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From: TSS (216-119-129-92.ipset9.wt.net)
Subject: Transmission of Prions from Mule Deer and Elk with Chronic Wasting [FULL TEXT]
Date: November 16, 2004 at 2:45 pm PST

-------- Original Message --------
Subject: Transmission of Prions from Mule Deer and Elk with Chronic Wasting [FULL TEXT]
Date: Tue, 16 Nov 2004 16:50:57 -0600
From: "Terry S. Singeltary Sr."
Reply-To: Bovine Spongiform Encephalopathy
To: BSE-L@UNI-KARLSRUHE.DE


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

JOURNAL OF VIROLOGY, Dec. 2004, p. 1334513350 Vol. 78, No. 23
0022-538X/04/$08.000 DOI: 10.1128/JVI.78.23.1334513350.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
NOTES
Transmission of Prions from Mule Deer and Elk with Chronic Wasting
Disease to Transgenic Mice Expressing Cervid PrP
Shawn R. Browning,1 Gary L. Mason,2 Tanya Seward,3 Mike Green,4 Gwyneth
A. J. Eliason,5
Candace Mathiason,5 Michael W. Miller,6 Elizabeth S. Williams,7 Ed
Hoover,5 and
Glenn C. Telling1,3,8*
Department of Microbiology, Immunology and Molecular Genetics,1 Sanders
Brown Center on Aging,3 Department of
Neurology,8 and University of Kentucky Transgenic Facility,4 University
of Kentucky, Lexington, Kentucky; Veterinary
Diagnostic Laboratory2 and Department of Microbiology, Immunology and
Pathology,5 Colorado State
University, and Colorado Division of Wildlife, Wildlife Research
Center,6 Fort Collins, Colorado;
and Department of Veterinary Sciences, University of Wyoming, Laramie,
Wyoming7
Received 6 May 2004/Accepted 3 August 2004
We generated mice expressing cervid prion protein to produce a
transgenic system simulating chronic
wasting disease (CWD) in deer and elk. While normal mice were resistant
to CWD, these transgenic mice
uniformly developed signs of neurological dysfunction 230 days
following intracerebral inoculation with four
CWD isolates. Inoculated transgenic mice homozygous for the transgene
array developed disease after 160
days. The brains of sick transgenic mice exhibited widespread spongiform
degeneration and contained abnormal
prion protein and abundant amyloid plaques, many of which were florid
plaques. Transmission studies
indicated that the same prion strain caused CWD in the analyzed mule
deer and elk. These mice provide a new
and reliable tool for detecting CWD prions.
The transmissible spongiform encephalopathies (TSEs), are
fatal neurodegenerative conditions which include human
Creutzfeldt-Jakob disease (CJD), scrapie of sheep and goats,
and bovine spongiform encephalopathy (BSE). Because of
their extraordinary biology and the unique properties of the
infectious agent, these diseases attracted interest well before
the advent and epizootic spread of BSE (40) and the subsequent
appearance of a variant of CJD (vCJD) (41). The time
between infection and disease is extremely long, and for this
reason, these diseases were originally thought to be caused by
slow viruses. However, while the molecular structure of the
agent still eludes definitive identification, it is widely accepted
that these diseases are caused by prions, which are defined as
proteinaceous infectious particles that lack informational nucleic
acid (25). Considerable evidence suggests that prions
consist largely, if not exclusively, of a disease-associated version
of the prion protein (PrP). This isoform, referred to as
PrPSc, is an abnormally folded, protease-resistant, -sheet-rich
version of a normally benign cellular protein referred to as
PrPC, which is protease sensitive and rich in -helix. According
to the prion hypothesis, the central event in the propagation of
prion infectivity is the coercion of cellular prion protein by
PrPSc to adopt the disease-associated conformation.
The various prion diseases share a number of characteristic
features, the most consistent being the neuropathologic
changes that accompany disease in the central nervous system
(CNS). These include neuronal vacuolation and degeneration,
which confer a spongiform appearance upon the cerebral grey
matter, and a reactive proliferation of astroglial cells. The lack
