Download Prion protein in cardiac muscle of elk (Cervus elaphus nelsoni) and

Journal of General Virology (2006), 87, 3443–3450
DOI 10.1099/vir.0.81777-0
Prion protein in cardiac muscle of elk (Cervus
elaphus nelsoni) and white-tailed deer
(Odocoileus virginianus) infected with chronic
wasting disease
Jean E. Jewell,1 Jeremy Brown,1 Terry Kreeger2 and Elizabeth S. Williams13
1
Department of Veterinary Sciences, University of Wyoming, Wyoming State Veterinary
Laboratory (WSVL), 1174 Snowy Range Road, Laramie, WY 82070, USA
Correspondence
Jean E. Jewell
2
[email protected]
Veterinary Services Branch, Wyoming Game and Fish Department (WGFD), Wheatland,
WY 82201, USA
Received 19 December 2005
Accepted 6 July 2006
To investigate the possible presence of disease-associated prion protein (PrPd) in striated muscle
of chronic wasting disease (CWD)-affected cervids, samples of diaphragm, tongue, heart and three
appendicular skeletal muscles from mule deer (Odocoileus hemionus), white-tailed deer
(Odocoileus virginianus), elk (Cervus elaphus nelsoni) and moose (Alces alces shirasi) were
examined by ELISA, Western immunoblot and immunohistochemistry (IHC). PrPd was detected in
samples of heart muscle from seven of 16 CWD-infected white-tailed deer, including one
free-ranging deer, and in 12 of 17 CWD-infected elk, but not in any of 13 mule deer samples, nor in
the single CWD-infected moose. For white-tailed deer, PrPd was detected by Western blot at
multiple sites throughout the heart; IHC results on ventricular sections of both elk and white-tailed
deer showed positive staining in cardiac myocytes, but not in conduction tissues or nerve ganglia.
Levels of PrPd in cardiac tissues were estimated from Western blot band intensity to be lower than
levels found in brain tissue. PrPd was not detected in diaphragm, triceps brachii, semitendinosus,
latissiumus dorsi or tongue muscles for any of the study subjects. This is the first report of PrPd in
cardiac tissue from transmissible spongiform encephalopathy-infected ruminants in the human food
chain and the first demonstration by immunological assays of PrPd in any striated muscle of
CWD-infected cervids.
INTRODUCTION
Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) that affects wild and captive
cervids, including mule deer (Odocoileus hemionus), whitetailed deer (Odocoileus virginianus), wapiti or Rocky
Mountain elk (Cervus elaphus nelsoni) and moose (Alces
alces shirasi) (Williams & Young, 1992; Williams, 2005;
Kreeger et al., 2006). In common with other TSEs, such as
scrapie of sheep and goats, bovine spongiform encephalopathy, transmissible mink encephalopathy and the human
TSEs, a diagnostic hallmark of CWD infection is accumulation of a disease-associated prion protein, PrPd, in the
central nervous system (CNS). PrPd, which is an aberrantly
refolded conformational isoform of a normal cellular
glycoprotein, PrPC (Prusiner, 2004), replicates during
infection by inducing the conversion of PrPC to the PrPd
conformation. PrPd is partially protease-resistant, unlike
PrPC, and this difference is the basis of many molecular
detection protocols currently used; it may also account for
3Deceased 29 December 2004.
0008-1777 G 2006 SGM
extracellular accumulation of PrPd in vivo and survival
through the digestive tract of exposed cervids.
