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Saturday, December 21, 2019

In vitro detection of haematogenous prions in white-tailed deer orally dosed with low concentrations of chronic wasting disease

JOURNAL OF GENERAL VIROLOGY

Research Article

In vitro detection of haematogenous prions in white-tailed deer orally dosed with low concentrations of chronic wasting disease 

No Access

Erin E. McNulty1​, Amy V. Nalls1​, Randy Xun1​, Nathaniel D. Denkers1​, Edward A. Hoover1​, Candace K. Mathiason1​ 

View Affiliations

First Published: 17 December 2019 https://doi.org/10.1099/jgv.0.001367

Infectivity associated with prion disease has been demonstrated in blood throughout the course of disease, yet the ability to detect blood-borne prions by in vitro methods remains challenging. We capitalized on longitudinal pathogenesis studies of chronic wasting disease (CWD) conducted in the native host to examine haematogenous prion load by real-time quaking-induced conversion (RT-QuIC) and protein misfolding cyclic amplification. Our study demonstrated in vitro detection of amyloid seeding activity (prions) in buffy-coat cells harvested from deer orally dosed with low concentrations of CWD positive (+) brain (1 gr and 300 ng) or saliva (300 ng RT-QuIC equivalent). These findings make possible the longitudinal assessment of prion disease and deeper investigation of the role haematogenous prions play in prion pathogenesis.

Received: 20/09/2019 Accepted: 19/11/2019 Published Online: 17/12/2019

Keyword(s): RT-QuIC , Haematogenous prions , prionemia , in vitro prion detection and PMCA

© 2019 The Authors

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Discussion 

Development of noninvasive, rapid and robust assays to detect prion infections throughout the course of disease are continuously being sought. Currently, the definitive diagnosis for prion diseases is by post-mortem examination of brain and lymphoid tissues for the presence of prion deposition (PrPSc) by immunohistochemistry. This process is expensive, time consuming and is most efficient in detecting high vs low prion burdens [25]. It has been confirmed that bodily fluids of prion-infected hosts harbour infectivity [1, 2, 4, 65–70]. In vitro amplification assays have been developed that recognize accumulated amyloid formation associated with prion infections [33, 34]. Further modifications to these assays have been instrumental in detecting the presence of low concentrations of the prion agent in biological tissues and fluids [52, 57, 59, 64]. We report in vitro prion detection in blood components harvested of cervids orally inoculated with ng (300ng) quantities of biologically relevant milieu (saliva).

We demonstrate the ability to detect prions in as few as 5×105 buffy-coat cells by lipase–iron-oxide bead–RT-QuIC performed at 42 °C (LIQ42) in 79% of CWD-biopsy positive WTD. We were able to detect prions in all LIQ42 negative buffy-coat samples when LIQ was performed at 55 °C (LIQ55), thus permitting prionemia detection by LIQ in 100% of WTD. RT-QuIC assessment of PMCA (PQ) round 5 product revealed haematogenous prions in 83% of the WTD. In vitro detection of haematogenous prions has been fraught with challenge. The use of amplification assays, PMCA and RT-QuIC, have steadily gained prowess in permitting detection of prions present in blood components of experimental and free-range naturally exposed cervids [48, 50, 71], scrapie-infected sheep [47, 72, 73] and humans infected with CJD [6, 13, 29, 51]. A variety of pre-amplification methods designed to concentrate or remove inhibitors associated with bodily fluids have been implemented prior to amplification by RT-QuIC [31, 57, 63] and PMCA [51, 52, 74, 75]. These modifications have been successful in revealing the presence of amyloid seeding activity in biological fluids harvested from prion-infected hosts.

We show that increasing the temperature at which LIQ is performed from 42 to 55 °C, and combined use of PMCA and RT-QuIC (PQ) provide enhanced sensitivity and detection confirmation of prionemia in CWD-infected WTD. The use of higher temperatures for the RT-QuIC assay, resulting in enhanced amyloid signal to noise ratios, has been pioneered by the Caughey laboratory [64]. Their use of RT-QuIC at higher temperatures has permitted the detection of disease associated tau, alpha synuclein and prion aggregates in CSF [76]. By incorporating RT-QuIC readout of PMCA product we demonstrate the presence of haematogenous prions in lymphoid biopsy positive, yet LIQ42 negative cervids, supporting combined assay use to confirm the presence of low circulating CWD prionemia. Combined use of RT-QuIC and PMCA has been reported to be a powerful platform to amplify minute quantities of amyloid present in tissues of CWD-infected WTD [25, 59].

The number of buffy-coat cells required for LIQ and PQ detection of haematogenous prions in this study represent that present in ~0.5–1ml of WTD blood. In our experience, collection of 10–15ml whole blood is sufficient to harvest adequate numbers of cells to perform either assay, making longitudinal assessment over the course of disease feasible. Adequate leukocyte numbers are present in similar volumes of human [77], sheep and cattle [78] whole blood. Of particular interest is the detection of blood-borne prions in cervids dosed with progressively lower concentrations of CWD; 1 g, 1mg or 300ng. It is suspected that prion exposures in nature are quite low [79]. Studies to define minimum infectious dose in native species have been undertaken. In sheep scrapie, sufficient infectivity to initiate disease has been described after intravenous inoculation of 105 white blood cells or 100µl whole blood [80]. Our own ongoing studies in WTD demonstrate that oral doses of 1mg and 300ng brain or 300ng saliva equivalent contain sufficient infectivity to initiate CWD infection (doses are 3–9 logs lower than our previous experimental exposures; manuscripts in preparation). Thus, the amplification methods described here show promise of sufficient sensitivity to detect low level CWD exposure as likely occurs in naturally infected cervids.

Current information indicates that cervids in the subclinical phase of disease carry and shed infectivity [9, 81, 82]. During the protracted subclinical phase of disease CWD burden in tissues and shed components are low and/or intermittent [48, 83, 84]. Corroborating our previous findings [48, 50], we provide further evidence that prionemia can be identified at all stages of the disease course (0–3). Circulating prion burdens in blood have been estimated to be in the 13–260 fg ml−1–0.5 pg ml−1 range [51, 85] and may be intermittent across the longitudinal course of disease [29, 49]. It is possible that we may have harvested blood from infected deer at opportune times resulting in detection across subclinical and clinical stages. Our ongoing work includes analysis of prionemia in blood samples collected throughout the disease course of individual infected WTD to determine the pattern and degree to which temporal fluctuations occur.

CWD continues its geographical, host range and strain expansion across North America, Korea and Scandinavia [14, 86]. Furthermore, new CWD strains have been reported in cervid populations [14, 87, 88]. It is unknown if new CWD strains are more or less infectious to cervid populations. Of considerable concern is whether new strains have increased propensity to cross-species barriers to humans and other species sympatric with CWD-infected cervids. One in 36 Americans (roughly 9million) hunt North American big game [89]. Estimates show that 7000 to 15000 conventional test CWD+ cervids are consumed per year and that this number increases by ~20% every year [90]. CWD-infected yet conventional test negative (subclinical) animals are prevalent in cervid populations [26, 38]. Bioassay has confirmed the presence of CWD infectivity in cervid muscle [91, 92]. Venison is shared among family and friends and is the mainstay protein for many indigenous populations [93, 94]. While no human cases of CWD have been detected, it was thought BSE would not become a human pathogen several years before vCJD was discovered [12, 18, 95, 96].

Our findings provide a path to assess prion pathogenesis throughout the disease process and can also be instrumental in the development of vaccines, therapeutics and management practices to mitigate CWD, and by extension, other prion and protein misfolding disorders.

Funding information

This study was supported by National Institutes of Health (NIH) R01- AI-112956, R01-AI-093634, NIH R01-NS-061902, Department of Microbiology, Immunology and Pathology, and the College of Veterinary Medicine and Biomedical Sciences, Colorado State University.

Acknowledgements

We especially thank the Warnell School of Forestry and Natural Resources at the University of Georgia, and Sallie Dahmes, David Osborn, and Karl Miller for producing the white-tailed deer used in these studies.

Conflicts of interest

The authors declare that there are no conflicts of interest.

Ethical statement

All animals were handled in strict accordance with guidelines for animal care and use provided by the United States Department of Agriculture (USDA), NIH and the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC), and all animal work was approved by Colorado State University Institutional Animal Care and Use Committee (IACUC protocol numbers 11-2622A, 12-3773A, 18-8396A and 18-7969A).


References

snip...see full text;


''Our study demonstrated in vitro detection of amyloid seeding activity (prions) in buffy-coat cells harvested from deer orally dosed with low concentrations of CWD positive (+) brain (1 gr and 300 ng) or saliva (300 ng RT-QuIC equivalent).''

