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From: TSS (
Subject: Re: Prion protein NMR structures of cats, dogs, pigs, and sheep
Date: January 18, 2005 at 12:10 pm PST

In Reply to: Prion protein NMR structures of cats, dogs, pigs, and sheep posted by TSS on January 17, 2005 at 1:07 pm:

Prion protein NMR structures of cats, dogs, pigs,
and sheep

Dominikus A. Lysek, Christian Schorn*, Lucas G. Nivon†, Vicent Esteve-Moya‡, Barbara Christen, Luigi Calzolai§,
Christine von Schroetter, Francesco Fiorito, Torsten Herrmann, Peter Gu¨ ntert¶, and Kurt Wu¨ thrich
Institut fu¨ r Molekularbiologie und Biophysik, Eidgeno¨ ssische Technische Hochschule-Zu¨ rich, CH-8093 Zu¨ rich, Switzerland

Contributed by Kurt Wu¨ thrich, December 6, 2004

The NMR structures of the recombinant cellular form of the prion
proteins (PrPC) of the cat (Felis catus), dog (Canis familiaris), and pig
(Sus scrofa), and of two polymorphic forms of the prion protein
from sheep (Ovis aries) are presented. In all of these species, PrPC
consists of an N-terminal flexibly extended tail with 100 amino
acid residues and a C-terminal globular domain of 100 residues
with three -helices and a short antiparallel -sheet. Although this
global architecture coincides with the previously reported murine,
Syrian hamster, bovine, and human PrPC structures, there are local
differences between the globular domains of the different species.
Because the five newly determined PrPC structures originate from
species with widely different transmissible spongiform encephalopathy
records, the present data indicate previously uncharacterized
possible correlations between local features in PrPC threedimensional
structures and susceptibility of different mammalian
species to transmissible spongiform encephalopathies.
mammalian species  feline transmissible spongiform encephalopathy 
The prion protein (PrP) in mammalian organisms has attracted
keen interest because of its relation to a group of
invariably fatal neurodegenerative diseases, the transmissible
spongiform encephalopathies (TSEs) or ‘‘prion diseases,’’ which
include bovine spongiform encephalopathy (BSE), Creutzfeldt–
Jakob disease in humans, feline spongiformencephalopathy, and
scrapie in sheep. It is well established that expression of the
host-encoded PrP is essential for TSE propagation (1, 2). In
transgenic mice lacking the gene that encodes PrP, TSEs could
not be observed, and the susceptibility toward TSE of these mice
could only be restored by reestablishing PrP expression (3). High
sequence conservation of PrP in mammalian species (4) indicates
that this protein is functionally important in the healthy
organism (1, 2), but the search for this unknown function is still
PrP was identified in the context of TSEs in an aggregated
‘‘scrapie’’ isoform of PrP (PrPSc) (5), which copurifies with the
infective agent (6). This osbservation, the apparent stability of
the infectious agent under DNARNA denaturing conditions
(7), and the unusual progression of the disease (8) led to the
‘‘protein-only hypothesis.’’ This hypothesis proposes that the
major component, if not the only one, of the infectious particle
causing TSE is a protein, i.e., presumably PrPSc (1, 7–9).
An early observation in TSE infections has been the species
barrier (10). Compared with transmission with infectious material
from the same species, the incubation time for onset of TSEs
is prolonged if a given species is challenged with infectious brain
homogenate originating from another species. The incubation
time may be reduced by consecutive passages within the new
host, whereby the adaptation to the new host can take several
generations for the disease to show clinical signs (11). In vivo and
in vitro experiments indicated that the species barrier for infectious
transmission of TSEs is somehow related to the extent of
PrP sequence homology between the species involved (12, 13)
(Fig. 1). Following the protein-only hypothesis, the compatibility
of the PrPs from the originating species to the new host should
actually be a decisive factor for the propagation of the disease
because the covalent structure of PrP in the PrPSc form is
assumed to be identical to that in the cellular isoform of PrP
(PrPC) present in healthy organisms (1). Overall, however,
inspection of the amino acid sequence of PrP has not been
conclusive to even qualitatively assess either the species barrier
for TSEs between different species or the susceptibility of a given
species to TSE (4, 14–16). For example, the species barrier for
transmission of BSE to feline species seems to be readily
overcome, as indicated by numerous cases of feline spongiform
encephalopathy during the BSE crisis in the United Kingdom,
whereas there have been no reports of TSE-infected dogs (17).
These observations present an apparent contrast with the facts
that the number of amino acid exchanges between bovines and
cats or dogs is almost equal, with 14 and 13, respectively (15, 18),
and that the sequences of the dog PrP (cPrP) and cat PrP (fPrP)
differ only in four positions within the fragment 121–230 (Fig. 1).
With the aim to obtain more detailed insight into possible
correlations between PrPC structure and PrPC function in health
and disease, we started years ago an investigation of the threedimensional
structures of recombinant PrPs (19). The relevancy
of this approach has recently been substantiated by the demonstration
that recombinant PrP has the same fold as PrPC (20, 21).
The lack of the posttranslational modifications in PrP expressed
in Escherichia coli thus has at most very limited local effects on
the protein molecular architecture (21). So far, threedimensional
structures in solution have been reported for recombinant
PrPC of the widely used laboratory animals mouse
PrP (19) and Syrian hamster PrP (22), and cattle PrP (bPrP) (23)
and human PrP (hPrP) (24). A crystal structure is available for
the globular domain of a sheep PrP (ovPrP) (25). This paper now
presents the prion proteinNMRstructures of the pig (scPrP), the
dog cPrP, the cat fPrP, and two variant ovPrPs. This selection of
three-dimensional PrPC structures includes the prion protein
from a species that has so far been resistant to the challenge with
BSE-infected food in the natural environment, i.e., cPrP (26).
Abbreviations: PrP, prion protein; bPrP, bovine PrP; BSE, bovine spongiform encephalopathy;
cPrP, dog PrP; fPrP, cat PrP; hPrP, human PrP; NOE, nuclear Overhauser enhancement;
ovPrP, sheep PrP; ovPrP[H168], recombinant ovPrP with histidine at position 168;
ovPrP[R168], recombinant ovPrP with arginine at position 168; PrPC, cellular isoform of PrP;
PrPSc, scrapie isoform of PrP; scPrP, pig PrP; TSE, transmissible spongiform encephalopathy.
Data deposition: The atomic coordinates for a bundle of 20 conformers of each of the five
NMRstructures have been deposited in the Protein Data Bank, [PDB ID codes
1XYQ for scPrP(121–231), 1XYJ for fPrP(121–231), 1XYK for cPrP(121–231), 1XYU for
ovPrP[H168](121–231), and 1Y2S for ovPrP[R168](121–231)].
*Present address: GlaxoSmithKline R&D Limited, Old Powder Mills, Tonbridge TN11 9AN,
United Kingdom.
†Present address: Department of Chemistry and Chemical Biology, Harvard University,
12 Oxford Street, Cambridge, MA 02138.
‡Present address: Universitat de Valencia, Dr. Moliner 50, 46100-Burjassot, Valencia, Spain.
§Present address: Department of Biotechnology and Molecular Sciences, University of
Insubria, Via J. Dunant 3, 21100 Varese, Italy.
¶Present address: RIKEN Genomic Sciences Center, 1-7-22 Suehiro, Tsurumi, Yokohama
231-0045, Japan.
To whom correspondence should be addressed. E-mail:
© 2005 by The National Academy of Sciences of the USA
640–645  PNAS  January 18, 2005  vol. 102  no. 3 www.pnas.orgcgidoi10.1073pnas.0408937102
For scPrP, observation of neurologic disorder after challenge
with BSE-infected brain homogenate (27) has so far not been
followed up with the standard procedures that would qualify the
disease as a TSE (1).

