DETECTION OF PrP^CWD IN FECES FROM NATURALLY EXPOSED ROCKY MOUNTAIN ELK (CERVUS ELAPHUS NELSONI) USING PROTEIN MISFOLDING CYCLIC AMPLIFICATION

Abstract

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy affecting captive and free-ranging cervids. Currently, tests for CWD in live animals involve relatively invasive procedures to collect lymphoid tissue biopsies and examine them for CWD-associated, protease-resistant cervid prion protein (PrP(CWD)) detected by immunohistochemistry (IHC). We adapted an ultrasensitive prion detection system, protein misfolding cyclic amplification (PMCA), to detect PrP(CWD) in Rocky Mountain elk (Cervus elaphus nelsoni) feces. Our PMCA reproducibly detected a 1.2 × 10(7) dilution of PrP(CWD) (a 10% infected brain homogenate diluted 1.2 × 10(6)-fold into 10% fecal homogenates), equivalent to approximately 100 pg of PrP(CWD)/g of feces. We developed a semiquantitative scoring system based on the first PMCA round at which PrP(CWD) was detected and fit a nonlinear regression curve to our serial dilutions to correlate PMCA scores with known PrP(CWD) concentrations. We used this PMCA scoring system to detect PrP(CWD) and estimate its concentration in feces from free-ranging elk from Rocky Mountain National Park, Colorado. We compared our results to PrP(CWD) IHC of rectoanal mucosa-associated lymphoid tissue and obex from the same animals. The PMCA successfully detected PrP(CWD) in feces from elk that were positive by IHC, with estimated prion loads from 100 to 5,000 pg PrP(CWD)/g of feces. These data show for the first time PrP(CWD) in feces from naturally exposed free-ranging elk and demonstrate the potential of PMCA as a new, noninvasive CWD diagnostic tool to complement IHC.

Can plants serve as a vector for prions causing chronic wasting disease?

“…5 Infected cervids have been reported to shed PrP TSE in bodily fluids, disseminating infectious prions in urine, feces, blood and saliva. [7][8][9][10] PrP TSE binds to whole soil and interaction with some soil minerals has been shown to increase the infectivity of PrP TSE . 11,12 Although PrP TSE binds to soil, it requires 60 d for CWD PrP TSE to become fully bound in sandy soil as compared with 30 d for prions associated with transmissible mink encephalopathy (TME).…”

Enhanced prion detection in biological samples by magnetic particle extraction and real-time quaking-induced conversion

“…These results could infer that IOME may preferentially bind prions from complex matrices, perhaps reducing potential assay inhibitors. We tested this theory on faeces, which has traditionally presented significant problems for in vitro prion-amplification assays (Pulford et al, 2012;Tamgüney et al, 2009). Despite higher false-positive rates compared with other excreta assayed, the IOME protocol resulted in a statistical significance difference between faecal samples from CWD-positive versus -negative deer.…”

“…Faecal samples have traditionally been difficult to assay by any method due to the complexity of the material present (Pulford et al, 2012;Krüger et al, 2009;Saborio et al, 2001). Blinded faecal samples from six CWD-positive and three CWDnegative deer were analysed, with or without IOME-RTQuIC.…”

Chronic Wasting Disease: Evolution of Diagnostic Testing for a Naturally Occurring Prion Disease

“…Using amplification approaches, evidence of CWD prion presence has been reported in a range of bodily fluids (19,74,78,(89)(90)(91), making them tempting targets for the development of novel diagnostic strategies. Studies in other model systems, including sheep and humans, have identified peripheral tissues which may also serve as a useful diagnostic sample and indicator of central nervous system infection (92-94).…”

“…to assess the potential for infectivity in body fluids and other excreta (19,74,78,79), and to detect low levels of misfolded protein in soil (80), water (81), and plant samples (82). Notably, the CWD seeds generated in vitro by sPMCA have proven infectious to some degree in susceptible hosts (83), indicating that the technique may accurately model what occurs in vivo; therein lays its advantage among amplification strategies.…”