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United States Department of Agriculture

Agricultural Research Service

2009 Annual Report

1a.Objectives (from AD-416)
Obj. 1. Assess the cross species transmissibility of transmissible spongiform encephalopathies (TSEs) in livestock and wildlife. Obj. 2. Investigate the pathobiology of TSEs in natural and secondary hosts. Obj. 3. Investigate pathogenesis and ante mortem detection of bovine spongiform encephalopathy (BSE). Obj. 4. Develop a method to detect central nervous system (CNS) tissue contamination on carcasses. Obj. 5. Discover effective methods to inactivate TSE agents in agricultural settings.

1b.Approach (from AD-416)
Studies are focused on the four animal Transmissible Spongiform Encephalopathy (TSE) agents found in the United States: bovine spongiform encephalopathy (BSE); scrapie of sheep and goats; chronic wasting disease (CWD) of deer, elk, and moose; and transmissible mink encephalopathy (TME). These agents will be tested for cross-species transmissibility into various livestock and cervid species using both oral and intracerebral inoculation. Sites of accumulation, routes of infection, methods of isolate differentiation, and in the case of BSE, genetics of susceptibility and ante-mortem diagnostics, will be investigated. Existing technology developed at the National Animal Disease Center and those used in the meat packing industry for the detection of fecal contamination on carcasses will be adapted to detect CNS tissue contamination on carcasses. Methods of TSE inactivation will be evaluated for efficacy in agricultural settings.

3.Progress Report
The project plan involves 5 objectives: .
1)Assess cross-species transmissibility of transmissible spongiform encephalopathies (TSEs) in livestock and wildlife;.
2)Investigate the pathobiology of TSEs in natural and secondary hosts;.
3)Investigate pathogenesis and ante mortem detection of bovine spongiform encephalopathy (BSE);.
4)Develop a method to detect CNS tissue contamination on carcasses; and.
5)Determine effective methods to inactivate TSE agents in agricultural settings. In meeting objective 1 of our project plan, Assess cross-species transmissibility of TSEs in livestock and wildlife, numerous experiments assessing the susceptibility of various TSEs in different host species have been initiated and are ongoing. At the initiation of such experiments they have a planned timeframe of 60 months unless disease develops prior to that time. Pertaining to objective 2, deer were previously inoculated with blood from chronic wasting disease affected deer at this time several animals have developed clinical signs consistent with CWD. Approaches to strain typing of various TSE agents by biochemical means continue to assess as to the applicability to livestock species. In order to meet research goals described in Objective 3, Investigate pathogenesis and ante mortem detection of BSE, work is ongoing to increase the number of animals containing the E211K polymorphism, a potential cause of genetic BSE. This will provide the only means by which to prove that the polymorphism may cause BSE. Objective 4, Develop a method to detect CNS tissue contamination on carcasses, has been completed. In order to accomplish Objective 5, Determine effective methods to inactivate TSE agents in agricultural settings, laboratory investigations of TSE inactivation have been initiated and the results utilized to design animal bioassays to confirm the preliminary results. In addition, a natural host model for use in TSE inactivation is being investigated. Such a model would provide a much-needed tool in the development of inactivants for TSEs.

1. PCR Based Detection of Spiroplasma Mirium in Animal Tissues. Spiroplasma mirum has been suggested in a causative role in transmissible spongiform encephalopathies (TSEs), but sensitive tests to detect spiroplasma in tissues were not available. A sensitive PCR based assay was developed to detect Spiroplasma mirium in brain homogenate and applied assay to experimental and naturally occurring TSE cases. This rapid and sensitive method did not detect Spiroplasma mirium in any of the cases studied indicating that Spiroplasma mirium is not the causative agent of TSEs.

2. Functionality of Electroretinagram for Detection of Prion Disease in Sheep. No widely accepted non-invasive antemortem diagnostic for transmissible spongiform encephalopathies (TSEs) exists for sheep. Based on affected cell types observed in the retina of animals affected with transmissible spongiform encephalopathies, it was anticipated that affected animals would show abnormal electroretinagrams. It was determined that animals exhibit electroretinagram abnormalities 6 months prior to the onset of clinical signs. These electroretinagram abnormalities correspond to morphologic change in retinal cells types described by our group. This is the first non-invasive TSE screening test developed. Because TSEs in various species cause similar retinal lesions, the potential for application of this technique is very broad.

