Chronic wasting disease (CWD) affects cervids in North America, Asia, and Scandinavia. CWD is unique in its efficient spread, partially because of contact with infectious prions shed in secreta. To assess temporal profiles of CWD prion shedding, we collected saliva, urine, and feces from white-tailed deer for 66 months after exposure to low oral doses of CWD-positive brain tissue or saliva. We analyzed prion seeding activity by using modified amyloid amplification assays incorporating iron oxide bead extraction, which improved CWD detection and reduced false positives. CWD prions were detected in feces, urine, and saliva as early as 6 months postinfection. More frequent and consistent shedding was observed in deer homozygous for glycine at prion protein gene codon 96 than in deer expressing alternate genotypes. Our findings demonstrate that improved amplification methods can be used to identify early antemortem CWD prion shedding, which might aid in disease surveillance of cervids.

Chronic wasting disease (CWD) has been identified in North America, Asia, and Scandinavia (1-4) and is the only known prion disease in wildlife. CWD is often considered the most infectious of all prion diseases, but how CWD spreads so efficiently remains unclear. It is generally accepted that facile CWD transmission results from robust prion replication in target tissues, leading to shedding of infectious prions into saliva, urine, and feces (i.e., secreta). Cervids are likely exposed to infectious prions via direct animal-to-animal contact or indirect contact with the agent shed into the environment.

Landscapes previously housing CWD-infected cervids are contaminated with sufficient infectious prions to initiate subsequent infections (5,6). Fomite-only exposure of uninfected deer to buckets and bedding from suites housing CWD-positive animals resulted in CWD infections in disease-naive deer within 19 months after exposure (7). Studies in white-tailed deer (WTD; Odocoileus virginianus) also revealed that large oral doses of saliva, urine, or feces contained adequate concentrations of the CWD agent to initiate infections (8,9). Additional studies in WTD established a minimum oral CWD infectious dose equivalent to 100-300 ng CWD-positive brain tissue (10). Yet, equivalent saliva doses (16.5-30 mL) seem large compared with a dose expected in nature. Nevertheless, taken together, those studies indicate doses of CWD prions in saliva and exposure to shed prions in the environment are sufficient for CWD transmission.

The temporal shedding profile of CWD prions in secreta remains poorly understood. Little is known about the onset and duration of the complete prion shedding profile during the asymptomatic phase of CWD infection. It is suspected that prion levels in shed secreta are low, and inhibitors or nonspecific substrate activators in secreta can constrain the use of sensitive in vitro amplification assays, such as serial protein misfolding amplification (sPMCA) and real-time quaking-induced conversion (RT-QuIC) (11-14). To enhance detection capabilities of those assays, sodium phosphotungstic acid or iron oxide bead capture techniques have been used to concentrate prions and reduce or bypass interfering factors (15-26). Thus, continued in vitro assay optimization has improved CWD antemortem detection.

In addition to the route and dose by which CWD prions are transmitted, the sequence of the prion protein gene PRNP is another factor affecting disease pathogenesis. The prion protein is highly conserved between cervids (27), although single-nucleotide polymorphisms within the gene sequence can lead to reduced susceptibility (28), slower disease progression (29,30), and prolonged survival (31,32), all of which might affect prion shedding (18,33). Specific codon polymorphisms known to influence cervid CWD susceptibility are codons 132 in elk (M→L) (28), 225 in mule deer (S→F) (31), and 96 in WTD (G→S) (34). As additional polymorphisms are identified (35), the role they play in CWD susceptibility, pathogenesis, and shedding will need to be explored.

We used iron oxide bead (IOB) capture in combination with sPMCA and RT-QuIC to profile longitudinal (66 months) prion shedding in urine, saliva, and feces collected from WTD exposed to low oral doses of CWD prions. Because dose and PRNP genotype affect initial detection of infection and disease onset, we correlated those factors with prion shedding consistency and duration. Our goal was to provide a more complete understanding of prion shedding onset and duration that contributes to CWD pathogenesis and transmission and aid in early antemortem detection by using minimally invasive methods.