Prion-Seeding Activity in Cerebrospinal Fluid of Deer with Chronic Wasting Disease

Nicholas J Haley,Edward Hoover,Candace Mathiason,Kristen Davenport,Alexandra Van De Motter,Davis M Seelig,Davin Henderson,Scott Carver

doi:10.1371/journal.pone.0081488

Abstract

Transmissible spongiform encephalopathies (TSEs), or prion diseases, are a uniformly fatal family of neurodegenerative diseases in mammals that includes chronic wasting disease (CWD) of cervids. The early and ante-mortem identification of TSE-infected individuals using conventional western blotting or immunohistochemistry (IHC) has proven difficult, as the levels of infectious prions in readily obtainable samples, including blood and bodily fluids, are typically beyond the limits of detection. The development of amplification-based seeding assays has been instrumental in the detection of low levels of infectious prions in clinical samples. In the present study, we evaluated the cerebrospinal fluid (CSF) of CWD-exposed (n=44) and naïve (n=4) deer (n=48 total) for CWD prions (PrPd) using two amplification assays: serial protein misfolding cyclic amplification with polytetrafluoroethylene beads (sPMCAb) and real-time quaking induced conversion (RT-QuIC) employing a truncated Syrian hamster recombinant protein substrate. Samples were evaluated blindly in parallel with appropriate positive and negative controls. Results from amplification assays were compared to one another and to obex immunohistochemistry, and were correlated to available clinical histories including CWD inoculum source (e.g. saliva, blood), genotype, survival period, and duration of clinical signs. We found that both sPMCAb and RT-QuIC were capable of amplifying CWD prions from cervid CSF, and results correlated well with one another. Prion seeding activity in either assay was observed in approximately 50% of deer with PrPd detected by IHC in the obex region of the brain. Important predictors of amplification included duration of clinical signs and time of first tonsil biopsy positive results, and ultimately the levels of PrPd identified in the obex by IHC. Based on our findings, we expect that both sPMCAb and RT-QuIC may prove to be useful detection assays for the detection of prions in CSF.

Highlights

Introduction across NorthAmerica, but can be as high as 30% in some areas of Colorado and up to 100% in captive populations [7,8].Identification of infected cervids requires a sensitive and specific detection assay
To evaluate the optimal volumes for seeded amplification in both sPMCAb and real-time quaking induced conversion (RT-QuIC), we evaluated a range of starting volumes and dilutions of cerebrospinal fluid (CSF) from six Chronic wasting disease (CWD)-positive deer in each assay: 20μl, 10μl and 1μl of CSF into 50μl of normal brain homogenate (NBH) with sPMCAb, and 10°, 10-1, and 10-2 dilutions of CSF in RT-QuIC
Over the course of multiple experiments, we found that RT-QuIC scores were fairly reproducible, with standard error of crossed the positive threshold (Ct) scores between separate experiments ranging from 0.01-0.07

Summary

Introduction

Identification of infected cervids requires a sensitive and specific detection assay. Immunohistochemistry (IHC) of designated target tissues is currently considered the gold standard diagnostic test for CWD and other prion diseases of animals and man. The true sensitivity and specificity of IHC in the detection of infected individuals is unknown, though it has been demonstrated that the assay may underestimate the level of abnormal pathogenic prion protein (PrPd) in a given sample due to the necessity of a proteolytic pre-treatment step to abolish cellular prion protein (PrPC) cross-reactivity [9,10,11]. Chronic wasting disease (CWD) is an efficiently transmitted prion disease of cervids (e.g. deer, elk, and moose), and the only known prion disease affecting free-ranging, non-domestic animals. While the origins of CWD are uncertain, the disease has been present in wild cervid populations of northern

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Conclusion