Thursday, August 29, 2013

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
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
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
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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.
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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 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. For information about publishing your research in BMC journals or any BioMed Central journal, go to
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