2020-21 Rapid Ag: Rapid Next-Generation Diagnostics for Chronic Wasting Disease
Veterinary Biomedical Sciences
Chronic Wasting Disease (CWD) is a highly contagious degenerative disease that is spreading throughout cervid populations (e.g., elk, mule deer, white-tailed deer, moose, etc.) in the United States (including Minnesota and Wisconsin), Canada, and Europe1,2. There is growing concern that CWD will spread to livestock and humans, as recent experiments show CWD pathogens can infect cattle, sheep, and primates3,4. Despite these observations, a reliable and easy-to-use antemortem diagnostic test for CWD remains to be developed. Available diagnostic assays for the detection of CWD are limited, with the most effective tests requiring tissue (e.g., brain or lymph nodes) from sacrificed animals. Moreover, existing diagnostics for detecting CWD in live animals are expensive, time-consuming, and require significant technical expertise. To combat the spread of CWD in Minnesota and elsewhere, an accurate, rapid, and easy-to-use antemortem diagnostic test that identifies infected animals in the preclinical stages of disease must be designed. Such a test would facilitate early detection of CWD and would help to reduce the spread of the disease. The goal of the research proposed herein is to design and experimentally validate a new and advanced diagnostic test for CWD. The diagnostic test will be capable of detecting CWD using biological samples collected from live cervids (e.g., saliva, blood, feces, mucus, urine).
CWD is spreading in both captive and wild cervid (elk and white-tailed deer) populations in Minnesota, with an ongoing outbreak occurring in wild white-tailed deer harvested from Fillmore County. The pathogenic agent of CWD is a malformed prion protein (PrPCWD), similar to the prion underlying mad cow disease (bovine spongiform encephalopathy). PrPCWD spreads through both direct (animal to animal) and indirect (environmental) routes (Fig 1). Upon infection, PrPCWD proteins colonize and amplify within lymph nodes, eventually spreading to the brain where they steadily begin to kill neurons5. The brains of CWD positive cervids deteriorate over time and the infected animals become thin, display cognitive impairment, and ultimately die. There is growing concern that CWD will spread beyond cervids, as new lab-based experiments suggest PrPCWD can infect livestock (cattle and sheep), wildlife, and non-human primates3. Recent data indicate particular PrPCWD variants can induce the conversion of human prion protein to a pathogenic form in vitro4. A critical observation concerning the spread of CWD is that it is nearly impossible to remove PrPCWD from natural habitats once an outbreak occurs6. Notably, CWD positive cervids shed misfolded prions into the soil (through urine, feces, saliva, and carcasses) for up to 88% of their infected life. As a result, infected prions contaminate vegetation and are further spread by rodents, thus facilitating indirect infection routes7. Knowing this, it is important to quickly identify CWD infections in order to minimize and control the spread of the disease. Despite these observations, available CWD diagnostic assays are limited and the most effective tests require tissue (e.g., brain or lymph nodes) from sacrificed animals8 while antemortem tests are expensive, time-consuming, and highly technical9. A rapid, reliable, and easy-to-use antemortem diagnostic test for CWD must be developed.
We have assembled a multi-disciplinary research team with expertise in the areas of prion disease, neurodegenerative disease, genomics, immunogenomics, and nanotechnology. The mission of this team is to design and experimentally validate an advanced CWD diagnostic assay that can be used to test biological samples collected from live cervids (e.g., saliva, blood, feces, mucus). To accomplish this, we will implement a two-pronged approach aimed at identifying key molecular signatures in CWD positive samples. First, we have identified specific genes that display unique RNA expression profiles during active prion infection10,11. These genes will be targeted for blood-based CWD biomarker development (Objective 1 below). Second, we have identified procedures within the protocols of currently available antemortem CWD assays (e.g., protein misfolding cyclic amplification12 [PMCA] and real-time quaking-induced conversion13 [RT-QuIC]) that can be improved upon using immunoprecipitation, microfluidics, and highly sensitive luminescent polymers within a nanoscale environment. These antemortem tests utilize prion amplification methods to identify PrPCWD in biological and environmental samples. We anticipate that improving upon these assays using nanotechnology will significantly reduce processing time and will increase sensitivity.