''Our own ongoing studies in WTD demonstrate that oral doses of 1mg and 300ng brain or 300ng saliva equivalent contain sufficient infectivity to initiate CWD infection (doses are 3–9 logs lower than our previous experimental exposures; manuscripts in preparation).''

98. Longitudinal comparison of real-time conversion and immunohistochemistry for detection of chronic wasting disease in orally exposed white-tailed deer

Nathaniel D. Denkersa, Davin M. Hendersona, Clare E. Hoovera,b*, Amy V. Nallsa, Erin McNultya, Candace K. Mathiasona and Edward A. Hoovera
aPrion Research Center, Department of Microbiology, Immunology, and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA
CONTACT Nathaniel D. Denkers 
*Present address: AstraZeneca, Waltham, NJ
ABSTRACT
Background: Chronic wasting disease (CWD) continues to expand across North America and Canada, and more recently was discovered in Scandinavia. While the exact mechanism of CWD transmission has yet to be elucidated, early diagnosis of infected animals remains a priority in curtailing its spread. While immunohistochemistry (IHC) and enzyme linked immunosorbent assay (ELISA) remain the gold standards for diagnosing CWD, real-time quaking induced conversion (RT-QuIC) is capable of detecting substantially lower concentrations of prions, which may translate into detection earlier in disease course. In this study, we sought to compare the sensitivity of RT-QuIC and IHC on a series of longitudinal tonsil and recto-anal mucosal lymphoid tissue (RAMALT) biopsies from white-tailed deer during the course of CWD progression.
Methods: White-tailed deer were inoculated via the per os (PO) route with CWD(+) [n = 20] and CWD(-) [n = 4] inocula (brain homogenate or saliva) and paired tonsil and RAMALT biopsies were collected every 3 months thereafter. Biopsies were assayed for prion seeding activity by RT-QuIC and for PrPCWD deposition by IHC.
Results: RT-QuIC detected seeding activity in 10 of 20 (50%) tonsil biopsies prior to IHC detection, on average of 5.1 months (3–15 months) earlier. In RAMALT biopsies, detectable seeding activity was observed in 13 of 20 (65%) samples prior to IHC detection, on average 4.6 months (3–15 months) earlier. In the remaining biopsies, detection was concurrent by both methods. At no sampling point did a positive IHC result precede a positive RT-QuIC result. Of note, for biopsy samples that had already been determined positive by both RT-QuIC and IHC in a previous collection, RT-QuIC detected seeding activity in seven (7) tonsil and four (4) RAMALT follow-up biopsies even though no lymphoid follicles were present in the paired samples to assess positivity by IHC. All biopsies from CWD(-) deer were negative by both assays throughout the study. Overall, these results demonstrate that RT-QuIC detected CWD positivity in both tonsil and RAMALT biopsies approximately 5 months prior to IHC. Moreover, seeding activity was detectable even when (due to poor biopsy sampling) lymphoid follicles were not present to permit meaningful IHC analysis.
Conclusions: These data indicate that RT-QuIC is more sensitive than IHC in detecting CWD infection, and could thereby be useful in determining future management strategies.
Funding
Supported by NIH R01-NS-061902, P01-AI-077774, F30-ODO-118,143, T32-OD0-10,437

124. Longitudinal studies of CWD after low-dose oral exposure in white-tailed deer

Clare E. Hoover*, Nathaniel D. Denkers, Kristen A. Davenport, Davin M. Henderson, Amy V. Nalls, Erin McNulty, Joanne Tennant, Sarah Cooper, Manci Li, Lauren Bracchi, Candace K. Mathiason and Edward A. Hoover
Prion Research Center, Department of Microbiology, Immunology, and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA
CONTACT Edward A. Hoover 
*Present address: AstraZeneca, Waltham, NJ
ABSTRACT
Background: The facile transmission of chronic wasting disease (CWD) in North America, Asia and Europe continues despite exposure to very low concentrations of prions shed by infected cervids in secreta and excreta. Explanations for this enigma could be that excreted prions may have enhanced infectivity and/or that the infectious prion dose is just quite low. Historical studies exploring CWD pathogenesis in deer have used exposures to CWD-positive brain homogenates containing from 0.5 to 10 g of brain – doses much higher than those measured in excreta, and thereby likely to ever occur in nature. We thereby sought to more closely emulate natural exposure context in our current experimental studies.
Methods: To explore how the origin or infectious dose of CWD prions may influence disease transmission or pathogenesis, we orally exposed cohorts of n = 4/group white-tailed deer to low doses of CWD presented as either CWD-positive brain homogenate (containing either 1 mg or 300 ng of brain) or to an amount of pooled saliva from CWD+ donors and containing prion seeding activity (by RT-QuIC) equivalent to 300 ng of CWD+ brain [1]. Inoculated deer were then longitudinally monitored for: (a) onset of prion infection in biopsied tonsil and recto-anal lymphoid tissues by RT-QuIC and immunohistochemistry; (b) prion shedding in saliva and faeces (by RT-QuIC); (c) onset of clinical signs (weekly observation and scoring); and (d) time to clinical disease.
Results: We first detected CWD infection by RT-QuIC seeding activity in tonsil biopsies at 6 or 9 months post exposure, in all inoculation cohorts. Prion shedding in saliva was detected (by RT-QuIC or PMCA-RT-QuIC [2]) concurrent with the first positive tonsil biopsy. Detection of prion seeding activity in faeces was less common overall and its onset observed after positivity in recto-anal lymphoid tissue biopsies. In comparison to historical studies, in which >1,000-fold higher inoculation doses were used, the time to first detection on infection (by tonsil biopsy) was prolonged by ~6 months. However, among the low-dose inoculation cohorts in this study, neither the attack rate, prion shedding profile, nor long-term disease course differed significantly, including the deer exposed to prions of saliva vs. brain origin.
Conclusions: These studies demonstrate that (a) much lower doses of CWD prions that have been used historically for point source exposure studies are sufficient to induce prion infection, shedding, and disease; and (b) although time to first detectable positivity is lengthened, the qualitative aspects of disease pathogenesis thereafter are indistinguishable by inoculum source or exposure magnitude.
Our future studies will aim at establishing the minimum oral infectious dose for CWD in deer and on exploring exposure cofactors.
Supported by NIH R01-NS-061902, P01-AI-077774, F30-ODO-118,143, T32-OD0-10,437

186. Serial detection of hematogenous prions in CWD-infected deer

Amy V. Nalls, Erin E. McNulty, Nathaniel D. Denkers, Edward A. Hoover and Candace K. Mathiason
Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO, USA
CONTACT Amy V. Nalls amy.nalls@colostate.edu
ABSTRACT
Blood contains the infectious agent associated with prion disease affecting several mammalian species, including humans, cervids, sheep, and cattle. It has been confirmed that sufficient prion agent is present in the blood of both symptomatic and asymptomatic carriers to initiate the amyloid templating and accumulation process that results in this fatal neurodegenerative disease. Yet, to date, the ability to detect blood-borne prions by in vitro methods remains difficult.
We have capitalized on blood samples collected from longitudinal chronic wasting disease (CWD) studies in the native white-tailed deer host to examine hematogenous prion load in blood collected minutes, days, weeks and months post exposure. Our work has focused on refinement of the amplification methods RT-QuIC and PMCA. We demonstrate enhanced in vitro detection of amyloid seeding activity (prions) in blood cell fractions harvested from deer orally-exposed to 300 ng CWD positive brain or saliva.
These findings permit assessment of the role hematogenous prions play in the pathogenesis of CWD and provide tools to assess the same for prion diseases of other mammalian species.
''Our study demonstrated in vitro detection of amyloid seeding activity (prions) in buffy-coat cells harvested from deer orally dosed with low concentrations of CWD positive (+) brain (1 gr and 300 ng) or saliva (300 ng RT-QuIC equivalent).'' 

