve University, Cleveland, Ohio, USA
Aims: Evaluate the zoonotic potential of the raccoon passaged chronic wasting disease (CWD) agent in humanized transgenic mice in comparison with the North American CWD agent from the original white-tailed deer host.
Material and Methods: Pooled brain material (GG96) from a CWD positive herd was used to oronasally inoculate two white-tailed deer with wild-type prion protein genotype and intracranially inoculate a raccoon. Brain homogenates (10% w/v) from the raccoon and the two white-tailed deer were used to intracranially inoculate separate groups of transgenic mice that express human prion protein with methionine (M) at codon 129 (Tg40h). Brains and spleens were collected from mice at experimental endpoints of clinical disease or approximately 700 days post-inoculation. Tissues were divided and homogenized or fixed in 10% buffered neutral formalin. Immunohistochemistry, enzyme immunoassay, and western blot were used to detect misfolded prion protein (PrPSc) in tissue.
Results: Humanized transgenic mice inoculated with the raccoon passaged CWD agent from white-tailed deer exhibited a 100% (12/12) attack rate with an average incubation period of 605 days. PrPScwas detected in brain tissue by enzyme immunoassay with an average optical density of 3.6/4.0 for positive brains. PrPScalso was detected in brain tissue by western blot and immunohistochemistry. No PrPScwas detected in the spleens of mice inoculated with the raccoon passaged CWD agent. Humanized mice inoculated with the CWD agent from white-tailed deer did not have detectable PrPScusing conventional immunoassay techniques.
Conclusions: The host range of the CWD agent from white-tailed deer was expanded in our experimental model after one passage through raccoons.
Funded by: This research was funded in its entirety by congressionally appropriated funds to the United States Department of Agriculture, Agricultural Research Service. The funders of the work did not influence study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Acknowledgement: We thank Quazetta Brown, Lexi Frese, Rylie Frese, Kevin Hassall, Leisa Mandell, and Trudy Tatum for providing excellent technical support to this project.
Stable and highly zoonotic cervid prion strain is possible
Manuel Camacho, Xu Qi, Liuting Qing, Sydney Smith, Jieji Hu, Wanyun Tao, Ignazio Cali, and Qingzhong Kong Department of Pathology, Case Western Reserve University, Cleveland, USA
Aims: Whether CWD prions can infect humans remains unclear despite the very substantial scale and long history of human exposure of CWD in some areas. Multiple in vitro conversion experiments and in vivo animal studies suggest that the CWD-to-human transmission barrier is not unbreakable. A major public health concern on CWD zoonosis is the emergence of highly zoonotic CWD strains. We aim to address the question of whether highly zoonotic CWD strains are possible.
Material and Methods: We inoculated a few sCJD brain samples into cervidized transgenic mice, which were intended as negative controls for bioassays of brain tissues from sCJD cases who had hunted or consumed vension from CWD-endemic states. Some of these mice became infected and their brain tissues were further examined by serial passages in humanized or cervidized mice.
Results: Passage of sCJDMM1 in transgenic mice expressing elk PrP (Tg12) resulted in a ‘cervidized’ CJD strain that we termed CJDElkPrP. We observed 100% transmission of CJDElkPrPin transgenic mice expressing human PrP (Tg40h). We passaged CJDElkPrPtwo more times in the Tg12 mice. We found that such second and third passage CJDElkPrPprions also led to 100% infection in the Tg40h mice. In contrast, we and others found zero or poor transmission of natural elk CWD isolates in humanized mice, despite that natural elk CWD isolates and CJDElkPrPshare the same elk PrP sequence.
Conclusions: Our data demonstrate that highly zoonotic cervid prion strains are not only possible but also can be stably maintained in cervids and that CWD zoonosis is prion strain-dependent.
Funded by: NIH
Grant number: R01NS052319, R01NS088604, R01NS109532
Acknowledgement: We want to thank the National Prion Disease Pathology Surveillance Center and Drs. Allen Jenny and Katherine O’Rourke for providing the sCJD samples and the CWD samples, respectively.
Adaptation of chronic wasting disease (CWD) prion strains in hosts with different PRNP genotypes
Camilo Duque Velasqueza,c, Elizabeth Triscotta,c, Chiye Kima,c, Diana Morenoa,c, Judd Aikenb,c, and Debbie McKenziea,c
aDepartment of Biological Science, University of Alberta, Edmonton, AB T6G 2G8, Canada; bDepartment of Agriculture, Food & Nutritional Science, University of Alberta, Edmonton, AB T6G 2G8, Canada; cCentre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, AB T6G 2M8, Canada
Aims: The contagious nature of CWD epizootics and the PrPCamino acid variation of cervids (and susceptible sympatric species) guarantee the expansion of prion conformational diversity and selective landscapes where new strains can arise. CWD strains can have novel transmission properties including altered host range that may increase zoonotic risk as circulating strains diversify and evolve. We are characterizing the host adaptability of characterized CWD strains as well as CWD isolates from different cervid species in various enzootic regions.
Material and Methods: Characterized CWD strains as well as a number of isolates from hunter-harvested deer were bioassayed in our rodent panel (transgenic mice expressing cervid alleles G96, S96 and H95-PrPC, elk PrPC, bovine PrPC, and both hamsters and non-transgenic laboratory mice). Strain characteristics were compared using computer based scoring of brain pathology (e.g. PrPCWDbrain distribution), western blot and protein misfolding cyclic amplification (PMCA).
Results: Transmission of various isolates resulted in the selection of strain mixtures in hosts expressing similar PrPC, particularly for polymorphic white-tailed deer and for Norwegian reindeer. As of the second passage, transmission of P153 moose prions from Norway has not resulted in emergence of strains with properties similar to any North American CWD strains in our taxonomic collection (Wisc-1, CWD2, H95+and 116AG).
Conclusions: Our data indicates polymorphic white-tailed deer can favor infection with more than one strain. Similar to transmission studies of Colorado CWD isolates from cervids expressing a single PrPCprimary structure, the isolate from Norway reindeer (V214) represents a strain mixture, suggesting intrinsic strain diversity in the Nordfjella epizootic. The diversity of CWD strains with distinct transmission characteristics represents a threat to wildlife, sympatric domestic animals and public health.
Funded by: Genome Canada and Genome Alberta (Alberta Prion Research Institute and Alberta Agriculture & Forestry); NSERC Grant number: #LSARP 10205; NSERC RGPIN-2017-05539
Acknowledgement: We would like to thank Margo Pybus (Alberta Environment and Parks) Trent Bollinger (University of Saskatchewan) for providing us with tissue samples from hunter-harvested deer and Sylvie Benestad for providing moose and reindeer samples.
Application of PMCA to understand CWD prion strains, species barrier and zoonotic potential
Sandra Pritzkowa, Damian Gorskia, Frank Ramireza, Fei Wanga, Glenn C. Tellingb, Justin J. Greenleec, Sylvie L. Benestadd, and Claudio Sotoa aDepartment of Neurology, University of Texas Medical School at Houston, Houston, Texas, USA; bDepartment of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, USA; cVirus and Prion Research Unit, United States Department of Agriculture, Ames, Iowa, USA; dNorwegian Veterinary Institute, OIE Reference Laboratory for CWD, Ås, Norway
Aims: Chronic wasting disease (CWD) is a prion disease affecting various species of cervids that continues to spread uncontrollably across North America and has recently been detected in Scandinavia (Norway, Sweden and Finland). The mechanisms responsible for the natural transmission of CWD are largely unknown. Furthermore, the risk of CWD transmission to other species, including humans, is also unknown and remains a dangerous enigma. In this study, we investigated the potential of CWD prions to infect several other animal species (sheep, cattle, pig, hamster, and mouse) including humans, by examining their capacity to convert the normal prion protein of distinct species in a PMCA reaction. Moreover, we also investigated whether the in vivo passage of CWD through intermediate species alters their capacity for zoonotic transmission, which may represent a major hazard to human health.
Material and Methods: For these studies, we used brain material from CWD-infected white-tailed deer (Odocoileus virginianus), elk (Cervus canadensis), and mule deer (Odocoileus hemionus) as species native to North America. We also used CWD-infected Moose (Alces alces), reindeer (Rangifer tarandus) and red deer (Cervus elaphus) as Norwegian cervids. We also used brains from cattle, sheep and pigs experimentally infected by CWD. To study interspecies-transmission and zoonotic potential, samples were tested via PMCA for the conversion of PrPCinto PrPScusing different combinations of inoculum and host species. Based on these analyses we estimated the spillover and zoonotic potential for different CWD isolates. We define and quantify spillover and zoonotic potential indices as the efficiency by which CWD prions sustain prion generation in vitro at the expense of normal prion proteins from various mammals and human, respectively.
Results: Our results show that prions from some cervid species, especially those found in Northern Europe, have a higher potential to transmit disease characteristics to other animals. Conversely, CWD-infected cervids originated in North America appear to have a greater potential to generate human PrPSc. We also found that in vivo transmission of CWD to cattle, but not to sheep or pigs substantially increases the ability of these prions to convert human PrPCby PMCA.
Conclusions: Our findings support the existence of different CWD prion strains with distinct spillover and zoonotic potentials. We also conclude that transmission of CWD to other animal species may increase the risk for CWD transmission to humans. Our studies may provide a tool to predict the array of animal species that a given CWD prion could affect and may contribute to understanding the risk of CWD for human health.
Funded by: National Institute of Health Grant number: P01 AI077774
Generation of human chronic wasting disease in transgenic mice
Zerui Wanga, Kefeng Qinb, Manuel V. Camachoa, Ignazio Cali a,c, Jue Yuana, Pingping Shena, Tricia Gillilanda, Syed Zahid Ali Shaha, Maria Gerasimenkoa, Michelle Tanga, Sarada Rajamanickama, Anika Yadatia, Lawrence B. Schonbergerd, Justin Greenleee, Qingzhong Konga,c, James A. Mastriannib, and Wen-Quan Zoua,c
aDepartment of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH, USA; bDepartment of Neurology and Center for Comprehensive Care and Research on Memory Disorders, the University of Chicago Pritzker School of Medicine, Chicago, USA; cNational Prion Disease Pathology Surveillance Center, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA; dDivision of High-Consequence Pathogens and Pathology, Centers for Disease Control and Prevention, 1600 Clifton Rd, Atlanta, GA, USA; eVirus and Prion Research Unit, National Animal Disease Center, USDA, Agricultural Research Service, 1920 Dayton Avenue, Ames, IA, USA
Aims: Chronic wasting disease (CWD) results from the accumulation of an infectious misfolded conformer (PrPSc) of cellular prion protein (PrPC) in the brains of deer and elk. It has been spreading rapidly throughout many regions of North America, exported inadvertently to South Korea, and more recently identified in Europe. Mad cow disease has caused variant Creutzfeldt-Jakob disease (vCJD) in humans and is currently the only known zoonotic prion disease. Whether CWD is transmissible to humans remains uncertain. The aims of our study were not only to confirm whether CWD prion isolates can convert human brain PrPCinto PrPScin vitro by serial protein misfolding cyclic amplification (sPMCA) but also to determine whether the sPMCA-induced CWD-derived human PrPScis infectious.
Material and Methods: Eight CWD prion isolates from 7 elks and 1 deer were used as the seeds while normal human brain homogenates containing either PrP-129 MM (n = 2) or PrP-129 VV (n = 1) were used as the substrates for sPMCA assay. A normal elk brain tissue sample was used as a negative control seed. Two lines of humanized transgenic (Tg) mice expressing either human PrP-129VV or −129 MM polymorphism were included for transmission studies to determine the infectivity of PMCA-amplified PrPSc. Wester blotting and immunohistochemistry and hematoxylin & eosin staining were used for determining PrPScand neuropathological changes of inoculated animals.
Results: We report here the generation of the first CWD-derived infectious human PrPScusing elk CWD PrPScto initiate conversion of human PrPCfrom normal human brain homogenates with PMCA in vitro. Western blotting with a human PrP selective antibody confirmed that the PMCA-generated protease-resistant PrPScwas derived from the human brain PrPCsubstrate. Two lines of humanized transgenic mice expressing human PrPCwith either Val or Met at the polymorphic codon 129 developed clinical prion disease following intracerebral inoculation with the PMCA-generated CWD-derived human PrPSc. Diseased mice exhibited distinct PrPScpatterns and neuropathological changes in the brain.
Conclusions: Our study, using PMCA and animal bioassays, provides the first evidence that CWD PrPSchas the potential to overcome the species barrier and directly convert human PrPCinto infectious PrPScthat can produce bona fide prion disease when inoculated into humanized transgenic mice.
Funded by: CJD Foundation and NIH
Mortality surveillance of persons potentially exposed to chronic wasting disease
R.A. Maddoxa, R.F. Klosb, L.R. Willb, S.N. Gibbons-Burgenerb, A. Mvilongoa, J.Y. Abramsa, B.S. Applebyc, L.B. Schonbergera, and E.D. Belaya aNational Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention (CDC), Atlanta, USA; bWisconsin Department of Health Services (WDHS), Division of Public Health, Madison, USA; cNational Prion Disease Pathology Surveillance Center (NPDPSC), Case Western Reserve University, Cleveland, USA
Aims: It is unknown whether chronic wasting disease (CWD), a prion disease of cervids, can infect people, but consumption of meat from infected animals would be the most likely route of transmission. Wisconsin Department of Health Services, Division of Public Health (WDHS) personnel maintain a database consisting of information collected from hunters who reported eating, or an intention to eat, venison from CWD-positive cervids. These data, collected since 2003, allow for the evaluation of causes of mortality in individuals potentially exposed to CWD.
Material and Methods: The WDHS database contains the name, date of birth, when available, year of CWD-positive deer harvest, and city and state of residence for each potentially exposed individual. The database also includes information on how the deer was processed (self-processed or by a commercial operator) and when applicable, names of others with whom the venison was shared. Duplicate entries (i.e., those who consumed venison from CWD-positive deer in multiple hunt years) are determined by first name, last name, and date of birth. All names in the database are cross-checked with reported cases of human prion disease in Wisconsin and cases in the National Prion Disease Pathology Surveillance Center (NPDPSC) diagnostic testing database. Persons with date of birth available are also cross-checked with prion disease decedents identified through restricted-use national multiple cause-of-death data via a data use agreement with the National Center for Health Statistics (NCHS).
Results: The database currently consists of 1561 records for hunt years 2003–2017 and 87 additional records for 2018–2019. Of these, 657 records have accompanying date of birth; 15 entries were removed as duplicates leaving 642 unique individuals. Of these individuals, 278 of 426 (66%) who ate venison from a CWD-positive deer and provided processing information reported self-processing. No matches were found among any persons in the database cross-checked with WDHS human prion disease surveillance data, NPDPSC data (February 2022 update), and NCHS data through 2020.
Conclusions: Because of the linkage of person and CWD-positive animal in the WDHS database, reviewing the cause of mortality in potentially exposed persons is possible. The number of individuals cross-checked so far is likely only a small percentage of those potentially exposed to CWD in Wisconsin, and many more years of vital status tracking are needed given an expected long incubation period should transmission to humans occur. Nevertheless, the findings of this ongoing review are thus far reassuring.
Prion disease incidence, United States, 2003–2020
R.A. Maddoxa, M.K. Persona, K. Kotobellib, A. Mvilongoa, B.S. Applebyb, L.B. Schonbergera, T.A. Hammetta, J.Y. Abramsa, and E.D. Belaya aNational Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention (CDC), Atlanta, USA; bNational Prion Disease Pathology Surveillance Center (NPDPSC), Case Western Reserve University, Cleveland, USA
Aims: Mortality data, in conjunction with neuropathological and genetic testing results, are used to estimate prion disease incidence in the United States.
Material and Methods: Prion disease decedents for 2003–2020 were identified from restricted-use U.S. national multiple cause-of-death data, via a data use agreement with the National Center for Health Statistics, and from the National Prion Disease Pathology Surveillance Center (NPDPSC) database. NPDPSC decedents with neuropathological or genetic test results positive for prion disease for whom no likely match was found in the NCHS multiple cause-of-death data were added as cases for incidence calculations, while those with negative neuropathology results but with cause-of-death data indicating prion disease were removed. Unmatched cases in the NPDPSC database lacking neuropathological testing but with a positive real-time quaking-induced conversion (RT-QuIC) test result were additionally assessed. Age-specific and age-adjusted average annual incidence rates were calculated from the combined data; the year 2000 as the standard population and the direct method were used for age-adjustment.
Results: A total of 7,921 decedents were identified as having prion disease during 2003–2020 for an age-adjusted average annual incidence of 1.2 per million population. The age-adjusted incidence between males and females (1.3 and 1.1 per million, respectively) differed significantly (p < 0.0001). The age-specific average annual incidence among those <55 and ≥55 years of age was 0.2 and 4.8 per million, respectively; incidence among those ≥65 was 6.1 per million. Eighteen cases were <30 years of age for an age-specific incidence of 8.0 per billion; only 6 of these very young cases were sporadic (3 sporadic CJD, 3 sporadic fatal insomnia), with the rest being familial (9), variant (2), or iatrogenic (1). The age-adjusted annual incidence for the most recent year of data, 2020, was 1.3 per million. However, assessment of RT-QuIC positive cases lacking neuropathology in the NPDPSC database suggested that approximately 20% more cases may have occurred in that year; the addition of a subset of these cases that had date of death information available (n = 44) increased the 2020 rate to 1.4 per million.
Conclusions: Mortality data supplemented with the results of neuropathological, CSF RT-QuIC, and genetic testing can be used to estimate prion disease incidence. However, the identification in the NPDPSC database of RT-QuIC-positive cases lacking date of death information suggests that this strategy may exclude a number of probable prion disease cases. Prion disease cases <30 years of age, especially those lacking a pathogenic mutation, continue to be very rare.
Shedding of Chronic Wasting Disease Prions in Multiple Excreta Throughout Disease Course in White-tailed Deer
Nathaniel D. Denkersa, Erin E. McNultya, Caitlyn N. Krafta, Amy V. Nallsa, Joseph A. Westricha, Wilfred Goldmannb, Candace K. Mathiasona, and Edward A. Hoovera
aPrion Research Center, College of Veterinary Medicine and Biological Sciences, Department of Microbiology, Immunology, and Pathology; Colorado State University, Fort Collins, CO, USA; bDivision of Infection and Immunity, The Roslin Institute and the Royal Dick School of Veterinary Studies, University of Edinburgh, Midlothian, UK
Aims: Chronic wasting disease (CWD) now infects cervids in South Korea, North America, and Scandinavia. CWD is unique in its efficient transmission and shedding of prions in body fluids throughout long course infections. Questions remain as to the magnitude of shedding and the route of prion acquisition. As CWD continues to expand, the need to better understand these facets of disease becomes more pertinent. The purpose of the studies described was to define the longitudinal shedding profile of CWD prions in urine, saliva, and feces throughout the course of infection in white-tailed deer.
Material and Methods: Twelve (12) white-tailed deer were inoculated with either 1 mg or 300ng of CWD. Urine, saliva, and feces were collected every 3-month post-inoculation (MPI) throughout the study duration. Cohorts were established based on PNRP genotype: codon 96 GG (n = 6) and alternate codons 96 GS (n = 5) & 103NT (n = 1). Urine and saliva were analyzed using iron-oxide magnetic extraction (IOME) and real-time quaking induced conversion (RT-QuIC)(IQ). Feces were subjected to IOME, followed by 4 rounds protein misfolding cyclic amplification (PMCA) with products analyzed by RT-QuIC (IPQ). To determine whether IPQ may be superior to IQ, a subset of urine and saliva were also tested by IPQ. Results were compared with clinical disease status.
Results: Within the 96 GG cohort, positive seeding activity was detected in feces from all deer (100%), in saliva from 5 of 6 (83%), and in urine from 4 of 6 (66%). Shedding in all excreta occurred at, or just after, the first positive tonsil biopsy result. In the 96 GS/103NT cohort, positive seeding activity could be detected in feces from 3 of 6 (50%) deer, saliva in 2 of 6 (33%), and urine in 1 of 6 (16%). Shedding in excreta was detected >5 months after the first tonsil positive result. Four of six 96 GG deer developed clinical signs of CWD, whereas only 2 of the 96 GS/103NT did. Shedding was more frequently detected in deer with clinical disease. The IPQ protocol did not significantly improve detection in saliva or urine samples, however, it significantly augmented detection in feces by eliminating non-specific background commonly experienced with IQ. Negative control samples remained negative in samples tested.
