Ante-mortem detection of chronic wasting disease in recto-anal mucosa-associated lymphoid tissues from elk (Cervus elaphus nelsoni) using real-time quaking-induced conversion (RT-QuIC) assay: A blinded collaborative study

Abstract

Prion diseases are transmissible spongiform encephalopathies (TSEs) characterized by fatal, progressive neurologic diseases with prolonged incubation periods and an accumulation of infectious misfolded prion proteins. Antemortem diagnosis is often difficult due to a long asymptomatic incubation period, differences in the pathogenesis of different prions, and the presence of very low levels of infectious prion in easily accessible samples. Chronic wasting disease (CWD) is a TSE affecting both wild and captive populations of cervids, including mule deer, white-tailed deer, elk, moose, muntjac, and most recently, wild reindeer. This study represents a well-controlled evaluation of a newly developed real-time quaking-induced conversion (RT-QuIC) assay as a potential CWD diagnostic screening test using rectal biopsy sections from a depopulated elk herd. We evaluated 69 blinded samples of recto-anal mucosa-associated lymphoid tissue (RAMALT) obtained from USDA Veterinary Services. The results were later un-blinded and statistically compared to immunohistochemical (IHC) results from the USDA National Veterinary Services Laboratories (NVSL) for RAMALT, obex, and medial retropharyngeal lymph node (MRPLN). Comparison of RAMALT RT-QuIC assay results with the IHC results of RAMALT revealed 92% relative sensitivity (95% confidence limits: 61.52-99.8%) and 95% relative specificity (95% confidence limits: 85.13-99%). Collectively, our results show a potential utility of the RT-QuIC assay to advance the development of a rapid, sensitive, and specific prion diagnostic assay for CWD prions.

Evaluation of Antemortem Diagnostic Techniques in Goats Naturally Infected With Scrapie

“…Two out of seven scrapie-positive goats had positive antemortem RAMALT biopsies at ~21 and ~23% of their incubation period (Animal IDs: P87 and B186, respectively). Several studies have described detecting PrP Sc in rectal mucosa of cervids that naturally acquired CWD, reporting a relatively high (75–91%) sensitivity of antemortem RAMALT biopsies tested by IHC and/or RT-QuIC ( 16 – 18 ). The efficacy of preclinical diagnosis using RAMALT biopsies has also been reported in sheep infected with scrapie.…”

“…Three of 14 (21%) antemortem rectal biopsies tested (2 of 6 goats) were determined negative by IHC and tested positive by RT-QuIC. Few studies have reported a higher sensitivity of RT-QuIC vs. IHC in detecting PrP Sc in rectal mucosa ( 16 , 19 ) and third eyelid ( 61 ) samples of cervids naturally and experimentally infected with CWD. We found that compared to IHC, RT-QuIC analysis of RAMALT biopsies identified two out of four scrapie-positive goats born to negative dams that were not yet showing clinical signs of scrapie, suggesting that RT-QuIC may serve as a useful method to follow preclinical goats over the course of their incubation periods and isolate them prior to the presence of clinical signs.…”

“…), prompting the development of biopsy tests for the antemortem diagnosis of scrapie using more accessible tissues, including recto-anal mucosa-associated lymphoid tissue (RAMALT) (12), third eyelid (13), or palatine tonsil (14) that are primarily analyzed after immunohistochemistry (IHC). Several studies have described successfully detecting PrP Sc in rectal mucosa in sheep infected with scrapie (15) and cervids infected with CWD (16)(17)(18)(19)(20). In infected goats, RAMALT samples collected in the preclinical stages demonstrate a lower percentage of positives as compared to sheep (21) despite accumulation of PrP Sc in other lymphoid tissues (22).…”

Progression of chronic wasting disease in white-tailed deer analyzed by serial biopsy RT-QuIC and immunohistochemistry

“…We and others have used RT-QuIC to detect and estimate levels of prion seeding activity in multiple tissues, excreta, and body fluids with the goal of increasing sensitivity and speed in CWD detection pre-and post-mortem [19][20][21][22][23][24][25][26]. Here we provide a dynamic longitudinal profile of progressing CWD infection in white-tailed deer after low dose oral exposure to CWD prions as assessed by serial lymphoid tissue biopsies examined by both the RT-QuIC and IHC methods.…”

Detection of Pathognomonic Biomarker PrPSc and the Contribution of Cell Free-Amplification Techniques to the Diagnosis of Prion Diseases

“…However, in sheep [159][160][161] and mainly in deer, another lymphoid tissue or its associated mucosa has been also widely used, namely the RAMALT [149,162]. For this tissue, cell-free prion propagation systems are being adapted in order to increase sensitivity and specificity [163][164][165][166][167]. Overall, the use of lymphoid tissues for the ante-mortem diagnosis of orally transmissible animal prion diseases is widely used together with brain samples for surveillance by government agencies.…”