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From: TSS ()
Subject: Re: Transmission of Elk and Deer Prions to Transgenic Mice
Date: November 28, 2006 at 9:42 am PST

In Reply to: Transmission of Elk and Deer Prions to Transgenic Mice posted by TSS on September 1, 2006 at 1:07 pm:

FURTHER INTO THIS STUDY, some might find interest ;

Prions are transmissible pathogens that accumulate in the
central nervous system (CNS) and cause fatal neurodegeneration
(34). Prions are composed of an alternatively folded isoform
of the prion protein (PrP), denoted PrPSc. The precursor
of PrPSc is a cellular protein designated PrPC that is encoded
by a chromosomal gene. Prion diseases afflict humans as well
as livestock, such as cattle, goats, and sheep; additionally, prions
cause CNS disease in captive and wild populations of deer
and elk (51, 53). In contrast to scrapie of sheep and goats,
bovine spongiform encephalopathy (BSE) in cattle has been
transmitted to humans and has killed more than 170 teenagers
and young adults as variant Creutzfeldt-Jakob disease (vCJD)
(44, 49, 51, 52). BSE prions have been experimentally transmitted
to sheep and appear to be transmitted naturally among
sheep (6). Recently, BSE prions were found in goats (14).
The transmission of BSE prions to humans has elevated
concern about the possibility of the zoonotic transmission of
chronic wasting disease (CWD) from deer and elk to humans
(5, 56). Hunters and other consumers of venison are potentially
at risk to acquire prion disease from infected deer and
elk. CWD was first observed in 1967 in cervids and was recognized
as a prion disease a decade later (54). CWD has been
reported in 14 U.S. states and 2 Canadian provinces.
The epidemiology of CWD is unclear. In contrast to BSE
and scrapie, CWD is highly transmissible among cervids. In
some captive mule deer (Odocoileus hemionus) herds, 90% of
the animals have been reported to be infected with CWD
prions (28). The prevalence of CWD cases in free-ranging deer
populations can be up to 30% (53). The number of cases of
CWD that arises spontaneously and then spreads horizontally,
however, is unknown. Commercial farming and trade with
cervids may foster horizontal transmission of CWD. It seems
likely that, as surveillance improves, the known geographic
distribution of CWD will increase. Cases of CWD have been
reported in South Korea where elk had been imported from
Canada (21). Furthermore, free-ranging elk and deer occasionally
share the same pastures with cattle and sheep. It is therefore
of concern whether CWD prions can be transmitted to
livestock and on to humans. The passage of prions into a new
host species can alter the host range: hamsters are resistant to
CWD prions from deer and elk but are susceptible to CWD
prions previously passaged in ferrets (4).
While our work was in progress, two reports appeared describing
the transmission of CWD prions to transgenic (Tg)
mice expressing cervid PrP (9, 23). The first study showed
transmission of CWD prions to Tg(CerPrP) mice expressing
the S2 PrP allele (GenBank accession no. AF009180) of mule
deer, and the second study reported transmission of CWD
prions to Tg12 mice expressing the eGMSE PrP allele (Gen-
Bank accession no. AF156183) of Rocky Mountain elk (Cervus
elaphus nelsoni). The respective alleles are expressed exclusively
in deer or elk and give rise to PrP molecules that differ
only at residue 226, which is glutamine in deer PrP and glutamate
in elk PrP. Transmission of a CWD brain sample from
Rocky Mountain elk to Tg(CerPrP) mice resulted in incubation
times of 240 days. Transmission times of several CWD
brain samples from mule deer were between 230 and 260 days.
On second passage, the incubation time was 160 days in mice

homozygous for the transgene (9). The second study showed
transmission of CWD prions from elk to Tg12 mice within 120
to 140 days; no change in incubation time was observed on
second passage (23).
In the findings reported here, we describe studies with Tg mice
expressing either elk PrP [Tg(ElkPrP)] or deer PrP [Tg(DePrP)].
The Tg(ElkPrP) mice used in this study express the same PrP
as Tg12 mice (23), and the Tg(DePrP) mice express the same
PrP as Tg(CerPrP) mice (9). We generated four lines of
Tg(ElkPrP) mice, one of which was bred to homozygosity, and
two lines of Tg(DePrP) mice. We inoculated all lines with
CWD prions from elk (n  1). Additionally, we inoculated
Tg(ElkPrP) and Tg(DePrP) lines with CWD prions from mule
deer (n  2) and white-tailed deer (Odocoileus virginianus; n 
2). Tg(ElkPrP) mice succumbed to prion disease within 180 to
200 days and Tg(DePrP) mice, with slightly lower DePrP transgene
expression levels, within 300 to 400 days. Neuropathologic
analysis showed spongiform degeneration, florid PrP
amyloid plaques, and astrocytic gliosis in ill mice.
Based on the data reported here and those of others cited
above, CWD prions from elk, mule deer, or white-tailed deer
seem to be equally transmissible among these three cervid
species. In addition, CWD prions do not readily transmit disease
to Tg mice expressing human, bovine, or ovine PrP.
Whether CWD prions cause disease in humans or livestock
remains uncertain.