of an inflammatory response is also an important trait. While
by no means a constant feature, some examples of prion disease
are characterized by the deposition of amyloid plaques
composed of insoluble aggregates of PrP.
Another important aspect of prion diseases is their transmissibility.
Inoculation of diseased brain material into individuals
of the same species will typically reproduce the disease. In
contrast, the passage of prions from one species to another is
generally inefficient and is referred to as the species barrier.
Expression of foreign and chimeric prion protein genes in
transgenic (Tg) mice has been an effective way to probe the
molecular basis of the species barrier (30, 31, 33, 37, 38).
Experiments in Tg mice demonstrated that the degree of homology
between PrP molecules in the host and inoculum was
an important determinant of the species barrier (26).
An equally important component affecting prion transmission
barriers is the strain of prion. Mammalian prion strains
are classically defined in terms of their incubation times in
susceptible animals and the profile of lesions they produce in
the CNS. Differences in the neuroanatomic distribution of
PrPSc are a parameter that has also been used to define prion
strains (11, 15, 18, 19). More recently attempts have been made
to use biochemical and/or immunological properties of PrPSc
as markers of prion strain differences (6, 24, 28, 36). Seminal
studies suggesting that PrPSc conformation was the basis of
prion strain diversity arose from investigations of transmissible
* Corresponding author. Mailing address: 332 Health Sciences Research
Building, University of Kentucky, 800 Rose St., Lexington, KY
40536. Phone: (859) 323-8564. Fax: (859) 257-6151. E-mail: gtell2
@uky.edu.
13345
mink encephalopathy, which, upon transmission, produced different
clinical symptoms and produced PrPSc with different
resistances to proteinase K digestion and altered amino-terminal
proteinase K cleavage sites (3). Evidence supporting the
concept that strain diversity is encoded in the tertiary structure
of PrPSc emerged from transmission studies of inherited and
sporadic human prion diseases in Tg mice (15, 17, 36). Banding
patterns of PrPSc forms with different glycosylation patterns
and sizes of PrPSc fragments following proteinase K treatment
have also been used to classify CJD strains (6, 12, 23).
Of all the prion diseases, chronic wasting disease (CWD) is
perhaps the least understood. CWD was first recognized as a
spongiform encephalopathy in captive mule deer (Odocoileus
hemionus hemionus) in north central Colorado in 1978 (42)
and subsequently was diagnosed in free-ranging deer and
Rocky Mountain elk (Cervus elaphus nelsoni) in southeastern
Wyoming and northeastern Colorado. The origins of the disease
are obscure, and, like scrapie in sheep, the natural route
of CWD transmission remains unknown. CWD was recently
detected in free-ranging white-tailed deer (Odocoileus virginianus)
in Wisconsin (14) and Illinois. Whether CWD in mule
deer, white-tailed deer, and elk is caused by the same or different
prion strains is unknown; whether different CWD prion
strains cause disease in captive and free-ranging mule deer and
elk in the original areas of endemicity in northern Colorado as
well as other areas of North America is also unknown.
While CWD is transmissible after intracerebral inoculation
of mule deer with incubation periods of up to 2 years (44),
experimental transmission of CWD to other species has had
mixed results. The inefficient primary transmission of CWD
prions to mice (M. Bruce, Neuropathogenesis Unit, Edinburgh,
personal communication) and to ferrets (2) is an example
of the species barrier. Based on previous studies demonstrating
that expression of foreign PrP in Tg mice is an
extremely efficient means of abrogating prion species barriers
(4, 5, 7, 30, 31, 3739), we hypothesized that expression of
cervid PrP (CerPrP) in Tg mice would eliminate the barrier to
CWD prion transmission, resulting in CWD susceptibility simulating
that in cervids. To produce Tg(CerPrP) mice, the open
reading frame (ORF) cassette of the CerPrP S2 allele (Gen-
