In vitro prion protein conversion suggests risk of bighorn sheep (Ovis canadensis) to transmissible spongiform encephalopathies

Abstract

BackgroundTransmissible 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.ResultsWe 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.ConclusionsOur 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.

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an <em>In Vitro</em> Prion Protein Conversion Assay

“…Disadvantages of the CER assay compared to PMCA include limited amplification of PrP res , additional steps in CER substrate preparation and no known infectivity in the PrP res produced by CER reactions. It is to be noted that in a previous study, however, we found the CER assay resulted in a similar pattern of PrP res formation as that of PMCA 15 . The results presented here also indicate that the CER assay correctly predicts the species barriers for transmission of various TSEs to laboratory mice (Figure 3) and suggest that bobcats may have susceptibility to CWD (Figure 4), in agreement with reports that domestic cats (Felis catus) can acquire prion disease after experimental CWD challenge 26,27 .…”

“…A number of in vitro assays, based on assessing the conversion of host PrP C to a proteinase K (PK PrP res in pH 7.4 substrate relative to that of the pH 3.5 substrate provides a measure of the species barrier. We have found that the CER assay predicts known species barriers of laboratory mice to various TSEs and have used the assay in efforts to predict the species barrier of numerous mammalian species, including bighorn sheep, to chronic wasting disease (CWD) and other TSEs 14,15 . Investigators interested in a tool allowing rapid screening of TSE species barriers or assessment of PrP C -to-PrP res conversion will find this methodology useful.…”

Predicting the spread-risk potential of chronic wasting disease to sympatric ungulate species

“…While our samples are from Canadian populations of these potentially at-risk species, the data likely represent the spread risk for many in North America. The PrP region has been sequenced in bighorn sheep in the United States from populations in Washington [29], and they found the single haplotype we identified. Similarly for bison, Seabury et al [30] sequenced individuals in four state parks, and found the same genetic variants we identified in the Canadian populations.…”

Opportunistic Surveillance of Captive and Free-Ranging Bighorn Sheep (Ovis Canadensis) in Colorado, Usa, for Transmissible Spongiform Encephalopathies

“…However, the potential susceptibility of other, more distantly related species has been suggested by intracerebral inoculation studies and protein conversion assays. Domestic sheep (Ovis aries) are susceptible to CWD by intracerebral inoculation (Hamir et al 2006), and both domestic and bighorn sheep (Ovis canadensis) are potentially susceptible to CWD on the basis of protein conversion assays (Raymond et al 2000;Morawski et al 2013). Species phylogeny and PRNP (prion protein) gene polymorphisms also predict susceptibility of bighorn sheep to CWD (Cullingham et al 2020).…”

“…Species phylogeny and PRNP (prion protein) gene polymorphisms also predict susceptibility of bighorn sheep to CWD (Cullingham et al 2020). Bighorn sheep overlap in range with deer, elk, and moose in parts of western North America, raising concerns for the transmission of CWD from cervids to bighorn sheep (Morawski et al 2013). Additionally, bighorn sheep may be exposed to scrapie, a naturally occurring TSE of domestic sheep and goats (Capra hircus; Greenlee 2019) that has also been recognized in captive mouflon (Ovis orientalis orientalis) sheep (Wood et al 1992a).…”