In vitro prion protein conversion suggests risk of bighorn sheep
(Ovis canadensis) to transmissible spongiform encephalopathies
In vitro prion protein conversion suggests risk of bighorn sheep (Ovis
canadensis) to transmissible spongiform encephalopathies
Aaron R Morawski124, Christina M Carlson23, Haeyoon Chang2 and Christopher
J Johnson2*
* Corresponding author: Christopher J Johnson cjjohnson@usgs.gov
Author Affiliations
1 Department of Bacteriology, University of Wisconsin, Madison, WI, USA
2 USGS National Wildlife Health Center, Madison, WI, USA
3 Program in Cellular and Molecular Biology, University of Wisconsin,
Madison, WI, USA
4 Present address: National Institutes of Health, 9000 Rockville Pike,
Bethesda 20892, Maryland, USA
For all author emails, please log on.
BMC Veterinary Research 2013, 9:157 doi:10.1186/1746-6148-9-157
Published: 9 August 2013 Abstract Background Transmissible spongiform
encephalopathies (TSEs) affect both domestic sheep (scrapie) and captive and
free-ranging cervids (chronic wasting disease; CWD). The geographical range of
bighorn sheep (Ovis canadensis; BHS) overlaps with states or provinces that have
contained scrapie-positive sheep or goats and areas with present epizootics of
CWD in cervids. No TSEs have been documented in BHS, but the susceptibility of
this species to TSEs remains unknown.
Results We acquired a library of BHS tissues and found no evidence of
preexisting TSEs in these animals. The prion protein gene (Prnp) in all BHS in
our library was identical to scrapie-susceptible domestic sheep (A136R154Q171
genotype). Using an in vitro prion protein conversion assay, which has been
previously used to assess TSE species barriers and, in our study appears to
recollect known species barriers in mice, we assessed the potential
transmissibility of TSEs to BHS. As expected based upon Prnp genotype, we
observed BHS prion protein conversion by classical scrapie agent and evidence
for a species barrier between transmissible mink encephalopathy (TME) and BHS.
Interestingly, our data suggest that the species barrier of BHS to white-tailed
deer or wapiti CWD agents is likely low. We also used protein misfolding cyclic
amplification to confirm that CWD, but not TME, can template prion protein
misfolding in A136R154Q171 genotype sheep.
Conclusions Our results indicate the in vitro conversion assay used in our
study does mimic the species barrier of mice to the TSE agents that we tested.
Based on Prnp genotype and results from conversion assays, BHS are likely to be
susceptible to infection by classical scrapie. Despite mismatches in amino acids
thought to modulate prion protein conversion, our data indicate that
A136R154Q171 genotype sheep prion protein is misfolded by CWD agent, suggesting
that these animals could be susceptible to CWD. Further investigation of TSE
transmissibility to BHS, including animal studies, is warranted. The lack of
reported TSEs in BHS may be attributable to other host factors or a lack of TSE
surveillance in this species.
Keywords: Bighorn sheep; Scrapie; Chronic wasting disease; Transmissible
mink encephalopathy; Species barrier
Discussion Using the CER assay, we found evidence that this method
recapitulates known species barriers of laboratory mice to TSEs and data to
suggest that BHS could be susceptible to classical scrapie and CWD, and less
susceptible to TME. Our present investigation and previous studies by others
[23,24] suggest that the CER assay can be a valuable addition to other in vitro
and in vivo measures of TSE species barriers such as cell-free conversions, PMCA
and animal bioassays. Advantages of the CER assay include its low cost, short
experimental timeframe and replacement of living animals with tissue samples
(which need not be from transgenic mice or perfused). Additionally, the assay
does not use radiation, reaction conditions are identical regardless of species
or strain of TSE agent and, in our hands, the CER assay is robust and forgiving.