Conclusions: These studies demonstrate: (a) CWD prion excretion occurs throughout infection; (2) PRNP genotype (GG≫GS/NT) influences the excreta shedding; and (3) detection sensitivity in excreta can vary with different RT-QuIC protocols. These results provide a more complete perspective of prion shedding in deer during the course of CWD infection.
Funded by: National Institutes of Health (NIH)
Grant number: RO1-NS061902-09 R to EAH, PO1-AI077774 to EAH, and R01-AI112956-06 to CKM
Acknowledgement: We abundantly thank Sallie Dahmes at WASCO and David Osborn and Gino D’Angelo at the University of Georgia Warnell School of Forestry and Natural Resources for their long-standing support of this work through provision of the hand-raised, CWD-free, white-tailed deer used in these studies
Large-scale PMCA screening of retropharyngeal lymph nodes and in white-tailed deer and comparisons with ELISA and IHC: the Texas CWD study
Rebeca Benaventea, Paulina Sotoa, Mitch Lockwoodb, and Rodrigo Moralesa
aDepartment of Neurology, McGovern Medical School, University of Texas Health Science Center at Houston, Texas, USA; bTexas Park and Wildlife Department, Texas, USA
Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy that affects various species of cervids, and both free-ranging and captive animals. Until now, CWD has been detected in 3 continents: North America, Europe, and Asia. CWD prevalence in some states may reach 30% of total animals. In Texas, the first case of CWD was reported in a free-range mule deer in Hudspeth and now it has been detected in additional 14 counties. Currently, the gold standard techniques used for CWD screening and detection are ELISA and immunohistochemistry (IHC) of obex and retropharyngeal lymph nodes (RPLN). Unfortunately, these methods are known for having a low diagnostic sensitivity. Hence, many CWD-infected animals at pre-symptomatic stages may be misdiagnosed. Two promising in vitro prion amplification techniques, including the real-time quaking-induced conversion (RT-QuIC) and the protein misfolding cyclic amplification (PMCA) have been used to diagnose CWD and other prion diseases in several tissues and bodily fluids. Considering the low cost and speed of RT-QuIC, two recent studies have communicated the potential of this technique to diagnose CWD prions in RPLN samples. Unfortunately, the data presented in these articles suggest that identification of CWD positive samples is comparable to the currently used ELISA and IHC protocols. Similar studies using the PMCA technique have not been reported.
Aims: Compare the CWD diagnostic potential of PMCA with ELISA and IHC in RPLN samples from captive and free-range white-tailed deer. Material and Methods: In this study we analyzed 1,003 RPLN from both free-ranging and captive white-tailed deer collected in Texas. Samples were interrogated with the PMCA technique for their content of CWD prions. PMCA data was compared with the results obtained through currently approved techniques.
Results: Our results show a 15-fold increase in CWD detection in free-range deer compared with ELISA. Our results unveil the presence of prion infected animals in Texas counties with no previous history of CWD. In the case of captive deer, we detected a 16% more CWD positive animals when compared with IHC. Interestingly, some of these positive samples displayed differences in their electroforetic mobilities, suggesting the presence of different prion strains within the State of Texas.
Conclusions: PMCA sensitivity is significantly higher than the current gold standards techniques IHC and ELISA and would be a good tool for rapid CWD screening.
Funded by: USDA
Grant number: AP20VSSPRS00C143
ATYPRION project: assessing the zoonotic potential of interspecies transmission of CWD isolates to livestock (preliminary results).
Enric Vidala,b, Juan Carlos Espinosac, Samanta Gilera,b, Montserrat Ordóñeza,b, Guillermo Canteroa,b, Vincent Béringued, Justin J. Greenleee, and Juan Maria Torresc
aUnitat mixta d’Investigació IRTA-UAB en Sanitat Animal. Centre de Recerca en Sanitat Animal (CReSA). Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra, Catalonia; bIRTA. Programa de Sanitat Animal. Centre de Recerca en Sanitat Animal (CReSA). Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra, Catalonia; cCentro de Investigación en Sanidad Animal, CISA-INIA-CSIC, Valdeolmos, Madrid, Spain; dMolecular Virology and Immunology, French National Research Institute for Agriculture, Food and Environment (INRAE), Université Paris-Saclay, Jouy-en-Josas, France; eVirus and Prion Research Unit, National Animal Disease Center, ARS, United States Department of Agriculture, Ames, IA, USA
Aims: Since variant Creutzfeldt-Jackob disease was linked to the consumption of bovine spongiform encephalopathy prions, the study of the pathobiological features of animal prions, particularly their zoonotic potential, is of great concern to the scientific community and public health authorities. Furthermore, interspecies transmission of prions has been demonstrated as a putative evolutionary mechanism for prions, that can lead to the emergence of new features including the ability to infect humans. For instance, small ruminants’ atypical scrapie prions, when propagated in a bovine or porcine host, can shift to a classical BSE phenotype thus posing a potential risk in case of human exposure. So far, no hard evidence of zoonotic transmission of cervids’ chronic wasting disease (CWD) to humans has been published, however experimental transmission to bovine, ovine and caprine hosts has been achieved. Our goal is to investigate if, once passaged through these domestic species, CWD prions might become infectious to humans.
Material and Methods: Different CWD isolates experimentally adapted to cattle, sheep and goat (Hamir et al, 2005, 2006, 2007, Greenlee et al 2012) have been intracerebrally inoculated to transgenic mouse models expressing the human cellular prion protein either homozygous for methionine or valine at codon 129 (Tg340-Met129 and Tg362-Val129). Additionally, inocula obtained from experimental transmission of elk CWD to ovinized (Tg501) and bovinized (BoTg110) transgenic mice, as well as white-tailed deer CWD to BoTg110 mice, are currently being bioassayed in both human PrPCtransgenic models.
Results and conclusions: No evidence of transmission has been found on first passage for bovine adapted elk and mule deer CWD to none of the humanized models. The remaining bioassays are ongoing without showing clinical signs yet, as well as second passages for the negative 1stpassages.
Funded by: La Marató de TV3 foundation. Grant number: ATYPRION (201,821–30-31-32)
PRION CONFERENCE 2022 ABSTRACTS CWD TSE PrP ZOONOSIS and ENVIRONMENTAL FACTORS
Chronic wasting disease detection in environmental and biological samples from a taxidermy site
Paulina Sotoa,b, J. Hunter Reedc, Mitch Lockwoodc, and Rodrigo Moralesa,b aDepartment of Neurology, McGovern Medical School, University of Texas Health Science Center at Houston, Texas, USA; bUniversidad Bernardo O’Higgins, Santiago, Chile; cTexas Parks and Wildlife Department, Texas, USA
Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy affecting captive and free-ranging cervids (e.g., mule deer, white-tailed deer, elk, reindeer, and moose). Nowadays, CWD is widely distributed in North America. It is suggested that CWD spreads due to direct animal contact or through exposure to contaminated environments previously inhabited by infected animals. CWD may also be spread through the movement of infected animals and carcasses. Taxidermy practices involve processing deer tissues (or whole animal carcasses). In many cases, the CWD status of processed animals is unknown. This can generate risks of disease spread and transmission. Taxidermy practices include different steps involving physical, chemical, and biological procedures. Without proper tissue handling or disposal practices, taxidermist facilities may become a focus of prion infectivity. Aims: In this study, we evaluated the presence of infectious prions in a taxidermy facility believed to be exposed to CWD. Detection was performed using the Protein Misfolding Cyclic Amplification (PMCA) technique in biological and inert environmental samples. Methods: We collected biological and environmental samples (plants, soils, insects, excreta, and others) from a taxidermy facility, and we tested these samples using the PMCA technique. In addition, we swabbed different surfaces possibly exposed to CWD-infected animals. For the PMCA reaction, we directly used a swab piece or 10 µL of 20% w/v homogenized samples. Results: The PMCA analysis demonstrated CWD seeding activity in some of the components of this facility, including insects involved in head processing, soils, and a trash dumpster. Conclusions: Different areas of this property were used for various taxidermy procedures. We were able to detect the presence of prions in i) soils that were in contact with the heads of dead animals, ii) insects involved in the cleaning of skulls, and iii) an empty dumpster where animal carcasses were previously placed. This is the first report demonstrating that swabbing is a helpful method to screen for prion infectivity on surfaces potentially contaminated with CWD. These findings are relevant as this swabbing and amplification strategy may be used to evaluate the disease status of other free-ranging and captive settings where there is a concern for CWD transmissions, such as at feeders and water troughs with CWD-exposed properties. This approach could have substantial implications for free-ranging cervid surveillance as well as in epidemiological investigations of CWD.
Funded by: USDA Grant number: AP20VSSPRS00C143
Large-scale PMCA screening of retropharyngeal lymph nodes and in white-tailed deer and comparisons with ELISA and IHC: the Texas CWD study
Rebeca Benaventea, Paulina Sotoa, Mitch Lockwoodb, and Rodrigo Moralesa aDepartment of Neurology, McGovern Medical School, University of Texas Health Science Center at Houston, Texas, USA; bTexas Park and Wildlife Department, Texas, USA
Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy that affects various species of cervids, and both free-ranging and captive animals. Until now, CWD has been detected in 3 continents: North America, Europe, and Asia. CWD prevalence in some states may reach 30% of total animals. In Texas, the first case of CWD was reported in a free-range mule deer in Hudspeth and now it has been detected in additional 14 counties. Currently, the gold standard techniques used for CWD screening and detection are ELISA and immunohistochemistry (IHC) of obex and retropharyngeal lymph nodes (RPLN). Unfortunately, these methods are known for having a low diagnostic sensitivity. Hence, many CWD-infected animals at pre-symptomatic stages may be misdiagnosed. Two promising in vitro prion amplification techniques, including the real-time quaking-induced conversion (RT-QuIC) and the protein misfolding cyclic amplification (PMCA) have been used to diagnose CWD and other prion diseases in several tissues and bodily fluids. Considering the low cost and speed of RT-QuIC, two recent studies have communicated the potential of this technique to diagnose CWD prions in RPLN samples. Unfortunately, the data presented in these articles suggest that identification of CWD positive samples is comparable to the currently used ELISA and IHC protocols. Similar studies using the PMCA technique have not been reported. Aims: Compare the CWD diagnostic potential of PMCA with ELISA and IHC in RPLN samples from captive and free-range white-tailed deer. Material and Methods: In this study we analyzed 1,003 RPLN from both free-ranging and captive white-tailed deer collected in Texas. Samples were interrogated with the PMCA technique for their content of CWD prions. PMCA data was compared with the results obtained through currently approved techniques. Results: Our results show a 15-fold increase in CWD detection in free-range deer compared with ELISA. Our results unveil the presence of prion infected animals in Texas counties with no previous history of CWD. In the case of captive deer, we detected a 16% more CWD positive animals when compared with IHC. Interestingly, some of these positive samples displayed differences in their electroforetic mobilities, suggesting the presence of different prion strains within the State of Texas. Conclusions: PMCA sensitivity is significantly higher than the current gold standards techniques IHC and ELISA and would be a good tool for rapid CWD screening.
Funded by: USDA Grant number: AP20VSSPRS00C143
Protein misfolding cyclic amplification (PMCA) as an ultra-sensitive technique for the screening of CWD prions in different sample types
Francisca Bravo‐Risia,b, Paulina Sotoa,b, Rebeca Benaventea, Hunter Reedc, Mitch Lockwoodc, Tracy Nicholsd, and Rodrigo Moralesa,b aDepartment of Neurology, The University of Texas Health Science Center at Houston, Houston, TX, USA; bCentro Integrativo de Biologia y Quimica Aplicada (CIBQA), Universidad Bernardo O’Higgins, Santiago, Chile; cTexas Park and Wildlife Department, Texas, USA; dVeterinary Services Cervid Health Program, United States Department of Agriculture, Animal and Plant Health Inspection Service, Fort Collins, Colorado, USA
Chronic wasting disease (CWD) is a prion disease that affects farmed and free-ranging cervids. The infectious agent in CWD is a misfolded form of the prion protein (PrPSc) that promotes conformational changes in the host’s cellular prion protein (PrPC). Currently, definitive CWD status is confirmed in the brain and lymphoid tissues by immunohistochemistry. The limitation of this technique is its poor sensitivity. Protein misfolding cyclic amplification (PMCA) and real-time quaking-induced conversion (RT- QuIC) are ultra-sensitive techniques that overcome these issues. PMCA mimics the self- propagation of infectious prions in vitro through multiple incubation/sonication cycles, increasing the number of prion particles present in a given sample. The detection of proteinase K (PK) -resistant PrPScby PMCA has been performed in experimental and natural samples that might harbor subclinical levels of prions. These samples include several tissues, bodily fluids, excreta, and different manmade and natural materials, including mineral licks, soils, and plants. Aims: In this study, we highlight recent advances and contributions that our group has performed in the detection of CWD prions from samples collected in farmed and free-ranging cervids, as well as other specimens involving the environment that contains CWD-infected deer. Material and Methods: A set of diverse samples analyzed in this study were collected by USDA and TPWD personnel in breeding and taxidermy facilities, and deer breeding facilities. These included animal and environmental samples. Additional samples from free-ranging animals were provided by hunters. Results: The diverse range of samples successfully detected for CWD prion infection in this study include blood, semen, feces, obex, retropharyngeal lymph node, fetuses (neural and peripheral tissues) and gestational tissues, parasites, insects, plants, compost/soil mixtures, and swabs from trash containers. Importantly, these results helped to identify seeding-competent prions in places reported to be free of CWD. The levels of prion infectivity in most of these samples are currently being investigated. Conclusions: Our findings contribute to the understanding of the transmission dynamics and prevalence of CWD. In addition, our data have helped to identify CWD in areas previously considered to be free of CWD. We also demonstrate that PMCA is a powerful technique for the screening of biological and environmental samples. Overall, our research suggests that PMCA may be a useful tool to implement for the surveillance and management of CWD. Funded by: NIH/NIAID and USDA Grant number: 1R01AI132695 (NIH) and AP20VSSPRS00C143 (USDA)
Nasal bot: an emerging vector for natural chronic wasting disease transmission
Paulina Sotoa,b, Francisca Bravo-Risia,b, Carlos Kramma, Nelson Pereza, Rebeca Benaventea, J. Hunter Reedc, Mitch Lockwoodc, Tracy A. Nicholsd, and Rodrigo Moralesa,b aDepartment of Neurology, McGovern Medical School, University of Texas Health Science Center at Houston, Texas, USA; bUniversidad Bernardo O’Higgins, Santiago, Chile; cTexas Park and Wildlife Department, Texas, USA; dVeterinary Services Cervid Health Program, United States Department of Agriculture, Animal and Plant Health Inspection Service, Fort Collins, Colorado, USA
Chronic wasting disease (CWD) is a fatal neurodegenerative disease that affects farmed and free-ranging cervids populations. The spread of CWD in cervids is thought to occur through the direct contact between cervids or through the exposure of naïve animals to contaminated environments. Parasites are known vectors of multiple diseases in animals. However, the potential role of parasites in CWD transmission remains unclear. Aims: The main objective of this study was to determine if CWD prions could be detected in the larvae of deer nasal bot flies, a common deer parasite, taken from CWD-infected white-tailed deer (Odocoileus virginianus). Methods: Bot fly larvae were collected from the nasal cavity of naturally infected CWD- positive or CWD non-detect white-tailed deer. The CWD seeding activity of the larvae was interrogated by PMCA. Prion infectivity was also evaluated in cervidized transgenic mouse bioassay (intra-cerebral administration in Tg1536 mice). Mice inoculated with bot larvae homogenate were sacrificed when they showed established signs of prion disease, or at extended periods after treatment (600 days). All inoculated mouse brains were evaluated for protease resistant prions to confirm clinical or sub-clinical infection. Bot larvae from CWD non-detect deer were used as controls. To further mimic environmental transmission, bot larvae homogenates were mixed with soils and plants were grown on them. Both plants and soils were tested for prion seeding activity. Results: PMCA analysis demonstrated CWD seeding activity in nasal bot larvae from captive and free-ranging white-tailed deer. CWD-contaminated bots efficiently infected transgenic mice, with attack rates and incubation periods suggesting high infectivity titers. Further analyses of treated animals (biochemical characterization of protease resistant prions and immunohistochemistry) confirmed prion infection. Analyses on dissected parts of the bot larvae demonstrate that the infectivity is concentrated in the larvae cuticle (outer part). Nasal bot larvae extracts mixed with
soils showed seeding activity by PMCA. Interestingly, plants grown in soil contaminated with the nasal bot larvae extract were found to produce seeding activity by PMCA. Conclusion: In this study we described for the first time that deer nasal bot larvae from CWD-infected deer carry high CWD infectivity titers. We also demonstrate that CWD prions in these parasites can interact with other environmental components relevant for disease transmission. Considering this information, we propose that deer nasal bot larvae could act as vectors for CWD transmission in wild and farming settings. Funded by: NIH/NIAID and USDA/APHIS Grant number: R01AI132695 and AP20VSSPRS00C143 PRION 2022 ABSTRACTS, AND A BIG THANK YOU TO On behalf of the Prion2020/2022 Congress Organizing Committee and the NeuroPrion Association, we heartily invite you to join us for the International Conference Prion2020/2022 from 13.-16. September 2022 in Göttingen.
Prion 2022 Conference abstracts: pushing the boundaries
Shedding of Chronic Wasting Disease Prions in Multiple Excreta Throughout Disease Course in White-tailed Deer
Nathaniel D. Denkersa, Erin E. McNultya, Caitlyn N. Krafta, Amy V. Nallsa, Joseph A. Westricha, Wilfred Goldmannb, Candace K. Mathiasona, and Edward A. Hoovera
aPrion Research Center, College of Veterinary Medicine and Biological Sciences, Department of Microbiology, Immunology, and Pathology; Colorado State University, Fort Collins, CO, USA; bDivision of Infection and Immunity, The Roslin Institute and the Royal Dick School of Veterinary Studies, University of Edinburgh, Midlothian, UK
Aims: Chronic wasting disease (CWD) now infects cervids in South Korea, North America, and Scandinavia. CWD is unique in its efficient transmission and shedding of prions in body fluids throughout long course infections. Questions remain as to the magnitude of shedding and the route of prion acquisition. As CWD continues to expand, the need to better understand these facets of disease becomes more pertinent. The purpose of the studies described was to define the longitudinal shedding profile of CWD prions in urine, saliva, and feces throughout the course of infection in white-tailed deer.
Material and Methods: Twelve (12) white-tailed deer were inoculated with either 1 mg or 300ng of CWD. Urine, saliva, and feces were collected every 3-month post-inoculation (MPI) throughout the study duration. Cohorts were established based on PNRP genotype: codon 96 GG (n = 6) and alternate codons 96 GS (n = 5) & 103NT (n = 1). Urine and saliva were analyzed using iron-oxide magnetic extraction (IOME) and real-time quaking induced conversion (RT-QuIC)(IQ). Feces were subjected to IOME, followed by 4 rounds protein misfolding cyclic amplification (PMCA) with products analyzed by RT-QuIC (IPQ). To determine whether IPQ may be superior to IQ, a subset of urine and saliva were also tested by IPQ. Results were compared with clinical disease status.
Results: Within the 96 GG cohort, positive seeding activity was detected in feces from all deer (100%), in saliva from 5 of 6 (83%), and in urine from 4 of 6 (66%). Shedding in all excreta occurred at, or just after, the first positive tonsil biopsy result. In the 96 GS/103NT cohort, positive seeding activity could be detected in feces from 3 of 6 (50%) deer, saliva in 2 of 6 (33%), and urine in 1 of 6 (16%). Shedding in excreta was detected >5 months after the first tonsil positive result. Four of six 96 GG deer developed clinical signs of CWD, whereas only 2 of the 96 GS/103NT did. Shedding was more frequently detected in deer with clinical disease. The IPQ protocol did not significantly improve detection in saliva or urine samples, however, it significantly augmented detection in feces by eliminating non-specific background commonly experienced with IQ. Negative control samples remained negative in samples tested.
Conclusions: These studies demonstrate: (a) CWD prion excretion occurs throughout infection; (2) PRNP genotype (GG≫GS/NT) influences the excreta shedding; and (3) detection sensitivity in excreta can vary with different RT-QuIC protocols. These results provide a more complete perspective of prion shedding in deer during the course of CWD infection.