The widespread involvement of both lymphoid and
nervous-system tissues as sites of accumulation of PrPd
during scrapie and CWD infection has been described
extensively (Hadlow et al., 1982; van Keulen et al., 1995,
1996; Sigurdson et al., 1999, 2001; Miller & Williams, 2002;
Spraker et al., 2002b), but recent investigations have also
demonstrated the presence of PrPd in skeletal muscles of
TSE-infected laboratory mice and hamsters, of sheep, deer
and humans (Bosque et al., 2002; Bartz et al., 2003; Glatzel
et al., 2003; Thomzig et al., 2003; Andréoletti et al., 2004;
Mulcahy et al., 2004; Ashwath et al., 2005; Casalone et al.,
2005; Angers et al., 2006; Thomzig et al., 2006). The levels of
PrPd that accumulated in muscles were lower than those
found in CNS or lymphoid tissue; however, in rodents,
muscle-derived PrPd was shown to be infectious (Bosque
et al., 2002; Angers et al., 2006). The detection of PrPd
(designated PrPSc in scrapie) in skeletal muscles of scrapieinfected sheep (Andréoletti et al., 2004) and of infectivity in
skeletal muscle of CWD-infected mule deer (Angers et al.,
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J. E. Jewell and others
2006) has raised the level of concern on the issue of potential
human health risks that might be encountered by consuming prion-containing meat.
Based on epidemiological evidence and transgenic mouse
bioassays, CWD seems not to be transmitted to humans
(Belay et al., 2004; Kong et al., 2005). However, because deer
and elk are food sources, hunters in areas where CWD is
known to occur are advised not to consume brain, spinal
cord or lymph nodes, not to kill and eat any animals that are
obviously unwell and to submit samples to state game
agencies for testing (for example, http://www.wildlife.utah.
gov/news/05-10/cwd.php; http://wildlife.state.co.us/CWD/
index.asp; http://www.dnr.state.wi.us/org/land/wildlife/
whealth/issues/CWD/index.htm; http://gf.state.wy.us/services/
education/cwd/index.asp; http://www.ngpc.state.ne.us/wildlife/
guides/cwd/faq.asp; http://www.sdgfp.info/Wildlife/hunting/
BigGame/CWD.htm). Information on PrPd in muscle tissue
of cervids is therefore of interest to hunters, as well as pertinent
to research questions on the pathogenesis of CWD in its
natural hosts. An extensive immunohistochemical analysis of
tissue samples from naturally infected mule deer (Spraker
et al., 2002a) and a report of two elk inoculated intracerebrally
(i.c.)withscrapie(Hamiretal.,2004)bothfailedtodetectPrPd
in muscle tissues. However, Angers et al. (2006) recently
reported successful TSE transmission to transgenic mice
inoculated i.c. with extracts of skeletal muscle from CWDinfected mule deer. We report here the detection of PrPd in
cardiac muscle of two species of cervid as a result of our
investigation of striated muscle samples from CWD-infected
and uninfected mule deer, white-tailed deer, elk and moose by
using ELISA, Western blots and immunohistochemistry
(IHC).
on high speed (setting 6?5), with 5 min room temperature cooling
between cycles. Homogenates were stored in the bead vials at
220 uC and reprocessed for 10 s at speed 4 in a FastPrep to resuspend tissue particles aggregated by freezing. Tissue slurries were
removed from large remaining particles of connective tissue with the
aid of a fine-tip transfer pipette (Samco Scientific). Aliquots
(0?1 ml) were treated with 100 mg PK ml21 (Invitrogen) and 2 units
DNase (Benzonase Nuclease; Novagen) in TBS100, 0?5 % each of
Nonidet P-40 (Pierce) and sodium deoxycholate, 1 % sodium Nlauroylsarcosine (SLS), 1 mM each of CaCl2 and MgCl2, for 30 min
at 45 uC. Digestion was terminated by addition of protease inhibitor
(Pefabloc; Fisher Scientific) to 4 mM. Samples were centrifuged at
1200 g for 3 min and supernatants, after transfer to clean 1?7 ml
microfuge tubes, were made up to 2 % in SLS and prewarmed to
37 uC. Methods for sodium phosphotungstic acid (NaPTA) precipitation were adapted from published protocols (Safar et al., 1998;
Wadsworth et al., 2001; McCutcheon et al., 2005). A 1/12 volume of
4 % (w/v) NaPTA, 0?17 M MgCl2, adjusted to pH 7?4 with NaOH,
was added at 37 uC and samples were incubated for 30 min with
shaking at 37 uC, followed by centrifugation at room temperature
for 30 min at 16 100 g. Supernatants were discarded and pellets were
resuspended in 16 NuPAGE LDS denaturing sample buffer
(Invitrogen) with 3 % SDS added and heated to 100 uC for 8 min
before gel electrophoresis. Samples were loaded on preformed 1 mm
4–12 % Bis/Tris SDS-PAGE denaturing, reducing, polyacrylamide
gradient gels in MOPS/SDS running buffer (NuPAGE gel system;
Invitrogen) and electrophoresed for 1 h at 200 V. Molecular mass
markers were MagicMark XP Western protein standard
(Invitrogen).