Materials and Methods
Cloning, Expression, and Purification of the Prion Proteins. The genes
for various ovPrP polymorphisms were provided to us by Dr. A.
Bossers (Central Institute for Animal Disease Control, Lelystad,
The Netherlands), and the constructs for cPrP(residues 121–
231), cPrP(23–231), fPrP(121–231), fPrP(23–231), scPrP(121–
231), and scPrP(23–231) were cloned from total DNA. All genes
were cloned into the vector pRSETA, and the proteins were
expressed in E. coli. For the purification of the recombinant
proteins, we followed procedures described in refs. 28 and 29.
NMR Measurements and Structure Calculations. NMR measurements
were performed at 20°C on Bruker DRX500, DRX600,
DRX750, and Avance900 spectrometers. The protein samples
used were uniformly 15N-labeled and 13C,15N-labeled
scPrP(121–231), cPrP(121–231), fPrP(121–231), ovPrP with histidine
at position 168 {ovPrP[H168](121–231)}, and ovPrP with
arginine at position 168 {ovPrP[R168](121–231)} and 15Nlabeled
scPrP(23–231), cPrP(23–231), fPrP(23–231), and
ovPrP[H168](23–231). The proteins were dissolved at concentrations
of 0.5–1.0 mM either in 95% H2O5% 2H2O or 99.9%
2H2O containing 5 mM sodium acetate at pH 4.5. The programs
PROSA (30) and XEASY (31) were used for data processing and
spectral analysis, respectively. Sequence-specific resonance assignments
for the proteins were obtained by using standard
triple-resonance NMR experiments (32).
Steady-state 15N{1H}-nuclear Overhauser enhancements
(NOEs) of 15N-labeled scPrP(23–231), cPrP(23–231), fPrP(23–
231), and ovPrP[H168](121–231) were measured with recovery
delays and proton saturation periods of 4 sec (33).
Distance constraints for the structure calculations were obtained
from three-dimensional 13C-resolved [1H,1H]-NOESY
and three-dimensional 15N-resolved [1H,1H]-NOESY spectra
recorded at a proton frequency of 750 or 900 MHz with mixing
times of 40 or 50 ms. For scPrP(121–231) and ovPrP[H168](121–
231), the automatic NOE assignment module CANDID (34),
implemented in the program DYANA (35), was used for the
structure calculation. For cPrP(121–231), fPrP(121–231), and
ovPrP[R168](121–231), automatic NOE identification was
added by using the program suite ATNOS (36)CANDID (34)
DYANA (35) for the structure calculation. The program DYANA
(35) was also used to convert NOE intensities into upper-limit
distance constraints according to a sixth power peak volume-to-
Table 1. Input for the structure calculation and characterization of the energy-minimized NMR structures of scPrP(121–231),
fPrP(121–231), cPrP(121–231), ovPrP[H168](121–231), and ovPrP[R168](121–231)
scPrP fPrP cPrP ovPrP[H168] ovPrP[R168]
NOE upper distance limits 1,922 1,454 1,479 2,064 1,622
Dihedral angle constraints 110 114 122 114 94
Residual target function value, Å2 0.99  0.19 1.77  0.28 1.93  0.21 0.98  0.21 1.61  0.23
Residual distance constraint violations
Number 0.1 Å 19 3 274 255 313 20 5
Maximum, Å 0.13  0.01 0.16  0.01 0.14  0.01 0.14  0.01 0.22  0.00
Residual dihedral angle constraint violations
Number 2.0° 1 1 11 11 11 0 1
Maximum, ° 1.8  0.8 1.94  0.45 2.90  0.85 2.3  0.8 1.45  0.78
AMBER energies (kcalmol)
Total 4,628  63 4,797  105 4,657  99 4,960  73 4,651  68
Van der Waals 300  12 280  15 283  16 341  14 123  14
Electrostatic 5,236  56 5,497  85 5,313  99 5,542  68 5,448  64
rms deviation to the averaged coordinates,* Å
bb (N, C, C) 0.78  0.13
0.74  0.14
(125–166, 172–225)
0.70  0.12
(125–166, 172–225)
0.76  0.10
0.94  0.18
(127–166, 173–225)
All heavy atoms 1.20  0.17 1.23  0.13 1.16  0.11 1.24  0.14 1.46  0.24
Except for the top two entries, the average for the 20 conformers with the lowest residual DYANA target function values and the standard deviation among
them are given.
*bb, backbone. The numbers in parentheses indicate the residues for which the rms deviation values were calculated.
Fig. 1. Amino acid sequence alignment of the polypeptide fragment 125–231 for the following prion proteins (numeration of hPrP by following ref. 15): cow,
bPrP; sheep, ovPrP; dog, cPrP; cat, fPrP; pig, scPrP; mouse, mPrP; Syrian hamster, shPrP; and human, hPrP. At the top, the locations of the regular secondary
structures in bPrP(121–231) are indicated, and the complete sequence of bPrP is given. For the other species, only the residue positions with amino acid exchanges
with respect to bPrP are indicated (and a deletion at position 230 of hPrP is indicated by F).
Lysek et al. PNAS  January 18, 2005  vol. 102  no. 3  641
distance relationship, to remove meaningless constraints, and to
derive constraints for the backbone torsion angles and from
C chemical shift values (37, 38). The final round of structure
calculation was started by using 100 randomized conformers.
The 20 conformers with the lowest residual DYANA target
function values were energy-minimized in a water shell with the
program OPALP (39, 40) by using the AMBER force field (41). The
program MOLMOL (42) was used to analyze the results of
the protein structure calculations (Table 1) and to prepare the
drawings of the structures (Figs. 2 and 3).