The overexpression of cervid PrPs in mice did not have any
deleterious effect on the Tg lines described here. Uninoculated
mice from one such Tg line was observed for 650 days (Table
1). The absence of spontaneous disease in these Tg mice allowed
us to use them to bioassay prions in the brains of elk and
deer that died of CWD.
Brainstem samples from elk, mule deer, and white-tailed
deer with CWD were inoculated into five Tg lines expressing
ElkPrP and two lines expressing DePrP. Bioassay of the Elk1
inoculum in the seven Tg cervid PrP lines showed that the
length of the incubation time is inversely proportional to the
level of cervid PrP expression in the brain (Fig. 5; Table 2).
When the level of cervid PrPC expression was similar to that of
MoPrPC in WT mice, it was designated 1. In Tg(DePrP) mice
expressing DePrP at 1, the incubation time was 300 days,
whether the CWD inoculum was from mule deer (MD1) or elk
(Elk1). Doubling the level of cervid PrPC to 2 resulted in a
reduction of the incubation time to 200 days for the Elk1 and
MD1 inocula while tripling the expression of cervid PrPC reduced
the incubation time to 100 days for the Elk1 inoculum.
A similar relationship was described earlier for Tg mice expressing
SHaPrPC (35); however, both MoPrP and SHaPrP
were coexpressed in those Tg lines. In the studies reported
here (Fig. 5), the MoPrP gene was disrupted (10) so that the
only PrP being expressed was cervid PrPC. In a recent study, Tg
mice expressing DePrP at 5 and 3 the level of PrP expressed
in WT FVB mice developed neurologic deficits at
235 days after intracerebral inoculation with CWD prions
from elk and at 225 to 264 days with CWD prions from mule
deer (9). In another study, two lines of Tg mice expressing
ElkPrP at 2 developed CNS disease 118 or 142 days after
inoculation with CWD prions from elk (23).
The CDI studies of the CWD inocula indicated that the Elk1
and MD1 inocula contained similar levels of PrPSc (Fig. 1A),
which is consistent with the indistinguishable incubation times
for these inocula in Tg(ElkPrP)12577, Tg(DePrP)10945, and
Tg(DePrP)10969 mice (Table 2). Interestingly, the levels of
PrPSc varied over a 100-fold range for the first nine cervid
brain specimens examined (Fig. 1A). Assessing the level of
PrPSc in brain samples in advance of our transmission studies
proved to have been quite useful in retrospect (Table 2).
Serial passage of CWD prions in the Tg(ElkPrP) mice resulted
in modest reductions in the incubation times, i.e., up to
70 days (Fig. 3A to D; Table 3). This shortening was seen
with prions from elk, mule deer, and white-tailed deer. These