Bank accession no.AF009180) was released from plasmid sequences
following digestion with SalI and XhoI and purified
ORF fragments were ligated to the SalI-cut cosSHa.Tet cosmid
expression vector. The cosSHa.Tet cosmid expression vector
contains a 49-kb DNA fragment encompassing the Syrian
hamster PrP gene (32) and has been used to produce numerous
Tg models of prion diseases (35), including mice in which
the species barriers to Syrian hamster, human, and bovine
prions are eliminated (1, 26, 30, 33, 37, 38). To increase CerPrP
expression in Tg mice, we modified the CerPrP S2 allele plasmid
nucleotide sequence by site-directed mutagenesis immediately
upstream of the initiating ATG to produce a consensus
Kozak translation initiation sequence. The isolation of recombinant
cosmid clones and production of Tg mice were achieved
by previously described methods (32). Two founders were generated
by microinjection of fertilized embryos from Prnp0/0
knockout mice on an FVB/N background (FVB/Prnp0/0). Brain
PrP expression was estimated by comparing serially diluted
brain extracts of F1 Tg mice and wild-type mice followed by
immuno-dot blotting or Western blotting with the monoclonal
antibody 6H4 (Prionics AG, Schlieren). By this approach, the
levels of CerPrP expression in brain extracts of Tg(CerPrP)
1536/ and Tg(CerPrP)1534/ mice, both hemizygous for
the transgene array, were estimated to be five- and threefold
higher, respectively, than the level of wild-type PrP expression
in FVB mice. Analysis of PrP expression in Tg mice by Western
blotting of extracts from brain, lung, spleen, muscle, liver,
kidney, and heart using monoclonal antibody 6H4 showed that
the cosSHa.Tet cosmid expression vector directed expression
exclusively to the CNS (data not shown).
Groups of Tg(CerPrP)1536/ mice were intracerebrally inoculated
with 30 l of 1% homogenate prepared in phosphatebuffered
saline (PBS) of a pooled collection of infected brains
from CWD-affected mule deer held captive at the Colorado
Division of Wildlife, Wildlife Research Center. We also compared
the transmission of CWD isolates from individual captive
mule deer and elk in Tg(CerPrP)1536/ mice. Samples
D10 and Db99 refer to captive mule deer does that developed
CWD at the Colorado Division of Wildlife, Wildlife Research
Center, and sample 7378 refers to an adult female captive elk
with natural clinical CWD from the Wyoming Game and Fish
Departments Sybille Wildlife Research Unit, Wheatland,
Wyo. Inoculated Tg(CerPrP)1536/ mice developed signs of
prion disease between 220 and 270 days after inoculation, and
the average incubation periods produced by all three CWD
isolates and the CWD pool were similar (Table 1). By mating
Tg(CerPrP)1536/ mice to each other, we produced offspring,
designated Tg(CerPrP)1536/, that were homozygous
for the CerPrP transgene array, which resulted in a doubling of
the level of expression of CerPrPC. Tg(CerPrP)1536/ mice
developed signs of prion disease between 153 and 169 days
after inoculation with CWD isolate D10 (Table 1). The pooled
CWD inoculum produced disease in the inoculated Tg(CerPrP)
1534/ mice between 261 and 273 days (Table 1). The neurologic
signs that accompanied prion disease in sick Tg mice
included truncal ataxia and slowed movement, increased tone
of the tail, dorsal kyphosis, head bobbing or tilting and roughened
coat. At the time of writing, Tg(CerPrP)1536/ mice
inoculated with PBS or the Rocky Mountain Laboratory
(RML) strain of mouse-adapted scrapie prions have not shown
signs of neurological dysfunction 360 and 380 days postinoculation,
respectively. Wild-type mice inoculated with the
CWD pool also failed to develop signs of neurological dysfunction
600 days postinoculation.
TABLE 1. Transmission of CWD prions to Tg(CerPrP) mice
Inoculum Species Recipient Mean  SE incubation
time in daysa
CWD pool Mule deer Tg(CerPrP)1536/ 264  3 (7/7)
Db99 Mule deer Tg(CerPrP)1536/ 259  4 (7/7)
D10 Mule deer Tg(CerPrP)1536/ 225  1 (8/8)
7378 Elk Tg(CerPrP)1536/ 235  2 (8/8)
RML Mouse-adapted
scrapie
Tg(CerPrP)1536/ 385 (0/8)
PBS Tg(CerPrP)1536/ 360 (0/8)
D10 Mule deer Tg(CerPrP)1536/ 160  3 (7/7)