For each of the four animal species for which the prion protein,
or in the case of the sheep two different polymorphisms of the
prion protein, were studied (Table 1), the mature full-length
polypeptide chain with residues 23–231 (the residue numeration
for human PrP (15) is used throughout this paper) and the stable
partial sequence 121–231 (19) were cloned and expressed in E.
coli. All nine recombinant proteins (ovPrP[R168](23–231) was
not studied) were prepared with uniform 15N-labeling, and the
five 121–231 fragments were also obtained with uniform13C,15Nlabeling.
The methods used for protein preparation and purification
are described in Materials and Methods.
Following up on the approach used previously for the global
characterization of other mammalian PrPs (23, 24, 43), heteronuclear
15N{1H}-NOEs were measured at 20°C for 15N-labeled
scPrP(23–231), fPrP(23–231), cPrP(23–231), and ovPrP[H168](23–
231). Each of the four proteins was thus shown to contain a
structured region extending approximately from residues 125–226,
with positive values for the 15N{1H}-NOEs, as expected for a
globular protein with the size of PrP (44). At both ends of the
globular domain, there are flexible peptide segments, as evidenced
by negative values of the steady-state 15N{1H}-NOEs (data not
shown). The C-terminal pentapeptide segment corresponds to a
flexible linker between the structured domain of PrPC and the
glycosylphosphatidylinositol anchor in the cell surface membrane
(1, 2). The N-terminal polypeptide segment 23–124 forms an
outstandingly long flexible tail, as evidenced by the observation in
all four species that the residues 23–121 all show negative values of
the 15N{1H}-NOEs for the backbone amide groups. This result
coincides with corresponding data on all of the mammalian PrPs
described in refs. 23, 24, 43, and 45. Because of different insertions
relative to the human prion protein sequence (hPrP), this tail
includes 103 residues for fPrP, 100 residues for cPrP and scPrP, and
101 residues for ovPrP (15).
In the remainder of this section and in Discussion, we focus
primarily on the NMR structure determination of the constructs
with residues 121–231 of the five aforementioned prion proteins
and on an analysis of the resulting structures for the globular
Resonance Assignments. For scPrP(121–231), complete resonance
assignments were obtained for the entire polypeptide backbone.
For cPrP(121–231), fPrP(121–231), ovPrP[H168](121–231), and
ovPrP[R168](121–231), nearly complete assignments were obtained
for the polypeptide backbone, the exceptions being the
amide protons and amide nitrogens of Gln-168 (fPrP), Tyr-169
Fig. 2. NMR structures of the globular domains in the five prion proteins
studied in this paper. Each structure is shown as a bundle of 20 energyminimized
conformers, with gray coloring of the backbone and speciesspecific
coloring of the amino acid side chains that are different from bPrP. For
each species, the amino acid replacements relative to bPrP are identified by
indication with the one-letter code of the amino acid in the species considered,
the sequence position, and the amino acid in bPrP. The conformers were
aligned for best fit of the backbone heavy atoms of the residues 128–166 and
172–220, and displayed are the residues 125–227. (A) scPrP(121–231) (side
chains pink); (B) cPrP(121–231) (side chains red); (C) fPrP(121–231) (side chains
blue); (D) ovPrP[H168](121–231) (side chains green); (E) ovPrP[R168](121–231)
(side chains yellow).
642  www.pnas.orgcgidoi10.1073pnas.0408937102 Lysek et al.
(cPrP and fPrP), Ser-170 (cPrP, ovPrP[H168], and ovPrP[R168]),
Asn-171 (fPrP, cPrP, ovPrP[H168], and ovPrP[R168]), and Phe-
175 (fPrP, cPrP, ovPrP[H168], and ovPrP[R168]). The amino
acid side chain assignments are nearly complete, including all
tyrosine, phenylalanine, and histidine ring resonances with the
sole exception of Phe-198 CH. The chemical shift lists of the five
proteins have been deposited in the BioMagResBank (www.bmrb. with the following entry codes: scPrP(121–231), 6380;
cPrP(121–231), 6378; fPrP(121–231), 6377; ovPrP[H168](121–231),
6381; and ovPrP[R168](121–231), 6403.
