Location: Virus and Prion Research
2024 Annual Report
Objectives
Objective 1: Develop highly sensitive detection tools to determine the distribution of CWD and scrapie prions in natural hosts (sheep, goats, cervids) and their environment.
Objective 2: Investigate the pathobiology of CWD, scrapie prion strains, and atypical TSEs in natural hosts including potential cross species transmission events.
Objective 3: Investigate the genetics of CWD susceptibility and resistance in white-tailed deer.
Objective 4: Evaluate the presence of and determine the appropriate methodology for CWD strain determination.
Approach
Eradication or control of a family of diseases is unlikely or impossible when an understanding of the basic mechanisms and influences on transmission are unknown and for which methods to evaluate disease status are lacking.
Scrapie and BSE represent the most thoroughly studied TSEs; however, significant knowledge gaps persist with regard to the atypical variants of these diseases. Further, much of the research emphasis to date on genetics of prion disease has focused on the recipient genotype rather than the source. Since both atypical BSE and atypical scrapie have been suggested to occur spontaneously, eradication of these diseases may not be possible unless we expand our understanding of the disease at both the source and recipient level. A better understanding of the tissue distribution and potential transmission of these atypical isolates is critical to understanding what risk these disease variants may pose to ongoing control and eradication efforts.
The European epizootic of BSE is waning and efforts to eradicate scrapie in the U.S. and abroad have progressed but are not complete. In the U.S., chronic wasting disease (CWD) presents the most serious challenge to regulatory efforts. CWD appears to be spreading unchecked in both free-ranging and farmed cervids. Methods for antemortem detection of TSEs in general and CWD in particular are needed to fulfill the goal of eradicating scrapie and controlling CWD. Performing these studies will allow us to address critical knowledge gaps that are relevant to developing measures to restrict further disease expansion beyond current, affected populations.
Understanding prion disease persistence in animal populations is challenging due to lack of tools for study and a less than complete understanding of transmission among animals within a flock or herd or in naturally occurring reservoirs. In addition to transmission between hosts of like species, free-ranging cervids may come in contact with numerous other species including cattle, sheep, and other susceptible hosts. Transmission of CWD to other species has been studied but limited with regard to the source genotype used.
The four primary objectives are inherently linked. Our focus is on developing tools needed for control and research, and using those tools to advance our understanding the complex disease process with the overall goal of eradication and control of disease in livestock, wildlife of economic importance, and potential wildlife reservoirs.
Progress Report
The goals of the project plan for fiscal year (FY) 2024 consisted of 11 milestones, ten of which were either fully or substantially met. The one milestone in this plan that was not met was due to insufficient animal availability and space constraints and cannot be initiated until those have been resolved.
In work toward addressing Objective 1, Develop highly sensitive detection tools to determine the distribution of chronic wasting disease (CWD) and scrapie prions in natural hosts (sheep, goats, cervids) and their environment, we have worked closely with ARS researchers in Pullman, Washington, as well as Animal and Plant Health Inspection Service (APHIS) and university partners to develop a unified protocol for the detection of CWD prions by real-time quaking induced conversion (RT-QuIC) that utilizes an enrichment step that is capable of detection of disease in antemortem samples. This protocol has been distributed to diagnostic laboratories for evaluation and published this year. We have also developed protocols for amplification-based TSE diagnosis using alternate choices of amyloid binding fluorescent dye. With studies address the utility of these alternative dyes ongoing.
Objective 2, Investigate the pathobiology of CWD, scrapie prion strains, and atypical TSEs in natural hosts including potential cross species transmission events, the studies in question have been initiated and observation of the animals is ongoing.
Objective 3, Investigate the genetics of CWD susceptibility and resistance in white-tailed deer, consists of two subobjectives: A) Investigate the susceptibility of white-tailed deer to CWD modeling direct contact exposure with infected deer, and B) Investigate the susceptibility of white-tailed deer to CWD after direct inoculation. The first of these has been initiated on schedule while the second has been delayed considerably (2 years at this point) due to insufficient animal space.
Objective 4, Evaluate the presence of and determine the appropriate methodology for CWD strain determination, is dependent upon obtaining a diverse set of CWD isolates. We have begun the acquisition of these samples. Thus far we have found sample availability quite limited and do not anticipate being able to acquire sufficient samples to draw conclusions as to the presence of CWD strains.