 ''Our own ongoing studies in WTD demonstrate that oral doses of 1mg and 300ng brain or 300ng saliva equivalent contain sufficient infectivity to initiate CWD infection (doses are 3–9 logs lower than our previous experimental exposures; manuscripts in preparation).''
8.CONSIDERING RECENT SCIENCE THAT CWD TSE PRION WILL TRANSMIT ORALLY TO PIGS AND ALSO SCRAPIE TO PIGS BY ORAL ROUTES, CONSIDERING CWD TRANSMIT EASILY TO CERVID BY ORAL ROUTE, CONSIDERING A NEW TSE PRION OUTBREAK IN A NEW LIVESTOCK SPECIES, THE CAMEL, CONSIDERING THE FACT THE USA THAT THE 1997 BSE feed regulation at 589.2000, which remains in effect but which applies only to feed for cattle and other ruminants, and specifically, the new section 589.2001, WAS AND STILL IS A TOTAL AND COLOSSAL FAILURE, AND PROVEN TO BE SO BY RECENT COMMENTS COMING FROM THE FDA, BUT FIRST, COMMENTS FROM DEFRA;
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.....

https://web.archive.org/web/20170404125557/http://webarchive.nationalarchives.gov.uk/20130822084033/http://www.defra.gov.uk/animal-diseases/files/qra_chronic-wasting-disease-121029.pdf

''Our own ongoing studies in WTD demonstrate that oral doses of 1mg and 300ng brain or 300ng saliva equivalent contain sufficient infectivity to initiate CWD infection (doses are 3–9 logs lower than our previous experimental exposures; manuscripts in preparation).''
FDA Reports on VFD Compliance 
John Maday 

August 30, 2019 09:46 AM VFD-Form 007 (640x427) 

Before and after the current Veterinary Feed Directive rules took full effect in January, 2017, the FDA focused primarily on education and outreach. ( John Maday ) Before and after the current Veterinary Feed Directive (VFD) rules took full effect in January, 2017, the FDA focused primarily on education and outreach to help feed mills, veterinarians and producers understand and comply with the requirements. Since then, FDA has gradually increased the number of VFD inspections and initiated enforcement actions when necessary. On August 29, FDA released its first report on inspection and compliance activities. The report, titled “Summary Assessment of Veterinary Feed Directive Compliance Activities Conducted in Fiscal Years 2016 – 2018,” is available online.


SUNDAY, SEPTEMBER 1, 2019 

***> FDA Reports on VFD Compliance 

https://bovineprp.blogspot.com/2019/09/fda-reports-on-vfd-compliance.html

''Our own ongoing studies in WTD demonstrate that oral doses of 1mg and 300ng brain or 300ng saliva equivalent contain sufficient infectivity to initiate CWD infection (doses are 3–9 logs lower than our previous experimental exposures; manuscripts in preparation).''

TUESDAY, APRIL 18, 2017 

*** EXTREME USA FDA PART 589 TSE PRION FEED LOOP HOLE STILL EXIST, AND PRICE OF POKER GOES UP *** 

http://camelusprp.blogspot.com/2018/04/dromedary-camels-algeria-prion-mad.html

I STRENUOUSLY URGE TEXAS FDA MODIFY THESE FEED BANS ASAP!
FRIDAY, DECEMBER 20, 2019 

Texas TAHC, Administrative Code, Title 4, Part 2, Chapter 40, Chronic Wasting Disease Amendments Open For Comment beginning December 20, 2019 thru January 20, 2020 Terry Singeltary Comments Submission


It is clear that the designing scientists must also have shared Mr Bradleys surprise at the results because all the dose levels right down to 1 gram triggered infection.


it is clear that the designing scientists must have also shared Mr Bradleys surprise at the results because all the dose levels right down to 1 gram triggered infection.


Evidence That Transmissible Mink Encephalopathy Results from Feeding Infected Cattle

Over the next 8-10 weeks, approximately 40% of all the adult mink on the farm died from TME.

snip...

The rancher was a ''dead stock'' feeder using mostly (>95%) downer or dead dairy cattle...

SEE;

Docket No. APHIS-2018-0011 Chronic Wasting Disease Herd Certification Program Standards Singeltary

View Attachment:View as format pdf

https://www.regulations.gov/contentStreamer?documentId=APHIS-2018-0011-0003&attachmentNumber=1&contentType=pdf

https://www.regulations.gov/document?D=APHIS-2018-0011-0003

http://chronic-wasting-disease.blogspot.com/2018/03/docket-no-aphis-2018-0011-chronic.html

''Our own ongoing studies in WTD demonstrate that oral doses of 1mg and 300ng brain or 300ng saliva equivalent contain sufficient infectivity to initiate CWD infection (doses are 3–9 logs lower than our previous experimental exposures; manuscripts in preparation).''
THURSDAY, DECEMBER 19, 2019 

The emergence of classical BSE from atypical/Nor98 scrapie


TUESDAY, OCTOBER 29, 2019 

America BSE 589.2001 FEED REGULATIONS, BSE SURVEILLANCE, BSE TESTING, and CJD TSE Prion


WEDNESDAY, AUGUST 15, 2018 

***> The agent of H-type bovine spongiform encephalopathy associated with E211K prion protein polymorphism transmits after oronasal challenge


MONDAY, JANUARY 09, 2017 

Oral Transmission of L-Type Bovine Spongiform Encephalopathy Agent among Cattle 

CDC Volume 23, Number 2—February 2017 

*** Consumption of L-BSE–contaminated feed may pose a risk for oral transmission of the disease agent to cattle.

*** Consumption of L-BSE–contaminated feed may pose a risk for oral transmission of the disease agent to cattle.


Detection of PrPBSE and prion infectivity in the ileal Peyer’s patch of young calves as early as 2 months after oral challenge with classical bovine spongiform encephalopathy 

Ivett Ackermann1 , Anne Balkema‑Buschmann1 , Reiner Ulrich2 , Kerstin Tauscher2 , James C. Shawulu1 , Markus Keller1 , Olanrewaju I. Fatola1 , Paul Brown3 and Martin H. Groschup1* 

Abstract 

In classical bovine spongiform encephalopathy (C-BSE), an orally acquired prion disease of cattle, the ileal Peyer’s patch (IPP) represents the main entry port for the BSE agent. In earlier C-BSE pathogenesis studies, cattle at 4–6 months of age were orally challenged, while there are strong indications that the risk of infection is highest in young animals. In the present study, unweaned calves aged 4–6 weeks were orally challenged to determine the earli‑ est time point at which newly formed PrPBSE and BSE infectivity are detectable in the IPP. For this purpose, calves were culled 1 week as well as 2, 4, 6 and 8 months post-infection (mpi) and IPPs were examined for BSE infectivity using a bovine PrP transgenic mouse bioassay, and for PrPBSE by immunohistochemistry (IHC) and protein misfolding cyclic amplifcation (PMCA) assays. For the frst time, BSE prions were detected in the IPP as early as 2 mpi by transgenic mouse bioassay and PMCA and 4 mpi by IHC in the follicular dendritic cells (FDCs) of the IPP follicles. These data indi‑ cate that BSE prions propagate in the IPP of unweaned calves within 2 months of oral uptake of the agent.

In summary, our study demonstrates for the frst time PrPBSE (by PMCA) and prion infectivity (by mouse bioassay) in the ileal Peyer’s patch (IPP) of young calves as early as 2 months after infection. From 4 mpi nearly all calves showed PrPBSE positive IPP follicles (by IHC), even with PrPBSE accumulation detectable in FDCs in some animals. Finally, our results confrm the IPP as the early port of entry for the BSE agent and a site of initial propagation of PrPBSE and infectivity during the early pathogenesis of the disease. Terefore, our study supports the recommendation to remove the last four metres of the small intestine (distal ileum) at slaughter, as designated by current legal requirements for countries with a controlled BSE risk status, as an essential measure for consumer and public health protection.


PLOS ONE Journal 

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 ***

http://www.plosone.org/annotation/listThread.action?root=86610

*** Singeltary reply ; Molecular, Biochemical and Genetic Characteristics of BSE in Canada Singeltary reply ;

P98 The agent of H-type bovine spongiform encephalopathy associated with E211K prion protein polymorphism transmits after oronasal challenge 

Greenlee 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 ABSTRACT


WEDNESDAY, OCTOBER 24, 2018 

Experimental Infection of Cattle With a Novel Prion Derived From Atypical H-Type Bovine Spongiform Encephalopathy


Sunday, March 20, 2016

Docket No. FDA-2003-D-0432 (formerly 03D-0186) Use of Material from Deer and Elk in Animal Feed Singeltary Submission

http://chronic-wasting-disease.blogspot.com/2016/03/docket-no-fda-2003-d-0432-formerly-03d.html

Singeltary previous submission to DOCKET-- 03D-0186 -- FDA Issues Draft Guidance on Use of Material From Deer and Elk in Animal Feed; Availability

DOCKET-- 03D-0186 -- FDA Issues Draft Guidance on Use of Material From Deer and Elk in Animal Feed; Availability Fri, 16 May 2003 11:47:37 0500 EMC 1 Terry S. Singeltary Sr. Vol #: 1

-------- Original Message --------

Subject: DOCKET-- 03D-0186 -- FDA Issues Draft Guidance on Use of Material From Deer and Elk in Animal Feed; Availability Date: Fri, 16 May 2003 11:47:37 -0500 From: "Terry S. Singeltary Sr." To: fdadockets@oc.fda.gov

Greetings FDA,

i would kindly like to comment on;

Docket 03D-0186

FDA Issues Draft Guidance on Use of Material From Deer and Elk in Animal Feed; Availability

Several factors on this apparent voluntary proposal disturbs me greatly, please allow me to point them out;

1. MY first point is the failure of the partial ruminant-to-ruminant feed ban of 8/4/97. this partial and voluntary feed ban of some ruminant materials being fed back to cattle is terribly flawed. without the _total_ and _mandatory_ ban of all ruminant materials being fed back to ruminants including cattle, sheep, goat, deer, elk and mink, chickens, fish (all farmed animals for human/animal consumption), this half ass measure will fail terribly, as in the past decades...