3. Differences in Expression Levels of the E211 and K211 Alleles in Cattle. Human genetic Creutzfeldt–Jakob disease (CJD) cases exhibit varied age of onset modulated by relative levels of allelic expression. Cattle may exhibit a modulation allelic ratios resulting in varied age of onset of bovine spongiform encephalopathy (BSE), modulated in a manner similar to that in humans. The relative expression levels of the E211 and K211 alleles were determined in the only K211 containing animal identified to date, and mechanisms of the regulation were investigated. K211 is expressed at a slightly lower level than E211 in a cow expression both E211 and K211. The magnitude of the expression level differences is substantially lower than that observed in late age of onset human genetic CJD cases indicating that the E211/K211 heterozygous cow is unlikely to exhibit delayed onset.

6.Technology Transfer

Number of Active CRADAs2
Number of Other Technology Transfer1

Review Publications
Greenlee, J.J., Nicholson, E.M., Hamir, A.N., Noyes, G.P., Holtzapple, M.T., Kehrli, Jr., M.E. 2008. Ablation of Prion Protein Immunoreactivity by Heating in Saturated Calcium Hydroxide. BMC Research Notes 2008 [online]. 1:99. Available:

Schonenbrucher, H., Gobel, K.A., Abdulmajood, A., Richt, J.A., Bulte, M. 2008. Comparative Studies of a Real-Time PCR Method and Three Enzyme-Linked Immunosorbent Assays for the Detection of Central Nervous System Tissues in Meat Products. Journal of Food Protection. 71(10):2059-2066.

Hamir, A.N., Picton, R., Blythe, L.L., Heidel, J.R. 2008. Astrocytoma with Involvement of Medulla Oblongata, Spinal Cord, and Spinal Nerves in a Raccoon (Procyon lotor). Veterinary Pathology. 45(6):949-951.

Hamir, A.N., Kunkle, R.A., Richt, J.A., Greenlee, J.J., Miller, J.M. 2009. Serial Passage of Sheep Scrapie Inoculum in Suffolk Sheep. Veterinary Pathology. 46(1):39-44.

Hamir, A.N., Kunkle, R.A., Greenlee, J.J., Richt, J.A. 2009. Experimental Oral Transmission of United States Origin Scrapie to Neonatal Sheep. Journal of Veterinary Diagnostic Investigation. 21(1):64-68.

Mukherjee, P., Bose, S., Hurd, A.A., Adhikary, R., Schonenbrucher, H., Hamir, A.N., Richt, J.A., Casey, T.A., Rasmussen, M.A., Petrich, J.W. 2009. Monitoring the Accumulation of Lipofuscin in Aging Murine Eyes by Fluorescence Spectroscopy. Photochemistry and Photobiology. 85(1):234-238.

Brunelle, B.W., Greenlee, J.J., Seabury, C.M., Brown II, C.E., Nicholson, E.M. 2008. Frequencies of Polymorphisms Associated with BSE Resistance Differ Significantly Between Bos taurus, Bos indicus, and Composite Cattle. BioMed Central (BMC) Veterinary Research. 4(1):36. Available:

Nicholson, E.M., Brunelle, B.W., Richt, J.A., Kehrli, Jr., M.E., Greenlee, J.J. 2008. Identification of a Heritable Polymorphism in Bovine PRNP Associated with Genetic Transmissible Spongiform Encephalopathy: Evidence of Heritable BSE. PloS ONE. 3(8):e2912.

Richt, J.A., Hall, S.M. 2008. BSE case associated with prion protein gene mutation. PLoS Pathogens. 4(9):e1000156. Available:

Hamir, A.N., Rupprecht, C.E. 2008. Trophoblast-like cells in tissues of porcupines (Erethizon dorsatum). Veterinary Pathology. 45:409-411.

Smith, J.D., Greenlee, J.J., Hamir, A.N., Greenlee, M.H. 2009. Altered Electroretinogram B-wave in a Suffolk Sheep Experimentally Infected with Scrapie. Veterinary Record. 165(6):179-181.

Smith, J.D., Greenlee, J.J., Hamir, A.N., Richt, J.A., Greenlee, M.H. 2009. Retinal Function and Morphology are Altered in Cattle Infected with the Prion Disease Transmissible Mink Encephalopathy. Veterinary Pathology. 46(5):810-816.

Last Modified: 8/25/2016
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