Sample preparation, gel electrophoresis, Western blot transfer and development. To prepare samples for gel electrophoresis
Samples from gels were transferred electrophoretically to Immobilon-P
PVDF membrane (Millipore) in 25 mM Tris, 192 mM glycine, 10 %
methanol by using a Hoefer TE22 Mightly Small Transphor tank
(Amersham Biosciences) at 400 mA for 1 h with stirring and cooling of
transfer buffer. Following transfer, membranes (blots) were rinsed
briefly in 100 % methanol and air-dried. Preceding Western
immunoblot development, dried blots were rinsed for 15 s in 100 %
methanol and for 2 min in distilled water and placed in 30 ml blocking
buffer [50 mM Tris/HCl (pH 7?5), 150 mM NaCl, 0?1 % Tween 20
(Bio-Rad) (TBS-T)], containing 4 % non-fat dry milk (NFDM)
(Carnation) and 2 mg normal goat serum ml21 (Jackson
ImmunoResearch Laboratories) for 30 min at room temperature.
Blots were subsequently incubated for 1 h with anti-PrP antibody
(mouse monoclonal F99/97.6.1; VMRD Inc.) diluted 1 : 200 in TBS-T/
NFDM, followed by three 10 min washes in 50 ml TBS-T per wash and
a second incubation in blocking buffer for 5 min, before incubating for
30 min with horseradish peroxidase-conjugated F(ab9)2 fragment of
goat anti-mouse IgG (Jackson ImmunoResearch Laboratories) diluted
1 : 30 000 in TBS-T/NFDM. Secondary antibody incubation was
followed by five 10 min washes in TBS-T, chemiluminescent detection
by using ECL Western blotting reagents (Amersham Biosciences)
according to the manufacturer’s directions and exposure of Kodak
BioMax ML film for times ranging from 1 min to overnight, followed
by manual film development. Digitized images for figures were
obtained by scanning developed films and photoediting with HP
Director software; photoediting was limited to cropping and labelling
images and adjustments in brightness and contrast.
and subsequent Western blot analysis, tissue homogenates (10 %,
w/v) from frozen tissues were prepared in 0?1 M Tris/HCl (pH 8?0),
0?1 M NaCl (TBS100). Frozen tissue was allowed to thaw partially at
room temperature in a biological safety cabinet and samples
(approx. 150–200 mg) were cut from the interior of the muscle
mass as described above for ELISA samples. Homogenates were prepared in 1?5 ml TBS100 by processing in FastPrep vials (Q-Biogene)
containing about 0?25 ml 1?0 mm zirconia/silica beads (BioSpec)
and one 0?25 inch ceramic bead (Q-Biogene) in a FastPrep FP120
bead mill homogenizer (ThermoSavant) for two cycles of 30 s each
IHC. Striated muscle tissues collected at necropsy were preserved in
10 % neutral-buffered formalin. Samples were trimmed into sections,
treated in formic acid, rinsed, dehydrated, paraffin-embedded, prepared for immunohistochemical staining and stained, all as
described previously (Miller & Williams, 2002), with the following
modifications: immersion in 98 % formic acid of sections on slides
to enhance epitope exposure was for 5 min, followed by a rinse in
50 mM Tris/HCl (pH 7?6), 0?15 M NaCl, then deionized water;
cooling after autoclaving was for 40 min. Primary antibody was
METHODS
ELISA. Muscle tissues were assayed for prion protein resistant to
digestion by proteinase K (PK) (PrPres) by using chronic wasting
disease antigen test kits (Bio-Rad). Frozen muscle tissue was allowed
to thaw partially at room temperature in a biological safety cabinet
and samples (approx. 200 mg) were cut from the interior of the
muscle mass. Care was taken to minimize potential contamination
of the sample with the surface of the muscle. Homogenates were
prepared and processed following manufacturer’s procedures.
