Longitudinal Detection of Prion Shedding in Saliva and Urine by Chronic Wasting Disease-Infected Deer by Real-Time Quaking-Induced Conversion

Abstract

Chronic wasting disease (CWD) is an emergent, rapidly spreading prion disease of cervids. Shedding of infectious prions in saliva and urine is thought to be an important factor in CWD transmission. To help to elucidate this issue, we applied an in vitro amplification assay to determine the onset, duration, and magnitude of prion shedding in longitudinally collected saliva and urine samples from CWD-exposed white-tailed deer. We detected prion shedding as early as 3 months after CWD exposure and sustained shedding throughout the disease course. We estimated that the 50% lethal dose (LD 50 ) for cervidized transgenic mice would be contained in 1 ml of infected deer saliva or 10 ml of urine. Given the average course of infection and daily production of these body fluids, an infected deer would shed thousands of prion infectious doses over the course of CWD infection. The direct and indirect environmental impacts of this magnitude of prion shedding on cervid and noncervid species are surely significant. IMPORTANCEChronic wasting disease (CWD) is an emerging and uniformly fatal prion disease affecting free-ranging deer and elk and is now recognized in 22 U.S. states and 2 Canadian provinces. It is unique among prion diseases in that it is transmitted naturally through wild populations. A major hypothesis to explain CWD's florid spread is that prions are shed in excreta and transmitted via direct or indirect environmental contact. Here we use a rapid in vitro assay to show that infectious doses of CWD prions are in fact shed throughout the multiyear disease course in deer. This finding is an important advance in assessing the risks posed by shed CWD prions to animals as well as humans. Chronic wasting disease (CWD) is an emergent transmissible spongiform encephalopathy affecting free-ranging populations of mule deer (Odocoileus hemionus), white-tailed deer (Odocoileus virginianus), elk (Cervus canadensis), and moose (Alces alces) in North America (1, 2). CWD is the only known prion disease to spread horizontally through wild populations, in which it continues to expand in prevalence and range in North America (3). As a prion disease, CWD is caused by a pathogenic, misfolded conformation of the normal, natively folded cellular protein PrP C to a pathogenic prion conformer (variously designated PrP CWD , PrP SC , or PrP D ) (2, 4-7). A leading hypothesis for the facile spread of CWD in wild populations is that the accumulation and excretion of CWD prions in bodily fluids facilitate both direct animal-to-animal transfer and substantial environmental contamination leading to indirect infection (8-10). Infectious CWD prions have been identified in urine, saliva, blood, and feces by bioassay of deer or cervid PrP Cexpressing transgenic mice (11-16). Prions bound to soil are remarkably stable, retaining infectivity even after a decade (9,(17)(18)(19)(20). Moreover, some evidence suggests that prions bound to soil may increase infectivity through an unknown mechanism (21). Understanding the kinetics and magnitude of C...

Pathways of Prion Spread during Early Chronic Wasting Disease in Deer

“…We have previously reported that the prion concentration in the sample used to seed the RT-QuIC reaction directly correlates with the rate of amyloid formation (26,27). To explore the kinetics of PrP CWD accumulation in lymphoid tissues over the course of early infection, we compared the average amyloid formation rates in 96GG WTD lymphoid tissues from the 1-to 4-MPE collections with those collected from 3 white-tailed deer with terminal disease (20 to 24 months postinfection).…”

“…RT-QuIC was performed as previously described (26,27). Briefly, RT-QuIC experiments were carried out in black, optical-bottom 96-well plates (Nunc), with each well containing 0.1 mg/ml SHrPrP substrate and RT-QuIC reaction buffer (20 mM NaH 2 PO 4 , 320 mM NaCl, 1.0 mM EDTA, 1 mM thioflavin T).…”

“…Real-time quaking-induced conversion (RT-QuIC) assays were performed with recombinant truncated Syrian hamster PrP C (SHrPrP) encompassing residues 90 to 231, which was expressed and purified as previously described (26,27). Briefly, SHrPrP was expressed in BL21 Rosetta Escherichia coli (Novagen).…”

Infectious Prions in the Pregnancy Microenvironment of Chronic Wasting Disease-Infected Reeves' Muntjac Deer

“…RT-QuIC provides a rapid methodology for analysis of tissues and fluids containing minute quantities of prions (85,86) and may have utility in quantitative and correlative investigations of early and carrier states of prion diseases and potentially other protein misfolding disorders (86)(87)(88)(89). There is little to no evidence that RT-QuIC generates, or represents, the presence of prion infectivity.…”

Specificity, Size, and Frequency of Spaces That Characterize the Mechanism of Bulk Transepithelial Transport of Prions in the Nasal Cavities of Hamsters and Mice

“…Deer, elk, moose, sheep, and cattle use their olfactory systems for reproductive, feeding, and exploratory purposes and thus frequently expose their NCs to environmental materials. There are a number of environmental sources of infectious prions, including decaying carcasses (34), placenta (35,36), antler velvet (37), blood (38)(39)(40)(41)(42), urine (43)(44)(45)(46), and feces (47)(48)(49)(50), that could be inhaled into the NC and horizontally transmit disease (7,8) in both captive and free-ranging animal populations. It is worth noting that soil-bound prions remain infectious (19,(51)(52)(53) and that the physical size of the intercellular spaces reported here would accommodate the particle size of silt and clay (between 2 and 50 m), allowing infection from inhaled contaminated soil.…”