Rapid Antemortem Detection of CWD Prions in Deer Saliva

Abstract

Chronic wasting disease (CWD) is an efficiently transmitted prion disease of cervids, now identified in 22 United States, 2 Canadian provinces and Korea. One hallmark of CWD is the shedding of infectious prions in saliva, as demonstrated by bioassay in deer. It is also clear that the concentration of prions in saliva, blood, urine and feces is much lower than in the nervous system or lymphoid tissues. Rapid in vitro detection of CWD (and other) prions in body fluids and excreta has been problematic due to the sensitivity limits of direct assays (western blotting, ELISA) and the presence of inhibitors in these complex biological materials that hamper detection. Here we use real-time quaking induced conversion (RT-QuIC) to demonstrate CWD prions in both diluted and prion-enriched saliva samples from asymptomatic and symptomatic white-tailed deer. CWD prions were detected in 14 of 24 (58.3%) diluted saliva samples from CWD-exposed white-tailed deer, including 9 of 14 asymptomatic animals (64.2%). In addition, a phosphotungstic acid enrichment enhanced the RT-QuIC assay sensitivity, enabling detection in 19 of 24 (79.1%) of the above saliva samples. Bioassay in Tg[CerPrP] mice confirmed the presence of infectious prions in 2 of 2 RT-QuIC-positive saliva samples so examined. The modified RT-QuIC analysis described represents a non-invasive, rapid ante-mortem detection of prions in complex biologic fluids, excreta, or environmental samples as well as a tool for exploring prion trafficking, peripheralization, and dissemination.

Quantitative assessment of prion infectivity in tissues and body fluids by real-time quaking-induced conversion

“…As no reactions were positive at 10 21 and 10 22 dilution of a 10 % brain homogenate, we tested whether the addition of PTA precipitations of dilutions of the same homogenate could eliminate presumed inhibitors as demonstrated in previous work (Elder et al, 2013;Henderson et al, 2013). Indeed, when a 10 22 dilution of CWD+ brain homogenate was subjected to PTA precipitation, we observed positive RT-QuIC reactions (Fig.…”

“…The ASA and RT-QuIC are generally analogous to sPMCA, but employ recombinant PrP C instead of brain homogenate as substrate for disease-specific, in vitro, amyloid formation (Atarashi et al, 2007(Atarashi et al, , 2011aColby et al, 2007). RT-QuIC offers the advantage of real-time assessment of amyloid fibril generation and has been successfully applied to many seed sources, some of which are available for antemortem sampling (Atarashi et al, 2011a, b;Elder et al, 2013;Haley et al, 2013;Henderson et al, 2013;McGuire et al, 2012;Orrú et al, 2011Orrú et al, , 2014Peden et al, 2011;Sano et al, 2013;Wilham et al, 2010).…”

“…2) (Elder et al, 2013;Henderson et al, 2013). However, the addition of the PTA did not result in positive reactions when a 10 21 dilution of brain homogenate was tested ( Fig.…”

Experimental Transmission of the Chronic Wasting Disease Agent to Swine after Oral or Intracranial Inoculation

“…The detection of PrP Sc in lymphoid tissues from the heads and guts of CWD-infected pigs raises the possibility that pigs are able to shed prions in excreta, as has been shown for saliva (48)(49)(50)(51) and feces (52-54) from CWD-affected cervids. Unfortunately, saliva and feces samples were not collected in the present study.…”

Challenges in managing the risks of chronic wasting disease

“…Ante-mortem CWD detection and surveillance detection methods (Haley et al, 2012;Haley and Hoover, 2015;Henderson et al, 2013;John et al, 2013) are under development. In the past, the main methods for CWD diagnosis have been immunohistochemistry or ELISA on post-mortem brain samples of deceased animals.…”