Funded by: National Institutes of Health (NIH)
Grant number: RO1-NS061902-09 R to EAH, PO1-AI077774 to EAH, and R01-AI112956-06 to CKM
Acknowledgement: We abundantly thank Sallie Dahmes at WASCO and David Osborn and Gino D’Angelo at the University of Georgia Warnell School of Forestry and Natural Resources for their long-standing support of this work through provision of the hand-raised, CWD-free, white-tailed deer used in these studies
Carrot plants as potential vectors for CWD transmission
Paulina Sotoa,b, Francisca Bravo-Risia,b, Claudio Sotoa, and Rodrigo Moralesa,b
aDepartment of Neurology, McGovern Medical School, University of Texas Health Science Center at Houston, Texas, USA; bUniversidad Bernardo O’Higgins, Santiago, Chile
Prion diseases are infectious neurodegenerative disorders afflicting humans and other mammals. These diseases are generated by the misfolding of the cellular prion protein into a disease-causing isoform. Chronic wasting disease (CWD) is a prevalent prion disease affecting cervids (captive and free-range). CWD is thought to be transmitted through direct animal contact or by indirect exposure to contaminated environments. Many studies have shown that infectious prions can enter the environment through saliva, feces, or urine from infected animals and decaying carcasses. However, we do not fully understand the specific contribution of each component to disease transmission events. Plants are logical environmental components to be evaluated since they grow in environments contaminated with CWD prions and are relevant for animal and human nutrition.
Aims: The main objective of this study is to study whether prions are transported to the roots and leaves of carrots, an edible plant commonly used in the human diet and as deer bait.
Methods: We have grown carrot plants in CWD-infected soils. After 90 days, we harvested the carrots and separated them from the leaves. The experiment was controlled by growing plants in soil samples treated with brain extracts from healthy animals. These materials were interrogated for their prion seeding activity using the Protein Misfolding Cyclic Amplification (PMCA) technique. Infectivity was evaluated in mouse bioassays (intracerebral injections in Tg1536 mice). The animals were sacrificed when they showed established signs of prion disease. Animals not displaying clinical signs were sacrificed at 600 days post-inoculation.
Results: The PMCA analysis demonstrated CWD seeding activity in soils contaminated with CWD prions, as well as in carrot plants (leaves and roots) grown on them. Bioassays demonstrated that both leaves and roots contained CWD prions in sufficient quantities to induce disease (92% attack rate). As expected, animals treated with prion-infected soils developed prion disease at shorter incubation periods (and complete attack rates) compared to plant components. Animals treated with soil and plant components exposed with CWD-free brain extracts did not display prion-associated clinical signs or evidence of sub-clinical prion infection.
Conclusions: We show that edible plant components can absorb prions from CWD contaminated soils and transport them to their aerial parts. Our results indicate that plants could participate as vectors of CWD transmission. Importantly, plants designated for human consumption represent a risk of introducing CWD prions into the human food chain.
Funded by: NIH
Grant number: R01AI132695
***Moreover, sporadic disease has never been observed in breeding colonies or primate research laboratories, most notably among hundreds of animals over several decades of study at the National Institutes of Health25, and in nearly twenty older animals continuously housed in our own facility.***
Even if the prevailing view is that sporadic CJD is due to the spontaneous formation of CJD prions, it remains possible that its apparent sporadic nature may, at least in part, result from our limited capacity to identify an environmental origin.
O.05: Transmission of prions to primates after extended silent incubation periods: Implications for BSE and scrapie risk assessment in human populations
Emmanuel Comoy, Jacqueline Mikol, Valerie Durand, Sophie Luccantoni, Evelyne Correia, Nathalie Lescoutra, Capucine Dehen, and Jean-Philippe Deslys Atomic Energy Commission; Fontenay-aux-Roses, France
Prion diseases (PD) are the unique neurodegenerative proteinopathies reputed to be transmissible under field conditions since decades. The transmission of Bovine Spongiform Encephalopathy (BSE) to humans evidenced that an animal PD might be zoonotic under appropriate conditions. Contrarily, in the absence of obvious (epidemiological or experimental) elements supporting a transmission or genetic predispositions, PD, like the other proteinopathies, are reputed to occur spontaneously (atpical animal prion strains, sporadic CJD summing 80% of human prion cases).
Non-human primate models provided the first evidences supporting the transmissibiity of human prion strains and the zoonotic potential of BSE. Among them, cynomolgus macaques brought major information for BSE risk assessment for human health (Chen, 2014), according to their phylogenetic proximity to humans and extended lifetime. We used this model to assess the zoonotic potential of other animal PD from bovine, ovine and cervid origins even after very long silent incubation periods.
*** We recently observed the direct transmission of a natural classical scrapie isolate to macaque after a 10-year silent incubation period,
***with features similar to some reported for human cases of sporadic CJD, albeit requiring fourfold long incubation than BSE. Scrapie, as recently evoked in humanized mice (Cassard, 2014),
***is the third potentially zoonotic PD (with BSE and L-type BSE),
***thus questioning the origin of human sporadic cases.
We will present an updated panorama of our different transmission studies and discuss the implications of such extended incubation periods on risk assessment of animal PD for human health.
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***thus questioning the origin of human sporadic cases***
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***our findings suggest that possible transmission risk of H-type BSE to sheep and human. Bioassay will be required to determine whether the PMCA products are infectious to these animals.
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PRION 2015 CONFERENCE
***Transmission data also revealed that several scrapie prions propagate in HuPrP-Tg mice with efficiency comparable to that of cattle BSE. While the efficiency of transmission at primary passage was low, subsequent passages resulted in a highly virulent prion disease in both Met129 and Val129 mice.
***Transmission of the different scrapie isolates in these mice leads to the emergence of prion strain phenotypes that showed similar characteristics to those displayed by MM1 or VV2 sCJD prion.
***These results demonstrate that scrapie prions have a zoonotic potential and raise new questions about the possible link between animal and human prions.
PRION 2016 TOKYO
Saturday, April 23, 2016
SCRAPIE WS-01: Prion diseases in animals and zoonotic potential 2016
Prion. 10:S15-S21. 2016 ISSN: 1933-6896 printl 1933-690X online
Taylor & Francis
Prion 2016 Animal Prion Disease Workshop Abstracts
WS-01: Prion diseases in animals and zoonotic potential
Transmission of the different scrapie isolates in these mice leads to the emergence of prion strain phenotypes that showed similar characteristics to those displayed by MM1 or VV2 sCJD prion.
These results demonstrate that scrapie prions have a zoonotic potential and raise new questions about the possible link between animal and human prions.
Title: Transmission of scrapie prions to primate after an extended silent incubation period)
*** In complement to the recent demonstration that humanized mice are susceptible to scrapie, we report here the first observation of direct transmission of a natural classical scrapie isolate to a macaque after a 10-year incubation period. Neuropathologic examination revealed all of the features of a prion disease: spongiform change, neuronal loss, and accumulation of PrPres throughout the CNS.
*** This observation strengthens the questioning of the harmlessness of scrapie to humans, at a time when protective measures for human and animal health are being dismantled and reduced as c-BSE is considered controlled and being eradicated.
*** Our results underscore the importance of precautionary and protective measures and the necessity for long-term experimental transmission studies to assess the zoonotic potential of other animal prion strains.
Research Project: Pathobiology, Genetics, and Detection of Transmissible Spongiform Encephalopathies
Location: Virus and Prion Research
Title: Transmission of the atypical/nor98 scrapie agent to suffolk sheep with VRQ/ARQ, ARQ/ARQ, and ARQ/ARR genotypes
Author item Cassmann, Eric item MAMMADOVA, JAJIBA - Orise Fellow item BENESTAD, SYLVIE - Norwegian Veterinary Institute item MOORE, SARA JO - Orise Fellow item Greenlee, Justin
Submitted to: PLoS ONE Publication Type: Peer Reviewed Journal Publication Acceptance Date: 1/21/2021 Publication Date: 2/11/2021
Citation: Cassmann, E.D., Mammadova, J., Benestad, S., Moore, S., Greenlee, J.J. 2021. Transmission of the atypical/nor98 scrapie agent to suffolk sheep with VRQ/ARQ, ARQ/ARQ, and ARQ/ARR genotypes. PLoS ONE. 16(2). Article e0246503. https://doi.org/10.1371/journal.pone.0246503. DOI: https://doi.org/10.1371/journal.pone.0246503
Interpretive Summary: Atypical scrapie is a prion disease that affects sheep. Unlike classical scrapie, atypical scrapie is thought to occur spontaneously, and it is unlikely to transmit between sheep under natural conditions. Another notable distinction between classical and atypical scrapie is the prion protein genotype of afflicted sheep and the locations in the brain where misfolded prions accumulate. Atypical scrapie generally occurs in sheep that are resistant to classical scrapie. Misfolded prions are predominantly found in the cerebellum for atypical scrapie and not in the brainstem as seen with classical scrapie. Atypical scrapie is a relevant disease because of its potential association with other prion diseases. Some research has shown that the atypical scrapie agent can undergo a transformation of disease forms that makes it appear like classical scrapie or classical bovine spongiform encephalopathy (mad cow disease). Therefore, atypical scrapie is thought to be a possible source for these prion diseases. We investigated the transmission of the atypical scrapie agent to sheep with three different prion protein genotypes. A diagnosis of atypical scrapie was made in all three genotypes of sheep. Misfolded prion protein was detected earliest in the cerebellum and the retina. This is the first report describing the early accumulation of misfolded prions in the retina of sheep with atypical scrapie. Understanding where misfolded prions accumulate in cases of atypical scrapie can lead to better detection earlier in the disease. Furthermore, the materials derived from this experiment will aid in investigating origins of other prion diseases.
Technical Abstract: Scrapie is a transmissible spongiform encephalopathy that occurs in sheep. Atypical/Nor98 scrapie occurs in sheep with that tend to be resistant to classical scrapie and it is thought to occur spontaneously. The purpose of this study was to test the transmission of the Atypical/Nor98 scrapie agent in three genotypes of Suffolk sheep and characterize the distribution of misfolded prion protein (PrPSc). Ten sheep were intracranially inoculated with brain homogenate from a sheep with Atypical/Nor98 scrapie. All sheep with the ARQ/ARQ and ARQ/ARR genotypes developed Atypical/Nor98 scrapie confirmed by immunohistochemistry, and one (1/3) sheep with the VRQ/ARQ genotype had detectable PrPSc consistent with Atypical/Nor98 scrapie at the experimental endpoint of 8 years. Sheep with mild early accumulations of PrPSc in the cerebellum had concomitant retinal PrPSc. Accordingly, large amounts of retinal PrPSc were identified in clinically affected sheep and sheep with dense accumulations of PrPSc in the cerebellum.
Research Project: Pathobiology, Genetics, and Detection of Transmissible Spongiform Encephalopathies Location: Virus and Prion Research
Title: Scrapie in white-tailed deer: a strain of the CWD agent that efficiently transmits to sheep?
Author item Greenlee, Justin item KOKEMULLER, ROBYN - US Department Of Agriculture (USDA) item MOORE, S - Oak Ridge Institute For Science And Education (ORISE) item WEST GREENLEE, M - Iowa State University
Submitted to: Meeting Abstract Publication Type: Abstract Only Publication Acceptance Date: 3/29/2019 Publication Date: N/A Citation: N/A
Interpretive Summary:
Technical Abstract: Scrapie is a transmissible spongiform encephalopathy of sheep and goats that is associated with widespread accumulation of abnormal prion protein (PrPSc) in the central nervous and lymphoid tissues. Chronic wasting disease (CWD) is the natural prion disease of cervid species, and the tissue distribution of PrPSc in affected cervids is similar to scrapie in sheep. There are several lines of evidence that suggest that multiple strains of CWD exist, which may affect the agent’s potential to transmit to hosts of the same or different species. We inoculated white-tailed deer with the scrapie agent from ARQ/ARQ sheep, which resulted in 100% attack rates by either the intracranial or oronasal route of inoculation. When examining tissues from the brainstems or lymphoid tissues by traditional diagnostic methods such as immunohistochemistry or western blots, it is difficult to differentiate tissues from deer infected with scrapie from those infected with CWD. However, there are several important differences between tissues from scrapie-infected white-tailed deer (WTD scrapie) and those infected with CWD (WTD CWD). First, there are different patterns of PrPSc deposition in the brains of infected deer: brain tissues from deer with WTD scrapie had predominantly particulate and stellate immunoreactivity whereas those from deer with WTD-CWD had large aggregates and plaque-like staining. Secondly, the incubation periods of WTD scrapie isolates are longer than CWD isolates in mice expressing cervid prion protein. Most notably, the transmission potential of these two isolates back to sheep is distinctly different. Attempts to transmit various CWD isolates to sheep by the oral or oronasal routes have been unsuccessful despite observation periods of up to 7 years. However, WTD scrapie efficiently transmitted back to sheep by the oronasal route. Upon transmission back to sheep, the WTD scrapie isolate exhibited different phenotypic properties when compared to the sheep receiving the original sheep scrapie inoculum including different genotype susceptibilities, distinct PrPSc deposition patterns, and much more rapid incubation periods in transgenic mice expressing the ovine prion protein. The scrapie agent readily transmits between sheep and deer after oronasal exposure. This could confound the identication of CWD strains in deer and the eradication of scrapie from sheep.
''The scrapie agent readily transmits between sheep and deer after oronasal exposure. This could confound the identication of CWD strains in deer and the eradication of scrapie from sheep.''
RT-QuIC detection of pathological prion protein in subclinical goats following experimental oral transmission of L-type BSE
Alessandra Favole1* , Maria Mazza1 , Antonio D’Angelo2 , Guerino Lombardi3 , Claudia Palmitessa1 , Luana Dell’Atti1 , Giulia Cagnotti2 , Elena Berrone1 , Marina Gallo1 , Tiziana Avanzato1 , Erika Messana1 , Loretta Masoero1 , Pier Luigi Acutis1 , Daniela Meloni1 , Franco Cardone4 , Maria Caramelli1 , Cristina Casalone1 and Cristiano Corona1*
Abstract
Objective: The spread of bovine spongiform encephalopathy (BSE) agent to small ruminants is still a major issue in the surveillance of transmissible spongiform encephalopathies (TSEs). L-type bovine spongiform encephalopathy (L-BSE) is an atypical form of BSE with an unknown zoonotic potential that is transmissible to cattle and small ruminants. Our current knowledge of bovine atypical prion strains in sheep and goat relies only on experimental transmission studies by intracranial inoculation. To assess oral susceptibility of goats to L-BSE, we orally inoculated five goats with cattle L-BSE brain homogenates and investigated pathogenic prion protein (PrPsc) distribution by an ultrasensitive in vitro conversion assay known as Real-Time Quaking Induced Conversion (RT-QuIC).
Results: Despite a prolonged observation period of 80 months, all these animals and the uninfected controls did not develop clinical signs referable to TSEs and tested negative by standard diagnostics. Otherwise, RT-QuIC analysis showed seeding activity in five out of five examined brain samples. PrPsc accumulation was also detected in spinal cord and lymphoreticular system. These results indicate that caprine species are susceptible to L-BSE by oral transmission and that ultrasensitive prion tests deserve consideration to improve the potential of current surveillance systems against otherwise undetectable forms of animal prion infections.
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Discussion and conclusions
Data here presented indicate that caprine species are susceptible to L-BSE after oral administration and are able to produce very low levels of prions in both lymphatic and central nervous tissues as demonstrated by optimized, high-sensitive, RT-QuIC assay.
At variance with goats intracerebrally infected with L-BSE [4], in this study, no animal developed clinical signs of disease despite prolonged periods of observation, suggesting a comparatively low efficiency of the oral route versus the intracerebral one in L-BSE, a feature that further distinguish this strain from classical BSE [14, 15].
Interestingly, all goats tested negative by standard diagnostics for PrPsc performed on brainstem. This finding, associated with the low amount of PrPsc detected in different brain areas, suggests a partial strain-specific transmission barrier. Indeed, inoculation of a prion into a new host species can produce prolonged incubation periods and/or subclinical infection [16, 17]. In addition, the lack of clinical signs suggests that naturally L-BSE-infected goats may be asymptomatic similarly to what proposed by Okada et al. for oral L-BSE in cattle [17].
In line with previous results [18], RT-QuIC detected lower levels of prions than traditional diagnostic tools. Rapid and confirmatory tests failed to identify any PrPsc in the subclinical animals, while RT-QuIC allowed us to detect misfolded prion protein in multiple brain regions, spinal cord and lymphoreticular system. Studies have established that the rate of fluorescence increase in RTQuIC, while not measuring infectivity, is directly related to the concentration of prions in the sample seeding the reaction [19, 20]. Prolonged lag phases of RT-QuIC reactions indicate relatively low amounts of PrPsc in the examined tissues and may reassure about the possibility of goat to play as silent L-BSE spreaders in natural conditions. However, we believe that prudence must be always adopted when dealing with the risk of prion spread in field conditions as also suggested by recent data by Denkers and colleagues, who showed that the oral route of infection for chronic wasting disease in deer, may be much more efficient than previously thought [21]. Furthermore, although the mere presence of PrPsc is not indicative of a possible infectivity of the tissue, the finding of positivity in the lymphoreticular tissue must alert to the potential distribution of PrPsc in peripheral body regions which may increase the risks for humans. Bioassay of infectivity by inoculation of susceptible animals with brains of these goats may help to clarify this issue.
Based on the results achieved with this prion form and also other animal strains, it would be useful to consider the possibility to enlarge current diagnostic criteria to include, in defined conditions (e.g. very limited amounts of source tissue, or preclinical testing), the application of ultrasensitive diagnostic methods. This will not only improve the sensitivity of our surveillance systems but will also help to protect food chain from accidental spillovers of the agent of L-BSE.
Limitations
Te primary limitation of this work is that infectivity was not demonstrated by bioassay and the infectious titre was not determined. Terefore, we cannot comment the degree of risk for human.
Despite these limitations, this work specifcally demonstrates prion-seeding activity in tissues of goats orally exposed to L-BSE and provide RT-QuIC as useful method to enhance surveillance of TSEs.
Keywords: Prion, L-BSE, RT-QuIC, Goat, Oral transmission, PrPsc, Ultrasensitive detection
snip...see full text;
Tuesday, May 31, 2022
89th General Session of the World Assembly of OIE Delegates image for WOAH General Summit 2022 Chronic Wasting Disease CWD TSE Prion Discussions and Concerns
WEDNESDAY, MARCH 16, 2022
SHEEP BY-PRODUCTS AND WHAT ABOUT Scrapie TSE PrP and Potential Zoonosis?
SO, WHO'S UP FOR SOME MORE TSE PRION POKER, WHO'S ALL IN $$$
SO, ATYPICAL SCRAPIE ROUGHLY HAS 50 50 CHANCE ATYPICAL SCRAPIE IS CONTAGIOUS, AS NON-CONTAGIOUS, TAKE YOUR PICK, BUT I SAID IT LONG AGO WHEN USDA OIE ET AL MADE ATYPICAL SCRAPIE A LEGAL TRADING COMMODITY, I SAID YOUR PUTTING THE CART BEFORE THE HORSE, AND THAT'S EXACTLY WHAT THEY DID, and it's called in Texas, TEXAS TSE PRION HOLDEM POKER, WHO'S ALL IN $$$
***> AS is considered more likely (subjective probability range 50–66%) that AS is a non-contagious, rather than a contagious, disease.
***> All sheep with the ARQ/ARQ and ARQ/ARR genotypes developed Atypical/Nor98 scrapie confirmed by immunohistochemistry, and one sheep with the VRQ/ARQ genotype had detectable PrPSc consistent with Atypical/Nor98 scrapie at the experimental endpoint of 8 years.