2. WHAT about sub-clinical TSE in deer and elk? with the recent findings of deer fawns being infected with CWD, how many could possibly be sub-clinically infected. until we have a rapid TSE test to assure us that all deer/elk are free of disease (clinical and sub-clinical), we must ban not only documented CWD infected deer/elk, but healthy ones as well. it this is not done, they system will fail...

3. WE must ban not only CNS (SRMs specified risk materials), but ALL tissues. recent new and old findings support infectivity in the rump or ass muscle. wether it be low or high, accumulation will play a crucial role in TSEs.

4. THERE are and have been for some time many TSEs in the USA. TME in mink, Scrapie in Sheep and Goats, and unidentified TSE in USA cattle. all this has been proven, but the TSE in USA cattle has been totally ignored for decades. i will document this data below in my references.

5. UNTIL we ban all ruminant by-products from being fed back to ALL ruminants, until we rapid TSE test (not only deer/elk) but cattle in sufficient numbers to find (1 million rapid TSE test in USA cattle annually for 5 years), any partial measures such as the ones proposed while ignoring sub-clinical TSEs and not rapid TSE testing cattle, not closing down feed mills that continue to violate the FDA's BSE feed regulation (21 CFR 589.2000) and not making freely available those violations, will only continue to spread these TSE mad cow agents in the USA. I am curious what we will call a phenotype in a species that is mixed with who knows how many strains of scrapie, who knows what strain or how many strains of TSE in USA cattle, and the CWD in deer and elk (no telling how many strains there), but all of this has been rendered for animal feeds in the USA for decades. it will get interesting once someone starts looking in all species, including humans here in the USA, but this has yet to happen...

6. IT is paramount that CJD be made reportable in every state (especially ''sporadic'' cjd), and that a CJD Questionnaire must be issued to every family of a victim of TSE. only checking death certificates will not be sufficient. this has been proven as well (see below HISTORY OF CJD -- CJD QUESTIONNAIRE)

7. WE must learn from our past mistakes, not continue to make the same mistakes...

REFERENCES

Six white-tailed deer fawns test positive for CWD

MADISON -- Six fawns in the area of south central Wisconsin where chronic wasting disease has been found in white-tailed deer have tested positive for the disease, according to Department of Natural Resources wildlife health officials. These are the youngest wild white-tailed deer detected with chronic wasting disease (CWD) to date.

Approximately 4,200 fawns, defined as deer under 1 year of age, were sampled from the eradication zone over the last year. The majority of fawns sampled were between the ages of 5 to 9 months, though some were as young as 1 month. Two of the six fawns with CWD detected were 5 to 6 months old. All six of the positive fawns were taken from the core area of the CWD eradication zone where the highest numbers of positive deer have been identified.

snip...

http://www.dnr.state.wi.us/org/caer/ce/news/on/2003/on20030513.htm#art4 

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Issued: Monday, 28 August 2000 NEW EVIDENCE OF SUB-CLINICAL PRION INFECTION: IMPORTANT RESEARCH FINDINGS RELEVANT TO CJD AND BSE

A team of researchers led by Professor John Collinge at the Medical Research Council Prion Unit1 report today in the Proceedings of the National Academy of Sciences, on new evidence for the existence of a 'sub-clinical' form of BSE in mice which was unknown until now.

The scientists took a closer look at what is known as the 'species barrier' - the main protective factor which limits the ability of prions2 to jump from one species to infect another. They found the mice had a 'sub-clinical' form of disease where they carried high levels of infectivity but did not develop the clinical disease during their normal lifespan. The idea that individuals can carry a disease and show no clinical symptoms is not new. It is commonly seen in conventional infectious diseases.

Researchers tried to infect laboratory mice with hamster prions3 called Sc237 and found that the mice showed no apparent signs of disease. However, on closer inspection they found that the mice had high levels of mouse prions in their brains. This was surprising because it has always been assumed that hamster prions could not cause the disease in mice, even when injected directly into the brain.

In addition the researchers showed that this new sub-clinical infection could be easily passed on when injected into healthy mice and hamsters.

The height of the species barrier varies widely between different combinations of animals and also varies with the type or strain of prions. While some barriers are quite small (for instance BSE easily infects mice), other combinations of strain and species show a seemingly impenetrable barrier. Traditionally, the particular barrier studied here was assumed to be robust.

Professor John Collinge said: "These results have a number of important implications. They suggest that we should re-think how we measure species barriers in the laboratory, and that we should not assume that just because one species appears resistant to a strain of prions they have been exposed to, that they do not silently carry the infection. This research raises the possibility, which has been mentioned before, that apparently healthy cattle could harbour, but never show signs of, BSE.

"This is a timely and unexpected result, increasing what we know about prion disease. These new findings have important implications for those researching prion disease, those responsible for preventing infected material getting into the food chain and for those considering how best to safeguard health and reduce the risk that theoretically, prion disease could be contracted through medical and surgical procedures."

ISSUED FRIDAY 25 AUGUST UNDER EMBARGO. PLEASE NOTE THAT THE EMBARGO IS SET BY THE JOURNAL.

FOR FURTHER INFORMATION CONTACT THE MRC PRESS OFFICE ON 020 7637 6011 (OFFICE HOURS) OR 07818 428297 OR 0385 774357 (OUT-OF-OFFICE-HOURS) OR PROFESSOR JOHN COLLINGE ON 020 7594 3760. PLEASE NOTE THAT OWING TO TRAVEL COMMITMENTS PROFESSOR COLLINGE WILL ONLY BE AVAILABLE UNTIL 16.30 ON FRIDAY 25 AUGUST AND CONTACTABLE AGAIN ON MONDAY 28 AUGUST VIA THE MRC PRESS OFFICE. DR ANDREW HILL (A CO-AUTHOR ON THE PAPER) FROM THE DEPARTMENT OF PATHOLOGY AT THE UNIVERSITY OF MELBOURNE WILL BE AVAILABLE ON 00 61 3 8344 3995 (DURING OFFICE HOURS) OR 00 61 3 9443 0009 (OUT-OF-OFFICE HOURS). PLEASE NOTE THAT AUSTRALIA IS TEN HOURS AHEAD OF UK TIME.

NOTES FOR EDITORS

Professor Collinge is a consultant neurologist and Director of the newly formed MRC Prion Unit based at The Imperial College School of Medicine at St Mary's Hospital. He is also a member of the UK Government's Spongiform Encephalopathy Advisory Committee (SEAC). The MRC prion unit is was set up in 1999, and its work includes molecular genetic studies of human prion disease and transgenic modelling of human prion diseases.

Prions are unique infectious agents that cause fatal brain diseases such as Creutzfeldt-Jakob disease (CJD) in humans and scrapie and BSE (mad cow disease) in animals. In some circumstances prions from one species of animals can infect another and it is clear that BSE has done this to cause the disease variant CJD in the UK and France. It remains unclear how large an epidemic of variant CJD will occur over the years ahead.

The strain of prion used here to infect the mice is the Sc237 strain (also known as 263K) which infects hamsters, and until now was assumed not to infect mice.

This research was funded by the Medical Research Council and Wellcome Trust.

The Medical Research Council (MRC) is a national organisation funded by the UK tax-payer. Its business is medical research aimed at improving human health; everyone stands to benefit from the outputs. The research it supports and the scientists it trains meet the needs of the health services, the pharmaceutical and other health-related industries and the academic world. MRC has funded work which has led to some of the most significant discoveries and achievements in medicine in the UK. About half of the MRC's expenditure of £345 million is invested in over 50 of its Institutes and Units, where it employs its own research staff. The remaining half goes in the form of grant support and training awards to individuals and teams in universities and medical schools.

The Wellcome Trust is the world's largest medical research charity with a spend of some £600 million in the current financial year 1999/2000. The Wellcome Trust supports more than 5,000 researchers, at 400 locations, in 42 different countries to promote and foster research with the aim of improving human and animal health. As well as funding major initiatives in the public understanding of science, the Wellcome Trust is the country's leading supporter of research into the history of medicine.

