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ARS Home Midwest Area Ames, Iowa National Animal Disease Center Virus and Prion Research Research





- Nicholson, Eric
- Cassmann, Eric
- Greenlee, Justin
- Bian, Jifeng
- 2023
- 2022
- Publications
- Animal Health (103)

Research Project: Elucidating the Pathobiology and Transmission of Transmissible Spongiform Encephalopathies **Location:
                            Virus and Prion Research** 2023 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**
                                

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, to develop a unified protocol for the detection of CWD prions byreal-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. We have also made significant progress on novel sampling procedures for detection of transmissible spongiform encephalopathies (TSEs) utilizing rectal brush sampling that does not require in depth training or knowledge of anatomy. We have also developed protocols for amplification-based TSE diagnosis using alternate choices of amyloid binding fluorescent dye. 

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 (two 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, but not completed the acquisition of these samples. In summary, the goals of the project plan for FY22 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 will be initiated when those have been resolved. **Accomplishments**
                                

1. Sheep scrapie agent can infect white-tailed deer after oronasal exposure. The origin of chronic wasting disease (CWD) is not known, but it has many similarities to the prion disease of sheep called scrapie. It has long been hypothesized that CWD could have arisen through transmission of sheep scrapie to deer. ARS researches in Ames, Iowa, conducted a study to determine if scrapie derived from sheep could be transmitted to white-tailed deer. This study reports that the deer inoculated with sheep scrapie developed clinical signs of TSE and that the abnormal prion protein could be detected in a wide range of neural and lymphoid tissues. These results indicate that deer may be susceptible to sheep scrapie if exposed to the disease in natural or agricultural settings. In addition, several strong similarities between CWD in white-tailed deer and the experimental cases of scrapie in white-tailed deer in this report suggest that it would be difficult to identify scrapie in deer were a case to occur.  This information should be considered when developing plans to reduce or eliminate TSEs or advising farmers that wish to keep their deer herds free from prion diseases. 2. A novel sampling method was developed for monitoring CWD in farmed cervids to help maintain a CWD free environment. While the transmissible spongiform encephalopathies (TSEs) scrapie and bovine spongiform encephalopathy (BSE) have been largely controlled through selective breeding and a ruminant feed ban respectively, neither approach is applicable to chronic wasting disease (CWD). At this point the only method for protecting farmed cervids is maintenance of a CWD free environment. To accomplish this highly sensitive antemortem diagnostic methods using a non-invasive sampling protocol are needed to ensure that animals leaving or being introduced to new herds are free of CWD. ARS researchers in Ames, Iowa, developed a method using a rectal brush procedure for sampling coupled with the highly sensitive test known as real time quaking induced conversion (RT-QuIC). The rectal brush eliminates the need for trained personnel in the sample collection and allows for repeat sampling without reduction of available lymphoid tissue as might occur for rectal biopsy. This method will assist in the monitoring of CWD status aiding producers in preventing the introduction of CWD into their herd. **Review Publications**
                            *Cassmann, E.D., Brown, Q.L., Frese, A.J., Lambert, Z.J., West Greenlee, H.M., Greenlee, J.J. 2022. Effect of inoculation with prion dilutions within the dynamic range of ELISA absorbance on prion incubation period. Veterinary Research Communications. 46(4):1377-1380. https://doi.org/10.1007/s11259-022-10013-w.*
                                *Greenlee, J.J., Moore, S.J., Cassmann, E.D., Lambert, Z.J., Kokemuller, R., Smith, J.D., Kunkle, R.A., Kong, Q., West Greenlee, H.M. 2022. Characterization of classical sheep scrapie in white-tailed deer after experimental oronasal exposure. Journal of Infectious Diseases. 227(12):1386-1395. Article jiac443. https://doi.org/10.1093/infdis/jiac443.*
                                *Silva, C.J., Cassmann, E.D., Greenlee, J.J., Erickson-Beltran, M.L., Requena, J.R. 2023. A mass spectrometry-based method of quantifying the contribution of the lysine polymorphism at position 171 in sheep PrP. Journal of American Society for Mass Spectrometry. 34(2):245-254. https://doi.org/10.1021/jasms.2c00277.*
                                *Harm, T.A., Smith, J.D., Cassmann, E.D., Greenlee, J.J. 2022. Combinatorial treatment of brain samples from sheep with scrapie using sodium percarbonate, sodium dodecyl sulfate, and proteinase K increases survival time in inoculated susceptible sheep. Research in Veterinary Science. 152:497-503. https://doi.org/10.1016/j.rvsc.2022.09.002.*


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