Transmission of the atypical/Nor98 scrapie agent to Suffolk sheep with VRQ/ARQ, ARQ/ARQ, and ARQ/ARR genotypes
Eric D. Cassmann, Najiba Mammadova, S. Jo Moore, Sylvie Benestad, Justin J. Greenlee
Published: February 11, 2021
Abstract
Scrapie is a transmissible spongiform encephalopathy that occurs in sheep. Atypical/Nor98 scrapie occurs in sheep that tend to be resistant to classical scrapie and it is thought to occur spontaneously. The purpose of this study was to test the transmission of the Atypical/Nor98 scrapie agent in three genotypes of Suffolk sheep and characterize the distribution of misfolded prion protein (PrPSc). Ten sheep were intracranially inoculated with brain homogenate from a sheep with Atypical/Nor98 scrapie. All sheep with the ARQ/ARQ and ARQ/ARR genotypes developed Atypical/Nor98 scrapie confirmed by immunohistochemistry, and one sheep with the VRQ/ARQ genotype had detectable PrPSc consistent with Atypical/Nor98 scrapie at the experimental endpoint of 8 years. Sheep with mild early accumulations of PrPSc in the cerebellum had concomitant retinal PrPSc. Accordingly, large amounts of retinal PrPSc were identified in clinically affected sheep and sheep with dense accumulations of PrPSc in the cerebellum.
***> These data confirm that ARR/ARR sheep cannot be considered to be fully resistant to classical scrapie.
Journal of General Virology header logoVolume 98, Issue 8
Classical scrapie transmission in ARR/ARR genotype sheep Free
Caroline Lacroux1,†, Hervé Cassard1,†, Hugh Simmons2, Jean Yves Douet1, Fabien Corbière1, Severine Lugan1, Pierette Costes1, Naima Aron1, Alvina Huor1, Cécile Tillier1, Francois Schelcher1, Olivier Andreoletti1
Published: 01 August 2017 https://doi.org/10.1099/jgv.0.000861 ;
ABSTRACT
The ARR allele is considered to provide a very strong resistance against classical scrapie infection in sheep. In this study, we report the occurrence of clinical transmissible spongiform encephalopathy in ARR/ARR sheep, following their inoculation by the intracerebral route with a classical scrapie isolate. On first passage, the disease displayed an incomplete attack rate transmission, with incubation periods exceeding 6 years. On second passage, the obtained prion did not display better abilities to propagate than the original isolate. These transmission results contrasted with the 100 % attack rate and the short incubation periods observed in ARQ/ARQ sheep challenged with the same isolate. These data confirm that ARR/ARR sheep cannot be considered to be fully resistant to classical scrapie. However, they also support the contention that classical scrapie has a very limited capacity to transmit and adapt to ARR/ARR sheep.
''These data confirm that ARR/ARR sheep cannot be considered to be fully resistant to classical scrapie. However, they also support the contention that classical scrapie has a very limited capacity to transmit and adapt to ARR/ARR sheep.''
Emerg Infect Dis. 2007 Aug; 13(8): 1201–1207.
doi: 10.3201/eid1308.070077
PMCID: PMC2828083
PMID: 17953092
Classic Scrapie in Sheep with the ARR/ARR Prion Genotype in Germany and France
Martin H. Groschup,corresponding author* 1 Caroline Lacroux,† 1 Anne Buschmann,* Gesine Lühken,‡ Jacinthe Mathey,† Martin Eiden,* Séverine Lugan,† Christine Hoffmann,* Juan Carlos Espinosa,§ Thierry Baron,¶ Juan Maria Torres,§ Georg Erhardt,‡ and Olivier Andreoletti†
Abstract
In the past, natural scrapie and bovine spongiform encephalopathy (BSE) infections have essentially not been diagnosed in sheep homozygous for the A136R154R171 haplotype of the prion protein. This genotype was therefore assumed to confer resistance to BSE and classic scrapie under natural exposure conditions. Hence, to exclude prions from the human food chain, massive breeding efforts have been undertaken in the European Union to amplify this gene. We report the identification of 2 natural scrapie cases in ARR/ARR sheep that have biochemical and transmission characteristics similar to cases of classic scrapie, although the abnormally folded prion protein (PrPSc) was associated with a lower proteinase-K resistance. PrPSc was clearly distinct from BSE prions passaged in sheep and from atypical scrapie prions. These findings strongly support the idea that scrapie prions are a mosaic of agents, which harbor different biologic properties, rather than a unique entity.
snip...
The discovery of these 2 cases clearly indicates that the genetic resistance of ARR/ARR sheep to the so-called classic scrapie agent is not absolute. It also provides evidence that, rather than being a single entity, scrapie is a mosaic of infectious agents harboring different biologic properties in its natural host. Finally, although many thousands of cases of classic scrapie have been reported in sheep of other PrP genotypes and hundreds of thousands of rapid tests have been performed in Europe since the implementation of active TSE surveillance in small ruminants began in 2001, the discovery of these 2 ARR/ARR cases supports the idea that such infections are extremely rare.
''The discovery of these 2 cases clearly indicates that the genetic resistance of ARR/ARR sheep to the so-called classic scrapie agent is not absolute.''
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.
A newly identified type of scrapie agent can naturally infect sheep with resistant PrP genotypes
Annick Le Dur*,?, Vincent Béringue*,?, Olivier Andréoletti?, Fabienne Reine*, Thanh Lan Laï*, Thierry Baron§, Bjørn Bratberg¶, Jean-Luc Vilotte?, Pierre Sarradin**, Sylvie L. Benestad¶, and Hubert Laude*,? +Author Affiliations
*Virologie Immunologie Moléculaires and ?Génétique Biochimique et Cytogénétique, Institut National de la Recherche Agronomique, 78350 Jouy-en-Josas, France; ?Unité Mixte de Recherche, Institut National de la Recherche Agronomique-Ecole Nationale Vétérinaire de Toulouse, Interactions Hôte Agent Pathogène, 31066 Toulouse, France; §Agence Française de Sécurité Sanitaire des Aliments, Unité Agents Transmissibles Non Conventionnels, 69364 Lyon, France; **Pathologie Infectieuse et Immunologie, Institut National de la Recherche Agronomique, 37380 Nouzilly, France; and ¶Department of Pathology, National Veterinary Institute, 0033 Oslo, Norway
***Edited by Stanley B. Prusiner, University of California, San Francisco, CA (received for review March 21, 2005)
Abstract
Scrapie in small ruminants belongs to transmissible spongiform encephalopathies (TSEs), or prion diseases, a family of fatal neurodegenerative disorders that affect humans and animals and can transmit within and between species by ingestion or inoculation. Conversion of the host-encoded prion protein (PrP), normal cellular PrP (PrPc), into a misfolded form, abnormal PrP (PrPSc), plays a key role in TSE transmission and pathogenesis. The intensified surveillance of scrapie in the European Union, together with the improvement of PrPSc detection techniques, has led to the discovery of a growing number of so-called atypical scrapie cases. These include clinical Nor98 cases first identified in Norwegian sheep on the basis of unusual pathological and PrPSc molecular features and "cases" that produced discordant responses in the rapid tests currently applied to the large-scale random screening of slaughtered or fallen animals. Worryingly, a substantial proportion of such cases involved sheep with PrP genotypes known until now to confer natural resistance to conventional scrapie. 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.
''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.''
CWD TSE PRION AND ZOONOTIC, ZOONOSIS, POTENTIAL
Subject: Re: DEER SPONGIFORM ENCEPHALOPATHY SURVEY & HOUND STUDY
Date: Fri, 18 Oct 2002 23:12:22 +0100
From: Steve Dealler
Reply-To: Bovine Spongiform Encephalopathy Organization: Netscape Online member
To: BSE-L@ References:
Dear Terry,
An excellent piece of review as this literature is desperately difficult to get back from Government sites.
What happened with the deer was that an association between deer meat eating and sporadic CJD was found in about 1993. The evidence was not great but did not disappear after several years of asking CJD cases what they had eaten. I think that the work into deer disease largely stopped because it was not helpful to the UK industry...and no specific cases were reported. Well, if you dont look adequately like they are in USA currenly then you wont find any!
Steve Dealler ===============
''The association between venison eating and risk of CJD shows similar pattern, with regular venison eating associated with a 9 FOLD INCREASE IN RISK OF CJD (p = 0.04).''
CREUTZFELDT JAKOB DISEASE SURVEILLANCE IN THE UNITED KINGDOM THIRD ANNUAL REPORT AUGUST 1994
Consumption of venison and veal was much less widespread among both cases and controls. For both of these meats there was evidence of a trend with increasing frequency of consumption being associated with increasing risk of CJD. (not nvCJD, but sporadic CJD...tss) These associations were largely unchanged when attention was restricted to pairs with data obtained from relatives. ...
Table 9 presents the results of an analysis of these data.
There is STRONG evidence of an association between ‘’regular’’ veal eating and risk of CJD (p = .0.01).
Individuals reported to eat veal on average at least once a year appear to be at 13 TIMES THE RISK of individuals who have never eaten veal.
There is, however, a very wide confidence interval around this estimate. There is no strong evidence that eating veal less than once per year is associated with increased risk of CJD (p = 0.51).
The association between venison eating and risk of CJD shows similar pattern, with regular venison eating associated with a 9 FOLD INCREASE IN RISK OF CJD (p = 0.04).
There is some evidence that risk of CJD INCREASES WITH INCREASING FREQUENCY OF LAMB EATING (p = 0.02).
The evidence for such an association between beef eating and CJD is weaker (p = 0.14). When only controls for whom a relative was interviewed are included, this evidence becomes a little STRONGER (p = 0.08).
snip...
It was found that when veal was included in the model with another exposure, the association between veal and CJD remained statistically significant (p = < 0.05 for all exposures), while the other exposures ceased to be statistically significant (p = > 0.05).
snip...
In conclusion, an analysis of dietary histories revealed statistical associations between various meats/animal products and INCREASED RISK OF CJD. When some account was taken of possible confounding, the association between VEAL EATING AND RISK OF CJD EMERGED AS THE STRONGEST OF THESE ASSOCIATIONS STATISTICALLY. ...
snip...
In the study in the USA, a range of foodstuffs were associated with an increased risk of CJD, including liver consumption which was associated with an apparent SIX-FOLD INCREASE IN THE RISK OF CJD. By comparing the data from 3 studies in relation to this particular dietary factor, the risk of liver consumption became non-significant with an odds ratio of 1.2 (PERSONAL COMMUNICATION, PROFESSOR A. HOFMAN. ERASMUS UNIVERSITY, ROTTERDAM). (???...TSS)
snip...see full report ;
http://web.archive.org/web/20090506050043/http://www.bseinquiry.gov.uk/files/yb/1994/08/00004001.pdf
http://web.archive.org/web/20090506050007/http://www.bseinquiry.gov.uk/files/yb/1994/10/00003001.pdf
http://web.archive.org/web/20090506050244/http://www.bseinquiry.gov.uk/files/yb/1994/07/00001001.pdf
Stephen Dealler is a consultant medical microbiologist deal@airtime.co.uk
BSE Inquiry Steve Dealler
Management In Confidence
BSE: Private Submission of Bovine Brain Dealler
snip...see full text;
MONDAY, FEBRUARY 25, 2019
***> MAD DOGS AND ENGLISHMEN BSE, SCRAPIE, CWD, CJD, TSE PRION A REVIEW 2019
***> ''The association between venison eating and risk of CJD shows similar pattern, with regular venison eating associated with a 9 FOLD INCREASE IN RISK OF CJD (p = 0.04).''
***> In conclusion, sensory symptoms and loss of reflexes in Gerstmann-Sträussler-Scheinker syndrome can be explained by neuropathological changes in the spinal cord. We conclude that the sensory symptoms and loss of lower limb reflexes in Gerstmann-Sträussler-Scheinker syndrome is due to pathology in the caudal spinal cord. <***
***> The clinical and pathological presentation in macaques was mostly atypical, with a strong emphasis on spinal cord pathology.<***
***> The notion that CWD can be transmitted orally into both new-world and old-world non-human primates asks for a careful reevaluation of the zoonotic risk of CWD. <***
***> All animals have variable signs of prion neuropathology in spinal cords and brains and by supersensitive IHC, reaction was detected in spinal cord segments of all animals.<***
***> In particular the US data do not clearly exclude the possibility of human (sporadic or familial) TSE development due to consumption of venison. The Working Group thus recognizes a potential risk to consumers if a TSE would be present in European cervids.'' Scientific opinion on chronic wasting disease (II) <***
***Moreover, sporadic disease has never been observed in breeding colonies or primate research laboratories, most notably among hundreds of animals over several decades of study at the National Institutes of Health25, and in nearly twenty older animals continuously housed in our own facility.***
Even if the prevailing view is that sporadic CJD is due to the spontaneous formation of CJD prions, it remains possible that its apparent sporadic nature may, at least in part, result from our limited capacity to identify an environmental origin.
TUESDAY, MAY 31, 2022
USA Bovine Spongiform Encephalopathy BSE: description of typical and atypical cases
TUESDAY, SEPTEMBER 13, 2022
BSE pathogenesis in the ileal Peyer’s patches and the central and peripheral nervous system of young cattle 8 months post oral BSE challenge
THURSDAY, OCTOBER 27, 2022
SEAFWA COMMITTEE Cervid Working Group Report August 16, 2022 CWD TSE Prion Report
FRIDAY, NOVEMBER 04, 2022
Texas CWD TSE Prion 409 Cases Confirmed To Date TPWD emergency rule adds two new surveillance zones located primarily in Gillespie and Limestone counties
TUESDAY, SEPTEMBER 07, 2021
Atypical Bovine Spongiform Encephalopathy BSE OIE, FDA 589.2001 FEED REGULATIONS, and Ingestion Therefrom
Bovine Spongiform Encephalopathy BSE TSE Prion Origin USA
WEDNESDAY, JANUARY 12, 2022
Bovine Spongiform Encephalopathy BSE TSE Prion Origin USA, what if?
PLOS ONE Journal
*** Singeltary reply ; Molecular, Biochemical and Genetic Characteristics of BSE in Canada Singeltary reply ;
IBNC Tauopathy or TSE Prion disease, it appears, no one is sure
Terry S. Singeltary Sr., 03 Jul 2015 at 16:53 GMT
***however in 1 C-type challenged animal, Prion 2015 Poster Abstracts S67 PrPsc was not detected using rapid tests for BSE.
***Subsequent testing resulted in the detection of pathologic lesion in unusual brain location and PrPsc detection by PMCA only.
*** IBNC Tauopathy or TSE Prion disease, it appears, no one is sure ***
MONDAY, SEPTEMBER 19, 2022
589.2001 BSE TSE regulations which prohibits the use of high-risk cattle material in feed for all animal species 2022
SATURDAY, SEPTEMBER 24, 2022
Transmission of CH1641 in cattle
FRIDAY, APRIL 1, 2022
USDA TAKES THE C OUT OF COOL, what's up with that?
MONDAY, JUNE 6, 2022
APHIS USDA History Highlight: APHIS Combats Bovine Spongiform Encephalopathy Published Jun 1, 2022
MONDAY, NOVEMBER 30, 2020
***> REPORT OF THE MEETING OF THE OIE SCIENTIFIC COMMISSION FOR ANIMAL DISEASES Paris, 9–13 September 2019 BSE, TSE, PRION
see updated concerns with atypical BSE from feed and zoonosis...terry
WEDNESDAY, DECEMBER 8, 2021
Importation of Sheep, Goats, and Certain Other Ruminants AGENCY: Animal APHIA, USDA, FINAL RULE [Docket No. APHIS–2009–0095] RIN 0579–AD10
WEDNESDAY, MARCH 24, 2021
USDA Animal and Plant Health Inspection Service 2020 IMPACT REPORT BSE TSE Prion Testing and Surveillance MIA
https://animalhealthreportpriontse.blogspot.com/2021/03/usda-animal-and-plant-health-inspection.html
SUNDAY, MARCH 21, 2021
Investigation Results of Texas Cow That Tested Positive for Bovine Spongiform Encephalopathy (BSE) Aug. 30, 2005 Singeltary's Regiew 2021
THURSDAY, AUGUST 20, 2020
Why is USDA "only" BSE TSE Prion testing 25,000 samples a year?
THURSDAY, JANUARY 23, 2020
USDA Consolidates Regulations for NAHLN Laboratory Testing USDA Animal and Plant Health Inspection Service
sent this bulletin at 01/23/2020 02:15 PM EST
WEDNESDAY, APRIL 24, 2019
USDA Announces Atypical Bovine Spongiform Encephalopathy Detection Aug 29, 2018 A Review of Science 2019
Saturday, July 23, 2016
BOVINE SPONGIFORM ENCEPHALOPATHY BSE TSE PRION SURVEILLANCE, TESTING, AND SRM REMOVAL UNITED STATE OF AMERICA UPDATE JULY 2016
Tuesday, July 26, 2016
Atypical Bovine Spongiform Encephalopathy BSE TSE Prion UPDATE JULY 2016
Monday, June 20, 2016
Specified Risk Materials SRMs BSE TSE Prion Program
*** PLEASE SEE THIS URGENT UPDATE ON CWD AND FEED ANIMAL PROTEIN ***
Sunday, March 20, 2016
Docket No. FDA-2003-D-0432 (formerly 03D-0186) Use of Material from Deer and Elk in Animal Feed ***UPDATED MARCH 2016*** Singeltary Submission
SEE MAD COW FEED VIOLATIONS AFER MAD COW FEED VIOLATIONS ;
Tuesday, April 19, 2016
Docket No. FDA-2013-N-0764 for Animal Feed Regulatory Program Standards Singeltary Comment Submission
17 years post mad cow feed ban August 1997
Monday, October 26, 2015
FDA PART 589 -- SUBSTANCES PROHIBITED FROM USE IN ANIMAL FOOD OR FEED VIOLATIONS OFFICIAL ACTION INDICATED OIA UPDATE October 2015
Tuesday, December 23, 2014
FDA PART 589 -- SUBSTANCES PROHIBITED FROM USE IN ANIMAL FOOD OR FEEDVIOLATIONS OFFICIAL ACTION INDICATED OAI UPDATE DECEMBER 2014 BSE TSE PRION
16 years post mad cow feed ban August 1997 2013
Sunday, December 15, 2013
FDA PART 589 -- SUBSTANCES PROHIBITED FROM USE IN ANIMAL FOOD OR FEED VIOLATIONS OFFICIAL ACTION INDICATED OIA UPDATE DECEMBER 2013 UPDATE
Saturday, August 29, 2009
FOIA REQUEST FEED RECALL 2009 Product may have contained prohibited materials Bulk Whole Barley, Recall # V-256-2009
Friday, September 4, 2009
FOIA REQUEST ON FEED RECALL PRODUCT 429,128 lbs. feed for ruminant animals may have been contaminated with prohibited material Recall # V-258-2009
Thursday, March 19, 2009
MILLIONS AND MILLIONS OF POUNDS OF MAD COW FEED IN COMMERCE USA WITH ONGOING 12 YEARS OF DENIAL NOW, WHY IN THE WORLD DO WE TO TALK ABOUT THIS ANYMORE $$$
MONDAY, FEBRUARY 25, 2019
***> MAD DOGS AND ENGLISHMEN BSE, SCRAPIE, CWD, CJD, TSE PRION A REVIEW 2019
SATURDAY, OCTOBER 8, 2022
Cattle with the EK211 PRNP polymorphism are susceptible to the H-type bovine spongiform encephalopathy agent from either E211K or wild type donors after oronasal inoculation
TUESDAY, NOVEMBER 01, 2022
SEAC Position statement Chronic wasting disease in UK deer January 2005 (updated July 2006) to 2021
TUESDAY, NOVEMBER 1, 2022
SEAC Scientific Steering Committee on TSE Prion
SATURDAY, NOVEMBER 5, 2022
EFSA Network on BSE-TSE Minutes of the 17th meeting Held on 13-14 October 2022
SPONGIFORM ENCEPHALOPATHY ADVISORY COMMITTEE
Minutes of the 99th meeting held on 14th December 2007
snip...
ITEM 8 – PUBLIC QUESTION AND ANSWER SESSION 40.
The Chair explained that the purpose of the question and answer session was to give members of the public an opportunity to ask questions related to the work of SEAC. Mr Terry Singeltary (Texas, USA) had submitted a question prior to the meeting, asking: “With the Nor-98 now documented in five different states so far in the USA in 2007, and with the two atypical BSE H-base cases in Texas and Alabama, with both scrapie and chronic 14 © SEAC 2007 wasting disease (CWD) running rampant in the USA, is there any concern from SEAC with the rise of sporadic CJD in the USA from ''unknown phenotype'', and what concerns if any, in relations to blood donations, surgery, optical, and dental treatment, do you have with these unknown atypical phenotypes in both humans and animals in the USA? Does it concern SEAC, or is it of no concern to SEAC? Should it concern USA animal and human health officials?” 41.