Collection of Conformational Constraints and Structure Calculation.
For scPrP(23–231) and ovPrP[H168](121–231), which were studied
earlier than the other proteins, peak picking of the threedimensional
15N-resolved and three-dimensional 13C-resolved
[1H,1H]-NOESY spectra was pursued interactively. The resulting
lists of NOESY cross peaks together with chemical shift lists
from the resonance assignments were used as input for automatic
NOE assignment and structure calculation by using the standard
protocol with the program package CANDID (34)DYANA (35).
For cPrP(121–231), fPrP(121–231), and ovPrP[R168](121–
231), the automation of the structure determination process
included the peak picking of the NOESY spectra by using the
program package ATNOSCANDIDDYANA with a standard protocol
(34–36). The input of NOE upper distance limits obtained
for the individual proteins (Table 1) shows that the interactive
peak picking resulted in an 25% higher total number of
constraints and in improved convergence of the structure calculation,
as evidenced by the lower residual DYANA target
function values.
The NMR Structures of the Globular Domains of scPrP, cPrP, fPrP,
ovPrP[H168], and ovPrP[R168]. Table 1 shows that four of the five
PrP structures were determined with comparable precision, as
documented by backbone rms deviation values of 0.70–0.78 Å.
The somewhat lower precision achieved for ovPrP[R168](121–
231) is due to the fact that the NOESY data sets had to be
recorded at 0.5 mM protein concentration as compared with
1.0 mM concentration for the other proteins.
In Fig. 2, the five structures are shown as bundles of 20
conformers (Table 1). The location of regular secondary structures
coincides nearly identically with bPrP (Fig. 1). In Fig. 2, the
amino acid exchanges relative to bPrP are indicated, which also
serves as a guide to follow the polypeptide fold. The drawings
start with residue 125 in the lower right, from where the
polypeptide goes through the first -strand 128–131 to the start
of helix 1, which is at residue 143 in the upper left corner of the
molecule. Following helix 1 from residues 144–154, the chain
winds through the -strand 161–164, which combines with
residues 128–131 to form an antiparallel -sheet, to the extreme
right. A loop of residues 166–173 connects to helix 2 with
residues 174–194, which leads to the lower left corner of the
structure. A five-residue loop then leads to helix 3 with residues
200–226, which ends in the top right corner of the structure.
Some local features in the structures of Fig. 2 can all be directly
related with the absence or very low intensity of theNMRsignals
for the backbone 15N-1H moieties of individual residues. First,
the loop of residues 166–173 is disordered; a complete set of
15N-1H NMR signals could be observed only in scPrP(121–231)
(Fig. 2A). Second, the start of helix 2 is poorly defined because
the amide proton NMR signal of Phe-175 could not be detected,
the sole exception being scPrP(121–231). Third, in all five
proteins the helix 3 is somewhat nonregular near residue 220,
which correlates with the observation that the 15N-1H NMR
signals for one or several residues in the segment 218–222 have
very low intensity. Local superposition of the residues 222–226
reveals the presence of two turns of well defined -helix, also in
ovPrP[R168](121–231) (Fig. 2E). Previously it was observed that
the distortion of the helix 3 is particularly pronounced in
murine PrP (19, 46).
Fig. 3. Surface views of the globular domains of the five proteins of Fig. 2.
Shown are the residues 125–229. The presentation in Right relates to the one
in Left through a 180° rotation around a vertical axis. The electrostatic surface
potential is indicated in red (negative charge), white (uncharged), and blue
(positive charge). The circles indicate charge differences relative to bPrP that
are discussed in the text, and the amino acid residues are identified from which
the charge differences originate. (A) scPrP(121–231), (B) cPrP(121–231), (C)
fPrP(121–231), (D) ovPrP[H168](121–231), and (E) ovPrP[R168](121–231).
Lysek et al. PNAS  January 18, 2005  vol. 102  no. 3  643


