From: TSS (216-119-162-55.ipset44.wt.net)
Subject: Comparison of Abnormal Prion Protein Glycoform Patterns from TSE Agent-Infected Deer, Elk, Sheep, and Cattle [FULL TEXT]
Date: December 17, 2002 at 7:19 pm PST
JOURNAL OF VIROLOGY, Dec. 2002, p. 12365-12368
0022-538X/02/$04.00+0 DOI: 10.1128/JV1.76.23.12365-12368.2002
Copyright © 2002, American Society for Microbiology.
All Rights Reserved. Vol. 76, No. 23
Comparison of Abnormal Prion Protein Glycoform Patterns from
Transmissible Spongiform Encephalopathy Agent-Infected
Deer, Elk, Sheep, and Cattle
Richard E. Race,l* Anne Raines,l Thierry G. M. Baron,2 Michael W.
Miller,3 Allen Jenny,4 and Elizabeth S. Williams5
Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories,
National Institute of Allergy and Infectious Diseases, Hamilton,
Montana1; Agence Francaise de Securite Sanitaire des Aliments, Lyon,
France2; Wildlife Research Center, Colorado Division of Wildlife, Fort
Collins, Colorado3; Veterinary Services Laboratories, Animal and Plant
Health Inspection Service, U.S. Department of Agriculture, Ames, Iowa4;
and Department of Veterinary Sciences, University of Wyoming, Laramie,
Wyoming5
Received 29 April 2002/Accepted 23 August 2002
Analysis of abnormal prion protein glycoform patterns from chronic
wasting disease (CWD)-affected deer and elk, scrapie-affected sheep and
cattle, and cattle with bovine spongiform encephalopathy failed to
identify patterns capable of reliably distinguishing these transmissible
spongiform encephalopathy diseases. However, PrP-res patterns sometimes
differed among individual animals, suggesting infection by different or
multiple CWD strains in some species.
Chronic wasting disease (CWD) of deer and elk is of particular concern
to scientists and the general public because the potential for
transmission to humans or livestock is unknown. Rapid methods by which
to identify the source of transmissible spongifimn encephalopathy (TSE)
following cross-species transmission of the causal agent would be
valuable in identifying expanding host ranges of known TSEs. Various TSE
diseases have been distinguished on the basis of brain-derived PrP-res
glycoform patterns (1, 2, 5, 8-10, 14). We therefore analyzed brain
tissue from CWD-affected deer and elk, scrapie-affected sheep and
cattle, and cattle with bovine spongiform encephalopathy (BSE) to
determine if distinct abnormal prion protein (PrP-res) profiles
capable of differentiating these diseases could be identified.
The PrP-res immunoblot profiles of brain tissues from six mule deer,
three white-tailed deer, and l0 elk, all with CWD, as well as 13 sheep
and seven cattle with scrapie and six cattle with BSE, were analyzed.
For all of the deer and elk, the strongest PrP-res signal was observed
in the uppermost diglycosylated (30-kDa) band and the weakest signal was
observed in the lowest nonglycosylated (22-kDa) PrP-res band. Some
animal-to-animal variation was observed, especially among the deer (Fig.
1 and 2). We also analyzed the proportions of the uppermost and lowest
PrP-res bands for each deer and elk and expressed the data on it
scattergraph (Fig. 3 a). The patterns for elk were more tightly grouped
than those for deer, suggesting that the elk and deer were possibly
infected by different CWD strains.
Because sheep scrapie is believed to be the cause of BSE in Europe (15)
and has been implicated as a potential source of CWD, we also determined
the PrP-res patterns of scrapie-affected sheep. Both Suffolk and
white-face sheep were analyzed. For all but one of the sheep (Fig. lC,
lane 12), the uppermost PrP-res band gave thc strongest PrP-res signal
although the relative amounts of PrP-res in the upper band varied
among individuals. The relative intensity of the lowest PrP-res band
also varied among the sheep, being weak for some but stronger for others
(Fig. lC and 2). One Suffolk sheep gave an overall pattern similar to
that of the white-face sheep, and one white-face sheep gave a pattern
similar to that of the Suffolk sheep (Suffolk sheep, lane 5; white-face
sheep, lane 13), making it unlikely that scrapie-affected Suffolk sheep
could be differentiated from affected white-face sheep on the basis of
the PrP-res glycoform pattern alone. The proportions of the uppermost
and lowest PrP-res bands of the Suffolk and white-face sheep were also
compared. Suffolk sheep segregated somewhat from the white-face sheep,
but there were several animals that were not clearly in a specific group
(Fig. 3b). Thus, on the basis of the range of the PrP-res glycoform
pattern possibilities, we concluded that the scrapie-affected Suffolk
sheep could not be distinguished from the white-face sheep and that, in
general, the scrapie-affected sheep could not be distinguished from
CWD-affected cervids (Fig. 1 to 3).