Duplicate wells were run for each processed sample and absorbance
results (A4502A620) for the two wells were averaged. Calculation of
the absorbance cut-off value for PrPres detection followed procedures
specified by the manufacturer for cervid retropharyngeal lymphnode assays.
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Journal of General Virology 87
Prion protein in cardiac muscle of CWD-infected cervids
mAb F99/97.6.1 [Anti-Prion (99); Ventana Medical Systems].
Digitized images for figures were obtained by using an Olympus
BX51 light microscope equipped with a MicroFire digital camera
and PictureFrame for Windows software to view and photograph
slides. Photoediting (Microsoft Office Picture Manager) was limited
to cropping and labelling images and adjustments in brightness,
contrast or colour balance.
Animals and tissues. Samples were of muscle tissues that had
been collected at necropsy at the WSVL and frozen. Animals
included free-ranging deer collected for CWD surveillance and submitted by WGFD to the WSVL for necropsy and testing, and captive
or free-ranging animals that were subjects in other CWD research
projects carried out in collaboration with WGFD and/or the
Colorado Division of Wildlife. All elk and moose were experimental
animals from WGFD research.
RESULTS
ELISAs
To investigate whether PrPd was present in striated muscle
of CWD-infected cervids, we first assayed muscle samples
from 15 mule deer (ten CWD-infected and five uninfected),
nine white-tailed deer (four infected, five uninfected) and 13
elk (ten infected, three uninfected) by ELISA (see Methods).
We used six muscle samples from each animal: diaphragm,
tongue, left ventricle (lv) of the heart and three appendicular
skeletal muscles (longissimus dorsi, triceps brachii and
semitendinosus). lv from two CWD-positive white-tailed
deer and seven CWD-positive elk resulted in positive ELISA,
but all other samples were negative (Fig. 1).
Western blots
We confirmed these results by Western blots using new
homogenates from the same tissues as were used for ELISA
for the 24 CWD-positive animals and seven of the negative
controls. The same six muscles were also sampled from one
additional CWD-positive white-tailed deer and from two
moose. In addition, lv only from seven CWD-infected elk,
three CWD-infected mule deer and eight CWD-infected
white-tailed deer was assayed by Western blot. The presence
of PK-resistant fragments of PrPd that exhibited the
characteristic electrophoretic pattern of three bands corresponding to the three glycosylated states of PrP and that
migrated between approximately 35 and 20 kDa was
demonstrated by Western blot in the cardiac lv of specimens
that were lv-positive by ELISA (Figs 2 and 4). For samples
tested only in lv, PK-resistant PrP was found in five of seven
elk, two of eight white-tailed deer and none of three mule
deer. Results are summarized in Table 1. The relative
concentration of PrPd in lv was estimated visually from band
intensity and comparison of tissue weights per sample lane to
be roughly 10- to 100-fold lower than that demonstrated in
brain tissue of the same animals (Fig. 3). Because both brain
and heart samples can exhibit locational sampling variation
as shown, more detailed estimates of concentrations were
Fig. 1. Summary of ELISA results for detection of PrPd in deer and elk striated muscles. Muscle tissues: tongue (TG), triceps
(TR), left ventricle (LV), diaphragm (DI), longissimus dorsi (LD) and semitendinosus (ST). Assays were performed as described
in Methods. Horizontal dashed lines mark cut-off values for positive ELISA. Symbols of the same geometric shape with interior
patterns are retests of the same specimen.
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J. E. Jewell and others
Fig. 2. Immunoblot assays for detection of PK-resistant PrP in
heart lv of CWD-infected white-tailed deer (WTD) and elk.
Comparison of PrPd from 10 mg wet tissue equivalents of lv of
WTD and elk. Film exposure to enhanced chemiluminescence
was 80 min.
not made. We did not detect PrPd by Western blot in any of
the other muscles tested in any of the animals, including
uninfected controls (data not shown).