Comparative NMR studies with natural bovine PrPC isolated
from calf brains showed that the three-dimensional structure of
recombinant PrP prepared with the methods used in this paper
corresponds to the polypeptide structure in natural PrPC containing
all of the posttranslational modifications (21). In the
following discussion, we therefore refer to the structures of Fig.
2 as the PrPC form of the prion protein.
All mammalian species studied so far (Fig. 2 and refs. 23, 24,
43, and 45) contain PrPC molecules with a flexibly extended
N-terminal tail of length 100 residues and a C-terminal globular
domain with100 residues. The architecture of the globular
domain is highly conserved in the different species, as was
expected from the high degree of sequence identity (Fig. 1).
Considering that the presently studied group (Fig. 2) includes
species with widely different susceptibilities toward TSEs, we
shall now search the preserved scaffold of the globular domain
for local structure variations that might relate to different
susceptibilities for developing TSE and, in particular, to variable
stringency of the species barrier against infection with BSE.
Two areas of the globular domain of PrPC have been suggested
to be important for the development of TSEs. First, the helix 1
has been implicated as a primary interaction site with the
TSE-associated isoform PrPSc (1). Second, an epitope comprising
the loop 166–172 and the C-terminal end of helix 3 has been
suggested to be recognized by a conversion chaperone, i.e.,
‘‘protein X’’ (47). Inspection of Fig. 2 then shows that species
variations of the amino acid sequence are predominately located
in or near these two molecular regions.
With regard to a possible role of helix 1 in TSE susceptibility,
the five previously uncharacterized structures of Fig. 2 do not
indicate any conclusive correlation. The helix has identical
length, location, and orientation in all of the structures. Furthermore,
species with or without a record of TSEs (cat and dog)
and sheep polymorphisms with high and low TSE susceptibility
all have identical sequences from residues 143–158 (Fig. 1).
Inspection of the amino acid substitutions in Fig. 2 indicates
that there should be surface charge variations at or near the
presumed protein X epitope (Fig. 3). A first intriguing observation
results for the different sheep PrPs. In vivo and in vitro
experiments that link BSE or scrapie susceptibility to the amino
acid sequence of ovPrP (48, 49), showed that sheep carrying
ovPrP[R168] are highly resistant to transmission of TSEs,
whereas ovPrP[Q168] has been linked with high susceptibility
and ovPrP[H168] with medium-high susceptibility to BSE or
scrapie transmission (50). Position 168 is surface-exposed in the
loop 166–173 and, therefore, has a dominant effect on the
surface charges distribution in this region (Fig. 3). The positive
charge of R168 in ovPrPC thus appears to protect healthy sheep
when challenged with BSE infectivity or scrapie infectivity.
All four amino acid substitutions between the globular domains
of cPrPC and fPrPC involve charged residues (Figs. 1 and
3). The presence of Asp-159 and Arg-177 in dogs causes unique
charge distribution patterns on the front as well as the back side
of cPrPC (Fig. 3), which might, from the presently available
evidence, correlate with protection of dogs against challenge
with BSE. fPrPC, in turn, shares the presence of a positive charge
near the C terminus (Fig. 3C) with other TSE-susceptible species
(Fig. 1).
Relative to bPrP, scPrP has a single charge-effective amino
acid substitution in position 223 (Fig. 3A), which it shares,
however, with both the dog and cat (Figs. 1 and 3), and which,
therefore, would not appear to be critical with regard to TSE
susceptibility. A charge-neutral amino acid replacement from
Asn-173 in bPrP to Ser-173 in scPrP (Fig. 1) stabilizes the loop
166–173 in scPrPC to the extent that completeNMRassignments
could be obtained. This amino acid substitution might thus affect
the presumed protein X epitope (1, 47).
In conclusion, the seminal observation by the Weissmann
group that expression of host-encoded PrP is a necessary condition
for the development of a TSE (3) implies that each
organism producing PrPC might be susceptible to spontaneous or
transmitted TSE. Thus, although PrPSc has an increasingly
prominent role in research on TSE diagnostics, it would appear
that independent of the nature of the TSE-causing agent, PrPC
will be a prime target for TSE prevention in healthy organisms
and TSE treatment in disease. Detailed knowledge of PrPC
three-dimensional structures will be an important part of the
platform for such future endeavors.
We thank the Tierspital of the University of Zu¨rich for the donation of
cat, dog, and pig blood; Dr. A. Bossers for providing the different ovPrP
genes; and Dr. G. Pioda for help with the structural determination of
ovPrP[H168](121–231). This work was supported by the Schweizerischer
Nationalfonds and the ETH Zu¨rich through the National Center of
Competence in Research ‘‘Structural Biology’’ and a Fannie and John
Hertz Foundation fellowship (to L.G.N.).
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Lysek et al. PNAS  January 18, 2005  vol. 102  no. 3  645