©2002 Medical Research Council Data Protection policy Contact the MRC

http://www.mrc.ac.uk/index/public_interest/public-press_office/public-press_releases_2000/public-mrc-43-00.htm

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Oral transmission and early lymphoid tropism of chronic wasting disease PrPres in mule deer fawns (Odocoileus hemionus) 

Christina J. Sigurdson1, Elizabeth S. Williams2, Michael W. Miller3, Terry R. Spraker1,4, Katherine I. O'Rourke5 and Edward A. Hoover1

Department of Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523- 1671, USA1 Department of Veterinary Sciences, University of Wyoming, 1174 Snowy Range Road, University of Wyoming, Laramie, WY 82070, USA 2 Colorado Division of Wildlife, Wildlife Research Center, 317 West Prospect Road, Fort Collins, CO 80526-2097, USA3 Colorado State University Veterinary Diagnostic Laboratory, 300 West Drake Road, Fort Collins, CO 80523-1671, USA4 Animal Disease Research Unit, Agricultural Research Service, US Department of Agriculture, 337 Bustad Hall, Washington State University, Pullman, WA 99164-7030, USA5

Author for correspondence: Edward Hoover.Fax +1 970 491 0523. e-mail ehoover@lamar.colostate.edu

Mule deer fawns (Odocoileus hemionus) were inoculated orally with a brain homogenate prepared from mule deer with naturally occurring chronic wasting disease (CWD), a prion-induced transmissible spongiform encephalopathy. Fawns were necropsied and examined for PrP res, the abnormal prion protein isoform, at 10, 42, 53, 77, 78 and 80 days post-inoculation (p.i.) using an immunohistochemistry assay modified to enhance sensitivity. PrPres was detected in alimentary-tract-associated lymphoid tissues (one or more of the following: retropharyngeal lymph node, tonsil, Peyer's patch and ileocaecal lymph node) as early as 42 days p.i. and in all fawns examined thereafter (53 to 80 days p.i.). No PrPres staining was detected in lymphoid tissue of three control fawns receiving a control brain inoculum, nor was PrPres detectable in neural tissue of any fawn. PrPres-specific staining was markedly enhanced by sequential tissue treatment with formic acid, proteinase K and hydrated autoclaving prior to immunohistochemical staining with monoclonal antibody F89/160.1.5. These results indicate that CWD PrP res can be detected in lymphoid tissues draining the alimentary tract within a few weeks after oral exposure to infectious prions and may reflect the initial pathway of CWD infection in deer. The rapid infection of deer fawns following exposure by the most plausible natural route is consistent with the efficient horizontal transmission of CWD in nature and enables accelerated studies of transmission and pathogenesis in the native species.

snip...

These results indicate that mule deer fawns develop detectable PrP res after oral exposure to an inoculum containing CWD prions. In the earliest post-exposure period, CWD PrPres was traced to the lymphoid tissues draining the oral and intestinal mucosa (i.e. the retropharyngeal lymph nodes, tonsil, ileal Peyer's patches and ileocaecal lymph nodes), which probably received the highest initial exposure to the inoculum. Hadlow et al. (1982) demonstrated scrapie agent in the tonsil, retropharyngeal and mesenteric lymph nodes, ileum and spleen in a 10-month-old naturally infected lamb by mouse bioassay. Eight of nine sheep had infectivity in the retropharyngeal lymph node. He concluded that the tissue distribution suggested primary infection via the gastrointestinal tract. The tissue distribution of PrPres in the early stages of infection in the fawns is strikingly similar to that seen in naturally infected sheep with scrapie. These findings support oral exposure as a natural route of CWD infection in deer and support oral inoculation as a reasonable exposure route for experimental studies of CWD.

snip...



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now, just what is in that deer feed? _ANIMAL PROTEIN_

Subject: MAD DEER/ELK DISEASE AND POTENTIAL SOURCES 

Date: Sat, 25 May 2002 18:41:46 -0700 

From: "Terry S. Singeltary Sr." 

Reply-To: BSE-L 

To: BSE-L

8420-20.5% Antler Developer For Deer and Game in the wild Guaranteed Analysis Ingredients / Products Feeding Directions

snip...

_animal protein_

http://www.surefed.com/deer.htm

BODE'S GAME FEED SUPPLEMENT #400 A RATION FOR DEER NET WEIGHT 50 POUNDS 22.6 KG.

snip...

_animal protein_

http://www.bodefeed.com/prod7.htm 

Ingredients

Grain Products, Plant Protein Products, Processed Grain By-Products, Forage Products, Roughage Products 15%, Molasses Products, __Animal Protein Products__, Monocalcium Phosphate, Dicalcium Pyosphate, Salt, Calcium Carbonate, Vitamin A Acetate with D-activated Animal Sterol (source of Vitamin D3), Vitamin E Supplement, Vitamin B12 Supplement, Riboflavin Supplement, Niacin Supplement, Calcium Panothenate, Choline Chloride, Folic Acid, Menadione Soduim Bisulfite Complex, Pyridoxine Hydorchloride, Thiamine Mononitrate, d-Biotin, Manganous Oxide, Zinc Oxide, Ferrous Carbonate, Calcium Iodate, Cobalt Carbonate, Dried Sacchoromyces Berevisiae Fermentation Solubles, Cellulose gum, Artificial Flavors added.

http://www.bodefeed.com/prod6.htm

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MORE ANIMAL PROTEIN PRODUCTS FOR DEER

Bode's #1 Game Pellets A RATION FOR DEER F3153

GUARANTEED ANALYSIS Crude Protein (Min) 16% Crude Fat (Min) 2.0% Crude Fiber (Max) 19% Calcium (Ca) (Min) 1.25% Calcium (Ca) (Max) 1.75% Phosphorus (P) (Min) 1.0% Salt (Min) .30% Salt (Max) .70%

Ingredients

Grain Products, Plant Protein Products, Processed Grain By-Products, Forage Products, Roughage Products, 15% Molasses Products, __Animal Protein Products__, Monocalcium Phosphate, Dicalcium Phosphate, Salt, Calcium Carbonate, Vitamin A Acetate with D-activated Animal Sterol (source of Vitamin D3) Vitamin E Supplement, Vitamin B12 Supplement, Roboflavin Supplement, Niacin Supplement, Calcium Pantothenate, Choline Chloride, Folic Acid, Menadione Sodium Bisulfite Complex, Pyridoxine Hydrochloride, Thiamine Mononitrate, e - Biotin, Manganous Oxide, Zinc Oxide, Ferrous Carbonate, Calcium Iodate, Cobalt Carbonate, Dried Saccharyomyces Cerevisiae Fermentation Solubles, Cellulose gum, Artificial Flavors added.

FEEDING DIRECTIONS Feed as Creep Feed with Normal Diet

http://www.bodefeed.com/prod8.htm 

INGREDIENTS

Grain Products, Roughage Products (not more than 35%), Processed Grain By-Products, Plant Protein Products, Forage Products, __Animal Protein Products__, L-Lysine, Calcium Carbonate, Salt, Monocalcium/Dicalcium Phosphate, Yeast Culture, Magnesium Oxide, Cobalt Carbonate, Basic Copper Chloride, Manganese Sulfate, Manganous Oxide, Sodium Selenite, Zinc Sulfate, Zinc Oxide, Sodium Selenite, Potassium Iodide, Ethylenediamine Dihydriodide, Vitamin E Supplement, Vitamin A Supplement, Vitamin D3 Supplement, Mineral Oil, Mold Inhibitor, Calcium Lignin Sulfonate, Vitamin B12 Supplement, Menadione Sodium Bisulfite Complex, Calcium Pantothenate, Riboflavin, Niacin, Biotin, Folic Acid, Pyridoxine Hydrochloride, Mineral Oil, Chromium Tripicolinate

DIRECTIONS FOR USE

Deer Builder Pellets is designed to be fed to deer under range conditions or deer that require higher levels of protein. Feed to deer during gestation, fawning, lactation, antler growth and pre-rut, all phases which require a higher level of nutrition. Provide adequate amounts of good quality roughage and fresh water at all times.

http://www.profilenutrition.com/Products/Specialty/deer_builder_pellets.html

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DEPARTMENT OF HEALTH & HUMAN SERVICES PUBLIC HEALTH SERVICE FOOD AND DRUG ADMINISTRATION

April 9, 2001 WARNING LETTER

01-PHI-12 CERTIFIED MAIL RETURN RECEIPT REQUESTED

Brian J. Raymond, Owner Sandy Lake Mills 26 Mill Street P.O. Box 117 Sandy Lake, PA 16145 PHILADELPHIA DISTRICT

Tel: 215-597-4390

Dear Mr. Raymond:

Food and Drug Administration Investigator Gregory E. Beichner conducted an inspection of your animal feed manufacturing operation, located in Sandy Lake, Pennsylvania, on March 23, 2001, and determined that your firm manufactures animal feeds including feeds containing prohibited materials. The inspection found significant deviations from the requirements set forth in Title 21, code of Federal Regulations, part 589.2000 - Animal Proteins Prohibited in Ruminant Feed. The regulation is intended to prevent the establishment and amplification of Bovine Spongiform Encephalopathy (BSE) . Such deviations cause products being manufactured at this facility to be misbranded within the meaning of Section 403(f), of the Federal Food, Drug, and Cosmetic Act (the Act).