Disadvantages of the assay include poor sensitivity compared to PMCA precluding
the use of CER as a means to detect PrPTSE, the PrPres product of conversion
reactions is not known to be infectious and, importantly, the CER assay is less
well-established than other methods of assessing species barriers which makes
interpreting reductions in conversion ratios in the absence of other
corroborating data difficult. For example, the correlation between a 50% CER and
TSE transmission parameters (e.g. disease penetrance, length of incubation
period) following experimental challenge remains undefined and further work is
needed to characterize this assay for use in species where bioassay data are not
available. Nonetheless, in our current study we did find a similar pattern of
PrPres formation when either CER or PMCA was used for conversion. Further
studies comparing the two techniques is an interesting future direction.
We are not aware of any studies examining natural transmission of scrapie
from domestic sheep to BHS, but in light of the sequence identity of BHS and
domestic A136R154Q171 sheep prion proteins, we must consider scrapie a potential
risk to BHS. Efforts to keep domestic sheep and BHS separated, as are prudent to
prevent transmission of other pathogens from domestic sheep to BHS [30], are
likely warranted around scrapie-infected farms. Further supporting the concept
that BHS are at risk for acquiring scrapie is a report of the disease in mouflon
(Ovis orientalis), another species of wild sheep [31].
In Figure 1, we show the overlap of BHS range with states and provinces
known to have had scrapie cases since 2008. Reduced numbers of scrapie outbreaks
in recent years, due to disease eradication efforts, may underrepresent the
exposure of BHS to scrapie in years prior to 2008. Long-term environmental
scrapie contamination may also still be contributing to BHS exposure to disease
agent many years after scrapie outbreaks. If incubation periods of scrapie in
BHS are greater than five years, exposure of BHS to pre-2008 scrapie flocks may
only now have the potential to manifest as disease in BHS. The lack of current
evidence for scrapie transmission to BHS could simply be due to insufficient
surveillance, but other explanations, such as different susceptibilities by
varying routes of exposure between domestic sheep and BHS or BHS being a
“dead-end” host for scrapie, should also be explored. Sheep with Prnp genotype
V136R154Q171 have been considered to be most -susceptible to classical scrapie
and selective breeding efforts have focused on reducing the numbers of these
animals in domestic sheep flocks across the U.S. as a scrapie risk reduction
measure. Recent research, however, by Gonzalez et al. strongly suggests that
Prnp genotype of the recipient sheep is not the sole factor determining its
scrapie susceptibility in vivo[32]. In carrying out a series of codon 136
homologous, semi-homologous, and heterologous transmissions of two different
natural scrapie isolates into domestic sheep, the study authors conclude that
Prnp genotype alone cannot account for the diversity of disease phenotypes
observed and that the “scrapie phenotype in sheep results from a complex
interaction between source, donor and recipient factors” [32]. The
susceptibility of BHS to scrapie is almost certainly dictated by this same
interplay. More work is needed to explore the role of scrapie genetics on
potential BHS disease transmission, as are analyses of BHS Prnp genetics using
more geographically disparate samples.
The finding that white-tailed deer CWD agent could convert sheep PrPC to
PrPres in either CER assays (Figure 6) or PMCA (Additional file 2) was notable
given the sequence variations found between BHS or domestic sheep and
white-tailed deer prion proteins (Figure 2), including serine to asparagine and
asparagine to threonine changes in the “rigid loop” portion of the protein
thought to control species susceptibility to CWD [33,34]. By CER assay, we
similarly found conversion of BHS PrPC by wapiti CWD heterozygous
methionine/leucine at position 132. In wapiti, animals heterozygous or
homozygous for leucine at prion protein amino acid 132 (L132) have a lengthened
CWD incubation period [35] and L132 appears to limit CWD, but not classical
scrapie, susceptibility in a transgenic mouse model [36]. Despite these amino
acid mismatches, including those in the “rigid loop”, the CWD agents were still
effective at misfolding PrPC from BHS. In previous studies, we have also found
that voles, which have mismatches in the “rigid loop” portion of the protein,
are susceptible PrP misfolding and infection by cervid CWD [37,38].