CWD pool Mule deer Tg(CerPrP)1534/ 268  2 (10/10)
PBS Tg(CerPrP)1534/ 300 (0/6)
CWD pool Mule deer Wild-type mice 596 (0/7)
a The number of mice developing clinical signs of prion disease divided
by the
original number of inoculated mice is shown in parentheses.
13346 NOTES J. VIROL.
Histopathologic findings were similar for all four inocula
and included multiple to coalescing foci of spongiform degeneration
of the perikaryon and neuropil. Foci of degeneration
were often severe, with a central focus of pale eosinophilic
reticulated material surrounded by vacuoles. Neurons adjacent
to foci of spongiform change often had shrunken scalloped
hyperchromatic nuclei. While spongiform change was widespread
in the brain, there was striking and severe vacuolation
of the hippocampus (Fig. 1A and B), piriform cortex, and
parenchyma adjacent to the ventricular and aqueduct system
throughout the brain. In all brains, spongiform degeneration
was present in many nuclei in the subcerebellar white matter
and brain stem. Patchy foci of degeneration were often
present in the middle lamina of the neocortex, within the
granular layer of the cerebellar cortex and within the olfactory
bulb. Amyloid plaque pathology, long recognized as a
pathognomonic feature in cervids with CWD (9, 10, 43), was
dramatically reproduced in Tg mice (Fig. 1C and D). All foci
of spongiform change had strong positive immunostaining
(Fig. 1C and D), often with large central stained plaques
FIG. 1. Neuropathology of Tg(CerPrP)1536/ mice inoculated with CWD
prions. Brains of sick animals from each study group were dissected
rapidly after sacrifice and immersion fixed in 10% buffered
paraformaldehyde. Tissue was embedded in paraffin, and sections were
prepared and
stained with hematoxylin and eosin for evaluation of spongiform
degeneration. (A and B) Hematoxylin-and-eosin staining of sections
through the
hippocampus of Tg(CerPrP)1536/ mice inoculated with brain tissue from
CWD-affected mule deer D10 showing spongiform degeneration.
Panel B is a higher magnification of an area in panel A. Note shrunken,
scalloped neuronal nuclei adjacent to foci of spongiform change. (C and
D) Immunohistochemistry of an adjacent section from the same
inoculated Tg(CerPrP)1536/ mouse showing amyloid plaque deposits. Panel D
is a magnification of the area indicated in panel C. Note large
immunoreactive plaques bordered by vacuoles. Slides were deparaffinized and
hydrated followed by immersion in 88% formic acid solution, treatment
with 25-mg/ml proteinase-K solution at 26°C for 10 min, followed by
autoclaving for 20 min at 121°C in Tris-buffered solution. Tissue
preparations were stained with anti-PrP polyclonal antibody R505 (8),
followed
by anti-rabbit immunoglobulin G-biotinylated secondary antibody
streptavidin conjugated to alkaline phosphatase, and then developed with
Fast
Red A, naphthol, and Fast Red B chromogen. Hematoxylin was used as
counterstain. Bar 100 m in all cases.
VOL. 78, 2004 NOTES 13347
partly bordered or surrounded by nonstaining vacuoles.
Such florid PrP plaque pathology has also been recognized
as a neuropathologic feature of CWD in mule deer (16).
Sham-inoculated mice analyzed in parallel had no histologic
lesions or positive immunostaining; neither was immunostaining
identified in CWD-positive deer brain or CWDinoculated
Tg mice when an irrelevant primary antibody was
used and when no primary antibody was applied (data not
shown). Brain tissue from a CWD-positive deer had excellent
positive immunostaining with the protocol used (data
not shown).
Biochemical analysis of prion proteins in brain extracts from
clinically sick Tg mice showed that protease-resistant PrPSc was
present in all inoculated groups. The diglycosylated form of
PrPSc predominated in the brains of sick Tg(CerPrP)1536/
mice (Fig. 2A). A similar PrPSc glycosylation pattern has been
observed in previous analyses of CWD-affected deer and elk
(27). Comparison of PrPSc profiles in brain extracts of sick
Tg(CerPrP)1536/ mice showed that the molecular weight
and glycosylation pattern of PrPSc were consistent among all