Our investigation found failure to label your swine feed with the required cautionary statement "Do Not Feed to cattle or other Ruminants" The FDA suggests that the statement be distinguished by different type-size or color or other means of highlighting the statement so that it is easily noticed by a purchaser.

In addition, we note that you are using approximately 140 pounds of cracked corn to flush your mixer used in the manufacture of animal feeds containing prohibited material. This flushed material is fed to wild game including deer, a ruminant animal. Feed material which may potentially contain prohibited material should not be fed to ruminant animals which may become part of the food chain.

The above is not intended to be an all-inclusive list of deviations from the regulations. As a manufacturer of materials intended for animal feed use, you are responsible for assuring that your overall operation and the products you manufacture and distribute are in compliance with the law. We have enclosed a copy of FDA's Small Entity Compliance Guide to assist you with complying with the regulation... blah, blah, blah...

http://www.fda.gov/foi/warning_letters/g1115d.pdf

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Subject: MAD DEER/ELK DISEASE AND POTENTIAL SOURCES 

Date: Sat, 25 May 2002 18:41:46 -0700 

From: "Terry S. Singeltary Sr." 

Reply-To: Bovine Spongiform Encephalopathy 

To: BSE-L@uni-karlsruhe.de

now, what about those 'deer scents' of 100% urine', and the prion that is found in urine, why not just pass the prion with the urine to other deer...

Mrs. Doe Pee Doe in Estrus Model FDE1 Mrs. Doe Pee's Doe in Estrus is made from Estrus urine collected at the peak of the rut, blended with Fresh Doe Urine for an extremely effective buck enticer. Use pre-rut before the does come into heat. Use during full rut when bucks are most active. Use during post-rut when bucks are still actively looking for does. 1 oz.

www.gamecalls.net/hunting...lures.html

ELK SCENT/SPRAY BOTTLE

Works anytime of the year *

100 % Cow Elk-in-Heat urine (2oz.) *

Economical - mix with water in spray mist bottle *

Use wind to your advantage

Product Code WP-ESB $9.95

www.elkinc.com/Scent.asp 

prions in urine? 

DEER & ELK URINE, LURES & SCENT CONTROL DEPARTMENT by MRS.DOE PEE'S Main Index

The Turkey Pro Sez... "Premium, fresh, top-quality, pure 100% undiluted deer lures from Mrs. Doe Pee really work. I won't trust anything else when I'm after big bucks. Sam Collora, owner of the company, proved how well his products work when he bagged this monster buck in 1996.............snip......end........CWD

http://www.turkeyhuntingsecrets.com/store/store-luresandscentcontroldept.htm 

 ''Our own ongoing studies in WTD demonstrate that oral doses of 1mg and 300ng brain or 300ng saliva equivalent contain sufficient infectivity to initiate CWD infection (doses are 3–9 logs lower than our previous experimental exposures; manuscripts in preparation).''

-------- Original Message -------- 

Subject: ON THE ORIGIN OF MINK TME MARSH/HANSON (Scrapie in USA sheep, to TSE in USA cattle, or BOTH) 

Date: Thu, 15 May 2003 15:23:46 -0500 

From: "Terry S. Singeltary Sr." Reply-To: Bovine Spongiform Encephalopathy To: BSE-L@uni-karlsruhe.de

######## Bovine Spongiform Encephalopathy #########

ABSTRACT--studies on mink susceptibility to sources of scrapie from the United States, but not from the United Kingdom, indicate that transmissible mink encephalopathy (TME) most likely originates from mink fed scrapie-infected sheep or goat tissues. Experiments further suggest that the shortest natural route of infection is via bite wounds inflicted by littermates rather than by the oral route per se. Other studies, on the biologic characterization of TME agent from Sawyer County, Wisconsin, indicate that this particular source of TME is composed of a mixture of subpopulations which include a hamster pathogen and a mink-monkey pathogen...

snip...

with so many disease features in common, it would seem a simple matter to demonstrate that TME results from feeding scrapie-infected tissue to mink. BUT such has not been the case. Epizootiologic studies of the 14 worldwide occurrences of TME have revealed probably exposure to scrapie in only one instance, a 1965 incidence in Finland in which the affected farm was the only one in the area feeding sheep heads (Kangas, personal communication). Experimentally, mink have been found to be susceptible to some sources of scrapie and the disease produces was indistinguishable from TME (6)...

snip...

The purpose of these present studies was to attempt to explain differences between field and experimental observations, and to further characterize the biologic properties of the Sawyer County, Wisconsin, isolate of TME. Our results indicate that mink are more susceptible to sources of scrapie present in the UNITED STATES that those found in the UK, and that BITE WOUNDS from littermates may represent a significant route of natural exposure...

snip...

This Nubian X Toggenburg buck was naturally infected via exposure to scrapie-contaminated pasture at Mission, TEXAS; the pasture being previously occupied by a flock of scrapie-affected Suffolk sheep. At 6 months of age, animal B-834 was removed from exposure and placed in a pen where he subsequently developed signs of scrapie at 40 months of age...

snip...

Therefore, it should be expected that the pathology of natural TME will vary depending on the source of scrapie to which mink are exposed. Johannsen and Hartung have reported an incidence of TME occuring in East Germany in 1967 in which affected mink had diffuse cerebral ''edema'' and widespread lesions in the spinal cord (10)...

snip...

Even though B-834 produced short incubation periods when inoculated intracerebrally, exposure by the oral route was ineffective during an observation period of two years. Thus, we once again seem to have a conflict between field and experimental data. However, Gajdusek has suggested that the main route of entry for these transmissible agents is not the oral route per se, but rather via breaks or abrasions of skin and mucosal surfaces (11).

full text;


years later Marsh finds out;

Part of the Proceedings of an International Roundtable on Bovine Spongiform Encephalopathy, Bethesda, Maryland, USA, June 27-28, 1989.

The possibility of infection with BSE in the United States, as defined by studies on the disease in Great Britain, is judged to be low on the basis of the following: (1) meat and bonemeals imported into the United States from Great Britain between 1980 and 1988 were used mainly in poultry, not ruminant feed; (2) the Scrapie Eradication Program had reduced the prevalence of scrapie in the United States compared with that in Great Britain; and (3) little, if any, rendered animal products are used for protein supplements in cattle feed in the United States. However, there is some evidence that there may already be a scrapie-like disease in cattle in the United States. This evidence comes from epidemiologic studies on an incident of transmissible mink encephalopathy (TME) in Stetsonville, Wis, in 1985. This mink farmer used no commercially available animal by-product mixtures in his feed, but instead slaughtered all animals going into the mink diet, which included mostly (>95%) "downer" dairy cows, a few horses, but never sheep. To examine the possibility that cattle may have been the source of this incident of TME, two 6-week-old Holstein bull calves were inoculated intracerebrally with mink brain from the affected farm. The bulls developed neurologic disease 18 and 19 months after inoculation. Both brains had spongiform degeneration at necropsy and both were transmissible back to mink by either intracerebral (incubation period of 4 months) or oral (incubation period of 7 months) inoculation Whereas TME has been thought to be caused by feeding scrapie-infected sheep to mink, this theory has no conclusive evidence. Experimental oral inoculation of mink with several different sources of sheep scrapie has never been successful, and an incubation period of less than 12 months has never (sic) produced by intracerebral inoculation. Transmissible mink encephalopathy can develop naturally by infection with incubation periods of less than 12 months. There is reason to believe that scrapie has not been transmitted in the United States from sheep to cattle by rendered protein concentrates as it was in Great Britain. However, some circumstantial evidence exists that cattle may be a source of some TME infections. It is recommended that we increase our surveillance for a BSE-like disease in American cattle by encouraging state diagnostic laboratories to formalin-fix specimens of midbrain and brain stem from bovine brains submitted for rabies testing. If results of these tests are negative, these fixed tissues can then be examined for evidence of spongiform degeneration of the gray matter.