May 26, 2003

Media Inquiries: 301-827-6242
Consumer Inquiries: 888-INFO-FDA

FDA BSE Update - Pet Food from Canadian Manufacturer

The Food and Drug Administration (FDA) has learned from the government
of Canada that rendered material from a Canadian cow that last week
tested positive for bovine spongiform encephalopathy (BSE, also known as
mad cow disease) may have been used to manufacture pet food,
specifically dry dog food, some of which was reported to have been
shipped to the United States. The Canadian government prevented the BSE
positive cow from being processed for human food. Therefore, consumers
can be assured that their food does not contain any remnants of the BSE
positive cow.

It is also important to stress that there is no scientific evidence to
date that dogs can contract BSE or any similar disease. In addition
there is no evidence that dogs can transmit the disease to humans.

FDA notified the U.S. pet food firm, The Pet Pantry International, of
Carson City, Nevada, when FDA learned that the pet food that the firm
received may have included rendered material from the BSE positive cow.
The manufacturer of the pet food is Champion Pet Food, Morinville,
Alberta. Even though there is no known risk to dogs from eating this dog
food, as a prudent measure to help assure that the U.S. stays BSE free
The Pet Pantry International is asking its customers who may have
purchased the suspect product to hold it for pickup by the distributor
so that the dog food will not mistakenly be mixed into cattle or other
feeds if any of the dog food is discarded or otherwise not used to feed
dogs. The suspect dog food was produced by Champion Pet Food between
February 4, 2003, and March 12, 2003.

The Pet Pantry products were packaged in 50 lb bags, distributed to
franchises around the country, and sold by home delivery only. There was
no retail distribution of the product. Consumers purchase Pet Pantry
products by phone or email orders. The product is then delivered by the
nearest franchisee directly to the consumers home.

The product subject to this notification includes Maintenance Diet
labeled with a use by date of 17FEB04 and Beef with Barley with a
use by date of 05MAR04. Consumers who have purchased dog food from The
Pet Pantry since February of this year are asked to check their present
supplies and see if any match the description of the product being
removed. If so, consumers are asked to contact The Pet Pantry at
1-800-381-7387 for further information on how to return the product to
The Pet Pantry for proper disposal. Consumers are asked not to destroy
or discard the product themselves. The Pet Pantry will also use its
sales records to contact consumers who purchased the affected product.