We prepared homogenates from other sites in the lv tissue of
the CWD-positive animals and assayed them by Western
blot to test the possibility of an inhomogeneous distribution
of PrPd (Glatzel et al., 2003). If PrPd were associated
primarily with cells of the cardiac conduction system or with
intramural cardiac ganglia, we reasoned that such arrangements could produce ‘hit-or-miss’ sampling and might
account for results that showed both lv-positive and lvnegative specimens among the CWD-positive animals. We
observed variable intensity of the PK-resistant PrPd bands
from lv-positive animals (Fig. 4), particularly some of the
elk samples, indicating uneven but dispersed distributions
of PrPd within the ventricular tissue. However, we were
unable to demonstrate the presence of PrPd in the lv tissue of
any mule deer, moose or three white-tailed deer and three
elk that had previously been negative.
IHC
Inspection of sections of fixed lv tissue by IHC revealed antiPrP-labelled groups of laterally adjacent muscle cells within
the ordinary myocardium, often surrounded by cells with no
detectable stain (Fig. 5a, b). Labelled antigen was more
Fig. 3. Comparison of PrPd from brain and lv of elk (lanes
1–8) and white-tailed deer (WTD) (lanes 9–16). Lanes: 1, elk
brain, 1 mg tissue equivalent; 2, elk lv, 10 mg tissue; 3, elk
brain, 0?1 mg (lanes 1, 2 and 3 are from the same blot; samples between lanes 1 and 2 were cut from image); 4, elk lv
”PK; 5, elk lv +PK; 6, elk lv +PK; 7, elk brain +PK; 8, elk
brain ”PK; 9, WTD brain +PK; 10, WTD brain sample 1,
1 mg; 11, WTD brain sample 2, 1 mg; 12, WTD brain sample
3, 1 mg; 13, WTD lv, 5 mg; 14, WTD lv, 1 mg; 15, WTD lv
”PK; 16, WTD lv +PK. M, Molecular mass markers (bottom to
top, approx. 20, 30 and 40 kDa).
readily visible in longitudinal than in transverse crosssections of cardiac myocytes. Staining generally consisted of
punctate granules of chromagen scattered in an apparently
cell-associated fashion (Fig. 5c), sometimes in linear
patterns along the edge of a muscle cell (Fig. 5d), but not
consistently so. Subcellular structures or nerve fibres with
which PrPd might have been associated were not identified.
Epicardial nerves and ganglia and subendocardial and
myocardial Purkinje fibres lacked detectable PrPd staining.
Staining of PrPd in elk myocardium was similar to that of
white-tailed deer (Fig. 5e).
We did not detect PrPd by IHC in striated muscles other
than heart. Staining was equivocal or lacking in the skeletal
muscles and diaphragm sections. Light staining in small
ganglia of the tongue was observed in a few specimens of
white-tailed deer and elk, and two small groups of
ventricular subendocardial Purkinje fibres in the heart
Table 1. Summary of heart tissue samples tested
Species
Mule deer
CWD-positive
CWD-negative
Elk
CWD-positive
CWD-negative
White-tailed deer
CWD-positive
CWD-negative
lv-positive*
0/13
0/5
12/17
0/3
7/16
0/6
*lv, Left ventricle; no. positive in lv/no. tested by ELISA, Western
blot or IHC.
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Fig. 4. Heterogeneous PrPd distribution in lv. Lanes a, b, c
and d were sampled from different sites in the lv tissue of the
same animal. WTD gel samples were 20 mg wet tissue equivalents; film exposure was 10 min. Elk gel samples were 10 mg
tissue equivalents; exposure time was 145 min.
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Journal of General Virology 87
Prion protein in cardiac muscle of CWD-infected cervids
(Fig. 6c). These results indicated that PrPd could be present
in atrial tissue without being detected in ventricular muscle.
Our results with lv tissues probably underestimated the
frequency of PrPd deposition in cardiac tissues of CWDaffected white-tailed deer. By using the complete heart from
a mule deer that died in a late stage of CWD (MD18), we
assayed 16 sites in atria and ventricles by Western blot for
PrPd. All samples were negative (Fig. 6d, e). No complete
hearts were available from CWD-affected elk.