A member considered that this question appeared to be primarily related to possible links between animal and human TSEs in the USA.
There is no evidence that sCJD is increasing in the USA and no evidence of any direct link between TSEs and CJD in the USA. Current evidence does not suggest that CWD is a significant risk to human health. There are unpublished data from a case of human TSE in the USA that are suggestive of an apparently novel form of prion disease with distinct molecular characteristics. However, it is unclear whether the case had been further characterised, if it could be linked to animal TSEs or if other similar cases had been found in the USA or elsewhere. In relation to the possible public health implications of atypical scrapie, H-type BSE and CWD, research was being conducted to investigate possible links and surveillance was in place to detect any changes in human TSEs. Although possible links between these diseases and human TSEs are of concern and require research, there is no evidence to suggest immediate public health action is warranted. The possible human health risks from classical scrapie had been discussed earlier in the meeting. Members noted that there are effective channels of discussion and collaboration on research between USA and European groups. Members agreed it is important to keep a watching brief on new developments on TSEs.
snip...
http://seac992007.blogspot.com/
>>>There is no evidence that sCJD is increasing in the USA and no evidence of any direct link between TSEs and CJD in the USA.<<<
https://seac992007.blogspot.com/2009/10/
TUESDAY, APRIL 05, 2022 2022
American Academy of Neurology Emerging Sciences
Abstract Website
Incidence of Creutzfeldt-Jakob Disease in the United States 1993-2014
SUNDAY, OCTOBER 30, 2022
Why is USDA "only" testing 25,000 samples a year?
https://bovineprp.blogspot.com/2022/10/why-is-usda-only-testing-25000-samples.htmlAPHIS Indemnity Regulations [Docket No. APHIS-2021-0010] RIN 0579-AE65 Singeltary Comment SubmissionComment from Singeltary Sr., Terry
Posted by the Animal and Plant Health Inspection Service on Sep 8, 2022
https://www.regulations.gov/comment/APHIS-2021-0010-0003
https://downloads.regulations.gov/APHIS-2021-0010-0003/attachment_1.pdf
August 2007HUMAN and ANIMAL TSE Classifications i.e. mad cow disease and the UKBSEnvCJD only theoryTSEs have been rampant in the USA for decades in many species, and they all have been rendered and fed back to animals for human/animal consumption. I propose that the current diagnostic criteria for human TSEs only enhances and helps the spreading of human TSE from the continued belief of the UKBSEnvCJD only theory in 2007. With all the science to date refuting it, to continue to validate this myth, will only spread this TSE agent through a multitude of potential routes and sources i.e. consumption, surgical, blood, medical, cosmetics etc. I propose as with Aguzzi, Asante, Collinge, Caughey, Deslys, Dormont, Gibbs, Ironside, Manuelidis, Marsh, et al and many more, that the world of TSE Transmissible Spongiform Encephalopathy is far from an exact science, but there is enough proven science to date that this myth should be put to rest once and for all, and that we move forward with a new classification for human and animal TSE that would properly identify the infected species, the source species, and then the route.This would further have to be broken down to strain of species and then the route of transmission would further have to be broken down. Accumulation and Transmission are key to the threshold from sub-clinical to clinical disease, and key to all this, is to stop the amplification and transmission of this agent, the spreading of, no matter what strain. In my opinion, to continue with this myth that the U.K. strain of BSE (one strain TSE in cows), and the nv/v CJD (one strain TSE humans) and that all the rest of human TSE are just one single strain i.e. sporadic CJD (when to date there are 6 different phenotypes of sCJD, and growing per Gambetti et al), and that no other animal TSE transmits to humans, to continue with this masquerade will only continue to spread, expose, and kill, who knows how many more in the years and decades to come. ONE was enough for me, My Mom, hvCJD i.e. Heidenhain Variant CJD, DOD 12/14/97 confirmed, which is nothing more than another mans name added to CJD, like CJD itself, Jakob and Creutzfeldt, or Gerstmann-Straussler-Scheinker syndrome, just another CJD or human TSE, named after another human.WE are only kidding ourselves with the current diagnostic criteria for human and animal TSE, especially differentiating between the nvCJD vs the sporadic CJD strains and then the GSS strains and also the FFI fatal familial insomnia strains or the ones that mimics one or the other of those TSE? Tissue infectivity and strain typing of the many variantsManuscriptof 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. ...Terry S. Singeltary Sr. P.O. Box Bacliff, Texas USA 77518 flounder9@verizon.net[Docket No. 03-025IFA] FSIS Prohibition of the Use of Specified Risk Materials for Human Food and Requirement for the Disposition of Non-Ambulatory Disabled Cattle
9/13/2005PDF]Freas, William TSS SUBMISSION File Format: PDF/Adobe Acrobat - Page 1. J Freas, William From: Sent: To: Subject: Terry S. Singeltary Sr. [flounder@wt.net] Monday, January 08,200l 3:03 PM freas ... http://web.archive.org/web/20170301223601/https://www.fda.gov/OHRMS/DOCKETS/AC/01/slides/3681s2_09.pdfFrom: Terry S. Singeltary Sr. [flounder9@verizon.net]Sent: Thursday, September 08, 2005 6:17 PMTo: fsis.regulationscomments@fsis.usda.govSubject: [Docket No. 03-025IFA] FSIS Prohibition of the Use of Specified Risk Materials for Human Food and Requirements for the Disposition of Non-Ambulatory Disabled CattleDocket No: 2002N-0273 (formerly Docket No. 02N-0273) Substances Prohibited From Use in Animal Food and Feed Scientists Comments December 20,2005''It is our opinion that the proposed rule falls woefully short in effective measures to minimize the potential for further transmissions of the disease.''Docket No: 2002N-0273 (formerly Docket No. 02N-0273) Substances Prohibited From Use in Animal Food and FeedDecember 20,2005Division of Dockets Management (HFA-305)Food and Drug Administration5630 Fishers LaneRoom 1061Rockville, MD 20852Re: Docket No: 2002N-0273 (formerly Docket No. 02N-0273) Substances Prohibited From Use in Animal Food and FeedDear Sir or Madame:As scientists and recognized experts who have worked in the field of TSEs for decades, we are deeply concerned by the recent discoveries of indigenous BSE infected cattle in North America and appreciate the opportunity to submit comments to this very important proposed rule We strongly supported the measures that USDA and FDA implemented to protect public health after the discovery of the case of bovine spongiform encephalopathy (BSE) found in Washington State in 2003. We know of no event or discovery since then that could justify relaxing the existing specified risk material (SRM) and non-ambulatory bans and surveillance that were implemented at that time. Further, we strongly supported the codification of those changes, as well as additional measures to strengthen the entire feed and food system. The discovery of additional cases of indigenous BSE in North America since that time has validated our position and strengthened OUT convictions.We caution against using the 18 month enhanced surveillance as a justification to relax or impede further actions. While this surveillance has not uncovered an epidemic, it does not clear the US cattle herd from infection. While it is highly likely that US and Canadian cattle were exposed to BSE prior to the 1997 feed ban, we do not know how many cattle were infected or how widely the infection was dispersed. BSE cases are most likely clustered in time and location, so while enhanced surveillance provides an 18 month snapshot, it does not negate the fact that US and Canadian cattle were exposed to BSE. We also do not know in any quantitative or controlled way how effective the feed ban has been, especially at the farm level. At this point we cannot even make a thorough assessment of the USDA surveillance as details such as age, risk category and regional distribution have not been released.A number of countries initially attempted to take partial steps in regard to feed controls only to face repeated disappointments in predicted downturns of the epidemic course. We in North America could do this experiment all over again, waiting for each new warning before adding more stringency to our control measures, or we can benefit from the experience of others and take decisive measures now to arrest any further development of underlying cases that is implicit in those already discovered to date. The discovery of 5 indigenous North American cases, including one born after the implementation of the current feed ban, should provide the necessary incentive to implement, monitor and enforce a comprehensive and protective feed ban that is more congruent with the measures that have been proven to be effective throughout the world. In particular, we urge the FDA to act without further delay to strengthen the animal feed regulations by implementing the program proposed by the Canadian Food Inspection Agency (CFIA) in the December 11, 2004 Gazette. This includes removing all specified risk materials (SRMs) and deadstock from all animal feed. We also urge that the FDA discontinues the legal exemptions which allow ruminant protein to be fed back to ruminants (with the exception of milk). Many of these exemptions do not exist in other countries.Bovine products and byproducts are used for both food and pharmaceuticals. These human uses require the highest level of safety. Because of the hardy nature of the BSE agent and its high potential for cross contamination, the most effective way to protect bovine products and bovine derived materials from contamination by BSE is to ensure that infected animals or carcasses never enter processing plants. The goal would be to discover and remove infected animals fi-om production as early as possible in the infection and long before they would be sent to slaughter. Until we have diagnostic tools powerful enough to allow us to discover the disease early in its prolonged pre-clinical incubation, we have to rely on the next best strategy which is to prevent any exposure through feed. The exemptions in the current ban as well as in the newly proposed rule make this difficult if not impossible, as they still provide legal avenues for ruminants to consume potentially contaminated ruminant protein.It is our opinion that the proposed rule falls woefully short in effective measures to minimize the potential for further transmissions of the disease. By the FDA’s own analysis, exempted tissues (such as distal ileum, DRGs, etc) contain approximately 10% of the infectivity in affected animals. Thus the proposed rule still allows the possibility for cattle to be exposed to BSE through:1. Feeding of materials currently subject to legal exemptions from the ban (e.g., poultry litter, plate waste)2. Cross feeding (the feeding of non-ruminant rations to ruminants) on farms; and3. Cross contamination of ruminant and non-ruminant feedWe are most concerned that the FDA has chosen to include a provision that would allow tissues from deadstock into the feed chain. We do not believe that down or dead stock should be allowed into the food or feed chain whatever the age of the animal and whether or not the CNS tissues are removed. We do not support the provision to allow removal of brain and spinal cord from deadstock over 30 months for a number of reasons. This category of animals contains the highest level of infectivity and that infectivity is in other tissues besides just brain and spinal cord. Recent improvements in the BSE bioassay, have now made it possible to detect BSE infectivity 1000 time more efficiently than before. This assay has revealed the presence of BSE infectivity in some but not all peripheral nerves and in one muscle. (Buschmann and Groschup, 2005) This published and peer reviewed work is consistent with other publicly reported studies in Japan where, by western blot testing, ,prions were found in the peripheral nerves of a naturally infected 94-month-old cow. We feel that the studies as reported above have merit. The current studies not only re-enforce the risk of down and deadstock but also appear to provide additional information that these animals may be a potential source of greater levels of infectivity into the feed system. We also doubt that brain and spinal cord can be completely removed especially during warmer weather. Given the biological composition of these tissues, they are predisposed to rapid autolysis.As world wide surveillance for BSE increases, several atypical cases of bovine TSE have been discovered. These cases either show no clinical signs, or present as ‘downers’, and have an atypical neuropathology with respect to lesion morphology and distribution, causing problems in both clinical and post-mortem diagnosis. The origin of the cases are unclear but they suggest that even should typical BSE be eliminated, there may be other TSE diseases of cattle that could result by “mutation” and selection. Refeeding of contaminated protein could potentially perpetuate transmission much like typical BSE. An effective feed ban could prevent the expansion of such strains. We also note that there are other species which are susceptible to BSE and the current regulations allow for SRMs to be included in feed for these animals.For BSE to be perpetuated, the animal production system must have a source of agent and a means by which cattle or other susceptible species are exposed to this agent. We feel that in North America, the source and routes of exposure still exist, hence allowing for the continued recycling of BSE. We have detailed the scientific justifications for our position below.Source of the agent: SRMs (Specified Risk Materials)SRMs, as defined by the USDA, are tissues which, in a BSE infected animal, are known to either harbor BSE infectivity or to be closely associated with infectivity. If SRMs are not removed, they may introduce BSE infectivity and continue tq provide a source of animal feed contamination. For example, the skull and vertebral column which encase the brain and spinal cord, respectively, can be assumed to have gross contamination. Rendering will reduce infectivity but it will not totally eliminate it. This is significant as research in the United Kingdom has shown that a calf may be infected with BSE by the ingestion of as little as .001 gram of untreated brain.The tissue distribution of infectivity in BSE infected cattle has primarily been determined by 3 studies conducted in the United Kingdom all of which had limitations. In two of the studies, bioassays were done in mice which are at least 1000 fold less sensitive to BSE infection than cattle themselves. Only higher titers of infectivity can be detected by this method. These investigations found infectivity in the brain, spinal cord, retina, trigeminal ganglia, dorsal root ganglia, distal ileum and bone marrow (the bone marrow finding was from one animal). Infectivity was found in distal ileum of experimentally infected calves beginning six months after challenge and continuing at other intervals throughout life. (Wells et. al., 1994; 1998). The bioassay study in calves has produced similar results and in addition infectivity has been found in tonsil. The study is still in progress. Another project has found infectivity in the lymphoid tissue of third eyelid from naturally infected animals. (Dr. Danny Matthews, UK DEFRA, personal communication).While bioassay in cattle is far preferable to mice in terms of sensitivity, cattle nevertheless present their own limitations in terms of the long incubation time and the limited number of animals that can be used for assay compared to rodents. As a consequence the significance of the negative finding for many tissues is questionable. In fact, by the end of 2004 there was increasing evidence in species other than cattle that peripheral nerves and muscle have infectivity. (Bosque et al., 2002; Glatzel et al., 2003;Bartz et al., 2002; Androletti et al., 2004; Mulcahy et al., 2004; Thomzig et al., 2003; Thomzig et al., 2004)In some of these species, studies indicate that the agent migrates to the brain and spinal cord, replicates to high levels in the CNS and then spreads centrifugally from the spinal cord back down through the spinal neurons to the junction of the nerves and muscle into the muscle cells themselves. A recent German study (Buschmann and Groschup, 2005) examined nerves and muscle from a cow naturally infected with BSE and found that infectivity was present in several peripheral nerves and one muscle. The method of detection was bioassay in bovinized transgenic mice that show the same or greater sensitivity to transmission of BSE as cattle. This research concurs with findings by Japanese scientists that BSE infectivity is present in peripheral nerves at least in the clinical stage of disease.It is our opinion that there is increasing evidence that the pathogenesis of BSE might not be entirely different from TSEs in other species at the point of clinical disease in that there is peripheral involvement. We feel that the studies as reported above have merit. The current studies not only re-enforce the risk of down and deadstock but also appear to provide additional information that these animals may be a potential source of greater levels of infectivity into the feed system.In the event that FDA may confer with USDA about the risks associated with peripheral nerves we want to point out one issue. In the recent publication of the final rule on the importation of whole cuts of boneless beef from Japan, 9 CFR Part 94 [Docket No. 05- 004-21 RIN 0579-AB93, we disagree with the interpretation provided by USDA, APHIS. APHIS seems to discount the studies conducted by Groschup et al. 2005. on the basis that the transgenic mouse bioassay that they used may be too sensitive. In taking this position they have failed to realize that the point of an assay is to reveal in which tissues the infectivity resides and its relative concentration to brain or spinal cord. For this purpose, no assay can be too sensitive. Of course, the probability of an actual infection will he affected by the efficiency of infection which will be a function of dose, route of exposure and any host barrier effects that are present.We would also like to point out a factual error in the conclusion. APHIS states, “Given these factors, APHIS has determined that the finding of l3SE infectivity in facial and sciatic nerves of the transgenic mice is nalt directly applicable to cattle naturally infected with BSE. Therefore, we do not consider it necessary to make any adjustments to the risk analysis for this rulemaking or to extend the comment period to solicit additional public comment on this issue.” It is incorrect that the infectivity was found in the peripheral nerves of transgenic mice. The peripheral nerves were harvested from a cow naturally infected with BSE. Transgenic mice were used as a bioassay model.From [Docket No. 05-004-21 RIN 0579-AB93:“Peripheral NervesIssue: Two commenters stated that the underlying assumption of the proposed rule. that whole cuts of boneless beef from #Japan will not contain tissues that may carry the BSE agent, is no longer valid because researchers have found peripheral nervous system tissues, including facial and sciatic nerves, that contain BSE infectivity. \2\ One of these commenters requested APHIS to explain whether and what additional mitigation measures are needed to reduce the risks that these tissues may be present in Japanese beef. This commenter further requested an additional comment period to obtain public comments to treat this new scientific finding.\2\ Bushmann, A., and Groschup, M.; Highly Bovine Spongiform Encephalopathy-Sensitive Transgenic Mice Confirm the Essential Restriction of Infectivity to the Nervous System in Clinically Diseased Cattle. The Journal of Infectious Diseases, 192: 93442, September 1, 2005.Response: APHIS is familiar with the results of the study mentioned by the commenters in which mice, genetically engineered to be highly susceptible to BSE and to overexpress the bovine prion protein, were inoculated with tissues from a BSE-infected cow. This study demonstrated low levels of infectivity in the mouse assay in the facial and sciatic nerves of the peripheral nervous system. APHIS has evaluated these findings in the context of the potential occurrence of infectivity in the peripheral nerves of cattle and the corresponding risks of the presence of infectivity in such tissues resulting in cattle or human exposure to the BSE agent. The results from these experiments in genetically engineered mice should be interpreted with caution, as the findings may be influenced by the overexpression of prion proteins and may not accurately predict the natural distribution of BSE infectivity in cattle. Further, the overexpression of prion proteins in transgenic mice may not accurately mimic the natural disease process because the transgenic overexpressing mice have been shown to develop spontaneous lethal neurological disease involving spongiform changes in the brain and muscle degeneration.\3\ In addition, the route of administration to the mice was both intraperitoneal and intracerebral, which are two very efficient routes of infection as compared to oral consumption. Given these factors, APHIS has determined that the finding of BSE infectivity in facial and sciatic nerves of the transgenic mice is not directly applicable to cattle naturally infected with BSE. Therefore, we do not consider it necessary to make any adjustments to the risk analysis for this rulemaking or to extend the comment period to solicit additional public comment on this issue.”Source of the agent: DeadstockThe total amount of TSE infectivity in a TSE infected animal increases steadily throughout the infection and exponentially once the infectivity reaches the brain. Infected individuals only exhibit recognizable clinical signs once infectivity titers have reached high levels in the brain. Surveillance data collected throughout Europe indicates there is a much greater likelihood for BSE to be detected in dead or down cattle than from healthy normal animals. This has so far also been borne out by the experience in North America. Animals that die of BSE harbor the greatest amount of agent that can be produced by the disease. Leaving the tissues from the highest risk category of cattle in the animal feed chain will effectively nullify the purported intent of this regulation. This point is supported by the 2001 Harvard risk assessment model that demonstrated that eliminating dead and downer, 4D cattle, from the feed stream was a disproportionately effective means of reducing the risk of re-infection.“The disposition of cattle thot die on the farm would also have a substantial influence on the spread of BSE if the disease were introduced ” The base case scenario showed that the mean total number of ID50.s (i.e., dosage sufficient to infect 50 percent of exposed cattle) from healthy animals at slaughter presented to the food/feed system was 1500. The mean total number of ID50s from adult cattle deadstock presented to the feed system was 37,000. This illustrates the risk of “4D cattle ” (i.e.. deadstock).From the Harvard Risk Assessment, 200 1, Appendix 3A Base Case and Harvard Risk Assessment, 200 1 Executive SummaryIt is likely that these numbers would have to be adjusted upwards, if the UK attack rate and Groschup data were considered.Inflammation and TSEsThere have been 3 recent peer reviewed publications which indicate that chronic inflammatory conditions in a host with a TSE may induce prion replication in, or distribution to organs previously thought to be low or no risk. They are as follows:1 Chronic Lymphocytic Inflammation Specifies the Organ Tropism of Prions (Heikenwalder et. al. 2005 >~xx .sci~:n~c\rpl-css.~~r~/~O .lunuarv 2005/ Parrc l/ &).I 1zois~icllcc.l lOh4hO)2. Coincident Scrapie Infection and Nephritis Lead to Urinary Priori Excretion (Seeger et al., Science 14 October 2005:Vol. 310. no. 5746, pp. 324 - 326 DOI: lO.l126/science. 1118829)3. PrPS” in mammary glands of sheep affected by scrapie and mastitis (Ligios C., et al. Nature Medicine, 11. 