Early detection of chronic wasting disease prions in urine of pre-symptomatic deer by real-time quaking-induced conversion assay

Abstract

Chronic wasting disease (CWD) is a prion disease of captive and free-ranging deer (Odocoileus spp), elk (Cervus elaphus nelsonii) and moose (Alces alces shirasi). Unlike in most other prion diseases, in CWD prions are shed in urine and feces, which most likely contributes to the horizontal transmission within and between cervid species. To date, CWD ante-mortem diagnosis is only possible by immunohistochemical detection of protease resistant prion protein (PrP (Sc) ) in tonsil or recto-anal mucosa-associated lymphoid tissue (RAMALT) biopsies, which requires anesthesia of animals. We report on detection of CWD prions in urine collected from pre-symptomatic deer and in fecal extracts by using real time quaking-induced conversion (RT-QuIC). This assay can be useful for non-invasive pre-symptomatic diagnosis and surveillance of CWD.

Keywords: RT-QuIC; chronic wasting disease; diagnosis; feces; prion; surveillance; urine.

Figure 1. Sensitive detection of CWD prions by RT-QuIC. (A) RT-QuIC reactions using cervid PrP as a substrate were seeded with serial dilutions of brain homogenate (10%) derived from a CWD-infected (CWD+) or non-infected (CWD-) mule deer. (B) Ten % CWD infected brain homogenate was diluted 10⁻³ or 10⁻⁴ with urine from a CWD-negative deer. Serial dilutions thereof were used to seed quadruplicate RT-QuIC reactions using cervid recombinant PrP as a substrate. The y-axis shows the mean ThT fluorescence. The x-axis depicts the reaction time.

Figure 2. Detection of CWD prions in urine of pre-clinical deer. Urine of orally infected animals (W804 = mule deer; W1004 = white-tailed deer) collected 5, 13 and 16 mo post infection, respectively, was diluted 20-fold in RT-QuIC buffer and was then used to seed RT-QuIC reactions as described using cervid PrP as a substrate. Urine of non-infected animals served as a negative control.

Figure 3. CWD prions are detectable in feces by RT-QuIC. Extracts of white-tailed deer (W1004) feces collected at 20 or 30 mo post oral inoculation were seeded either undiluted or in a 2 × 10⁻¹ dilution into quadruplicate reactions. Fecal extracts from non-infected deer served as a negative control.