Early and Non-Invasive Detection of Chronic Wasting Disease Prions in Elk Feces by Real-Time Quaking Induced Conversion

Abstract

Chronic wasting disease (CWD) is a fatal prion disease of wild and captive cervids in North America. Prions are infectious agents composed of a misfolded version of a host-encoded protein, termed PrPSc. Infected cervids excrete and secrete prions, contributing to lateral transmission. Geographical distribution is expanding and case numbers in wild cervids are increasing. Recently, the first European cases of CWD have been reported in a wild reindeer and two moose from Norway. Therefore, methods to detect the infection early in the incubation time using easily available samples are desirable to facilitate effective disease management. We have adapted the real-time quaking induced conversion (RT-QuIC) assay, a sensitive in vitro prion amplification method, for pre-clinical detection of prion seeding activity in elk feces. Testing fecal samples from orally inoculated elk taken at various time points post infection revealed early shedding and detectable prion seeding activity throughout the disease course. Early shedding was also found in two elk encoding a PrP genotype associated with reduced susceptibility for CWD. In summary, we suggest that detection of CWD prions in feces by RT-QuIC may become a useful tool to support CWD surveillance in wild and captive cervids. The finding of early shedding independent of the elk’s prion protein genotype raises the question whether prolonged survival is beneficial, considering accumulation of environmental prions and its contribution to CWD transmission upon extended duration of shedding.

The ecology of chronic wasting disease in wildlife

“…These procedures enable specific detection of very small quantities of PrP Sc in tissues and biological fluids, likely approaching the levels of single particles of PrP Sc . Both PMCA and RT-QuIC have been used to detect CWD prions at high sensitivity and specificity in various tissues, fluids, and excreta (Pritzkow, Morales & Soto, 2014;Cheng et al, 2016;Kramm et al, 2017). Moreover, both PMCA (Saborio et al, 2001) and RT-QuIC (Orrú et al, 2017) have been reproduced extensively by many investigators around the world, and these technologies are currently being used in the diagnosis of human prion diseases in the USA and Europe.…”

Pathogen-mediated selection in free-ranging elk populations infected by chronic wasting disease

“…This suggests that greater PrP variability could interact with and overcome selection for alleles that reduce susceptibility to prion infection or are associated with prolonged incubation times of prion diseases. Second, recent captive work has found that elk with an L allele can shed PrP res well before the signs of disease, suggesting that prolonged survival and selection for such alleles may lead to greater transmission potential and environmental contamination (30). Together, these factors emphasize that managers should be cautious when trying to assess the future long-term impacts of prion genotype frequency on elk survival and population growth, particularly given that CWD has been found to decrease elk survival and population growth even in the presence of greater L allele frequency (10).…”

Amplification Techniques for the Detection of Misfolded Prion Proteins in Experimental and Clinical Samples

“…Only rarely have both of these assays been used to evaluate the same sample sets (Haley et al., 2013), so direct comparisons between the sensitivities and specificities of the two have been problematic. A number of sample types have been analyzed using both the PMCA and the RT‐QuIC assays, including blood (Orru et al., 2011; Saa et al., 2006), cerebrospinal fluid (Nichols et al., 2012; Orru et al., 2009) urine (Gonzalez‐Romero, Barria, Leon, Morales, & Soto, 2008; John, Schatzl, & Gilch, 2013), feces (Cheng et al., 2016; Pulford et al., 2012), brain (Atarashi et al., 2008; Saborio et al., 2001), and various peripheral tissues (Haley et al., 2011; Henderson et al., 2015). When deciding which assay would be most practical for a given laboratory, it may be useful to consider factors unrelated to the assays’ published performance.…”

“…Substrate selection is likewise an important factor to consider for successful amplification using the RT‐QuIC assay. One of the most widely employed substrates is a truncated form of the Syrian‐hamster prion protein (Cramm et al., 2016; Franceschini et al., 2017; Henderson et al., 2013; Orru et al., 2009; Schmitz et al., 2016; Wilham et al., 2010), although a number of other truncated and full‐length prion proteins have been used to successfully amplify a range of infectious prions (Cheng et al., 2016; Haley et al., 2017). Although careful handling of the recombinant protein is important for effective amplification, it is equally important to appropriately handle all buffers involved in the protein purification process (Support Protocol 2).…”

“…Tissues like brain and lymph node can be effectively amplified using crude homogenates in readily prepared buffers. Other tissues, and more importantly bodily fluids or environmental samples, may require more advanced preparatory steps (Cheng et al., 2016; Denkers et al., 2016; Henderson et al., 2013; Saunders et al., 2011), including concentration methods or techniques meant to reduce the level of supposed assay inhibitors. For either assay, one of these suggested inhibitors is blood, which may explain why there have been relatively few publications describing prion amplification from blood samples (Castilla et al., 2005; Elder et al., 2015; Orru et al., 2011).…”