3 137 - 1138, 2005)These studies from the Aguzzi laboratory warn that concurrent chronic inflammatory disease could dramatically alter the distribution of BSE infectivity in infected cattle. Down and dead stock are at higher risk for both BSE and other systemic conditions. If the results reported above are also applicable to cattle, the carcasses of dead and down stock affected by BSE might contain even higher levels of infectivity, or contribute infectivity via tissues thai. are not ordinarily at risk in normal animals.Exposure: Industry Practices or Exemptions which may pose a riskPoultry LitterIn the United States poultry litter can be fed to cattle. There are two potential sources of risk from poultry litter. Poultry litter not only consists of digested feed but also of feed which spills from the cages. As a consequence, the practice of feeding litter back to cattle is by its nature non--compliant with the current feed ban if the poultry themselves are being fed ruminant protein. Given that ruminant protein can no longer be fed to ruminants in the United States and that most. if not all. countries will no longer import North American ruminant MBM, an even larger part of poultry diets is now ruminant MBM. Spillage provides a direct link to back to cattle but feces are also likely to contain infectivity.There is no reason to expect that TSE infectivity would be inactivated by passage through the poultry gut, and only a slim possibility that composting would reduce infectivity at all. Thus poultry feces are another potential route of transmission back to cattle. Evidence for this comes from rodent experiments where infectivity was demonstrated in the feces after being fed: “Laboratory experiments show that mice orally challenged with scrapie have detectable infectivity that passes through the gut. Gut contents and fecal matter may therefore contain infectivity, and it is noted that in experimental oral challenges in cattle conducted in the UK, feces must be treated as a medical waste for one month following the challenge. It is concluded that digestive contents and fecal material from livestock or poultry currently being fed with MBM potentially contaminated with BSE should not be used as a feed ingredient for animal feed.” [Proceedings: Joint WHO/FAO/OIE/ Technical Consultation on BSE: public health, animal health and trade. Paris, lo-14 June 2001; and Alan Dickinson, personal communication].It may be possible to remove the risk from poultry litter by sterilization. However, unless or until a method can be developed and validated, poultry litter should be banned from ruminant feed.Ruminant BloodIn contrast with humans, sheep, monkeys, mice and hamsters, including sheep and mice infected with BSE and humans infected with vCJD considered identical to BSE, no infectivity has so far been demonstrated in the blood of BSE infected cattle. However, we consider it unlikely that cattle are the sole outlier to what has been a consistent finding in all other TSE diseases where the measurement has been made with sufficient sensitivity to detect the low levels of infectivity that are present in blood. Rather, this failure is more likely the result of the very small volumes of blood that were used for the inoculations (less than 1 ml), whereas whole transfusions were administered to assay animals in the published .sheep scrapie/BSE experiments. If blood is infected then all vascularized tissues can bc expected to contain some infectivity in proportion to the content of residual blood..Micro emboli are a possible source of blood-borne agent that could be at much higher titer than blood itself, in slaughtered cattle carrying BSE infection. Stunning can release micro emboli of brain tissue into the circulatory system from where they can be distributed to other tissues in the few moments before the exsanguination and death. (Anil, et al, 2001a & b; Anil et al, 2002; Love, et al, 2000). This source of infection could extend a higher infectivity risk to tissues that would otherwise be at low risk, thereby allowing exposure of cattle through any of the legal exemptions and potentially producing a feed and food risk. Blood-borne contamination may be a special problem where spray-dried blood is being used as a milk replacer for calves, as it is thought that young animals are especially susceptible to infection.Certainly, blood and blood proteins should not be used as feed without conclusive evidence that they are safe.Unfiltered TallowRuminant tallow is exempted from the current feed ban. Tallow contains protein impurities (i.e. MBM) that could be a source of TSE infectivity. There are no impurity level requirements for this tallow. It has been reported that it is standard practice to produce tallow which has an impurity level of .15% or below, but it is not clear that this is fully adequate to remove the risk of transmission and there is no requirement to meet even this standard. We urge that protein contaminants be excluded from tallow and that SRMs also be removed.Plate WastePlate waste is not limited to meat (muscle tissue). For example, cuts that include a portion of the spinal cord or that are contaminated by cord or ganglia during preparation could contain high levels of infectivity if derived from a TSE infected animal late in the preclinical stage of infection. At best this material would only be exposed to normal cooking temperatures. USDA, APHIS experience with the Swine Health Protection Act has revealed that plate waste also includes uncooked trimmings and bones. Although the current FDA regulation requires the plate waste be treated again, there are no specifications which would render a TSE agent inactive. Of greatest risk would be any bovine source of infectivity but also sheep scrapie, although not known to be a risk for human consumption, is one of the possible origins of BSE. The sheep scrapie agent is known to be widely dispersed including relatively high titers in lymphoid as well as nervous tissue. We support the USDA’s opposition to the exemption of “plate waste” as stated in written comments since 1997.Exposure: Cross Feeding and Cross ContaminationThe UK epidemiology has clearly shown that BSE contaminated feed is the primary if not sole vehicle for the transmission of BSE between cattle. Moreover, results from the United Kingdom’s attack: rate study indicate that it does not take much exposure to transmit BSE to cattle. Recent results from the attack rate study which is still in progress have found that .1 g of brain transmitted BSE by the oral route to 3 cows out of 15 thus far, and .01 and .001 gr of brain have transmitted BSE (1 cow out of 15). (Danny Matthews, DEFRA presentation at TAFS meeting, Washington, DC April 2004). Rendering may reduce infectivity but it does not eliminate it. (Taylor et al, 1995; Taylor et al, 1997; Schreuder et al, 1998). Given that BSE can be transmitted to cattle via an oral route with just .001 gram of infected tissue, it may not take much infectivity to contaminate feed and keep the disease recycling. This is especially true in countries like the US and Canada which do not have dedicated lines and equipment to manufacture and process feed for ruminants and non-ruminants.In addition, epidemiological investigations in European countries have shown that cross feeding and cross contamination on farm can be a significant vehicle for continued BSE transmission even after feed bans are well established. Cross feeding is the practice of feeding meal for poultry or pigs or pet food (which can legally contain ruminant MBM) to cattle on the same farm. This is usually due to simple human error or negligence. (Hoinville, 1994; Hoinville et al, 1995; Doherr et al, 2002% Stevenson et al, 2000) FDA, CVM reports that compliance with the existing feed ban is high. For the most part this does not include the compliance level on the farm. There are hundreds of thousands of farms in the US. Many of these have multiple species- That is, they raise cattle, pigs, chickens etc., on the same premises. The sheer numbers of farms make it very difficult to assure compliance on farm and to adequately cover all farms by inspection- Even if the rendering industry and feed industry can maintain 100% compliance at their facilities, if a producer inadvertently feeds chicken feed containing bovine MBM to their cattle, they negate a perfect compliance rate higher in the chain. Recent data from the Harvard BSE risk assessment suggest that the level of misfeeding on farms plays a significant role in the ability of the agent to recycle. In fact George Gray, principal investigator for the study, stated that if, in the United States, misfeeding were to occur at a level of 15%, the RO would be over 1, indicating that the BSE level would not be declining. (George Gray presentation at the Meeting on BSE Prevention in North America: An Analysis of the Science and Risk; January 27,2005, Washington, DC.)The May 2003 Canadian BSE case illustrates the difficulty of on farm enforcement and its serious ramifications. The BSE positive cow was rendered and the MBM distributed to various locations. Two of these locations were poultry farms which mixed their own feed. The farms also had cattle. The subsequent investigation could not eliminate the possibility that the cattle had been fed the same feed as the poultry. The cattle on these farms were completely depopulated.Human error is extremely difficult to prevent, and managing the risk through enforcement is problematical when confronted with the extreme logistical challenges of on farm monitoring. By eliminating the highest risk materials (SRMs and deadstock) which could introduce infectivity into the feed stream, the MBM resulting from processing becomes inherently safer. If mistakes are then made on farm, they no longer contribute to the recycling of BSE.Exposure: Susceptibility of other SpeciesFelinesA transmissible spongiform encephalopathy has been diagnosed in eight species of captive wild ruminants as well as exotic felines (cheetahs, pumas, a tiger and an ocelot) and domestic cats (Wyatt 1991). There have been over 80 domestic cat cases of Feline Spongiform Encephalopathy (FSE) in Great Britain, and cats in Norway, Northern Ireland, Lichtenstein and Switzerland. The agent isolated from several of these cases is indistinguishable from BSE in cattle using strain typing in mice, suggesting that FSE is actually BSE in exotic and domestic cats. Epidemiological evidence suggests BSE contaminated feed to be the probable source of infection in these species. (MAFF Progress Report, June 1997), thus providing additional supporting evidence for the dangers of BSE contaminated feed and reinforcing the necessity of removing all sources of potential contamination from the feed streamOther speciesStudies conducted at the National Institutes of Health Rocky Mountain Laboratory caution against assuming that animals which do not become clinically ill are not infected. It is unknown if certain animals may become carriers, i.e., become infected, shed agent but do not progress to clinical disease. Infection of certain rodent species with different TSE strains suggests the possibility of a carrier state (Race and Chesebro, 1998; Race et. al, 2001, Race et al., 2002). In the more recent studies, mice were inoculated with 263K hamster scrapie. There was a prolonged period (approximately one year) where there was no evidence of replication of infectivity. Furthermore, there was no evidence of PrPres during this phase of inactive persistence, which was followed by a period of active replication of infectivity and agent adaptation. In most cases, PrPres was not detected in the active phase as well. It is important to determine if this persistence and adaptation occurs in other species exposed to TSEs as it may have significance in feeding programs which continually expose other species to BSE infectivity. For example, if BSE infected brain and spinal cord are continually fed to certain species, it may be possible for the agent to persist and adapt in these new species. Over time, the ‘resistant’ species may become a source of agent. The results of Race and colleagues, warns that an inactive persistent phase might not produce detectable PrPres, yet there would be infectivity (Race et. al., 2001).Pigs displayed evidence of TSE infection after exposure to BSE by 3 distinct parenteral routes. Evidence of infectivity was found in the CNS, stomach, intestine and pancreas (Dawson et. al., 1990). CIral transmission has also been attempted in swine, but after an observation period of 84 months there was neither clinical nor pathological evidence of infection (Dawson et. al., 1990). Parenteral and oral transmission has also been attempted in chickens with no evidence of disease. Tissues from the BSE-challenged pigs and chickens were inoculated into susceptible mice to look for residual infectivity, but to date none has been found. In both instances the detection sensitivity was limited by the use of mice for bioassay instead of same species transmissions into cattle (or pigs and chickens).If any of these scenarios played out and inapparent infections became established in commercial species, those species could become reservoirs for reinfection of cattle and perpetuation or reintroduction of the epidemic. We also do not know if atypical cases of BSE are more pathogenic for other species and if chronic inflammation may influence the susceptibility of other species. We offer these possibilities to re-enforce the need to eliminate all possible sources of infectivity from the feed stream.In January 2005, the European Union announced that BSE had been confirmed in a goat in France illustrating that the disease can be naturally transmitted to one of the small ruminants. The potential ramifications of this and the logistical challenges associated with controlling BSE in sheep or goats also provides a justification for removing SRMs from all animal feed. Although these species are covered under the current regulations the cross contamination and cross feeding aspects stated for cattle are applicable. The need to remove high risk material from all animal feed is also supported by other bodies with expertise in the field of TSEs:Recommendations of the World Health Organization (WHO) The World Health Organization (WHO) has issued the following recommendations for countries with BSE or those where a known exposure exists:No part or product of any animal which has shown signs of a TSE should enter any food chain (human or animal). In particular:All countries must ensure the killing and safe disposal of all parts or products of such animals so that TSE infectivity cannot enter any food chain.Countries should not permit tissues that are likely to contain the BSE agent to enter any food chain (human or animal).From the report of a WHO Consultation on Public Health Issues related to Human and Animal Transmissible Spongiform Encephalopathies WHO/EMC/DIS 96.147, Geneva, 2-3 April 1996.Office of International Epizooties OIEThe OIE is recommending that a list of SRMs which include brain, spinal cord, eyes, skull and vertebral column be removed from preparations used for food, feed, fertilizer, etc. If these tissues should not be traded we feel that they should not be used in domestic products either.BSE Code Article 2.3.13.18“From cattle, originating from a country or zone with a minimal BSE risk, that were at the time of slaughter over 30 months of age, the following commodities, and any commodity contaminated by them, should not be traded for the preparation of Food, feed, fertilizers, cosmetics, pharmaceuticals including biologicals, or medical devices: brains, eyes and spinal cord, skull, vertebral column and derived protein products. Food, feed, fertilizers, cosmetics, pharmaceuticals or medical devices prepared using these commodities should also not be traded.”ConclusionIn conclusion we urge the: FDA to implement, monitor and enforce a comprehensive and protective feed ban that is more congruent with the measures that have been proven to be effective in other countries that have experienced BSE. We do not feel that we can overstate the dangers from the insidious threat from these diseases and the need to control and arrest them to prevent any possibility of spread.We also wish to emphasize that as scientists who have dedicated substantive portions of our careers to defining the risks from TSEs as well as developing strategies for managing those risks, we are confident that technical solutions will be found for many of the challenges posed by these diseases. Thus, we urge the FDA to frame its regulations in terms that allow for the future use of any banned material if it can be proven safe for a given application.Signatories:Paul W. Brown, M.D. Medical Director, USPH[S, and Senior Investigator, NIH (retired) Consultant, TSE Risk Management 78 15 Exeter Rd. Bethesda, MD 208 14 Fax 301-652-43 12 Email: paLII\\ hr-c~~~rl~~/‘~c)m~as~.rlct -----Neil R Cashman MD Professor, Department 0-C Medicine (Neurology) Diener Chair of Neurode:generaGve Diseases Centre for Research in Neurodegenerative Diseases 6 Queen’s Park Crescent West Toronto Ontario M5S3H2 Ph: 416-978-1875 Fax: 4 16-978- 1878 e-mail: neil.cashman@utoronto.caLinda A. Detwiler, DVM Consultant, TSE Risk Management 225 Hwy 35 Red Bank, NJ 07701 Ph 732-74 l-2290 Fax 732-741-775 1 Email: l.~\Vc~92’rr’ac,l.c0111.Laura Manuelidis, MD Professor and Head of Neuropathology, Department of Surgery and Faculty of Neurosciences Yale Medical School 333 Cedar St. New Haven, CT 065 10 email: I~IL~ra.~~~ar~clclirli~~~~~~alc.cdi~ Tel: 203-785-4442Jason C. Bar-k, Ph.D. Assistant Professor Department of Medical Microbiology and Immunology Creighton University 2500 California Plaza Omaha, NE 68178 (402) 280- 18 11 voice (402) 280-l 875 fax jbartz@creighton .eduRobert B. Petersen, Ph.D. Associate Professor of Pathology and Neuroscience Case Western Reserve University 5- 123 Wolstein 13~1ilding 2 103 Cornell Road Cleveland, OH 44 106-26122 Phone 216-368-6709 FAX 360-838-9226 Email rhp~,-c\\~~.c~!t~ Robert G. Rohwer, Ph.D. Director, Molecular Neurovirology Laboratory Veterans Affairs Medicall Center Medical Research Service 151 Assoc. Professor of Neurology School of Medicine University of Maryland ;at Baltimore 10 N. Greene St. Baltimore, MD 21201 ph. 4 1 O-605-7000 x6462 Fax 4 1 o-605-7959 email: rrohwer@maryland.eduREFERENCESSignatories: ...snip...see full text with references and submitters names;FDA PROPOSED RULE DECEMBER 20, 2005December 20, 2005Division of Dockets Management (HFA-305) Food and Drug Administration5630 Fishers LaneRoom 1061Rockville, MD 20852Re: Docket No: 2002N-0273 (formerly Docket No. 02N-0273)Substances Prohibited From Use in Animal Food and FeedDear Sir or Madame:As scientists and recognized experts who have worked in the field of TSEs for decades, we are deeply concerned by the recent discoveries of indigenous BSE infected cattle in North America and appreciate the opportunity to submit comments to this very important proposed rule We strongly supported the measures that USDA and FDA implemented to protect public health after the discovery of the case of bovine spongiform encephalopathy (BSE) found in Washington State in 2003. We know of no event or discovery since then that could justify relaxing the existing specified risk material (SRM) and non-ambulatory bans and surveillance that were implemented at that time. Further, we strongly supported the codification of those changes, as well as additional measures to strengthen the entire feed and food system. The discovery of additional cases of indigenous BSE in North America since that time has validated our position and strengthened our convictions.We caution against using the 18 month enhanced surveillance as a justification to relax or impede further actions. While this surveillance has not uncovered an epidemic, it does not clear the US cattle herd from infection. While it is highly likely that US and Canadian cattle were exposed to BSE prior to the 1997 feed ban, we do not know how many cattle were infected or how widely the infection was dispersed. BSE cases are most likely clustered in time and location, so while enhanced surveillance provides an 18 month snapshot, it does not negate the fact that US and Canadian cattle were exposed to BSE. We also do not know in any quantitative or controlled way how effective the feed ban has been, especially at the farm level. At this point we cannot even make a thorough assessment of the USDA surveillance as details such as age, risk category and regional distribution have not been released.9 December 2005Division of Dockets Management (RFA-305)Food and Drug Administration5630 Fishers LaneRoom 1061Rockville, MD 20852Re: Docket No: 2002N-0273 (formerly Docket No. 02N-0273)Substances Prohibited From Use in Animal Food and FeedDear Sir or Madame:Serologicals Corporation is a global provider of biological products to life science companies. The Company’s products are essential for the research, development and manufacturing of biologically based diagnostic, pharmaceutical and biological products. customers include many of the leading research institutions, diagnostic and pharmaceutical companies throughout the world. The Company’s products and technologies are used in a wide variety of applications within the areas of neurobiology, cell signaling, oncology, angiogenesis, apoptosis, developmental biology, cellular physiology, hematology, immunology, cardiology, infectious diseases and molecular biology.A number of our products are derived from bovine blood or other bovine tissues sourced in the United States, hence the overall health of the national herd is extremely important to our company as well as to our customers and their patients. Some of our bovine based products are used in the manufacture of vaccines and drugs for humans, hence it is critical that all measures are taken to assure these are safe and free from disease especially Bovine Spongiform Encephalopathy (BSE). The most effective way to insure this is to create a system which processes cattle that are BSE free. As a company there are a number of precautions that we can take by our strict specifications but many of the needed precautions require the force of federal regulation, hence we appreciate the opportunity to submit comments to this very important proposed rule.After the identification of bovine spongiform encephalopathy (BSE) in indigenous North American cattle, the U.S. Department of Agriculture (USDA) responded rapidly to implement measures to protect public health in regard to food. Our company recognizes and supports the importance of the current feed ban which went into effect in August 1997. However, given what is known about the epidemiology and characteristically long incubation period of BSE, we urgeDivision of Dockets Management (HFA-305) Page 2 9 December 2005the FDA to act without further delay and implement additional measures which will reduce the risk of BSE recycling in the US cattle herd.We feel that for the FDA to provide a more comprehensive and protective feed ban, specified risk materials (SRMs) and deadstock must be removed from all animal feed and that legal exemptions which allow ruminant protein to be fed back to ruminants (with the exception of milk) should be discontinued.SRMs, as defined by the USDA, are tissues which, in a BSE infected animal, are known to either harbor BSE infectivity or to be closely associated with infectivity. If SRMs are not removed, they may introduce BSE infectivity and continue to provide a source of animal feed contamination. Rendering will reduce infectivity but it will not totally eliminate it. This is significant as research in the United Kingdom has shown that a calf may be infected with BSE by the ingestion of as little as .OOl gram of untreated brain.The current proposed rule falls short of this and would still leave a potential source of infectivity in the system. In fact by the FDA’s own statement the exempted tissues which are known to have infectivity (such as distal ileum, DRGs, etc) would cumulatively amount to 10% of the infectivity in an infected animal, This proposed rule would still allow for the possibility that cattle could be exposed to BSE through:1. Feeding of materials currently subject to legal exemptions from the ban (e.g., poultry litter, plate waste)2. Cross feeding (the feeding of non-ruminant rations to ruminants) on farms; and3. Cross contamination of ruminant and non-ruminant feedWe are most concerned that the FDA has chosen to include a provision which would allow tissues from deadstock into the feed chain. We do not support the provision to allow the removal of brain and spinal cord from down and deadstock over 30 months of age for several reasons. These are the animals with the highest level of infectivity in tissues which include more than brain and spinal cord. We do not feel that there can be adequate removal and enforcement of this regulation especially during warmer weather. In addition there is emerging information that at end stage disease, infectivity may also be included in additionai tissues such as peripheral nerves (Buschmann and Groschup, 2005).Leaving the tissues from these cattle in the animal feed chain will effectively nullify the intent of this regulation. This point is illustrated by the 2001 Harvard risk assessment model which demonstrated that eliminating dead and downer, 4D cattle, from the feed stream was a disproportionately effective means of reducing the risk of re-infection “The disposition of c&e that die on the farm would also have a substantial influence on the spread of BSE if the disease were in traduced. ” The base case scenario showed that the mean total number ofID.50~ (i.e., dosage sufficient to infect SO percent of exposed cattte) from healthy animals at slaughter presented to the food/feed system was 1500, The mean total number of ID.50.s from adult cattleDivision of Dockets Management (HFA-305) Page 3 9 December 2005deadstock presented to the feed system was 37,000. deadstock).This illustrates the risk of “4D cattle” (i.e., Deadstock).From the Harvard Risk Assessment, 2001, Appendix 3A Base Case and Harvard Risk Assessment, 2001 Executive SummarySerologicals and companies like ours which supply components of drugs and biologicals have a responsibility to the manufacturers of these products, the medical community and their patients as well as regulatory agencies throughout the world to provide the safest products as possible. Since there is no test for BSE in live cattle or for product, the regulatory agencies throughout the world expect us to reduce or eliminate risk via sourcing criteria, These parameters may include but not be limited to country of origin, herd of origin, age of the animal, etc. The United States is no longer a country with negligible risk, hence individual animal criteria has become more important. In fact other Centers of the FDA have stated that more attention should be given to sourcing from herds likely to be a source of BSE free animals. The exemptions in the current ban as well as in the newly proposed rule make this difficult if not impossible as there are still legal avenues for ruminants to consume potentially contaminated ruminant protein. In addition, the USDA still has not implemented a system of identification and traceability.Serologicals urges agencies of the US government to work with academia and industry on research in the following areas:Methods to inactivate TSEs agents which then may allow a product to be used and even fed to animals without riskAlternative uses for animal byproducts which would maintain valueSerologicals will continue to work with the FDA and other government agencies to implement a strong BSE risk control program, Serologicals would like to reiterate our opinion that for the FDA to provide a more comprehensive and protective feed ban, specified risk materials (SRMs) and deadstock must be removed from all animal feed and that legal exemptions which allow ruminant protein to be fed back to ruminants (with the exception of milk) should be discontinued. Thank you for the opportunity to submit these comments to the public record.Respectfully, SEROLOGICALS CORPORATION James J. Kramer, Ph.D. Vice President, Corporate Operations9 December 2005 Division of Dockets Management (RFA-305)
SEROLOGICALS CORPORATION James J. Kramer, Ph.D. Vice President, Corporate Operations
http://www.fda.gov/ohrms/dockets/dockets/02n0273/02n-0273-c000383-01-vol35.pdf2002N-0273 Animal Proteins Prohibited in Ruminant FeedFDA Comment Number : EC240Submitter : Mr. Masahiro Mori Date & Time: 01/04/2006 05:01:26Organization : Embassy of JapanCategory : International GovernmentIssue Areas/CommentsGENERALComments of Japan on the United States? WTO/SPS Notification (G/ https://www.fsis.usda.gov/sites/default/files/media_file/2020-07/BSE_Risk_Assess_Response_Public_Comments.pdf SPS/N/USA/1141)The Government of Japan welcomes the opportunity to comment on the United States? notification (G/SPS/N/USA/1141) on substances prohibited from use in Animal food or feed.The Food safety risk assessment related to the import of beef and beef offal from the U.S.A. and Canada by the Food Safety Commission of Japan (FSC) was completed on December 8, 2005. Regarding the feed ban, the following was noted as an addendum to the conclusion on the risk assessment report of FSC:To prevent BSE exposure and amplification in U.S.A and Canada, the use of SRM must be prohibited completely. The ban must be applied not only to cattle feed but also to all other animal food/feed that may cause cross-contamination.?To accomplish the effectively enforced feed ban requested in the OIE Terrestrial Animal Health Code (CHAPTER 2.3.13, Bovine Spongiform Encephalopathy), whole SRM should be excluded as high risk material from animal feed chain as the above-mentioned addendum points out. The U.S. Government should also carry out continuous BSE surveillance sufficient to verify the efficacy of U.S. feed ban and make necessary revision of its feed regulations on a basis of its results.The Government of Japan would like to request that the U.S. Government take account of the above comments in implementing its animal food and feed regulations.