Goals and Objectives
The primary goal of the proposed research is to develop a robust next-generation antemortem test for the rapid detection of CWD in biological samples secured from cervids using minimally invasive techniques. To accomplish this goal, we will utilize cutting-edge genomic and nanotechnology with the following objectives:
- Identify blood-based molecular biomarkers that will facilitate the pre-clinical diagnosis of CWD in live cervids.
- Improve upon existing antemortem CWD tests by developing a microfluidic assay enhanced by nanotechnology capable of identifying CWD-causing prions in a variety of biological samples (e.g., saliva, blood, feces, mucus, urine).
- Experimentally validate both blood-biomarker and prion-detection assays within the University of Minnesota Veterinary Diagnostic Laboratory.
- Hannaoui, S., Schatzl, H. M. & Gilch, S. Chronic wasting disease: Emerging prions and their potential risk. PLOS Pathog. 13, e1006619 (2017).
- Joly, D. O. et al. Chronic wasting disease in free-ranging Wisconsin White-tailed Deer. Emerg. Infect. Dis. 9, 599–601 (2003).
- Kurt, T. D. & Sigurdson, C. J. Cross-species transmission of CWD prions. Prion 10, 83–91 (2016).
- Barria, M. A., Libori, A., Mitchell, G. & Head, M. W. Susceptibility of Human Prion Protein to Conversion by Chronic Wasting Disease Prions. Emerg. Infect. Dis. 24, 1482–1489 (2018).
- Hoover, C. E. et al. Pathways of Prion Spread during Early Chronic Wasting Disease in Deer. J. Virol. 91, JVI.00077-17 (2017).
- Miller, M. W., Williams, E. S., Hobbs, N. T. & Wolfe, L. L. Environmental sources of prion transmission in mule deer. Emerg. Infect. Dis. 10, 1003–6 (2004).
- Saunders, S. E., Bartelt-Hunt, S. L. & Bartz, J. C. Occurrence, Transmission, and Zoonotic Potential of Chronic Wasting Disease. Emerg. Infect. Dis. 18, 369–376 (2012).
- Blasche, T. et al. Rapid Detection of CWD PrP: Comparison of Tests Designed for the Detection of BSE or Scrapie. Transbound. Emerg. Dis. 59, 405–415 (2012).
- Ricci, A. et al. Scientific opinion on chronic wasting disease (II). EFSA J. 16, (2018).
- Skinner, P. J. et al. Gene expression alterations in brains of mice infected with three strains of scrapie. BMC Genomics 7, 114 (2006).
- Kim, H. O. et al. Prion disease-induced alterations in gene expression in spleen and brain prior to clinical symptoms. Adv. Appl. Bioinform. Chem. 1, 29–50 (2008).
- Saborio, G. P., Permanne, B. & Soto, C. Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding. Nature 411, 810–813 (2001).
- Atarashi, R., Sano, K., Satoh, K. & Nishida, N. Real-time quaking-induced conversion: a highly sensitive assay for prion detection. Prion 5, 150–3 (2011).
- Paramithiotis, E. et al. A prion protein epitope selective for the pathologically misfolded conformation. Nat. Med. 9, 893–899 (2003).
- Orru, C. D. et al. Prion Disease Blood Test Using Immunoprecipitation and Improved Quaking-Induced Conversion. MBio 2, e00078-11 (2011).
- Makarava, N., Savtchenko, R. & Baskakov, I. V. Purification and fibrillation of full-length recombinant PrP. in Methods in Molecular Biology (2017). doi:10.1007/978-1-4939-7244-9_1
- Sigurdson, C. J. et al. Prion strain discrimination using luminescent conjugated polymers. Nat. Methods 4, 1023–1030 (2007).
- Henderson, D. M. et al. Rapid Antemortem Detection of CWD Prions in Deer Saliva. PLoS One 8, e74377 (2013).