FDA is working closely with the Pet Pantry International to assure for
proper disposal of the recovered product.

FDA will continue to provide updates on this case of BSE in Canada as
additional information becomes available.

It was thought likely that at least some, and probably all, of the cases
in zoo animals were caused by the BSE agent. Strong support for this
hypothesis came from the findings of Bruce and others (1994)
( Bruce, M.E., Chree, A., McConnell, I., Foster, J., Pearson, G. &
Fraser, H. (1994) Transmission of bovine spongiform encephalopathy and
scrapie to mice: strain variation and species barrier. Philosophical
Transactions of the Royal Society B 343, 405-411: J/PTRSL/343/405
), who demonstrated that the pattern of variation in incubation period
and lesion profile in six strains of mice inoculated with brain
homogenates from an affected kudu and the nyala, was similar to that
seen when this panel of mouse strains was inoculated with brain from
cattle with BSE. The affected zoo bovids were all from herds that were
exposed to feeds that were likely to have contained contaminated
ruminant-derived protein and the zoo felids had been exposed, if only
occasionally in some cases, to tissues from cattle unfit for human


cases have been reported in domestic cats), are characterised by
long asymptomatic incubation periods followed by progressive
symptoms and signs of degeneration of the brain, leading
eventually to death.


worse still, there is serious risk the media could get
to hear of such a meeting...


Crushed heads (which inevitably involve brain and spinal cord material)
are used to a limited extent but will also form one of the constituent
raw materials of meat and bone meal, which is used extensively in
pet food manufacturer...

2. The Parliamentary Secretary said that he was concerned
about the possibility that countries in which BSE had not
yet been detected could be exporting raw meat materials
(in particular crushed heads) contaminated with the disease
to the UK for use in petfood manufacture...


YOU explained that imported crushed heads were extensively used in the
petfood industry...

In particular I do not believe one can say that the levels of
the scrapie agent in pet food are so low that domestic animals are
not exposed...

some 100+ _documented_ TSE cats of all types later...tss

on occassions, materials obtained from slaughterhouses
will be derived from sheep affected with scrapie or
cattle that may be incubating BSE for use in petfood manufacture...

Meldrum's notes on pet foods and materials used


Confidential BSE and __________________

1st case natural FSE

FSE and pharmaceuticals

confidential cats/dogs and unsatisfactory posture
MAFFs failure to assure key research

can't forget about the mad man and his mad cat;

Deaths of CJD man and cat linked

In October 1998 the simultaneous occurrence of spongiform encephalopathy
in a man and his pet cat was reported. The report from Italy noted that
the cat did not display the same clinical features as FSE cases
previously seen. Indeed, the presence of a new type of FSE was
suggested. The man was diagnosed as having sporadic CJD, and neither
case (man nor cat) appeared to be affected by a BSE-related condition.

indeed there have been 4 documented cases of TSE in Lions to date.

Lion 32 December 98 Born November 86

Lion 33 May 1999 (euthanased) Born November 81.

Lion 36 Euthanased August 2000 Born July 87. Deteriorating hind limb

Lion 37 Euthanased November 2001 Male, 14 years. Deteriorating hind
limb ataxia since September 2001. (Litter mate to Ref. 36.)

go to the url above, on the bar at the top, click on _statistics_,
then in middle of next page, click on _other TSEs_.

or go here;



Reports on the clinical symptoms presented by these cats give a
relatively homogeneous picture: Affected cats show a lack of
coordination with an ataxia mainly of the hind limbs, they often fall
and miss their target when jumping. Fear and increased aggressiveness
against the owner and also other animals is often seen. They do not
longer tolerate to be touched (stroked) and start hiding. These
behavioural chances might be the result of a hypersensibility to touch
and noise, but also to increased fear. Excessive salivation is another
more frequently seen symptom. Cats with FSE in general show severe
behavioural disturbances, restlessness and depression, and a lack of
coat cleaning. Symptoms in large cats in general are comparable to those
in domestic cats. A
report on FSE (in german) has been presented in 2001 in the Swiss FVO
Magazin. A paper on the first FSE case in a domestic cat in Switzerland
is currently in press in the Journal Schweizer Archiv für Tierheilkunde

Date: Thu, 17 Oct 2002 17:04:51 -0700
From: "Terry S. Singeltary Sr."
Reply-To: Bovine Spongiform Encephalopathy

Greetings BSE-L,

is there any other CWD surveys/testing in the UK on their deer?
what sort of testing has been done to date on UK/EU deer?
any input would be helpful... thank you


hope they did not go by the wayside as the hound study;

37.Putative TSE in hounds - work started 1990 -(see para 41)