inoculated groups. However, while the amounts of diglycosylated
and unglycosylated PrPSc in CWD-affected cervids and
CWD-affected Tg(CerPrP)1536/ mice remained constant,
the amount of monoglycosylated PrPSc was consistently lower
following transmission of Db99, D10, and 7378 brain extracts
to Tg(CerPrP)1536/ mice (Fig. 2B). Similar differences in
glycoform ratios of the same prion strain propagated in mice
and human brain have been observed previously (12).
The neuroanatomic distribution of PrPSc was assessed by
histoblotting as described previously (34). The most notable
feature of histoblotted Tg(CerPrP)1536/ mouse brains inoculated
with CWD prions from D10, Db99 mule deer, and
7378 elk was the widespread punctate deposition of PrPSc (Fig.
3), which likely corresponds to the PrPSc-containing plaques
detected by immunohistochemistry (Fig. 1). The concordant
patterns of PrPSc deposition in coronal sections of Tg(CerPrP)
1536/ mice inoculated with prions from the D10 CWDpositive
mule deer and the 7478 CWD-positive elk, along with
the similar incubation times, histopathologic findings, and biochemical
properties of PrPSc, indicate that the same CWD
prion strain caused disease in these analyzed mule deer and
elk. Although the incubation time in Tg(CerPrP)1536/ mice
of the Db99 CWD mule deer isolate was similar to that of the
D10/7378 strain, the difference in the neuroanatomic distribution
of PrPSc in Db99-inoculated Tg(CerPrP)1536/ mice
(Fig. 3) suggests that a different prion strain caused CWD in
the Db99-infected mule deer. Additional passaging studies are
required to further characterize the strain properties of these
CWD isolates.
The simulation of CWD in deer and elk following transmission
to Tg(CerPrP) mice represents a breakthrough in CWD
research. Tg(CerPrP) mice should find broad use in the future
to study the biology of CWD prions and CWD pathogenesis.
There is currently no quantitative information available regarding
the infectivity of any CWD prion preparations, and
Tg(CerPrP) mice promise to be a reliable experimental host in
which to bioassay CWD prions. Using Tg(CerPrP) mice, it will
be possible to expand these preliminary investigations of CWD
prion strain prevalence in captive and wild populations of mule
deer, white-tailed deer, and Rocky Mountain elk and to assess
the effect of cervid PrP polymorphisms on CWD susceptibility
(13, 22). In the long term, it should also be possible to gain
FIG. 2. Western blots of PrP in brains from Tg(CerPrP)1536/ mice
inoculated with prions from mule deer and elk with CWD. (A) The brains
of Tg(CerPrP)1536/ mice inoculated with D10, 7378, Db99, and the CWD
pool were analyzed for the presence of protease-resistant PrPSc. Brain
extracts of three individual brains from each inoculated group were
treated () or not treated () with 40-g/ml proteinase K (PK) in the
presence
of 2% Sarkosyl for 1 h at 37°C. Uninoc., uninoculated. In panel B, PrPSc
in brain homogenates of Tg(CerPrP)1536/ mice was directly compared
with the corresponding CWD inocula from deer and elk. Immunoblots were
probed with recombinant Fab Hum-P, which recognizes an epitope
on PrP between amino acid residues 96 and 105 (29). The positions of
protein molecular mass markers at 28.7 and 21.3 kDa (from top to bottom)
are shown to the left of the immunoblots.
13348 NOTES J. VIROL.
insights into the origins and mode of transmission of CWD
using Tg(CerPrP) mice. Efficient horizontal rather than maternal
transmission has been shown to be important in sustaining
CWD epidemics (21). The most plausible natural routes of
CWD transmission are via ingestion of forage or water contaminated
by secretions, excretions, or other sources of
agentfor example, carcasses (20). Using CWD-susceptible
Tg(CerPrP) mice, it will be possible to bioassay CWD prions in
blood and other tissues, body fluids, and secretions of deer and
elk that may provide insights into the mode of transmission of
CWD and ultimately lead to better disease control in wild
cervids.
This work was supported in part by grants from the U.S. Public
Health Service RO1 NS/AI40334 from the National Institute of Neurological
