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United States Department of Agriculture

Agricultural Research Service

Research Project: Mitigating the Risk of Transmission and Environmental Contamination of Transmissible Spongiform Encephalopathies

Location: Animal Disease Research

Title: Sensitive and specific detection of classical scrapie prions in the brain of goats by real-time quaking-induced conversion

Author
item Dassanayake, Rohana
item Orru, Christina
item Hughson, Andrew
item Caughey, Byron
item Graça, Telmo
item Zhuang, Dongyue
item Madsen-bouterse, Sally
item Knowles, Donald - Don
item Schneider, David

Submitted to: Journal of General Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/9/2015
Publication Date: 3/1/2016
Citation: Dassanayake, R.P., Orru, C.D., Hughson, A.G., Caughey, B., Graça, T., Zhuang, D., Madsen-Bouterse, S.A., Knowles Jr, D.P., Schneider, D.A. 2016. Sensitive and specific detection of classical scrapie prions in the brain of goats by real-time quaking-induced conversion. Journal of General Virology. (97):803-812.

Interpretive Summary: Classical scrapie is a naturally transmissible, fatal neurologic disease of goats and sheep that is caused by scrapie prions. Diagnosis is currently limited to immunoassays that detect significant tissue accumulation of the prion protein that became misfolded during the course of infection. Recent studies demonstrate that alternative use of novel protein misfolding amplification assays greatly improves detection sensitivity for misfolded forms of the prion protein. In this study, we report the successful application and optimization of one such assay—the real-time quaking-induced conversion (RT-QuIC) assay—to the highly sensitive detection of misfolded forms of the prion protein in the hindbrain of goats with naturally acquired classical scrapie infection.

Technical Abstract: The real-time quaking-induced conversion (RT-QuIC) is a rapid, specific, and sensitive prion seeding activity detection assay that uses recombinant prion protein (rPrPSen) to detect sub-infectious levels of the abnormal isoforms of the prion protein (PrPSc). Although RT-QuIC has been successfully used to detect PrPSc in various tissues from humans and animals, including sheep, tissues from goats infected with classical scrapie have not yet been tested. Therefore, aims of the present study were to (a) evaluate whether prion seeds in brain tissues of goats with scrapie could be detected using RT-QuIC, (b) further optimize RT-QuIC conditions to improve scrapie detection, and (c) compare the performance of the RT-QuIC for the detection of PrPSc to the more commonly used western blot and enzyme-linked immunosorbent assays (ELISA). We further optimized RT-QuIC conditions for sensitive and specific detection of goat scrapie seeding activity in brain tissue from clinical animals. When used in the presence of a 200 mM final concentration of sodium chloride, both full-length sheep rPrPSen substrates (PrP genotypes A136R154Q171 and V136R154Q171) provided good discrimination between scrapie infected and normal goat brain samples within 15 h. Our findings indicate that the RT-QuIC is at least 10,000-fold more sensitive than the ELISA and western blot assays for the detection of scrapie seeding activity in goat brains samples. Taken together, these findings suggest that RT-QuIC sensitively detects prion seeding activity in classical scrapie-infected goat brain samples.

Last Modified: 06/28/2017
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