DISCUSSION
Fig. 5. IHC staining of PrPd in lv of white-tailed deer and elk.
(a) Low-power magnification showing scattered groups of PrPd
IHC labelled myocytes in lv myocardium, WTD1. (b) Enlarged
view of boxed area from (a). (c) Anti-PrPd IHC showing punctate, granular staining in cardiomyocytes, WTD1. (d) Linear
staining patterns (arrows), WTD11. (e) PrPd staining in elk cardiomyocytes, E20. (f) Negative lv from CWD-infected mule
deer, MD8. Bars, 160 mm (a); 40 mm (b); 20 mm (c–f).
of the CWD-positive moose were stained for PrPd (data not
shown).
Distribution of PrPd in heart
By using complete hearts collected and frozen at necropsy
from two captive CWD-affected white-tailed deer (the
generous gift of Dr L. L. Wolfe, Colorado Division of
Wildlife, Denver, CO, USA) and from a CWD-affected freeranging white-tailed deer (the generous gift of D. Edmunds,
University of Wyoming, Laramie, WY, USA), we tested
other areas of the heart by Western blot to investigate how
restricted or widespread PrPd accumulation might be. One
specimen (WTD21) was positive for PrPd throughout the
right atrium and strongly so in the free wall of both
ventricles (Fig. 6a); another (WTD22) was weakly positive
only in atrial tissue taken from the terminal crest between
the right auricle and atrium, and in the interatrial septum
adjacent and cranial to the opening of the coronary sinus in
the area of the atrioventricular node (Fig. 6b). Heart of the
free-ranging white-tailed deer (WTD20) was positive in all
atrial areas sampled, strongly so in the region of the
sinoatrial node, but negative in the free wall of the lv
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We have demonstrated by three immunological assays that
PrPd is found in cardiac tissue of CWD-infected cervids. We
failed to detect PrPd by the same assays in the five other
striated muscles tested, which suggested that PrPd did not
accumulate there, but which by the nature of negative
evidence is inconclusive. We cannot rule out the possibility
that some PrPd is present in other striated muscles not
examined or might be detectable by other assays, including
bioassay by inoculation of conspecific animals or of
transgenic mice. Recently, Angers et al. (2006) reported
the observation of TSE in transgenic mice following i.c.
inoculation with extracts of semitendinosus muscle from
CWD-infected mule deer. Similar tests of skeletal muscles
from CWD-infected white-tailed deer or elk have not yet
been reported.
The negative evidence in the case of mule deer heart is
somewhat more convincing, because positive heart samples
from white-tailed deer and elk are essentially positive
controls by which the lack of PrPd in mule deer heart can be
validated for each assay. In addition, our negative results for
mule deer heart are in agreement with an earlier investigation using IHC (Spraker et al., 2002a). Results of
experiments to demonstrate the infectivity of cardiacassociated PrPd by bioassays, as well as to test for infectivity
in mule deer heart, are not yet completed.
It was not known previously that PrPd accumulated in
cardiac muscle of any TSE-infected animals and several
questions are raised by these findings. One question is
whether impairment of cardiac function is associated with
prion deposition in myocardium. For the human spongiform encephalopathies, one case has been reported of PrPd
detected by IHC in an endomyocardial biopsy sample from a
Creutzfeld–Jakob disease patient with dilated cardiomyopathy (DCM) (Ashwath et al., 2005). The authors of that
report suggested the possibility of prion-induced DCM on
the basis of finding no other causes for the condition after an
exhaustive search. Neither DCM nor other gross lesions in
heart of CWD-infected deer and elk have been commonly
observed at necropsy (E. S. Williams, unpublished observations; T. E. Cornish, personal communication).
Disturbances in heart rate have been identified in BSEinfected cattle (Austin et al., 1997; Pomfrett et al., 2004), but
were attributed possibly to associated neuropathology in the
parasympathetic centres of cardiac innervation; neither
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3447
J. E. Jewell and others
Fig. 6. Immunoblot analysis of PrPd in tissues from various locations in heart. (a)
WTD21, 5–8 mg tissue equivalents per lane,
15 h film exposure; (b) WTD22, 13 mg per
lane, 15 h exposure; (c) WTD20, 7–13 mg
per lane, 15 h exposure; (d, e) MD18,
11–20 mg per lane, 3 h exposure. PC
(positive control)=7 mg WTD21 RV-FW.
