Standardization of Data Analysis for RT-QuIC-Based Detection of Chronic Wasting Disease.

Chronic wasting disease (CWD) is a disease affecting cervids and is caused by prions accumulating as pathogenic fibrils in lymphoid tissue and the central nervous system. Approaches for detecting CWD prions historically relied on antibody-based assays. However, recent advancements in protein amplification technology provided the foundation for a new class of CWD diagnostic tools. In particular, real-time quaking-induced conversion (RT-QuIC) has rapidly become a feasible option for CWD diagnosis. Despite its increased usage for CWD-focused research, there lacks a consensus regarding the interpretation of RT-QuIC data for diagnostic purposes. It is imperative then to identify a standardized and replicable method for determining CWD status from RT-QuIC data. Here, we assessed variables that could impact RT-QuIC results and explored the use of maxpoint ratios (maximumRFU/backgroundRFU) to improve the consistency of RT-QuIC analysis. We examined a variety of statistical analyses to retrospectively analyze CWD status based on RT-QuIC and ELISA results from 668 white-tailed deer lymph nodes. Our results revealed an MPR threshold of 2.0 for determining the rate of amyloid formation, and MPR analysis showed excellent agreement with independent ELISA results. These findings suggest that the use of MPR is a statistically viable option for normalizing between RT-QuIC experiments and defining CWD status.

COMPARISON OF CHRONIC WASTING DISEASE DETECTION METHODS AND PROCEDURES: IMPLICATIONS FOR FREE-RANGING WHITE-TAILED DEER (ODOCOILEUS VIRGINIANUS) SURVEILLANCE AND MANAGEMENT.

Throughout North America, chronic wasting disease (CWD) has emerged as perhaps the greatest threat to wild cervid populations, including white-tailed deer (WTD; Odocoileus virginianus). White-tailed deer are the most sought-after big game species across North America with populations of various subspecies in nearly all Canadian provinces, the contiguous US, and Mexico. Documented CWD cases have dramatically increased across the WTD range since the mid-1990s, including in Minnesota, US. CWD surveillance in free-ranging WTD and other cervid populations mainly depends upon immunodetection methods such as immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) on medial retropharyngeal lymph nodes and obex. More recent technologies centered on prion protein amplification methods of detection have shown promise as more sensitive and rapid CWD diagnostic tools. Here, we used blinded samples to test the efficacy of real-time quaking-induced conversion (RT-QuIC) in comparison to ELISA for screening tissues collected in 2019 from WTD in southeastern Minnesota, where CWD has been routinely detected since 2016. Our results support previous findings that RT-QuIC is a more sensitive tool for CWD detection than current antibody-based methods. Additionally, a CWD testing protocol that includes multiple lymphoid tissues (e.g., medial retropharyngeal lymph node, parotid lymph node, and palatine tonsil) per animal can effectively identify a greater number of CWD detections in a WTD population than a single sample type (e.g., medial retropharyngeal lymph nodes). These results show that the variability of CWD pathogenesis, sampling protocol, and testing platform must be considered for the effective detection and management of CWD throughout North America.

RT-QuIC detection of CWD prion seeding activity in white-tailed deer muscle tissues.

Chronic wasting disease (CWD) is a prion disease circulating in wild and farmed cervid populations throughout North America (United States and Canada), Europe (Finland, Norway, Sweden), and South Korea. CWD is a long-term threat to all cervid populations and to cervid hunting heritage, with the potential to cause substantial economic losses across multiple sectors. In North America, hunting and farming industries focused on the processing and consumption of white-tailed deer (WTD) venison are particularly vulnerable to CWD prion contamination, as millions of WTD are consumed annually. Real-time quaking-induced conversion (RT-QuIC) is a highly sensitive assay amplifying misfolded CWD prions in vitro and has facilitated CWD prion detection in a variety of tissues and excreta. To date, no study has comprehensively examined CWD prion content across bulk skeletal muscle tissues harvested from individual CWD infected WTD. Here, we use RT-QuIC to characterize prion-seeding activity in a variety of skeletal muscles from both wild and farmed CWD-positive WTD. We successfully detected CWD prions in muscles commonly used for consumption (e.g., backstrap, tenderloin, etc.) as well as within tongue and neck samples of WTD. Our results suggest that CWD prions are distributed across the skeletal muscles of infected WTD. We posit that RT-QuIC will be a useful tool for monitoring CWD prions in venison and that the method (with additional protocol optimization and high-throughput functionality) could be used to reduce and/or prevent CWD prions from entering animal and human food chains.

Morphometric and genetic variation in 8 breeds of Ethiopian camels (Camelus dromedarius).

Dromedary camels (Camelus dromedarius) are a domesticated and closely guarded economic staple of indigenous people located throughout Ethiopian territorial states. Seventeen morphometric variables were examined to determine intraspecific variation among 8 pastoralist-designated breeds of camels. Additionally, DNA sequences from mitochondrial cytochrome-b gene and genotyping of 6 nuclear microsatellite loci were examined to assess genetic diversity and phylogenetic relationship of Ethiopian camels. Examination of 525 individuals revealed significant morphometric differentiation in Afar as compared with the remaining 7 breeds. Analysis of cytochrome-b sequences failed to recover monophyletic groups associated with pastoralist-recognized breeds. Analysis of 6 microsatellite loci from 104 individuals depicted no resolution of distinct genetic lineages in accordance to geographical or designated breeds. Overall, separation of 2 ecotypes based on the morphometric data was supported; however, genetic analysis of cytochrome-b and microsatellite data failed to support any unique genetic lineage or statistically significant population structure.