We reasoned that if sheep scrapie is the source of BSE, one might expect
sheep with scrapie, cattle with BSE, and scrapie-affected cattle to give
similar PrP-res glycoform patterns, further supporting this hypothesis.
We therefore determined the PrP-res glycoform pattern of cattle with
sheep scrapie or BSE and compared the patterns to those previously
determined for scrapie-affected sheep. The PrP-res profiles for the
scrapie-affected cattle were uniform (Fig. 1D and 2), possibly
reflecting the fact that the cattle were infected at the same time with
the same inoculum, exposed to the same environment, and sacrificed at
similar stages of clinical disease. The PrP-res patterns of these
animals were not different enough to allow differentiation of
individual animals from CWD-affected cervids or scrapie-affected sheep.
However, the PrP-res pattern was clearly different from that of
BSE-affected cattle (Fig. 2 and 3c). Furthermore, the PrP-res patterns
of BSE-affected cattle were different from those of scrapie-affected
cattle and white-face sheep, especially when the ratios of the uppermost
and lowest PrP-res bands were compared (Fig. 2 and 3). BSE-affected
cattle also tended to cluster away from the other species, but there was
overlap with individual deer, elk, and Suffolk sheep, which precluded
differentiation of the cervid TSE diseases and scrapie from BSE (Fig. 2
and 3).
There is no direct evidence that CWD has been transmitted to humans (3).
However, because CWD is a relatively new TSE, it is unlikely that enough
people have consumed enough CWD-affected cervids to result in a
clinically or pathologically recognizable disease attributable to CWD,
especially considering the very long incubation periods characteristic
of TSE diseases. Also unknown is whether sheep or cattle can be infected
with CWD under natural circumstances or whether people might be
susceptible to CWD passaged through sheep or cattle. In the past,
differences in PrP-res glycoform profiles have reliably differentiated
certain TSE agent strains from others (1, 2, 4, 5, 8, 9, 14). However,
inconsistent results were reported when sheep scrapie and BSE were
analyzed. One study stated that scrapie-affected and BSE-affected sheep
could be differentiated on the basis of differences in brain-associated
PrP-res glycoforms (7), whereas a second study concluded that they could
not be distinguished (2). In the study reported here, brain-derived
PrP-res glycoform patterns sometimes varied among individual deer, elk,
or sheep. Variation primarily involved differences in the ratio of the
three PrP-res protein bands relative to each other in individuals rather
than differences in the molecular weights of the various bands. The
basis for the PrP-res glycoform variation that we observed among
individual animals is not clcar. Variation could simply reflect the
range expected from randomly selected, heterogeneous populations of
TSE-affected ruminants. Alternatively, it is possible that the different
PrP-res patterns represent infection by one or more specific TSE strains
not yet characterized in these species. For example, the relatively
tight grouping of CWD-affected elk, compared to that of mule deer, might
indicate that elk are infected with a single strain of CWD agent
whereas mule deer are infected with multiple or different strains. The
brain region could influence the glycoform pattern. However, we observed
no differences in PrP-res glycoform patterns when six distinct brain
regions of clinically affected deer or elk were analyzed
(data not shown).
The general similarity of PrP-res glycoform patterns in scrapie-affected
sheep and CWD-affected cervids seems to support the hypothesis that CWD
arose from sheep scrapie, as do in vitro analyses (13). Sheep scrapie
has been present in the United States since at least 1947, and in many
geographical areas, sheep, deer, and elk share pastures and rangeland.
If scrapie-affected sheep were present in these situations, then
cross-species transmission might have occurred. Sheep scrapie is not
thought to cause disease in humans, although passage through cattle
appears to have changed this characteristic. It remains to be determined
if the same will be true of CWD.
