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United States Department of Agriculture

Agricultural Research Service

Related Topics


Location: Virus and Prion Research

2012 Annual Report

1a. Objectives (from AD-416):
1. Investigate the pathobiology of atypical transmissible spongiform encephalopathies (TSEs) in natural hosts. A. Investigate the pathobiology of atypical scrapie. B. Investigate the pathobiology of atypical bovine spongiform encephalopathy (BSE). 2. Investigate the horizontal transmission of TSEs. A. Assess the horizontal transmission of sheep scrapie in the absence of lambing. B. Determine routes of transmission in chronic wasting disease (CWD) infected premises. C. Assess oral transmission of CWD in reindeer. 3. Investigate determinants of CWD persistence. A. Determine CWD host range using natural routes of transmission. B. Investigate the pathobiology of CWD.

1b. Approach (from AD-416):
The studies will focus on three animal transmissible spongiform encephalopathy (TSE) agents found in the United States: bovine spongiform encephalopathy (BSE); scrapie of sheep and goats; and chronic wasting disease (CWD) of deer, elk, and moose. The research will address sites of accumulation, routes of infection, environmental persistence, and ante mortem diagnostics with an emphasis on controlled conditions and natural routes of infection. Techniques used will include clinical exams, histopathology, immunohistochemistry and biochemical analysis of proteins. The enhanced knowledge gained from this work will help mitigate the potential for unrecognized epidemic expansions of these diseases in populations of animals that could either directly or indirectly affect food animals.

3. Progress Report:
In research directed toward meeting objective 1 of our project plan, Investigate the pathobiology of atypical TSEs in natural hosts, we have inoculated animals for studies designed to address the pathobiology of atypical scrapie, atypical bovine spongiform encephalopathy (BSE), as well as a genetic version of BSE. In addition, we have begun investigating the possibility that atypical scrapie was present earlier than previously detected in the national flock by analyzing archived field isolates using methods that were unavailable at the time of original diagnosis. In research pertaining to objective 2, Investigate the horizontal transmission of TSEs, we have initiated a study to determine if cohousing non-lambing scrapie inoculated sheep is sufficient to transmit scrapie to neonatal lambs. At this time, scrapie inoculated sheep are being co-housed with pregnant ewes. For objective 3, all work planned for FY12 has been delayed until FY13 due to limited availability of animals of the required genotypes.

4. Accomplishments
1. Pathologic and biochemical characterization of a genetic form of bovine spongiform encephalopathy (BSE). The complete pathologic and biochemical features of a genetic form of BSE were defined and reported for the first time by ARS scientists at the National Animal Disease Center, Ames, IA. The genetic form of BSE is analogous to the most prevalent hereditary form of human TSE. Heritable BSE along with spontaneous BSE forms are also referred to as atypical BSE cases which have important implications in that they are not associated with the feedborne epidemic of classical BSE first recognized in the United Kingdom in the 1980s. Atypical BSE cases emphasize the need to maintain the specified risk material ruminant feed ban as a science-based policy to prevent a feedborne epidemic from developing; the feedborne nature of the classical BSE epidemic has been demonstrated to negatively impact export markets in various countries around the world, whereas atypical BSE does not connote the same concern.

2. Defined the relationship between PrPSc stability and incubation time for U.S. scrapie strains in sheep. In order to complete this accomplishment we developed a novel protease free version of the fibril stability assay. In this work we demonstrated the utility of the method in characterizing 2 different strains as well as being able to differentiate a mixture of strains. Showing the correlation between stability and incubation time holds in livestock also advances our understanding of the disease process in Transmissible spongiform encephalopathy (TSE) strains. Furthermore, this work results in a rapid and robust alternative to TSE strain differentiation that until now has relied largely on a time consuming and costly bioassay in rodents. TSE strains have disease implications with regard to host range, tissue distribution, and resistance to inactivation as well as significant regulatory implications when a TSE is identified in a herd. This work conducted by ARS scientists at the National Animal Disease Center, Ames, IA is significant not only in the enhancement of our understanding of TSE strains but also represents a rapid and robust method developed to differentiate TSE strains.

3. Development of purification and refolding methods for recombinant bovine prion. A method of recombinant bovine prion protein purification and refolding was developed using a commercially available expression system. This work, by scientists at the National Animal Disease Center, Ames, IA, is significant to researchers studying how abnormally folded prions cause bovine spongiform encephalopathy (BSE) and other prion diseases. Previously, the quality and yield of recombinant protein varied across production methods resulting in limitations for some research applications of purified prion proteins. This method uses a standard commercially available expression system available to anyone and highlights the preferred approach for optimal quality and yield.

4. Development of a rapid method for detection of disease-associated prions (PrPSc) in formalin-fixed paraffin-embedded tissue by ELISA. A method for the detection of PrPSc in formalin-fixed paraffin-embedded tissue by ELISA has been developed and described by ARS scientists at the National Animal Disease Center, Ames, IA. Methods for diagnosis of transmissible spongiform encephalopathies (TSEs) in cattle, sheep and cervids have traditionally depended on the availability of both frozen fresh and formalin-fixed tissues. However, in many diagnostic sample submissions only formalin-fixed samples have been available for TSE diagnosis, a situation that previously precluded analysis by rapid diagnostic procedures such as ELISA. This work describes a method suitable for extraction of the PrPSc from formalin-fixed paraffin-embedded tissue for detection by ELISA. This represents a significant advancement for diagnostic laboratories and provides a rapid alternative method for TSE detection beyond immunohistochemistry (IHC).