http://web.archive.org/web/20120128115225/http://www.fda.gov/ohrms/dockets/dockets/02n0273/02N-0273-EC240.htm
Embassy of JapanSingeltary Comment Docket No: 2002N-0273 (formerly Docket No. 02N-0273)MY comments/questions are as follows ;1. SINCE the first Harvard BSE Risk Assessment was so flawed and fraught with error after the PEER REVIEW assessment assessed this fact, how do you plan on stopping this from happening again, will there be another peer review with top TSE Scientist, an impartial jury so-to-speak, to assess this new and updated Harvard BSE/TSE risk assessment and will this assessment include the Atypical TSE and SRM issues ?*** Suppressed peer review of Harvard study October 31, 2002 ***2. WITH A RECENT NATION WIDE MAD COW FEED BAN RECALL in the past few months that consisted of some 10,878.06 TONS, then another Mad Cow feed ban warning letter in May, IT should seem prudent to ask why our feed bans continue to fail in 2006, and continue to fail today ?snip...see full text;Singeltary Full Comments Submissions;Linda A. Detwiler, DVM225 Hwy 35Red Bank, New Jersey 07701 cfPhone: 732-741-2290 LZCell: 732-580-9391 l,Fax: 732-741-7751May 7, 2004FSIS Docket Clerk 03-025IFU.S. Department of Agriculture 03-025IF-634 Food Safety and Inspection Service Linda A. Detwiler300 12th Street, SW. Room 102 Cotton Annex Washington, DC 20250RE: DEPARTMENT OF AGRICULTURE Food Safety and Inspection Service9 CFR Parts 301, 309, 310, 311, 313, 318, 319 and 320Prohibition of the Use of Specified Risk Materials for Human Food and Requirements for the Disposition of Non-Ambulatory Disabled Cattle; Meat Produced by Advanced Meat/Bone Separation Machinery and Meat Recovery (AMR) Systems; Prohibition of the Use of Certain Stunning Devices Used To Immobilize Cattle During Slaughter; Bovine Spongiform Encephalopathy (BSE) Surveillance ProgramThe USDA is to be commended for taking the much needed actions outlined in the interim final rules. Current evidence indicates that the risk to humans from BSE is extremely low. However, these actions are essential for the further protection of public health in the United States as the detection of the Canadian case of BSE in May 2003, provided evidence that the BSE agent had indeed been introduced into the North American cattle production system. USDA must continue to monitor new scientific findings as well as other world events and adjust the regulations accordingly.Docket Number 01-033IF: Prohibition of the Use of Certain Stunning Devices Used To Immobilize Cattle During SlaughterI am in full support of the prohibition of all devices which may cause the release of brain macro- emboli into the circulatory system of stunned cattle. Given there has been consistent evidence that stunning methods utilizing air-injection have resulted in the circulation of macro-emboli from Central Nervous System (CNS) tissue, USDA should make this interim final rule permanent. (Garland, et al, 1996; personal observation) Docket Number 03-025IF: Prohibition of the Use of Specified Risk Materials for Human Food and Requirements for the Disposition of Nonambulatory Disabled CattleSpecified Risk Materials (SRMs)Lam in full support of the interim final rule which prohibits SRMs from being included in food for human consumption. In addition to the list of tissues published in this rule, I am requesting that additional tissues be added to the list. These would include the dura (“sheath”) covering the spinal cord and the entire intestine (from pylorus to rectum). The scientific justification is provided below. These SRMs should also be prohibited from ANY FDA regulated food or product intended for human consumption, including but not limited to flavorings, extracts, etc.Evidence from the United Kingdom indicates that BSE is the most likely cause of the vCJD (Will et al., 1996). The UK’s Spongiform Encephalopathy Advisory Committee (SEAC) concluded that although there was no direct scientific evidence of a link between BSE and vCJD, based on current data and in the absence of any credible alternative, the most likely explanation at that time was that the cases were linked to exposure to BSE before the introduction of control measures, in particular, the specified bovine offal (SBO) ban in 1989. Given that it has been postulated that one of the most likely routes of exposure is through the consumption of meat contaminated with infected CNS tissue (Will, 1999), SRM bans are one of the most important measures for the protection of human health.The list of tissues to be considered by USDA as SRM includes those bovine tissues where actual infectivity has been identified (based on the research outlined below) or tissues which have a close association to the infected tissues.A study was conducted to examine the pathogenesis of BSE in cattle, i.e. the replication (tissue distribution) of the agent during the incubation period. This study first identified the agent via mouse bioassay in the distal ileum of the experimentally infected calves. It is thought that the agent may be associated with the lymphoid tissue of the intestines. The calves were four months of age at the time of oral dosing. First isolation of the agent in the distal ileum was made at six months after challenge. Subsequent isolations from the distal ileum were made at ten, 14 and 18 months after dosing. (Wells et. al., 1994) This study has also identified infectivity in spinal cord, brain, dorsal root ganglia, and trigeminal ganglia beginning at 32 months post challenge. (Wells, et. al. 1998). The study was repeated using calves as the bioassay model. Results of the calf bioassay study are very similar to the mouse bioassays. However, this study has also identified apparently low levels of infectivity in tonsil (Update of the Opinion on TSE Infectivity Distribution in Ruminant Tissues adopted by the EU Scientific Steering Committee on November 7-9, 2002). Lymphoid tissue of the 3" eyelid from naturally infected cattle has also demonstrated a low level of infectivity (Presentation by Danny Matthews, UK VLA at the TAFS Workshop, Washington, DC; April 6, 2004). The calf bioassay study is still in progress.It should be pointed out that the data on the distribution of BSE infectivity in the bovine was derived primarily from 2 studies, one of which has not been completed. The studies conducted are very logistically challenging and expensive. Not every tissue could undergo bioassay. Some tissues were assumed to have a risk due to a close association or the identification of PrPres and are considered as SRM. Such is the case with dura and the remaining sections of intestine.DuraDura was harvested but not tested in the pathogenesis study. Its close association with the brain and spinal cord and the documented evidence of its role in the human to human transmission of ' Creutzfeldt-Jakob Disease (CJD) has prompted scientists to designate bovine dura as a high risk tissue.Dura may become separated from the vertebral column during the fabrication process allowing the possibility that it be included in ground beef products or contaminate surfaces where deboning occurs. | recommend that USDA amend the definition of SRM to include dura and that it be removed along with spinal cord on the kill floor.IntestineThe scenario described above is essentially true for the intestine. Infectivity was readily detectable in the distal ileum of cattle infected with BSE. While certain additional sections of the intestine were tested with no infectivity identified, not every section of the intestine was included in the bioassays. Positive immunostaining for PrPres was identified along the length of the intestine providing evidence for the entire intestine to be considered as SRM per EU regulations. (personal communication Danny Matthews, UK, VLA). The International Advisory Committee appointed by Secretary Veneman also recommended that the SRM ban in the US be amended to the entire intestine from duodenum to rectum. I recommend that the USDA adjust the definition of SRM to include the entire intestine from duodenum to rectum.The removal of SRMs to protect public health is also supported by the WHO and the Harvard Risk Assessment.The World Health Organization (WHO) has issued the following recommendations for countries with BSE or those where a known exposure exists:"No part or product of any animal which has shown signs of a TSE should enter any food chain (human or animal). In particular:- All countries must ensure the killing and safe disposal of all parts or products of such animals so that TSE infectivity cannot enter any food chain.- Countries should not permit tissues that are likely to contain the BSE agent to enter any food chain (human or animal)citation:Report of a WHO Consultation on Public Health Issues related to Human and Animal Transmissible Spongiform Encephalopathies WHO/EMC/DIS 96.147 Geneva 2-3 April 1996The Harvard Study also made recommendations similar to the WHO, “Our evaluation of potential risk mitigation actions highlights potential measures to further reduce the already low likelihood that BSE could spread to cattle or contaminate human food if it were to arise...... Implementation of a UK-style ban on specified risk material (e.g., spinal cords, brains, vertebral columns) from both human food and animal feed reduces the predicted number of BSE cases in cattle by 80% and the potential human exposure by 95%.”Cross contamination between SRMs and edible tissue can add risk to products intended for human consumption. Therefore, USDA, FSIS is urged to assure that each slaughterplant which processes cattle have systems in place which prevent cross contamination between edible tissue and SRMs. This should include separate equipment, such as knives, blades, etc where appropriate and utilize effective (for TSE agents) disinfection procedures for equipment used to handle SRMs.The Update of the Opinion on TSE Infectivity Distribution in Ruminant Tissues adopted by the EU Scientific Steering Committee on November 7-9, 2002 provides some guidelines to avoid cross contamination. Under separate cover I have submitted a CD of practices used throughout Europe and in some plants in the United States. The CD also includes effective disinfection methods that are recommended by the WHO. These practices assist in the reduction of cross contamination.Nonambulatory Disabled CattleAs appropriately described in the interim final rule, nonambulatory disabled cattle constitute the majority of the BSE high risk population. I agree that they should be prohibited from being included in the supply of food for human consumption. I also agree with the definition as written in the Interim Final Rule. I urge the USDA to not alter this definition and to continue to prohibit for human food any bovine which cannot walk to the “knock box” regardless of reason.Downers accounted for over half of the detected BSE cases in both the EU and Switzerland in 2003. In Switzerland 21 cases of BSE were confirmed. Two were from normal slaughter, eleven were from the fallen stock and emergency slaughter (equivalent to US downers) and eight were clinical BSE suspects (ref . http://www.bvet.admin.ch/0_navigation-e/0_index-intern htm] ) In the European Union (January - September 2003) a total of 947 cases were confirmed. Of these confirmed cases 189 (20%) were from normal slaughter, 525 (55.4%) were from the fallen stock/emergency slaughter (US Downers) and 233 (24.6%) were clinical BSE suspects (ref http://europa.eu.int/comm/food/food/biosafety/bse/annual_reps_en.htm).The previous system of clinical examination of the nonambulatory bovine was not adequate for determining a disposition regarding BSE. This was clearly illustrated by the two native cases of BSE in North America. Both cases of BSE (May and December) were observed by veterinarians prior to slaughter. Neither was specifically set aside as a BSE clinical suspect. The Washington State case was passed for human consumption because she was determined to have a calving injury (which apparently masked signs of BSE or other neurological disease) and the May 2003 case detected in Canada was slaughtered in January and went to rendering. The testing of this cow was completed in May.Neurological, metabolic or other diseases which affect coordination and other aspects of gait often predispose an animal to injuries such as broken limbs or soft tissue damage. If the animal is then down because of a broken leg, or torn ligament, the injury may be the prominent or sole presenting sign. Without a complete diagnostic work up and history of disease progression the true underlying cause of the nonambulatory condition may be impossible to ascertain.I have listed the clinical signs of BSE below. This is the list provided on the UK’s DEFRA website. In reviewing the list, the vast majority of signs would be difficult if not impossible to observe once an anima! is down.The clinical symptoms of BSE are varied. Most cattle with BSE show a gradual development of symptoms over a period of several weeks or even months, although some can deteriorate very rapidly. Only a small proportion of affected cattle show what would be considered typical "mad cow" signs. Most suspects show several (but not all) of the following symptoms if they are observed closely enough:apprehensivenessnervousnessreluctance to cross concrete, turn corners, enter yards, go through doorways or permit milkingoccasional aggression directed at other cattle or humans manic kicking when milkedhead shyness, with head held lowhigh stepping gait, particularly hind legsdifficulties in risingtremorsloss of condition, weight or milk yieldIn addition, per the list, difficulty in rising is a clinical sign of BSE. Eventually cattle with BSE progress and become recumbent. Per the interim rule testing is not absolute and cannotguarantee absence of infectivity. In fact in at most countries beef from tested animals cannot be labeled as BSE free.I do suggest that this regulation not prohibit any individual or family from using one of their own nonambulatory animals for personal consumption. This can be done at their risk through custom slaughter.Docket Number 03-0381F: Meat Produced by Advanced Meat/Bone Separation Machinery and Meat Recovery (AMR) Systems and Docket Number 03-025IF: Prohibition of the Use of Specified Risk Materials for Human FoodThe restrictions on AMR are the most important regulations put into place by the USDA. I fully support all of the prohibitions placed on the production of meat from AMR systems. J also urge that for the reasons outlined in the SRM section above, dura be prohibited from being included in AMR product.In the original Harvard risk assessment completed in 2001 as well as the update in 2003, in the US system it was illustrated that AMR could deliver more 1D50s for potential human consumption than the direct consumption of brain and spinal cord together.The 2001 Harvard risk assessment’s base case scenario found that out of 1000 simulation runs the mean number of 1D50s (infectious doses) available for human consumption would be 35. Almost 11 1D50s (10.9) would be derived from brain and spinal cord. Twenty (more than half) are derived from AMR products. (Harvard Risk Assessment, 2001 Section 3.1.2.7 - 3.1.2.9; Appendix 3A Base Case).Statistics from the Harvard Risk Assessment (2003) showing ID50s from brain and spinal cord vs. AMR product:Worse case scenarioMean 5% percentile 95" percentileTotal 1D50s available for humans - 6000 2000 12,000*[D50s from brain - 770 5.2 20001D50s from spinal cord - 280 2 760ID50s from AMR - 2900 960 5600Best case scenarioTotal IDS0s available for humans - 10 0 36*1D50s from brain - 1.6 0 01DSQs from spinal cord - 69 0 01D50s from AMR - 3.7 0 10* Additional tissues included but not listed here.Lalso agree that Mechanically Separated Product should be completely prohibited for human consumption.SurveillanceThe proposed increased surveillance program announced by USDA APHIS is essential. In order to estimate the amount of BSE in the US national herd it is imperative that as many of the high risk population be tested as possible. This is also true for Canada. Without this surveillance the US will not be able to monitor the effects of past and present control efforts. It is important for the industry, academia and government to work together to achieve this goal. We need to know if there is any regionality to cases, what ages are affected and if the current feed ban has failed.Animal FeedEpidemiological evidence in Europe and results from the attack rate study indicate that it does not take much exposure to transmit BSE to cattle. Recent results from the attack rate study, which is still in progress, has found that .001 gr of raw infected brain can transmit BSE (1 cow out of 15) through the oral route. The role of cross contamination was under estimated throughout Europe. Experience in other countries has also shown that human error especially at the farm level is difficult to control. It is imperative that the feed ban be effective. There are a number of actions which still need to be taken by the FDA to prevent any potential recycling of the BSE agent in the US cattle population. The FDA is urged to act immediately and put these measures in place.Per the 2001 Harvard risk assessment, “Our evaluation of potential risk mitigation actions highlights potential measures to further reduce the already low likelihood that BSE could spread to cattle or contaminate human food if it were to arise. Prohibiting the rendering of animals that die on the farm, possibly of BSE, removes a great deal of potential contamination in the animal feed chain and reduces average predicted cases of BSE following introduction of ten infected cattle by 77%. Implementation of a UK-style ban on specified risk material (¢.g., spinal cords, brains, vertebral columns) from both human food and animal feed reduces the predicted number of BSE cases in cattle by 80% and the potential human exposure by 95%.”Thank you for the opportunity to comment on these rules.Linda A. Detwiler, DVMDrlucele Detus | OP vee REFERENCESGarland, T., Bauer, N., Bailey, M. (1996) Brain emboli in the lung of cattle after stunning. The Lancet, 348: 610.Wells G.A.H., Dawson M., Hawkins, S.A.C., Green R. B., Dexter I., Francis M. E., Simmons M. M., Austin A. R., & Horigan M. W. (1994) Infectivity in the ileum of cattle challenged orally with bovine spongiform encephalopathy. Vet. Rec., 135, 40-41.Wells G.A.H., Hawkins, $.A.C., Green R. B., Austin A. R., Dexter I., Spencer, Y. I., Chaplin, M. J., Stack, M. J., & Dawson, M. (1998) Preliminary observations on the pathogenesis of experimental bovine spongiform encephalopathy (BSE): an update. Vet. Rec., 142, 103-106. Will, R. (1999) New variant Creutzfeldt-Jakob Disease. Biomed & Pharmacother, 53, 9-13.Will, R. G., Ironside, J. W., Zeidler, M., Cousens, S. N., Estibeiro, K., Alperovitch, A., Poser, S., Pocchiari, M., Hofman, A. & Smith, P. G. (1996) A new variant of Creutzfeldt-Jakob disease in the UK. Lancet., 347, 921-925,Linda A. Detwiler, DVM 225 Hwy 35 Red Bank, New Jersey 07701 L Phone: 732-741-2290 lj Cell: 732-580-9391Fax: 732-741-7751May 6, 200403-025IF 03-025IF-631 Linda A. DetwilerFSIS Docket ClerkU.S. Department of Agriculture Food Safety and Inspection Service 300 12th Street, SW.Room 102 Cotton Annex Washington, DC 20250RE: DEPARTMENT OF AGRICULTURE Food Safety and Inspection Service[Docket No. 03-025N] 9 CFR Parts 301, 309, 310, 311, 313, 318, 319 and 320Prohibition of the Use of Specified Risk Materials for Human Food and Requirements for the Disposition of Non-Ambulatory Disabled Cattle; Meat Produced by Advanced Meat/Bone Separation Machinery and Meat Recovery (AMR) Systems; Prohibition of the Use of Certain Stunning Devices Used To Immobilize Cattle During Slaughter; Bovine Spongiform Encephalopathy (BSE) Surveillance ProgramThe USDA’s Food Safety Inspection Service requested any additional comments or information on practices which would reduce or eliminate cross contamination betweenI am submitting a CD with a presentation;snip...see full submission with presentation from Dr. Detwiler;Public Comments on Prohibition of the Use of Specified Risk Materials for Human Food and Requirements for the Disposition of Non-Ambulatory Disabled Cattle:========Title: Prohibition of the Use of Specified Risk Materials for Human Food and Requirements for the Disposition of Non-Ambulatory Disabled Cattle FR Document Number: 05-17683 Legacy Document ID: RIN:Publish Date: 09/07/2005 00:00:00 Submitter Info:First Name: Jason Last Name: Frost Mailing Address: 37 Observatory Circle, NW City: Washington Country: United States State or Province: DC Postal Code: 20008Organization Name: New Zealand EmbassyCOMMENTS ON FEDERAL REGISTER 9 CFR Parts 309 et al [Docket No. 03- 025IF] Prohibition of the Use of Specified Risk Materials for Human Food and Requirements for the Disposition of Non-Ambulatory