(please note, pictures, charts, and graphs not available...TSS)
FIG. 1. Immunoblot analysis of brain derived PrP-res from CWD-affected
deer and elk and scrapie-affected sheep and cattle. PrP-res was purified
as previously described (11). Immunoblot assays were done as previously
described (12), except that a primary antibody designated R35, made
against PrP peptide 5'-CGQGGTHGQWNKPSK-3', was used. This antibody has
broad reactivity against PrP from deer, elk, sheep, cattle, mice,
hamsters, and possibly other species. Gels were developed with an ECF
kit (Amersham-Pharmacia) that allows quantitation of individual protein
bands with a phosphorimager (Molecular Dynamics). Panel A shows PrP-res
in brain tissue from nine (CWD)-affected mule or white-tailed deer.
Lanes contain 5- to 25-mg equivalents of brain tissue. No bands were
seen when 100 mg of brain tissue from normal deer was analyzed (data
not shown). Panel B shows 5- to 25-mg equivalents of brain tissue from
each of 10 CWD-affected elk. One hundred milligrams of brain tissue from
normal elk gave no PrP bands (data not shown). Panel C shows PrP-res
derived from brain tissue of scrapie-affected sheep. Lanes 1 to 7
contain 5- to 25-mg equivalents of brain tissue from naturally infected
Suffolk sheep of U.S. origin, and lanes 8 to 13 contain brain tissue
from white-faced breeds of sheep, including Leicester (lane 8),
Shropshire (lanes 9 and 10), Rambouillet (lane 11), and Cheviot (lanes
12 and 13). Panel D shows PrP-res from 5 to 25 mg of brain tissue from
scrapie-affected cattle. Details relating to those animals have been
published before (6). kd, kilodaltons.
FIG. 2. Brain samples from 10 elk, six mule deer, and three white-tailed
(wt) deer, all affected with CWD, and seven scrapie-affected cattle were
each immunoblotted four times. Brain samples from 13 scrapie-affected
sheep were each immunoblotted twice, and brain samples from six
BSE-affected cattle were immunoblotted five times. The values shown are
the average amounts of PrP-res per protein band (bars) for all
individuals (points) within the indicated species. Panel a shows a
species comparison of the uppermost fully glycosylated PrP-res band.
Mule deer and elk were statistically significantly different from
white-face (wf) sheep (P < 0.05) and from scrapie-affected cattle
(P < 0.05). BSE-affected cattle were different from white-face sheep and
scrapie-affected cattle (P < 0.05). Panel b shows species comparisons of
the middle diglycosylated band. Elk differed statistically
significantly from white-face sheep (P < 0.05), and BSE-affected cattle
differed from white-face sheep and scrapie-affected cattle (P < 0.05).
Panel c shows species comparisons of the lowest unglycosylated PrP-res
band. Mule deer differed statistically significantly from white-face
sheep and scrapie-affected cattle (P < 0.05). Suffolk sheep differed
from white-face sheep and scrapie-affected cattle (P < 0.05), while
BSE-affected cattle were different from white-face sheep and
scrapie-affected cattle (P < 0.05).
FIG. 3. Glycoform ratios of PrP-res from TSE-affected ruminants.
Glycoform ratios are presented as plots of the percentage of the total
PrP-res found in the upper diglycosylated PrP-res band versus the
percentage found in the lowwest nonglycosylated band. Each point
represents the average value for multiple immunoblots of each brain.
Panel a compares mule deer, white-tailed (wt) deer, and elk. Panel b
compares Suffolk and white-face (wf) sheep. Panel c compares
scrapie-affected and BSE-affected cattle, and panel d compares all of
the above.
We thank Anita Mora for graphic-art assistance, Irene Cook Rodriguez and
Cam Ruark for helping with the preparation of the manuscript, and Bruce
Chesebro, Byron Caughey, and Sue Priola for discussions relating to the
manuscript. Personnel of the Wyoming Game and Fish Department and the
Colorado Division of Wildlife are also appreciated and were critical to
collection of samples.
* Corresponding author. Mailing address: Laboratory of Persistent Viral
Diseases, Rocky Mountain Laboratories, National Institute of Allergy and
Infectious Diseases, 903 S. Fourth St. Hamilton, MT 59840. Phone: (406)
363-9358. Fax: (406) 363-9286. E-mail: rrace@niaid.nih.gov.
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TSS