Disorders and Stroke and N01-AI-25491 from the National
Institute of Allergy and Infectious Diseases.
We thank Katherine O Rourke, U.S. Department of Agriculture,
Agricultural Research Service, Animal Disease Research Unit, 3003
ADBF, Pullman, Wash., for supplying the S2 mule deer allele, and
Stanley Prusiner, Institute for Neurodegenerative Diseases, Department
of Neurology, University of California, San Francisco, for supplying
Prnp0/0 knockout mice and the cosSHa.Tet vector.
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24. Peretz, D., R. A. Williamson, G. Legname, Y. Matsunaga, J. Vergara,
D. R.
Burton, S. J. DeArmond, S. B. Prusiner, and M. R. Scott. 2002. A change in
the conformation of prions accompanies the emergence of a new prion
strain. Neuron 34:921932.
25. Prusiner, S. B. 1982. Novel proteinaceous infectious particles cause
scrapie.
Science 216:136144.
26. Prusiner, S. B., M. Scott, D. Foster, K.-M. Pan, D. Groth, C.
Mirenda, M.
Torchia, S.-L. Yang, D. Serban, G. A. Carlson, P. C. Hoppe, D. Westaway,
and S. J. DeArmond. 1990. Transgenetic studies implicate interactions
between
homologous PrP isoforms in scrapie prion replication. Cell 63:673
686.
27. Race, R. E., A. Raines, T. G. M. Baron, M. W. Miller, A. Jenny, and
E. S.
Williams. 2002. Comparison of abnormal prion protein glycoform patterns
from transmissible spongiform encephalopathy agent-infected deer, elk,
sheep, and cattle. J. Virol. 76:1236512368.
28. Safar, J., H. Wille, V. Itri, D. Groth, H. Serban, M. Torchia, F. E.
Cohen, and
S. B. Prusiner. 1998. Eight prion strains have PrPSc molecules with
different
conformations. Nat. Med. 4:11571165.
29. Safar, J. G., M. Scott, J. Monaghan, C. Deering, S. Didorenko, J.
Vergara,
H. Ball, G. Legname, E. Leclerc, L. Solforosi, H. Serban, D. Groth, D. R.
Burton, S. B. Prusiner, and R. A. Williamson. 2002. Measuring prions causing
bovine spongiform encephalopathy or chronic wasting disease by immunoassays
and transgenic mice. Nat. Biotechnol. 20:11471150.
30. Scott, M., D. Foster, C. Mirenda, D. Serban, F. Coufal, M. Wa¨lchli, M.
Torchia, D. Groth, G. Carlson, S. J. DeArmond, D. Westaway, and S. B.
Prusiner. 1989. Transgenic mice expressing hamster prion protein produce
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DeArmond, and
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32. Scott, M. R., R. Ko¨hler, D. Foster, and S. B. Prusiner. 1992.
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Prusiner. 1997. Identification of a prion protein epitope modulating
transmission
of bovine spongiform encephalopathy prions to transgenic mice.
Proc. Natl. Acad. Sci. USA 94:1427914284.
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35. Telling, G. C. 2000. Prion protein genes and prion diseases: studies in
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Gabizon, J. Mastrianni, E. Lugaresi, P. Gambetti, and S. B. Prusiner. 1996.
Evidence for the conformation of the pathologic isoform of the prion protein
enciphering and propagating prion diversity. Science 274:20792082.
37. Telling, G. C., M. Scott, K. K. Hsiao, D. Foster, S. L. Yang, M.
Torchia, K. C.
Sidle, J. Collinge, S. J. DeArmond, and S. B. Prusiner. 1994.
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Creutzfeldt-Jakob disease from humans to transgenic mice expressing chimeric
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38. Telling, G. C., M. Scott, J. Mastrianni, R. Gabizon, M. Torchia, F.
E. Cohen,
S. J. DeArmond, and S. B. Prusiner. 1995. Prion propagation in mice
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human and chimeric PrP transgenes implicates the interaction of
cellular PrP with another protein. Cell 83:7990.
39. Vilotte, J.-L., S. Soulier, R. Essalmani, M.-G. Stinnakre, D. Vaiman, L.
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Groschup, S. Petit, M.-F. Madelaine, S. Rakatobe, A. Le Dur, D. Vilette, and
H. Laude. 2001. Markedly increased susceptibility to natural sheep
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Hancock,
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13350 NOTES J. VIROL.
==================