results contrast with those for the serial passaging of BSE
prions in Tg(BoPrP) mice and the serial passaging of CWD
prions in Tg12 mice, for which no changes in the incubation
times were observed (23, 42, 44). There are several possible
explanations for the shortening of the incubation times upon
serial passage in Tg(ElkPrP)12577 mice. First, the level of
prions in the brains of cervids may be lower than in Tg(Elk
PrP)12577 mice. If that were the case, then the first Tg mouseto-
Tg mouse passages would be expected to exhibit shorter
incubation times than those found with passages from the
cervids to Tg mice. A corollary to this scenario is that the
incubation times upon subsequent passage in a given Tg line
should remain constant. Second, the cervid brain inocula may
be composed of a mixture of strains, and one strain may
emerge as the predominant strain over the length of the incubation
time. In this case, the predominant strain in the Tg
mouse brain exhibits a shorter incubation time during the next
passage, because it exists at a higher titer in the mouse brain
than in the cervid brain sample. Third, within a mixture of
prion strains, some slow strains may be inhibitory for faster
ones as previously reported (13, 22). If this were the case and
transmission to Tg mice resulted in the elimination of the
slower strain, then on subsequent passage in Tg mice, the
incubation time would shorten. Fourth, a posttranslational
modification in cervid PrP, such as the N-linked oligosaccharides
or the glycosylphosphatidylinositol anchor, might slow
replication of cervid prions in the Tg mice. If this were the
case, then on subsequent passage, CWD prions formed in a
mouse would exhibit shorter incubation times.
Except for the first possibility, for which endpoint titrations
can be used to establish the titers of CWD prions in cervid and
Tg mouse brains, distinguishing among the possibilities listed
above may be difficult. Interpreting such a titration study will
be facilitated if endpoint titrations in cervids give results similar
to those obtained with the Tg mice. It is notable that
endpoint titrations performed with cattle resulted in a titer of
106 50% infective dose units/g of brain tissue from the obexes
of BSE-infected cattle, whereas endpoint titrations performed
with Tg(BoPrP) mice resulted in a titer of 106.9 50% infective
dose units/g of brain tissue (39, 42, 50).
Both the glycoform abundance patterns and the distribution
of neuropathologic lesions in CWD-inoculated Tg(ElkPrP)
and Tg(DePrP) mice argue for a single prion strain. The molecular
masses of the di-, mono-, and unglycosylated PrPSc
fragments from all CWD isolates were similar before (Fig. 2A)
and after passaging in Tg(ElkPrP)12577 (Fig. 2B and D) and
Tg(DePrP)10945 (Fig. 2C) mice. Moreover, the relative abundance
of these glycoforms did not change upon passage in the
Tg mice (see Fig. S1 in the supplemental material).
Neuropathologic examination of ill Tg(ElkPrP) and Tg(DePrP)
mice demonstrated similar CNS lesions in all of the mice inoculated
with CWD prions from elk, mule deer, and whitetailed
deer (Fig. 6). The deposition patterns and neuroanatomic
distribution of both PrPSc deposition (Fig. 7) and florid
PrP amyloid plaques (Fig. 6) were similar for all inocula but
differed somewhat in intensity. Upon serial passage in Tg(Elk
PrP) mice, the CNS lesions remained unchanged (data not
shown). Overall, our neuropathologic findings for CWD-infected
Tg mice expressing DePrP or ElkPrP did not differ
substantially from those reported by others (9, 23).
In Tg(MoPrP)4053 mice inoculated with CWD prions, both
the morphology of the lesions and distribution of PrP amyloid
plaques (Fig. 6I) were different from those found in Tg(Elk
PrP) and Tg(DePrP) mice. In contrast to RML prions that
produce finely granular deposits of PrPSc in the absence of
amyloid plaques, the CWD prion strain was amyloidogenic in
the brains of Tg(MoPrP)4053 mice.
In contrast to Tg mice expressing cervid PrP, Tg mice expressing
human, bovine, or ovine PrP did not succumb to prion
disease after inoculation with CWD prions after 500 days
(Table 4). Our results agree with those of others who reported
that Tg mice expressing human PrP were resistant to CWD
prions but susceptible to sCJD prions (23). Despite our con-
firmation of an earlier study demonstrating that Tg mice expressing
HuPrP(M129) do not develop prion disease when
inoculated with CWD prions, any conclusions from such negative
data need to be tempered (7), especially in light of a
recent study with squirrel monkeys. Intracerebral inoculation
of CWD prions into squirrel monkeys (Saimiri sciureus) demonstrated
transmission to a nonhuman primate, arguing that
humans might be susceptible to CWD prions (27).
While our studies of Tg(BoPrP) mice inoculated with CWD
prions also gave negative results, a recent study reported that
five of 13 cattle inoculated with CWD prions produced PrPSc,
based on limited proteolysis and immunohistochemical staining
of brain sections (16). These studies were terminated 6
years after intracerebral inoculation, before any of the cattle
developed neurologic dysfunction. In other studies, sheep
scrapie prions were inoculated into elk (17). After more than
three years, PrPSc was found in the brains of three elk that
developed neurologic deficits before dying. Our negative results
with CWD prions inoculated into Tg(HuPrP), Tg(Bo
PrP), and Tg(OvPrP) mice might reflect low levels of prion
replication that are insufficient to produce disease during the
500-day observation period. Several investigators have described
situations in which prions from one species replicate
too slowly in another species to cause signs of neurologic
dysfunction but do produce disease with serial passage (19, 36).
In the studies reported here, we did not choose to passage
serially the brains of asymptomatic, CWD-inoculated Tg(Hu
PrP), Tg(BoPrP), and Tg(OvPrP) mice (Table 4). An alternative
explanation for our negative results may reside in the
strain(s) of CWD prions that we used for inoculation. While
our CWD prions were unable to initiate the conversion of
HuPrPC, BoPrPC, or OvPrPC into PrPSc, some other CWD
strains might be able to do so. Precedent for the latter has been
seen with human prions: human vCJD prions replicate well in
Tg(BoPrP) mice but multiply slowly in Tg(HuPrP) mice and in
Tg(MHu2M) mice expressing a chimeric human-mouse PrP (2,
24, 42, 44).
Whether hunters, cervid farmers, and aficionados of venison
are at increased risk for prion disease remains to be established.
Recently, CWD prions were also reported in the skeletal
muscles of infected deer, indicating a possible hazard for
the oral transmission of CWD prions (1). Tg(ElkPrP) and
Tg(DePrP) mice provide a sensitive and specific bioassay for
determining the levels of CWD prion infectivity in cervid tissues
and for studying the biology of these particular prions.
These Tg mice make it possible to determine the levels of
CWD prion titers in brain, muscle, liver, pancreas, kidney, and

other tissues as well as in the blood, urine, feces, and saliva of
both elk and deer. Elucidating the mode of CWD prion spread
among elk, deer, and moose will be important for understanding
why CWD prions are so contagious for domesticated and
free-ranging cervids. Such information may prove useful in
learning how to restrict the epizootic spread of CWD prions to
humans and livestock. ......snip.......end

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