Recipient Organization
UNIVERSITY OF WYOMING
1000 E UNIVERSITY AVE DEPARTMENT 3434
LARAMIE,WY 82071-2000
Performing Department
Veterinary Sciences
Non Technical Summary
Chronic wasting disease (CWD) is a fatal neurodegenerative disease of cervids (i.e., deer, elk, moose, and reindeer) first identified in Colorado and now detected in 26 additional states, three Canadian provinces, Scandinavia, and the Republic of Korea. The disease is caused by a misfolded protein known as a prion.Conventional diagnostic methodsrequire brain or lymphoid tissue for prion detection, and are limited in the capacity for detection in the early stages of disease. Efforts to develop better methods for prion detection are therefore in progress in many laboratories across the United States.One approach to prion detection involves amplification ofsmall amounts of prions to detectable levels using a method known as real-time quaking induced conversion (RT-QuIC). Using this method, researchers are able to detect CWD earlier in the course of disease. Further, some researchers have been successful in detecting prions in non-invasive sample types(i.e., feces, urine, saliva,), providing an opportunity for live animal testing. Here we propose development and optimization of RT-QuIC protocols to support CWD research in Wyoming.CWD has been identified as a significant threat to Wyoming cervid populations. The critical need for research to inform control strategies is highlighted by models that predict local population extinctions due to CWD, possibly within the next few decades. The ability to minimize the disease depends on increased understanding of many important variables, such as prion shedding and genetic susceptibility of various animals and populations. The RT-QuIC assay will aid the investigation of these variables as related to CWD transmission dynamics, andimprove Wyoming's ability to implement appropriate CWD management practicesto temper or slow the spread of disease across the state.
Animal Health Component
20%
Research Effort Categories
Basic
60%
Applied
20%
Developmental
20%
Goals / Objectives
Advance diagnostic testing for CWD with a focus on facilitating adoption of the RT-QuIC assay and improved sourcing for the recombinant prion protein substrate.
Project Methods
Real-time quaking induced conversion relies on a recombinant form of the cellular prion protein (rPrPc) as a substrate, and a process of incubation with periodic shaking to amplify low levels of prions to detectable levels. Infectious prions in a sample act as "seeds" to convert the recombinant protein substrate to the misfolded, amyloid isoform. This conversion is detectable in real time by the incorporation of a fluorescent dye (thioflavin T) into the growing prion amyloid. This process requires reagents and materials that are not widely available commercially, and dedicated equipment that is not traditionally owned by most government agencies. Successful independent laboratory development and optimization of RT-QuIC depends on the ability to procure such equipment, and either produce materials in-house or source them from an experienced collaborator. Further, all prions should be considered a zoonotic risk and must be handled in laboratories with a minimum of Biosafety Level 2 (BSL-2) certification, by trained personnel using adequate personal protective equipment. Other potential barriers to success include technical challenges associated with sample preparation, including removal of inhibitors or targeted prion concentration.The proposed program will follow an attainable, supported approach to RT-QuIC development, involving collaboration with leading experts in this investigative technique. Adequate BSL-2 laboratory space is available for the proposed assay development within the Wyoming State Veterinary Laboratory, and technical support and materials will be procured from experienced collaborators. Initial development will be based on the detailed protocol provided in Haley 2020.Personnel from the Malmberg Research Laboratorywill be dedicated to development of this assay. Accuracy and consistency of prion detection will be ensured based on cross-comparisons with IHC and ELISA results, as well as cross-laboratory verification (Haley Laboratory, Midwestern University).Specifically, RT-QuIC will be developed using dilutions of retropharyngeal lymph node tissue from mule deer. All tissues will be procured from the Wyoming Game & Fish Department tissue archive.Reactions will include positive control tissue (from mule deer with CWD, as detected by IHC), negative control tissue (from mule deer testing negative by ELISA and IHC), and "unknown" samples that have been previously tested by ELISA and IHC. Assay development will be performed as a "blind" experiment to confirm ELISA and IHC results upon completion. Each sample will be amplified in triplicate using a truncated form of recombinant prion protein in a 24-hr amplification process with fluorescent emission readings collected every 15 min. Incubation, shaking, and fluorscent readout will be performed using a microplate shaker with a fluorescense reader (FLUOstar Omega, BMG LABTECH). Each reaction will include homogenized tissue sample (0.1 g of retropharyngeal lymph node diluted 1:10 in sterile PBS with 0.1% SDS), truncated recombinant protein "substrate" (procured from CWD Evolution LLC (https://www.cwdevolution.com/rt-quic-rprp-substrate)), the fluorescent molecule thioflavin T, and RT-QuIC master mix (1.25 M NaCl (Fisher Scientific, cat. no. S271),100 mM Na2HPO4 (dibasic, 7H2O; Millipore-Sigma, cat. no. S9390), 5 mM EDTA (Millipore-Sigma, cat. no. ED2SS) and sterile, distilled H2O to 200 ml, with pH adjustment to 7.4). Reactions will be conducted in a sealed 96 well plate. Following completion of the amplification protocol, MARS software will be using to calculate the threshold, determine time to fluorescence, and generate amplification curves for all samples.Once these methods are developed, accuracy will be ensured based on previous ELISA and IHC results, as well as results produced using the same method and same samples in an external laboratory (Haley Laboratory, Midwestern University). The assay will then be applied to research projects to better assess CWD status in free-ranging cervids of Wyoming.