Early and Pre-Clinical Detection of Prion Seeding Activity in Cerebrospinal Fluid of Goats using Real-Time Quaking-Induced Conversion Assay

Abstract

Since 2005, two cases of natural bovine spongiform encephalopathies (BSE) have been reported in goats. Furthermore, experimental transmissions of classical (C-BSE) and atypical (L-BSE) forms of BSE in goats were also reported. To minimize further spreading of prion diseases in small ruminants the development of a highly sensitive and specific test for ante-mortem detection of infected animals would be of great value. Recent studies reported high diagnostic value of a second generation of cerebrospinal fluid (CSF) Real-Time Quaking-Induced Conversion (RT-QuIC) assay across a wide spectrum of human prions. Here, we applied this improved QuIC (IQ-CSF) for highly efficient detection of TSEs prion protein in goat cerebrospinal fluid. IQ-CSF sensitivity and specificity were evaluated on CSF samples collected at disease endpoint from goats naturally and experimentally infected with scrapie or bovine isolates of C-BSE and L-BSE, respectively. Next, CSF samples collected from L-BSE infected goats during pre-symptomatic stage were also analysed. PrP L-BSE associated seeding activity was detected at early time points after experimental inoculation, with an average time of 439 days before clinical symptoms appeared. Taken together these data are indicative of the great potential of this in vitro prion amplification assay as ante-mortem TSE test for live and asymptomatic small ruminants.

Quaking-induced conversion of prion protein on a thermal mixer accelerates detection in brains infected with transmissible spongiform encephalopathy agents

“…We investigated the performance of the three assay platforms at 55°C because the amplification reaction accelerates at higher temperatures [16–19]. We compared brain homogenates infected with CWD, sCJD and vCJD, three naturally occurring infections, tested with QuIC-C and RT-QuIC using truncated recombinant substrates.…”

“…In some cases, assaying biological materials containing minute amounts of PrP TSE by RT-QuIC can take 40–50 hours or longer [13–15]. Recent modifications of the method conducting the reaction at 55°C and using truncated prion protein as the substrate, have markedly reduced lag times [16–19]. However, RT-QuIC assay time is a function of PrP TSE concentration in the test materials, requiring longer times to detect low levels of the abnormal protein.…”

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

“…On the contrary, although atypical scrapie could be detected in brain tissue [42], no report has been found on atypical scrapie detection in CSF. An improved version of RT-QuIC, also known as second generation RT-QuIC, has been similarly applied to CSF from classical BSE and L-BSE infected goat, in which PrP Sc was detected in preclinical and clinical stage [88]. Finally, it is worth mentioning the detection of Chronic Wasting Disease (CWD) prions in CSF of affected cervids, for which both PMCA and RT-QuIC have been used.…”

RT-QuIC detection of pathological prion protein in subclinical goats following experimental oral transmission of L-type BSE

“…Experimental transmission by the intracerebral route to sheep and goats showed that both species are susceptible to L-BSE with an efficiency of infection as high as 100% and preferential accumulation of PrP sc in the central nervous system (CNS) and to a lesser extent in the peripheral nervous system, in the cerebrospinal fluid and in the lymphoreticular system (LRS) [ 1 – 5 ].…”