Robert Higgins, a Veterinary Investigation Officer at Thirsk,
had been working on a hound survey in 1990. Gerald Wells
and I myself received histological sections from this survey
along with the accompanying letter (YB90/11.28/1.1) dated
November 1990. This letter details spongiform changes found
in brains from hunt hounds failing to keep up with the rest of
the pack, along with the results of SAF extractions from
fresh brain material from these same animals. SAFs were not
found in brains unless spongiform changes were also present.
The spongiform changes were not pathognomonic (ie.
conclusive proof) for prion disease, as they were atypical,
being largely present in white matter rather than grey matter in
the brain and spinal cord. However, Tony Scott, then head of
electron microscopy work on TSEs, had no doubt that these
SAFs were genuine and that these hounds therefore must have
had a scrapie-like disease. I reviewed all the sections
myself (original notes appended) and although the pathology
was not typical, I could not exclude the possibility that this was
a scrapie-like disorder, as white matter vacuolation is seen
in TSEs and Wallerian degeneration was also present in the
white matter of the hounds, another feature of scrapie.

38.I reviewed the literature on hound neuropathology, and
discovered that micrographs and descriptive neuropathology from
papers on 'hound ataxia' mirrored those in material from
Robert Higgins' hound survey. Dr Tony Palmer (Cambridge) had
done much of this work, and I obtained original sections
from hound ataxia cases from him. This enabled me provisionally to
conclude that Robert Higgins had in all probability detected
hound ataxia, but also that hound ataxia itself was possibly a
TSE. Gerald Wells confirmed in 'blind' examination of single
restricted microscopic fields that there was no distinction
between the white matter vacuolation present in BSE and
scrapie cases, and that occurring in hound ataxia and the hound
survey cases.

39.Hound ataxia had reportedly been occurring since the 1930's,
and a known risk factor for its development was the feeding
to hounds of downer cows, and particularly bovine offal.
Circumstantial evidence suggests that bovine offal may also be
causal in FSE, and TME in mink. Despite the inconclusive
nature of the neuropathology, it was clearly evident that this
putative canine spongiform encephalopathy merited further

40.The inconclusive results in hounds were never confirmed,
nor was the link with hound ataxia pursued. I telephoned Robert
Higgins six years after he first sent the slides to CVL.
I was informed that despite his submitting a yearly report to the
CVO including the suggestion that the hound work be continued,
no further work had been done since 1991. This was
surprising, to say the very least.

41.The hound work could have provided valuable evidence
that a scrapie-like agent may have been present in cattle offal long
before the BSE epidemic was recognised. The MAFF hound
survey remains unpublished.

Histopathological support to various other published
MAFF experiments

42.These included neuropathological examination of material
from experiments studying the attempted transmission of BSE to
chickens and pigs (CVL 1991) and to mice (RVC 1994).

nothing to offer scientifically;

maddogs and Englishman

kind regards,

###########bse-l ############

Date: Fri, 18 Oct 2002 23:12:22 +0100
From: Steve Dealler
Reply-To: Bovine Spongiform Encephalopathy
Organization: Netscape Online member
To: BSE-L@
References: <>

Dear Terry,
An excellent piece of review as this literature is desparately difficult
to get
back from Government sites.

What happened with the deer was that an association between deer meat
eating and
sporadic CJD was found in about 1993. The evidence was not great but did not
disappear after several years of asking CJD cases what they had eaten.
I think that the work into deer disease largely stopped because it was
not helpful
to the UK industry...and no specific cases were reported.
Well, if you dont look adequately like they are in USA currenly then you
wont find

Steve Dealler

Incubation periods for BSE are proportional to the life expectancy of
the animal affected. The disease's incubation period is 18% of a cow's
life expectancy and would be expected to about double when crossing to
another species [---] that is, to 36% of 70 years in humans.

Steve Dealler, consultant in medical microbiology.
Burnley General Hospital, Burnley BB10 2PQ


########### ############

Docket Management Docket: 02N-0273 - Substances Prohibited From Use in
Animal Food or Feed; Animal Proteins Prohibited in Ruminant Feed
Comment Number: EC -10
Accepted - Volume 2 [PART 1]

Docket Management Docket: 02N-0273 - Substances Prohibited From Use in
Animal Food or Feed; Animal Proteins Prohibited in Ruminant Feed
Comment Number: EC -11
Accepted - Volume 2 [PART 2]

FDA BSE Update - Pet Food from Canadian Manufacturer & MAD DOG DATA



August 22, 2003 5:11 PM

Mad cat disease

A second case of feline spongiform encephalopathy (FSE), a disease
affecting the brain tissue of cats, has been recorded in Switzerland.
The veterinary authorities said the likely cause of the infection, which
is similar to mad cow disease, was contaminated pet food.
A first case of FSE was reported two years ago.
Experts say the disease poses no health risk for people.



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