Previous work on the species barrier of sheep to CWD has been equivocal.
Using cell-free conversion assays, Raymond et al. found that A136Q171 domestic
sheep PrPC was not especially-well converted by CWD agent, but was the
non-cervid substrate, among six tested species, that yielded the most
PrPres[21]. In an animal study, Hamir et al. intracerebrally challenged eight
domestic sheep of various Prnp genotypes with mule deer CWD [39]. One
clinically-positive (heterozygous A/V136R154Q171) and one preclinical sheep
(homozygous A136R154Q171) were identified at the conclusion of the study,
indicating that sheep can be infected by CWD, although transmission is not
especially facile.
The results of our conversion assays appear to be supported by in vivo work
by Béringue et al. which indicate that V136R154Q171 ovinized transgenic mice
challenged with wapiti CWD harbor high levels of splenic PrPres, indicating that
sheep PrPC is susceptible to misfolding by CWD agent [40]. At least one group
has failed to observe clinical TSE signs in BHS when they were housed with a
facility with CWD-infected animals [7]. Our results in combination with those of
Béringue et al., suggest that the lack of CWD transmission to BHS was not due to
inability of BHS PrPC to be misfolded by CWD agent, but must derive from other
factors.
In our investigation, we used white-tailed deer and wapiti CWD, but have
not yet investigated BHS prion protein conversion by mule deer CWD. Given the
sequence similarity among cervid Prnp genes and our evidence that white-tailed
deer and wapiti CWD can convert BHS PrPC to PrPres, we expect CWD from the
various species to behave similarly. In a previous report, Li et al. found less
PrPres generation in domestic sheep substrates when templated by wapiti CWD [23]
than we found for BHS in our study. The genotype of the domestic sheep substrate
in the previous study is unclear and differences between the sheep prion protein
sequences or other species-specific differences could explain the limited
conversion that they observed. Alternatively, differences in the genotypes of
the wapiti CWD isolates used in the two studies could also explain variations in
PrPres levels in sheep substrates.
Conclusion The results from our study suggest that the CER assay has the
potential to be a useful tool to screen TSE species barriers. Further
comparisons with PMCA and bioassays will clarify the best uses of the assay and
help to define CER that are < 100%. We found that BHS are unlikely to have
resistance to domestic sheep classical scrapie due to their Prnp genotype. Our
conversion reactions suggest that the species barrier protecting BHS from CWD
may not be large and further studies, including in vivo experiments, are
warranted. These animal challenge studies need not necessarily be performed in
BHS, but could rather use Prnp genotype A136R154Q171 domestic sheep or existing
transgenic mouse models [41]. Additionally, investigation into the
susceptibility of BHS to atypical forms of scrapie is also an interesting future
direction.
Deadly Diseases Could Strike Bighorn Sheep
Released: 8/28/2013 1:18:09 PM
Contact Information: U.S. Department of the Interior, U.S. Geological
Survey Office of Communications and Publishing 12201 Sunrise Valley Dr, MS 119
Reston, VA 20192 Gail Moede Rogall 1-click interview Phone: 608-270-2438
Marisa Lubeck 1-click interview Phone: 303-202-4765
Declining bighorn sheep populations may be vulnerable to some of the fatal
diseases, including chronic wasting disease (CWD), that are found in their
western U.S. habitats, according to a new U.S. Geological Survey study.
USGS National Wildlife Health Center (NWHC) research showed that bighorn
sheep are likely susceptible to the deadly neurological diseases scrapie and
CWD, which are occurring in or near natural bighorn sheep environments. These
fatal diseases are caused by mysterious proteins called prions, and are known to
infect domestic sheep (scrapie) and non-domestic deer, elk, and moose (CWD). The
USGS study is published in the journal BMC Veterinary Research, and is available
online.
"Bighorn sheep are economically and culturally important to the western
U.S.," said Dr. Christopher Johnson, USGS scientist and senior author of the
report. "Understanding future risks to the health of bighorn sheep is key to
proper management of the species."
