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

Alessandra Favole,Byron Caughey,Elena Berrone,Claudia Palmitessa,Cristina Casalone,Marina Gallo,Maria Mazza,Cristiano Corona,Maria Caramelli,Christina D Orrù,Paola Crociara,Guerino Lombardi,Elena Vallino Costassa,Pier L Acutis,Paola Marconi,Antonio D’Angelo

doi:10.1038/s41598-019-42449-7

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. PrPL-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.

Highlights

Transmissible Spongiform Encephalopathies (TSEs) or prion diseases are fatal neurodegenerative diseases that include Creutzfeldt-Jakob disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep and chronic wasting disease (CWD) in cervids
The three goat prion strains exhibited distinct Real-Time Quaking Induced Conversion (RT-QuIC) seeded www.nature.com/scientificreports polymerization kinetics, our results show that Ha-S rPrPSen supports detection of goat C-BSE, L-BSE- and scrapie-associated seeding activity
A recent study by Madsen-Bouterse et al.[37] revealed that the polymorphisms in caprine PRNP can affect the sensitivity of PrPSc detection in brain samples by anti-prion mAb-based immunoassays such as IHC and Western blot analysis

Summary

Introduction

Transmissible Spongiform Encephalopathies (TSEs) or prion diseases are fatal neurodegenerative diseases that include Creutzfeldt-Jakob disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep and chronic wasting disease (CWD) in cervids. An important goal to minimize further spreading of small ruminant TSEs is the development of an assay for highly sensitive and intra-vitam detection of prions in infected, but not clinically sick animals. RT-QuIC assays can rapidly detect sub-infectious levels of prion seeding activity and have been used successfully to detect multiple human, cervid, ovine, hamster, and mouse prion strains in a variety of biological tissues, such as skin[13], cerebrospinal fluid[14,15,16,17,18], saliva[19], blood[20,21,22,23], and nasal brushings[24], showing that this test has the potential of being used for ante-mortem TSE diagnosis. Initial evaluation of the IQ-CSF assay indicated greater analytical and diagnostic sensitivity, and markedly shorter testing times[25,26,27,28,29]

Methods

Results

Conclusion