Comparative analysis of CWD prion infectivity and in vitro detection in tissues and fluids using bioassays, PMCA, and RT-QuIC

Chronic wasting disease (CWD) is a prion disorder affecting cervids and characterized by the presence of infectious prions (PrP^CWD) in the brain, peripheral tissues, bodily fluids, and excreta. Understanding the tissue-specific prion burden and optimizing detection strategies are key for effective surveillance. In this study, we evaluated the infectivity and amplification efficacy of various tissues from a terminally ill, CWD-infected white-tailed deer. The biological samples tested included obex, retropharyngeal lymph node (RPLN), recto anal mucosa (RAMALT), eye, blood, ear, and feces. Blood and sample homogenates were intracerebrally inoculated into Tg1536 transgenic mice (overexpressing deer PrP) to assess the infectivity titers in each sample. All samples were analyzed by the PMCA and RT-QuIC techniques. In bioassays, RPLN, RAMALT, obex, and eye resulted in 100% attack rates, with incubation periods ranging from 322 to 397 days (indicating high infectivity titers). Blood induced efficient transmission with longer incubation periods, while ear showed lower infectivity (22.9%). Feces did not transmit disease under these conditions. PMCA results closely matched bioassay findings, with a maximum detection at tissue dilutions ranging 10^-8 to 10⁻⁹ in RPLN, RAMALT, obex, and eye. Moderate PMCA amplification was achieved in blood, ear, and feces (10⁻⁴ to 10⁻⁸). The same samples were analyzed using RT-QuIC, obtaining similar results compared to those in PMCA. Together, these findings suggest that prion distribution is tissue-dependent and influenced by both prion concentration and potential inhibitors. This study contributes to understanding prion tissue tropism and supports the use of complementary approaches for improved CWD diagnostics.