Letter to the Editor, Journal of the American Veterinary Medical Association, August 15, 1990 In my article, "Bovine spongiform encephalopathy in the United States" (JAVMA, May 15, 1990, p 1677), I stated that "little, if any, rendered animal products are used for protein supplements in cattle feed in the United States." I have since learned that this is incorrect, because of the recent trend of using less assimilated "by-pass" proteins in cattle feed. A large amount of meat-and-bone meal is being fed to American cattle, and this change in feeding practice has greatly increased the risk of bovine spongiform encephalopathy (BSE) developing in the United States. Epidemiologic studies on BSE in Great Britain have indicated that the disease originated in cattle by exposure to the heat-resistant transmissible agent in compounded feed containing rendered animal protein. The most likely source of infection was assumed to be meat-and-bone meal prepared from scrapie-infected sheep, but it is also possible that a heretofore unrecognized scrapie-like infection of cattle could have been spread in the same manner. Because of concern for the possible development of BSE in the United States, the American rendering industry discontinued the processing of fallen and sick sheep last December. In my opinion, this was a prudent policy, but one that will not prevent the possible transmission of BSE from cattle to cattle. As emphasized in my article, there is some evidence that BSE-like infection may already exist in American cattle. The current practice of feeding meat-and-bone meal to cattle solidifies the most important means to perpetuate and amplify the disease cycle. In Great Britain, BSE has produced a great economic and emotional burden. We must take all reasonable measures to prevent BSE from developing in the United States. Therefore, the practice of using animal protein in cattle feed should be discontinued as soon as possible. Waiting until the first case of BSE is diagnosed in the United States will certainly be "closing the barn door after the horse is gone." With a disease having a 3- to 6-year incubation period, thousands of animals would be exposed before we recognize the problem and, if that happens, we would be in for a decade of turmoil. R. F. Marsh, DVM, PhD Madison, Wis

To be published in the Proceedings of the Fourth International Scientific Congress in Fur Animal Production. Toronto, Canada, August 21-28, 1988

Evidence That Transmissible Mink Encephalopathy Results from Feeding Infected Cattle

_ - R.F. Marsh* and G.R. Hartsough

"Department of Veterinary Science, University of Wisconsin-Madison, Madison, Wisconsin 53706; and ^Emba/Creat Lakes Ranch Service, Thiensville, Wisconsin 53092

ABSTRACT Epidemiologic investigation of a new incidence of transmissible mink encephalopathy (TME) in Stetsonville, Wisconsin suggests that the disease may have resulted from feeding infected cattle to mink. This observation is supported by the transmission of a TME-like disease to experimentally inoculated cattle, and by the recent report of a new bovine spongiform encephalopathy in England.

INTRODUCTION

Transmissible mink encephalopathy (TME) was first reported in 1965 by Hartsough and Burger who demonstrated that the disease was transmissible with a long incubation period, and that affected mink had a spongiform encephalopathy similar to that found in scrapie-affecied sheep (Hartsough and Burger, 1965; Burger and Hartsough, 1965). Because of the similarity between TME and scrapie, and the subsequent finding that the two transmissible agents were indistinguishable (Marsh and Hanson, 1969), it was concluded that TME most likely resulted from feeding mink scrapie-infecied sheep. The experimental transmission of sheep scrapie to mink (Hanson et al., 1971) confirmed the close association of TME and scrapie, but at the same time provided evidence that they may be different. Epidemiologic studies on previous incidences of TME indicated that the incubation periods in field cases were between six months and one year in length (Harxsough and Burger, 1965). Experimentally, scrapie could not be transmitted to mink in less than one year. To investigate the possibility that TME may be caused by a (particular strain of scrapie which might be highly pathogenic for mink, 21 different strains of the scrapie agent, including their sheep or goat sources, were inoculated into a total of 61 mink. Only one mink developed a progressive neurologic disease after an incubation period of 22 mon..s (Marsh and Hanson, 1979). These results indicated that TME was either caused by a strain of sheep scrapie not yet tested, or was due to exposure to a scrapie-like agent from an unidentified source.

OBSERVATIONS AND RESULTS

A New Incidence of TME. In April of 1985, a mink rancher in Stetsonville, Wisconsin reported that many of his mink were "acting funny", and some had died. At this time, we visited the farm and found that approximately 10% of all adult mink were showing typical signs of TME: insidious onset characterized by subtle behavioral changes, loss of formal habits of cleanliness, deposition of droppings throughout the pen rather than in a single area, hyperexcitability, difficulty in chewing and swallowing, and tails arched over their _backs like squirrels. These signs were followed by progressive deterioration of neurologic function beginning with locomoior incoordination, long periods of somnolence in which the affected mink would stand motionless with its head in the corner of the cage, complete debilitation, and death. Over the next 8-10 weeks, approximately 40% of all the adult mink on the farm died from TME. Since previous incidences of TME were associated with common or shared feeding practices, we obtained a careful history of feed ingredients used over the past 12-18 months. The rancher was a "dead stock" feeder using mostly (>95%) downer or dead dairy cattle and a few horses. Sheep had never been fed.

Experimental Transmission. The clinical diagnosis of TME was confirmed by histopaihologic examination and by experimental transmission to mink after incubation periods of four months. To investigate the possible involvement of cattle in this disease cycle, two six-week old castrated Holstein bull calves were inoculated intracerebrally with a brain suspension from affected mink. Each developed a fatal spongiform encephalopathy after incubation periods of 18 and 19 months.

DISCUSSION

These findings suggest that TME may result from feeding mink infected cattle and we have alerted bovine practitioners that there may exist an as yet unrecognized scrapie-like disease of cattle in the United States (Marsh and Hartsough, 1986). A new bovine spongiform encephalopathy has recently been reported in England (Wells et al., 1987), and investigators are presently studying its transmissibility and possible relationship to scrapie. Because this new bovine disease in England is characterized by behavioral changes, hyperexcitability, and agressiveness, it is very likely it would be confused with rabies in the United Stales and not be diagnosed. Presently, brains from cattle in the United States which are suspected of rabies infection are only tested with anti-rabies virus antibody and are not examined histopathologically for lesions of spongiform encephalopathy. We are presently pursuing additional studies to further examine the possible involvement of cattle in the epidemiology of TME. One of these is the backpassage of our experimental bovine encephalopathy to mink. Because (here are as yet no agent-specific proteins or nucleic acids identified for these transmissible neuropathogens, one means of distinguishing them is by animal passage and selection of the biotype which grows best in a particular host. This procedure has been used to separate hamster-adapted and mink-udapted TME agents (Marsh and Hanson, 1979). The intracerebral backpassage of the experimental bovine agent resulted in incubations of only four months indicating no de-adaptation of the Stetsonville agent for mink after bovine passage. Mink fed infected bovine brain remain normal after six months. It will be essential to demonstrate oral transmission from bovine to mink if this proposed epidemiologic association is to be confirmed.

ACKNOWLEDGEMENTS These studies were supported by the College of Agricultural and Life Sciences, University of Wisconsin-Madison and by a grant (85-CRCR-1-1812) from the United States Department of Agriculture. The authors also wish to acknowledge the help and encouragement of Robert Hanson who died during the course of these investigations.

REFERENCES Burger, D. and Hartsough, G.R. 1965. Encephalopathy of mink. II. Experimental and natural transmission. J. Infec. Dis. 115:393-399. Hanson, R.P., Eckroade, R.3., Marsh, R.F., ZuRhein, C.M., Kanitz, C.L. and Gustatson, D.P. 1971. Susceptibility of mink to sheep scrapie. Science 172:859-861. Hansough, G.R. and Burger, D. 1965. Encephalopathy of mink. I. Epizoociologic and clinical observations. 3. Infec. Dis. 115:387-392. Marsh, R.F. and Hanson, R.P. 1969. Physical and chemical properties of the transmissible mink encephalopathy agent. 3. ViroL 3:176-180. Marsh, R.F. and Hanson, R.P. 1979. On the origin of transmissible mink encephalopathy. In Hadlow, W.J. and Prusiner, S.P. (eds.) Slow transmissible diseases of the nervous system. Vol. 1, Academic Press, New York, pp 451-460. Marsh, R.F. and Hartsough, G.R. 1986. Is there a scrapie-like disease in cattle? Proceedings of the Seventh Annual Western Conference for Food Animal Veterinary Medicine. University of Arizona, pp 20. Wells, G.A.H., Scott, A.C., Johnson, C.T., Cunning, R.F., Hancock, R.D., Jeffrey, M., Dawson, M. and Bradley, R. 1987. A novel progressive spongiform encephalopathy in cattle. Vet. Rec. 121:419-420.