Abbreviations: AVB, area of atrioventricular
bundle; AVN, interatrial septum in region of
atrioventricular node adjacent to coronary
sinus opening; FW, free wall; IV, interventricular septum; LA, left atrium; LV, left ventricle; MM, molecular mass markers (top to
bottom, approx. 40, 30 and 20 kDa); PM,
pectinate muscle from inner surface of right
auricle; RTA, right atrium; RV, right ventricle;
SAN, area of sinoatrial node in right atrial
wall; SM, septomarginal band of right ventricle; -S, septal end; -M, midsection; -FW,
free wall end; TC, terminal crest of right
auricle/atrium junction.
infectivity nor PrPSc has been detected in heart of BSEinfected cattle (Wells et al., 1996; European Commission
Scientific Steering Committee, 2002; Buschmann &
Groschup, 2005; Wells et al., 2005). Similar data from
cardiac monitoring of CWD-infected cervids are not
available, but could be informative.
(McKibben & Getty, 1969a, b, 1970; Cabot, 1990). Both
autonomic cardiac-control centres are among neural sites
that accumulate PrPd during CWD infection following
initial deposition and propagation in the dorsal motor
nucleus of the vagus and in the spinal cord (Sigurdson et al.,
1999, 2001; Peters et al., 2000; Spraker et al., 2002b).
It remains to be determined whether the prions that we
detected in cardiac tissue were localized to nerve fibres or
subcellular structures within myocytes. For this study, our
IHC examination of cardiac tissues from CWD-infected
animals was restricted to lv. The grouped arrangement of
PrP-labelled myocytes that we found in ventricular muscle
might be the result of prion movement along nerve fibres
that correspond to a particular subpopulation of neurons
innervating the heart. Further investigation, such as colabelling studies, will be required for a better understanding
of the structural arrangements underlying our observations.
Finally, determining what differential factors in these closely
related species are responsible for the appearance of PrPd in
heart of elk and white-tailed deer, but not of mule deer, may
provide important insights into variables that promote or
inhibit prion trafficking along neural conduits during a TSE
infection.
It will also be necessary to examine by IHC sites throughout
the heart such as were analysed in frozen tissues by Western
blot, in order to find out what the distribution of PrPd is
and, possibly, where it appears first. Many of the heartpositive animals were in clinical stages of disease, but the
time during infection at which PrPd appears in heart tissue
has not been determined. Although discussions of routes by
which PrPd reaches the heart are speculative in nature at this
time, if it appears only after having progressed to the brain
and spinal cord, this would suggest secondary dissemination
from CNS to the heart, possibly via efferent parasympathetic
and sympathetic neurons that innervate the heart from
parasympathetic nuclei of the ventrolateral medulla of the
brainstem (nucleus ambiguus primarily) (Loewy & Spyer,
1990; Standish et al., 1994) and from sympathetic
preganglionic neurons that arise in the intermediolateral
cell column of rostral sections of the thoracic spinal cord
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ACKNOWLEDGEMENTS
We would like to thank S. Ezidinma and L. DeLauter for performance
of the ELISAs, P. Jaeger and M. Thelen for excellent histology and
immunohistochemical work, T. E. Cornish and W. Cook for
performing some of the necropsies, M. W. Miller of the Colorado
Division of Wildlife for providing heart tissue from animals used in
unpublished collaborative projects with E. S. W., D. L. Montgomery for
invaluable assistance and guidance on questions of histopathology and
anatomy following the loss of Dr Williams and K. Loughney for helpful
advice on the manuscript. This work was supported by the Department
of Veterinary Sciences, the National Cattlemen’s Beef Association, the
International Association of Fish and Wildlife Agencies through the US
Geological Survey and the Wyoming Wildlife/Livestock Disease
Partnership.
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PrPSc accumulation in myocytes from sheep incubating natural
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