GREETINGS,

i must comment please;

> The most plausible natural routes of
> CWD transmission are via ingestion of forage or ...


FORAGE = Food suitable for cattle or other domestic animals,
TAINTED WITH TSE AGENT daaa...TSS

PRODUCT
Product is custom made deer feed packaged in 100 lb. poly bags. The
product has no labeling. Recall # V-003-5.
CODE
The product has no lot code. All custom made feed purchased between June
24, 2004 and September 8, 2004.
RECALLING FIRM/MANUFACTURER
Farmers Elevator Co, Houston, OH, by telephone and letter dated
September 27, 2004. Firm initiated recall is ongoing.
REASON
Feed may contain protein derived from mammalian tissues which is
prohibited in ruminant feed.
VOLUME OF PRODUCT IN COMMERCE
Approximately 6 tons.
DISTRIBUTION
OH.

END OF ENFORCEMENT REPORT FOR October 20, 2004

============================================

-------- 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

snip...
======================================

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...

http://vir.sgmjournals.org/cgi/content/full/80/10/2757
===================================

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
===================================

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
===================================================

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
==================================

snip...END

J Infect Dis 1980 Aug;142(2):205-8


Oral transmission of kuru, Creutzfeldt-Jakob disease, and scrapie to
nonhuman primates.

Gibbs CJ Jr, Amyx HL, Bacote A, Masters CL, Gajdusek DC.

Kuru and Creutzfeldt-Jakob disease of humans and scrapie disease of
sheep and goats were transmitted to squirrel monkeys (Saimiri
sciureus) that were exposed to the infectious agents only by their
nonforced consumption of known infectious tissues. The asymptomatic
incubation period in the one monkey exposed to the virus of kuru was
36 months; that in the two monkeys exposed to the virus of
Creutzfeldt-Jakob disease was 23 and 27 months, respectively; and
that in the two monkeys exposed to the virus of scrapie was 25 and
32 months, respectively. Careful physical examination of the buccal
cavities of all of the monkeys failed to reveal signs or oral
lesions. One additional monkey similarly exposed to kuru has
remained asymptomatic during the 39 months that it has been under
observation.

PMID: 6997404

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6997404&dopt=Abstract

TSS

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