Accomplishments
1. Neurofilament light chain identified as a potential biomarker for human prion disease, is not a reliable biomarker for pre-clinical detection of scrapie. Scrapie is a prion disease that affects sheep and goats. There is not an easily accessible and rapid antemortem diagnostic test for prion diseases in livestock. Based on previous research in humans that indicated Neurofilament light chain (Nf-L) may elevate in the blood years prior to symptom development, ARS scientists at Ames, Iowa, assessed the feasibility of using a blood sample to measure Nf-L as an indicator of brain damage. This study characterized the timeframe of Nf-L increases in sheep relative to the incubation period of scrapie. Serum Nf-L concentrations in experimental sheep increased as scrapie progressed; however, Nf-L blood levels did not increase significantly before the animals showed neurologic symptoms. The Nf-L levels increased substantially throughout the final 10% of the animals’ scrapie incubation period when other clinical signs were present placing the increase at a point in the incubation where established diagnostic tests are already suitable for detection. This study concluded that serum Nf-L is not a reliable biomarker for pre-clinical detection of scrapie. This information may be useful to researchers and veterinary diagnosticians.
2. The naturally occurring lysine to glutamic acid substitution results in short incubation periods for H-type Bovine Spongioform Encephalopathy. Classical Bovine Spongioform Encephalopathy (C-BSE) is a prion disease of cattle that was responsible for the “mad cow disease” epizootic in Europe in the 1980’s. C-BSE was determined to cause the human prion disease vCJD. Since then, atypical spontaneous strains of BSE were identified. H -BSE is one of those strains. Much research has explored the origins of C-BSE, and strain emergence from atypical H- BSE is one hypothesis. An H-BSE case was determined to have a germline mutation E211K substitution in the prion protein gene, which is analogous to a hereditary human prion disease. ARS scientists in Ames, Iowa reported the transmission of H-BSE from cattle, with and without the germline prion protein amino acid substitution, to cattle with various prion genotypes: EE211 (wild-type), EK211, and KK211. Results indicated a significantly shorter incubation period in K containing cattle compared to prion wild-type cattle. The scientists also explored the possibility that the C-BSE strain might have occurred after serial passages of K211 containing H-BSE in cattle; but results did not support this concept. This information may be important to prion researchers, veterinary diagnostic laboratories, and those involved with establishing regulatory guidelines.
Review Publications
Zhang, W., Orru, C.D., Foutz, A., Ding, M., Yuan, J., Shah, S., Zhang, J., Kotobelli, K., Gerasimenko, M., Gilliland, T., Chen, W., Tang, M., Cohen, M., Safar, J., Xu, B., Hong, D., Ciu, L., Hughson, A.G., Schonberger, L.B., Tatsuoka, C., Chen, S.G., Greenlee, J.J., Wang, Z., Appleby, B.S., Caughey, B., Zou, W. 2024. Large-scale validation of skin prion seeding activity as a biomarker for diagnosis of prion diseases. Acta Neuropathologica. https://doi.org/10.1007/s00401-023-02661-2.
Kokemuller, R., Moore, S., Bian, J., West Greenlee, H.M., Greenlee, J.J. 2023. Disease phenotype of classical sheep scrapie is changed upon experimental passage through white-tailed deer. PLoS Pathogens. https://doi.org/10.1371/journal.ppat.1011815.
Cassmann, E.D., Freese, L.J., Becker, K.A., Greenlee, J.J. 2023. Short incubation periods of atypical H-type BSE to cattle with EK211 and KK211 prion protein genotypes after intracranial inoculation. Frontiers in Veterinary Science. https://doi.org/10.3389/fvets.2023.1301998.
Lathrop, Q.L., Nicholson, E.M., Wang, C., Greenlee, J.J., Seger, H., Veneziano, S.E., Cassmann, E.D. 2024. Temporal serum neurofilament light chain concentrations in sheep inoculated with the agent of classical scrapie. PLOS ONE. https://doi.org/10.1371/journal.pone.0299038.
Alexis, F.J., West Greenlee, H.M., Bian, J., Greenlee, J.J. 2024. Transmission of classical scrapie using lymph node inoculum. Research in Veterinary Science. https://doi.org/10.1016/j.rvsc.2024.105348.
Ding, M., Teruya, K., Zhang, W., Lee, H., Yuan, J., Oguma, A., Foutz, A., Camacho, M., Mitchell, M., Greenlee, J.J., Kong, Q., Doh-Ura, K., Cui, L., Zou, W. 2021. Decrease in skin prion-seeding activity of prion-infected mice treated with a compound against human and animal prions: a first possible biomarker for prion therapeutics. Molecular Neurobiology. https://doi.org/10.1007/s12035-021-02418-6.