Disabled Cattle: Interim final rule and request for commentsOn 12 January 2004, the Food Safety Inspection Service (FSIS) published an Interim Final Rule titled Prohibition of the Use of Specified Risk Materials for Human Food and Requirements for the Disposition of Non-Ambulatory Disabled Cattle: Interim final rule and request for comments.This rule prohibited the use of certain cattle material, to address the potential risk of bovine spongiform encephalopathy (BSE), in human food, including dietary supplements, and cosmetics. On 7 September 2005 FSIS issued an amendment to this interim final rule to permit collection of the small intestine excluding the distal ileum and sought further comments.New Zealand welcomes the opportunity to comment on the amendment to FSIS Docket No.03-0251FA. This submission, while supporting and commending FSIS on the amendment, reiterates the position presented in New Zealand’s original comments on the interim final rule when first issued, that there is no scientific justification for applying the interim rule to New Zealand given its widely acknowledged BSE-free risk status.New Zealand has closely followed the events in the United States and around the world since the United States announced its first case of BSE late in 2003. New Zealand continues to strongly advocate that the world take a more rational risk-based approach to dealing with this disease of cattle, noting that it has only infected consumers in countries where the epidemic resulted in thousands of cattle cases and precautions to protect the human population had not yet been implemented effectively. Regulatory reactions and decisions around the world need to be commensurate with the real risk selected hazards pose to our consumers relative to the other diseases with which we are battling. This is a position New Zealand has firmly supported. There is a continued need for such a principled approach to ensure we can more appropriately focus and apportion resources on those areas most likely to significantly improve and protect the health of our populations.A science and risk-based response from the United States in respect of domestic BSE measures is also crucial in underpinning appropriate international standards. It is unlikely that the international community will be prepared to adopt risk-based standards if the United States itself does not demonstrate this approach. Accordingly, New Zealand remains concerned that the measures introduced by the interim final rule, which are directly in response to the discovery of BSE in North America, continue to be applied to New Zealand bovine products. New Zealand is not aware of any risk assessment of our BSE status being conducted by the United States. The measures applied by FSIS are in excess of the relevant international standard (OIE). Most of our trading partners acceptance of New Zealand’s BSEfree status has meant that there are no SRMs associated with cattle born, raised and slaughtered in New Zealand.While the FSIS advised that the interim final rule was in response “to the finding of an adult cow that tested positive for BSE in the State of Washington”, it is important to note that New Zealand’s disease status has not changed. Nor does our cattle population share a common risk profile with that of the United States. With the exception of very small volumes from Australia, which is also widely recognised as being BSE free, New Zealand has not imported meat and bone meal from any country since the early part of the last century. Nor have we imported animal feeds containing such ruminant protein from any BSE affected country (including the United States and Canada). New Zealand has imported very few live cattle from the US and Canada, all of which are identified and officially monitored and controlled. As a consequence of the integrated nature of international trade the continuing application of the FSIS’s interim final rule (specifically the declaring of certain bovine tissues from any country as adulterants regardless of whether they are truly SRMs), to demonstrably BSE free countries such as New Zealand, is having substantial adverse economic effects on New Zealand industries. These inappropriate negative impacts could easily be avoided.BackgroundWhile the interim final rule’s stated intent was to protect the food supply from materials that may carry the risk of transmitting BSE, New Zealand remains concerned that the burden of many of the measures imposed are disproportionate to the actual risks involved. The US has only detected one indigenous case of BSE to date, hence there is an extremely low risk posed to US consumers by this disease. This raises the question of the extent of the measures introduced within the United States in response to such an extremely low risk. We acknowledge the linkage between BSE of cattle and vCJD of humans, but note that evidence that has accumulated since 1996, when vCJD was first reported, strongly indicates that it is not easy for humans to become infected with vCJD. At the peak of the British BSE epidemic well over 700 clinical cases of the disease were being reported each week. Since 1986, nearly 200,000 British cows have been confirmed with BSE and epidemiological modeling suggests that perhaps 1 to 2 million additional BSE-infected animals may have entered the human food supply in the United Kingdom. Despite that level of exposure, fewer than 160 cases of vCJD have been recorded in that country. That is, fewer than 18 cases per year, on average, and the evidence continues to suggest that the vCJD epidemic has peaked and is in decline.The United States has applied substantial BSE-measures. It is extremely unlikely that a country such as the United States, which has applied anti-BSE measures with increasing stringency for several years, could experience a BSE epidemic as seen in the United Kingdom. In addition any possible exposure of the United States cattle population would at least be two or three orders of magnitude less than in the United Kingdom. The results to date of the United States Department of Agriculture surveillance and testing regimes support the fact that the United States is highly resistant to any proliferation of BSE and confirm the position formerly acknowledged by the Harvard-Tuskegee study of BSE.Specific risk materialsNew Zealand continues to support the United States decision to classify as SRMs brain, skull, eyes, trigeminal ganglia, spinal cord, vertebral column and dorsal root ganglia from animals of age 30 months and over, and the tonsils and distal ileum of all cattle in populations where a case of BSE has been reported. This response fully reflects the SPS principle that measures put in place to mitigate a food-borne risk should be proportionate to the risks involved. Current scientific knowledge indicates that exclusion of these tissues from animals of younger age would provide very little further mitigation of what is already an extremely low level of risk. New Zealand again urges the United States to take a scientific approach in recognizing that the exclusion of these tissues, from cattle of any age, is completely unwarranted in a country, such as New Zealand, demonstrated to be free from BSE.ConclusionIt is essential that the United States takes a science and risk-based response in respect of domestic BSE measures as this is going to be crucial in securing appropriate international standards. It is unlikely that the international community will be prepared to adopt risk-based standards if the United States itself does not demonstrate this approach. Accordingly, New Zealand is concerned to see that the measures, which are directly in response to the discovery of BSE in North America, continue to be applied to New Zealand origin bovine products some 21 months after the interim final rule was issued. There is a continuing failure to apply the international standard (OIE) for BSE to exporting countries.New Zealand requests that any subsequent measures adopted by the United States recognise the different BSE status or risk profile of bovine products from exporting countries. In doing so the United States would be giving appropriate regard to its obligations under the WTO Agreement on the Application of Sanitary and Phytosanitary Measures (the SPS Agreement).New Zealand has a well established and widely acknowledged freedom from BSE and other TSEs. The existing measures achieve the same level of human and/or animal health protection anticipated by this interim final rule. The imposition of unnecessarily prescriptive trade requirements are impediments to legitimate trade and create unnecessary and burdensome compliance costs.USDA, FSIS, APHIS, BSE, Harvard Study Reply to Singeltary et alFDA PROPOSED RULE DECEMBER 20, 2005''It is our opinion that the proposed rule falls woefully short in effective measures to minimize the potential for further transmissions of the disease.''Subject: SEAC Draft minutes of the open session of the 93rd meeting held on 6th July 2006 (atypical BSE USA)Date: August 22, 2006 at 3:03 pm PST SPONGIFORM ENCEPHALOPATHY ADVISORY COMMITTEEDraft minutes of the open session of the 93rd meeting held on 6th July 2006snip...The Chair noted that recent reports described two cases of BSE in cattle in the United States of America (USA) as being similar to atypical cases of BSE found in a number of European countries. The Chair suggested that the term "atypical BSE", used in the USA report, is potentially confusing and that this would be discussed under any other business. Dr Danny Matthews (Veterinary Laboratories Agency [VLA]) explained that data from western blots of the USA cases resembled that of a small number of atypical cases of BSE in France. A study of the French cases had shown the condition to be transmissible to mice by intracerebral (ic) inoculation with the neuropathological phenotype maintained on transmission3. Claims have been made about the existence of atypical cases of BSE in other countries but these have yet to be confirmed. No study has yet examined the tissue distribution of abnormal prion protein (PrPSc) or infectivity in such atypical cases of BSE.3 Baron et al. (2006) Transmission of new bovine prion to mice. Emerging. Infect. Diseases. 12, 1125-1128.snip...However, based on analysis of molecular features of priondiseases in cattle, this situation is similar to that in humans(5), in which different subtypes of sporadic Creutzfeldt-Jakob disease agents are found.DISPATCHES1126 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 7, July 2006Medical Sciences Identification of a second bovine amyloidotic spongiform encephalopathy: Molecular similarities with sporadic Creutzfeldt-Jakob diseaseSnip...end2022Cattle with the EK211 PRNP polymorphism are susceptible to the H-type bovine spongiform encephalopathy agent from either E211K or wild type donors after oronasal inoculationResearch Project: Elucidating the Pathobiology and Transmission of Transmissible Spongiform Encephalopathies Location: Virus and Prion ResearchTitle: Cattle with the EK211 PRNP polymorphism are susceptible to the H-type bovine spongiform encephalopathy agent from either E211K or wild type donors after oronasal inoculationAuthor item Greenlee, Justin item Cassmann, Eric item MOORE, SARA JO - Oak Ridge Institute For Science And Education (ORISE) item WEST GREENLEE, HEATHER - Iowa State UniversitySubmitted to: Meeting AbstractPublication Type: Abstract OnlyPublication Acceptance Date: 6/24/2022Publication Date: 9/16/2022Citation: Greenlee, J.J., Cassmann, E.D., Moore, S., West Greenlee, H.M. 2022.Cattle with the EK211 PRNP polymorphism are susceptible to the H-type bovine spongiform encephalopathy agent from either E211K or wild type donors after oronasal inoculation.Prion 2022 Conference abstracts: pushing the boundaries. 16(1):150. https://doi.org/10.1080/19336896.2022.2091286.Interpretive Summary:Technical Abstract:In 2006, a case of H-type bovine spongiform encephalopathy (H-BSE) was reported in a cow with a previously unreported prion protein polymorphism (E211K). The E211K polymorphism is heritable and homologous to the E200K mutation in humans that is the most frequent PRNP mutation associated with familial Creutzfeldt-Jakob disease. Although the prevalence of the E211K polymorphism is low, cattle carrying the K211 allele develop H-type BSE with a rapid onset after experimental inoculation by the intracranial route. The purpose of this study was to investigate whether the agents of H-type BSE or H-type BSE associated with the E211K polymorphism transmit to wild type cattle or cattle with the K211 allele after oronasal exposure. Wild type (EE211) or heterozygous (EK211) cattle were oronasally inoculated with the H-BSE agent from either the US 2004 case (wild type donor; n=3) or from the US 2006 case with the E211K polymorphism (n=4). Cattle were observed daily throughout the course of the experiment for the development of clinical signs. When signs were noted, animals were euthanized and necropsied. Cattle were confirmed positive for abnormal BSE prions by enzyme immunoassay (EIA; Idexx HerdChek BSE Ag Test), anti-PrP immunohistochemistry (IHC) on brainstem, and microscopic examination for vacuolation. Three-out-of-four (75%) calves with the EK211 genotype developed clinical signs of H-BSE including inattentiveness, loss of body condition, weakness, ataxia, and muscle fasciculations and were euthanized. Two of the positive EK211 steers received H-BSE US 2004 inoculum (Incubation Period (IP): 59.3 and 72.3 months) while the other positive steer received the E211K H-BSE inoculum (IP: 49.7 months). EIA confirmed that abundant misfolded protein (O.D. 2.57-4.0) in the brainstem, and IHC demonstrated PrPSc throughout the brain. All cattle in the EE211 recipient group remain asymptomatic for the duration of the experiment (approximately 7 years post-inoculation). This study demonstrates that the H-type BSE agent is transmissible by the oronasal route. Cattle with the EK211 genotype are oronasally susceptible to small doses of the H-BSE agent from either EK211 or EE211 (wild type) donors. Wild-type EE211 cattle remained asymptomatic for the duration of the experiment with this small dose (0.1g) of inoculum. These results reinforce the need for ongoing surveillance for classical and atypical BSE to minimize the risk of potentially infectious tissues entering the animal or human food chains.''This study demonstrates that the H-type BSE agent is transmissible by the oronasal route. Cattle with the EK211 genotype are oronasally susceptible to small doses of the H-BSE agent from either EK211 or EE211 (wild type) donors. Wild-type EE211 cattle remained asymptomatic for the duration of the experiment with this small dose (0.1g) of inoculum. These results reinforce the need for ongoing surveillance for classical and atypical BSE to minimize the risk of potentially infectious tissues entering the animal or human food chains.''Cattle with the EK211 PRNP polymorphism are susceptible to the H-type bovine spongiform encephalopathy agent from either E211K or wild type donors after oronasal inoculationJustin J. Greenleea, Eric D. Cassmanna, S. Jo Moorea,b, and M. Heather West GreenleecaVirus and Prion Research Unit, National Animal Disease Center, ARS, United States Department of Agriculture, Ames, IA, USA; bOak Ridge Institute for Science and Education (ORISE), U.S. Department of Energy, Oak Ridge, TN, US; cDepartment of Biomedical Sciences, Iowa State University College of Veterinary Medicine, Ames, IA, USAims: In 2006, a case of H-type bovine spongiform encephalopathy (H-BSE) was reported in a cow with a previously unreported prion protein polymorphism (E211K). The E211K polymorphism is heritable and homologous to the E200K mutation in humans that is the most frequent PRNP mutation associated with familial Creutzfeldt-Jakob disease. Although the prevalence of the E211K polymorphism is low, cattle carrying the K211 allele develop H-type BSE with a rapid onset after experimental inoculation by the intracranial route. The purpose of this study was to investigate whether the agents of H-type BSE or H-type BSE associated with the E211K polymorphism transmit to wild type cattle or cattle with the K211 allele after oronasal exposure.Material and Methods: Wild type (EE211) or heterozygous (EK211) cattle were oronasally inoculated with the H-BSE agent from either the US 2004 case (wild type donor; n = 3) or from the US 2006 case with the E211K polymorphism (n = 4). Cattle were observed daily throughout the course of the experiment for the development of clinical signs. When signs were noted, animals were euthanized and necropsied. Cattle were confirmed positive for abnormal BSE prions by enzyme immunoassay (EIA; Idexx HerdChek BSE Ag Test), anti-PrP immunohistochemistry (IHC) on brainstem, and microscopic examination for vacuolation.Results: Three-out-of-four (75%) calves with the EK211 genotype developed clinical signs of H-BSE including inattentiveness, loss of body condition, weakness, ataxia, and muscle fasciculations and were euthanized. Two of the positive EK211 steers received H-BSE US 2004 inoculum (Incubation Period (IP): 59.3 and 72.3 months) while the other positive steer received the E211K H-BSE inoculum (IP: 49.7 months). EIA confirmed that abundant misfolded protein (O.D. 2.57–4.0) in the brainstem, and IHC demonstrated PrPScthroughout the brain. All wild type recipient cattle and a single EK211 steer remained asymptomatic for the duration of the experiment (approximately 7 years post-inoculation) and no abnormal prion protein was detected in these cattle by EIA.Conclusions: This study demonstrates that the H-type BSE agent is transmissible by the oronasal route. Cattle with the EK211 genotype are oronasally susceptible to small doses of the H-BSE agent from either EK211 or EE211 (wild type) donors. Wild-type EE211 cattle remained asymptomatic for the duration of the experiment with this small dose (0.1 g) of inoculum. These results reinforce the need for ongoing surveillance for classical and atypical BSE to minimize the risk of potentially infectious tissues entering the animal or human food chains.Funded by: US Department of AgricultureAcknowledgement: This research was funded in its entirety by congressionally appropriated funds to the United States Department of Agriculture, Agricultural Research Service. The funders of the work did not influence study design, data collection and analysis, decision to publish, or preparation of the manuscript. This research was supported in part by an appointment to the Agricultural Research Service (ARS) Research Participation Program administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the U.S. Department of Energy (DOE) and the U.S. Department of Agriculture (USDA). ORISE is managed by ORAU under DOE contract number DE-SC0014664. All opinions expressed in this paper are the author’s and do not necessarily reflect the policies and views of USDA, ARS, DOE, or ORAU/ORISE.WEDNESDAY, AUGUST 15, 2018The agent of H-type bovine spongiform encephalopathy associated with E211K prion protein polymorphism transmits after oronasal challengePRION 2018 CONFERENCEP98 The agent of H-type bovine spongiform encephalopathy associated with E211K prion protein polymorphism transmits after oronasal challengeGreenlee JJ (1), Moore SJ (1), and West Greenlee MH (2) (1) United States Department of Agriculture, Agricultural Research Service, National Animal Disease Center, Virus and Prion Research Unit, Ames, IA, United States (2) Department of Biomedical Sciences, Iowa State University College of Veterinary Medicine, Ames, IA, United States.reading up on this study from Prion 2018 Conference, very important findings ;***> This study demonstrates that the H-type BSE agent is transmissible by the oronasal route.***> These results reinforce the need for ongoing surveillance for classical and atypical BSE to minimize the risk of potentially infectious tissues entering the animal or human food chains.PRION 2018 CONFERENCE ABSTRACTWEDNESDAY, OCTOBER 24, 2018Experimental Infection of Cattle With a Novel Prion Derived From Atypical H-Type Bovine Spongiform EncephalopathyWEDNESDAY, OCTOBER 24, 2018Experimental Infection of Cattle With a Novel Prion Derived From Atypical H-Type Bovine Spongiform Encephalopathysnip...see full text;MONDAY, OCTOBER 10, 2022Docket No: 2002N-0273 (formerly Docket No. 02N-0273) Substances Prohibited From Use in Animal Food and Feed Scientists Comments December 20, 2005SUNDAY, OCTOBER 16, 2022USDA Transmissible Spongiform Encephalopathy TSE Prion Action Plan National Program 103 Animal Health 2022-2027
MONDAY, SEPTEMBER 19, 2022589.2001 BSE TSE regulations which prohibits the use of high-risk cattle material in feed for all animal species 2022Terry S. Singeltary Sr.
TERRY S. SINGELTARY SR.- retired
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