Is there a Scrapie-like disease in cattle in USA


Date: Fri, 16 May 2003 11:47:37 0500 EMC 1 Terry S. Singeltary Sr. Vol #: 1



http://madcowfeed.blogspot.com/2008/07/docket-03d-0186-fda-issues-draft.html

 ''Our own ongoing studies in WTD demonstrate that oral doses of 1mg and 300ng brain or 300ng saliva equivalent contain sufficient infectivity to initiate CWD infection (doses are 3–9 logs lower than our previous experimental exposures; manuscripts in preparation).''

***> These findings support oral exposure as a natural route of CWD infection in deer

***> 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.



cwd scrapie pigs oral routes

***> However, at 51 months of incubation or greater, 5 animals were positive by one or more diagnostic methods. Furthermore, positive bioassay results were obtained from all inoculated groups (oral and intracranial; market weight and end of study) suggesting that swine are potential hosts for the agent of scrapie. <*** 

 >*** Although the current U.S. feed ban is based on keeping tissues from TSE infected cattle from contaminating animal feed, swine rations in the U.S. could contain animal derived components including materials from scrapie infected sheep and goats. These results indicating the susceptibility of pigs to sheep scrapie, coupled with the limitations of the current feed ban, indicates that a revision of the feed ban may be necessary to protect swine production and potentially human health. <*** 

***> 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 5="" 6="" at="" by="" detected="" eia.="" examined="" group="" in="" intracranial="" least="" lymphoid="" month="" months="" of="" one="" pigs="" positive="" prpsc="" quic="" the="" tissues="" was="">6 months group, 5/6 pigs in the oral <6 4="" and="" group="" months="" 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. 




TUESDAY, APRIL 18, 2017 

*** EXTREME USA FDA PART 589 TSE PRION FEED LOOP HOLE STILL EXIST, AND PRICE OF POKER GOES UP ***


FRIDAY, APRIL 22, 2016 

Texas Scrapie Confirmed in a Hartley County Sheep where CWD was detected in a Mule Deer April 22, 2016


THURSDAY, JUNE 09, 2016


Scrapie Field Trial Experiments Mission, Texas, The Moore Air Force Base Scrapie TSE Prion Experiment 1964

Scrapie Field Trial Experiments Mission, Texas, The Moore Air Force Base Scrapie Experiment 1964

How Did CWD Get Way Down In Medina County, Texas?

Confucius ponders...

Could the Scrapie experiments back around 1964 at Moore Air Force near Mission, Texas, could this area have been ground zero for CWD TSE Prion (besides the CWD cases that have waltzed across the Texas, New Mexico border near WSMR Trans Pecos region since around 2001)?

Epidemiology of Scrapie in the United States 1977

snip...

Scrapie Field Trial Experiments Mission, Texas

A Scrapie Field Trial was developed at Mission, Texas, to provide additional information for the eradication program on the epidemiology of natural scrapie. The Mission Field Trial Station is located on 450 acres of pastureland, part of the former Moore Air Force Base, near Mission, Texas. It was designed to bring previously exposed, and later also unexposed, sheep or goats to the Station and maintain and breed them under close observation for extended periods to determine which animals would develop scrapie and define more closely the natural spread and other epidemiological aspects of the disease.

The 547 previously exposed sheep brought to the Mission Station beginning in 1964 were of the Cheviot, Hampshire, Montadale, or Suffolk breeds. They were purchased as field outbreaks occurred, and represented 21 bloodlines in which scrapie had been diagnosed. Upon arrival at the Station, the sheep were maintained on pasture, with supplemental feeding as necessary. The station was divided into 2 areas: (1) a series of pastures and-pens occupied by male animals only, and (2) a series of pastures and pens occupied by females and young progeny of both sexes. ...

snip...see full text ;


Mission, Texas Scrapie transmission to cattle study 

Wilbur Clarke (reference the Mission, Texas scrapie transmission transmission to cattle study) is now the State Veterinarian for Montana based at Helena. 

I was given confidential access to sections from the Clarke scrapie-cattle transmission experiment. Details of the experimental design were as supplied previously by Dr. Wrathall (copy of relevant information appended). Only 3 animals (2 inoculated with 2nd pass Suffolk scrapie and 1 inoculated with Angora goat passaged scrapie) showed clinical signs. Clinical signs were characterised by weakness, ''a stilted hindlimb gait'', disorientation, ataxia and, terminally, lateral recumbency. The two cattle from which I examined material were inocluated at 8 months of age and developed signs 36 months pi (goat scrapie inoculum) and 49 months pi (one of the Suffolk scrapie inoculated) respectively. This latter animal was killed at 58 months of age and so the clinical duration was only 1 month. The neuropathology was somewhat different from BSE or the Stetsonville TME in cattle. Vacuolar changes were minimal, to the extent that detection REQUIRED CAREFUL SEARCHING. Conversely astrocyte hypertrophy was a widespread and prominent feature. The material requires DETAILED NEUROPATHOLOGICAL ASSESSMENT BUT WHETHER OR NOT THIS WILL BE DONE REMAINS A QUESTION. 

Transmission Studies 

Mule deer transmissions of CWD were by intracerebral inoculation and compared with natural cases {the following was written but with a single line marked through it ''first passage (by this route)}...TSS 

resulted in a more rapidly progressive clinical disease with repeated episodes of synocopy ending in coma. One control animal became affected, it is believed through contamination of inoculum (?saline). Further CWD transmissions were carried out by Dick Marsh into ferret, mink and squirrel monkey. Transmission occurred in ALL of these species with the shortest incubation period in the ferret. 

snip... 

Appendix 3 

VISIT TO USA - DR A E WRATHALL - INFO ON BSE AND SCRAPIE 

1. Dr Clark lately of the Scrapie Research Unit, Mission Texas has successfully transmitted ovine and caprine scrapie to cattle. The experimental results have not been published but there are plans to do this. This work was initiated in 1978. 

A summary of it is:- 

Expt A 

6 Her x Jer calves born in 1978 were inoculated as follows with 

a 2nd Suffolk scrapie passage:- 

i/c 1ml; i/m, 5ml; s/c 5ml; oral 30ml. 

1/6 went down after 48 months with a scrapie/BSE-like disease. 

Expt B 

6 Her or Jer or HxJ calves were inoculated with angora Goat 

virus 2/6 went down similarly after 36 months. 

Expt C 

Mice inoculated from brains of calves/cattle in expts A • B were resistant, only 1/20 going down with scrapie and this was the reason given for not publishing. 

Diagnosis in A, B, C was by histopath. No reports on SAT were given. 

2. Dr Warren Foote indicated success so far in eliminating scrapie in offspring from experimentally- (and naturally) infected sheep by ET. He had found difficulty in obtaining embryos from naturally infected sheep (cf SPA). 

3. Prof. A Robertson gave a brief account of BSE. 

The US approach was to accord it a very low profile indeed. 

Dr A Thiermann showed the picture in the "Independent" with cattle being incinerated and thought this was a fanatical incident to be avoided in the US at all costs. BSE was not reported in USA. 

4. Scrapie incidents (ie affected flocks) have shown a dramatic increase since 1978. 

In 1953 when the National Control Scheme was started there were 10-14 incidents, in 1978 - 1 and in 1988 so far 60. 

5. Scrapie agent was reported to have been isolated from a solitary fetus. 

6. A western blotting diagnostic technique (? on PrP) shows some promise. 

7. Results of a questionnaire sent to 33 states on the subject of the national sheep scrapie programme survey indicated 

17/33 wished to drop it 

6/33 wished to develop it 

9/13/2005 33 Page 15 of 17 

8/33 had few sheep and were neutral Information obtained from Dr Wrathall's notes of a meeting of the U.S. Animal Health Association at Little Rock, Arkansas Nov. 1988. end...TSS 



Spongiform Encephalopathy in Captive Wild ZOO BSE INQUIRY


Mission, Texas Scrapie transmission to cattle study


THURSDAY, DECEMBER 19, 2019 

The emergence of classical BSE from atypical/Nor98 scrapie


MONDAY, DECEMBER 09, 2019 

Preclinical Detection of Prions in Blood of Nonhuman Primates Infected with Variant Creutzfeldt-Jakob Disease


Saturday, November 23, 2019 

Prion disease incidence in the United States, 2003–2015


THURSDAY, DECEMBER 12, 2019 

Heidenhain Variant Creutzfeldt Jakob Disease hvCJD, sporadic spontaneous CJD and the TSE Prion December 14, 2019

22 years, rip mom dod 12/14/97 confirmed hvcjd, just made a promise to mom, and you don't break those promises, never forget, and never let them forget, before we all do...this pearl's for you! love terry


Terry S. Singeltary Sr.

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