USGS laboratory tests found evidence that bighorn sheep could be vulnerable
to CWD from either white-tailed deer or elk, and to a domestic sheep prion
disease known as scrapie. However, none of a small number of bighorn sheep
sampled in the study showed evidence of infection.
"Our results do not mean that bighorns get, or will eventually get, prion
diseases," Johnson said. "However, wildlife species like bighorn sheep are
increasingly exposed to areas where CWD occurs as the disease expands to new
geographical areas and increases in prevalence."
The laboratory test results could be useful to wildlife managers because
bighorn sheep habitats overlap with farms and ranches with scrapie-infected
sheep and regions where CWD is common in deer, elk, and moose.
Bighorn sheep populations in western North America have declined from
habitat loss and, more recently, epidemics of fatal pneumonia thought to be
transmitted to them from domestic sheep. Prion diseases are another possible
threat to this valuable species.
For more information on prion diseases such as CWD, please visit the USGS
NWHC website.
Friday, July 26, 2013
Voluntary Scrapie Program USA UPDATE July 26, 2013 increase in FY 2013 is
not statistically meaningful due to the sample size
Thursday, March 29, 2012
atypical Nor-98 Scrapie has spread from coast to coast in the USA 2012
NIAA Annual Conference April 11-14, 2011San Antonio, Texas
Wednesday, February 16, 2011
IN CONFIDENCE
SCRAPIE TRANSMISSION TO CHIMPANZEES
IN CONFIDENCE
Sunday, December 12, 2010
EFSA reviews BSE/TSE infectivity in small ruminant tissues News Story 2
December 2010
Sunday, April 18, 2010
SCRAPIE AND ATYPICAL SCRAPIE TRANSMISSION STUDIES A REVIEW 2010
Saturday, July 6, 2013
Small Ruminant Nor98 Prions Share Biochemical Features with Human
Gerstmann-Sträussler-Scheinker Disease and Variably Protease-Sensitive
Prionopathy
Research Article
Thursday, August 15, 2013
The emergence of novel BSE prions by serial passages of H-type BSE in
bovinized mice
snip...
Stability comparison of BSE to other cattle-passaged TSEs
In addition to comparing the different BSE strains, we also used the
stability assay to characterize the biochemical properties of other TSEs
passaged into cattle. Scrapie and CWD are both transmissible into cattle by IC
inoculation, leading to PrPSc accumulation--but not significant spongiform
changes--in the brain [29,30]. Transmissible mink encephalopathy has been
hypothesized to have originated from the feeding of downer cattle, possibly
carrying atypical, L-type BSE, to farm-raised mink [37]. We wanted to determine
if the profiles of PrPSc from these TSEs passaged in cattle brain were
distinguishable from each other or from other BSE strains, with potential
implications for understanding strain origins and/or improving (non-BSE) TSE
diagnosis in cattle.
snip...
Keywords
Bovine spongiform encephalopathy, BSE, ELISA, Prion, PrP, Scrapie,
Stability, Transmissible spongiform encephalopathy, TSE
ISSN 1746-6148 Article type Research article Submission date 12 April 2013
Acceptance date 12 August 2013 Publication date 15 August 2013 Article URL http://www.biomedcentral.com/1746-6148/9/167
Like all articles in BMC journals, this peer-reviewed article can be downloaded,
printed and distributed freely for any purposes (see copyright notice below).
Articles in BMC journals are listed in PubMed and archived at PubMed Central.
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Central journal, go to http://www.biomedcentral.com/info/
snip...see full text and more here ;
Thursday, August 15, 2013
Stability properties of PrPSc from cattle with experimental transmissible
spongiform encephalopathies: use of a rapid whole homogenate, protease-free
assay
Sunday, July 21, 2013
Welsh Government and Food Standards Agency Wales Joint Public Consultation
on the Proposed Transmissible Spongiform Encephalopathies (Wales) Regulations
2013 Singeltary Submission WG18417
TSS
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