5. The amino acid, lysine, at position 171 of the sheep prion protein delays development of scrapie. Demonstrated the affect of the amino acid lysine at position 171 of the sheep prion protein on susceptibility to scrapie, a transmissible spongiform encephalopathy of sheep. Amino acid differences in the prion protein are known to play a major role in scrapie susceptibility in sheep and these genetic differences are utilized in the strategy to remove scrapie from our nation's sheep flock. Natural scrapie had previously only been described in one sheep with lysine at position 171 of the prion protein, hence not enough information was available from natural cases to determine the affect of lysine at position 171 on scrapie susceptibility. ARS scientists at the National Animal Disease Center, Ames, IA demonstrated that sheep with a prion protein containing lysine at position 171 are susceptible to scrapie but have a prolonged scrapie incubation period, and that the abnormal prion protein accumulates throughout the central nervous system and lymphoid organs. Because sheep with lysine at prion amino acid position 171 develop scrapie at a slower rate than other known susceptible genotypes this information is critical to sheep breeders that want to eradicate genotypes susceptible to scrapie.

Review Publications
Greenlee, J.J., Smith, J.D., Kunkle, R.A. 2011. White-tailed deer are susceptible to the agent of sheep scrapie by intracerebral inoculation. Veterinary Research. 42(1):107.

Greenlee, J.J., Smith, J.D., West Greenlee, M.H., Nicholson, E.M. 2012. Clinical and pathologic features of H-type bovine spongiform encephalopathy associated with E211K prion protein polymorphism. PLoS ONE. 7(6):e38678.

Porcario, C., Hall, S.M., Martucci, F., Corona, C., Iulini, B., Perazzini, A.Z., Acutis, P., Hamir, A.N., Loiacono, C.M., Greenlee, J.J., Richt, J.A., Caramelli, M., Casalone, C. 2011. Evaluation of two sets of immunohistochemical and Western blot confirmatory methods in the detection of typical and atypical BSE cases. BMC Research Notes. 4:376.

Hamir, A.N. 2011. Pathology of neurologic disorders of raccoons (Procyon lotor). Journal of Veterinary Diagnostic Investigation. 23(5)873-884.

Nicholson, E.M., Greenlee, J.J., Hamir, A.N. 2011. PrPSc detection in formalin-fixed paraffin-embedded tissue by ELISA. BMC Research Notes. 4(1):432.

Greenlee, J.J., Zhang, Xia, Nicholson, E.M., Kunkle, R.A., Hamir, A.N. 2012. Prolonged incubation time in sheep with prion protein containing lysine at position 171. Journal of Veterinary Diagnostic Investigation. 24(3):554-558.

Tamguney, G., Richt, J.A., Hamir, A.N., Greenlee, J.J., Miller, M.W., Wolfe, L.L., Sirochman, T.M., Young, A.J., Glidden, D.V., Johnson, N.L., Giles, K., Dearmond, S.J., Prusiner, S.B. 2012. Salivary prions in sheep and deer. Prion. 6(1):52-61.

Sharma, V.K., Sacco, R.E., Kunkle, R.A., Bearson, S.M., Palmquist, D.E. 2012. Correlating levels of type III secretion and secreted proteins with fecal shedding of Escherichia coli O157:H7 in cattle. Infection and Immunity. 80(4):1333-1342.

Smith, J.D., Greenlee, J.J., Foster, G.H., Nicholson, E.M. 2012. Acetone precipitation of the scrapie agent results in successful recovery of PrPSc but decreased infectivity. Journal of Agricultural and Food Chemistry. 60(18):4758-4762.

Vrentas, C.E., Onstot, S., Nicholson, E.M. 2012. A comparative analysis of rapid methods for purification and refolding of recombinant bovine prion protein. Protein Expression and Purification. 82(2):380-388.

Wilson, R., Plinston, C., Hunter, N., Casalone, C., Corona, C., Tagliavini, F., Suardi, S., Ruggerone, M., Moda, F., Graziano, S., Sbriccoli, M., Cardone, F., Pocchiari, M., Ingrosso, L., Baron, T., Richt, J., Andreoletti, O., Simmons, M., Lockey, R., Manson, J.C., Barron, R.M. 2012. Chronic wasting disease and atypical forms of bovine spongiform encephalopathy and scrapie are not transmissible to mice expressing wild-type levels of human prion protein. Journal of General Virology. 93(Pt 7):1624-1629.

Vrentas, C.E., Greenlee, J.J., Tatum, T.L., Nicholson, E.M. 2012. Relationships between PrPSc stability and incubation time for United States scrapie isolates in a natural host system. PLoS ONE. 7(8):e43060.

Last Modified: 06/25/2017
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