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United States Department of Agriculture

Agricultural Research Service

Research Project: TRANSMISSION, DIFFERENTIATION, AND PATHOBIOLOGY OF TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES
2008 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 central nervous system (CNS) tissue contamination on carcasses; and.
5)Determine effective methods to inactivate TSE agents in agricultural settings. To meet objective 1 of our project plan, Assess cross-species transmissibility of TSEs in livestock and wildlife, we initiated experiments assessing the susceptibility of sheep of different genetic background to elk chronic wasting disease and the susceptibility of white-tailed deer to scrapie. At the initiation of such experiments they have a planned timeframe of 60 months unless disease develops prior to that time. As part of addressing both objective 1 and objective 2, we applied the polymerase chain reaction to various TSEs in natural and secondary hosts. Also pertaining to objective 2, deer were orally inoculated with blood from chronic wasting disease affected deer. The affects of hormonal regulation of intestinal permeability were studied. Approaches to enrich samples for abnormal prion protein were assessed for feasibility to be used in conjunction with labeling approaches for in vivo studies. Approaches to strain typing of various TSE agents by biochemical means were assessed 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, the prevalence of promoter region polymorphisms associated with BSE resistance was determined in rare U.S. cattle breeds. Further, an animal containing the E211K polymorphism, a potential cause of genetic BSE, has been identified and brought on site for embryo production purposes. This provides not only proof that the polymorphism is heritable but also provides a unique resource for the study of BSE. It affords us the opportunity to address if the polymorphism is indeed sufficient to result in a genetic form of BSE. In order to meet Objective 4, Develop a method to detect CNS tissue contamination on carcasses, a ruminant CNS tissue bank from a broad set of animals including both fresh and frozen tissues was established. Non-CNS tissues were also collected. The spectroscopic properties of CNS tissues were established, and the properties of the CNS tissue was compared to that of the adjacent non-CNS tissue. This has established the experimental basis for the development of a prototype device allowing real-time monitoring of CNS tissue contamination on bovine carcasses and meat cuts. This research addresses National Program 103–Animal Health Action Plan Component 2: Genetic and Biological Determinants of Disease Susceptibility; and Component 8: Countermeasures to Prevent and Control Transmissible Spongiform Encephalopathies.


4.Accomplishments
1. Real-time detection of central nervous system (CNS) tissue. Central nervous system tissue must be removed during slaughterhouse processing of bovine carcasses. Established experimental basis for development of a prototype device allowing real-time monitoring of CNS tissue contamination on bovine carcasses and meat cuts. CNS tissue contamination can now be detected in real-time on site in meat processing facilities. National Program 103 - Animal Health Action Plan: Component 8: Countermeasures to Prevent and Control Transmissible Spongiform Encephalopathies; Problem Statement 8C: Bovine Spongiform Encephalopathy (BSE).

2. Functionality of electroretinogram for detection of prion disease in cattle. No non-invasive antemortem diagnostic for transmissible spongiform encephalopathies (TSEs) exists for cattle. Based on affected cell types observed in the retina of animals affected with a transmissible spongiform encephalopathy it was anticipated that affected animals would show abnormal electroretinograms. It was determined that cattle exhibit electroretinogram abnormalities 6 months prior to the onset of consistent clinical signs of a prion disease. These electroretinogram abnormalities correspond to morphologic change in retinal cell 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 application of this technique is very broad. National Program 103 - Animal Health Action Plan: Component 8: Countermeasures to Prevent and Control Transmissible Spongiform Encephalopathies; Problem Statement 8C: Bovine Spongiform Encephalopathy (BSE).

3. Identification of the E211K polymorphism in a cow with bovine spongiform encephalopathy (BSE). Previously no polymorphisms in cattle had been identified with any similarity to those known to result in human prion disease. A cow positive for atypical BSE was identified as having a polymorphism analogous to one in humans that results in an inherited form of Creutzfeldt-Jakob disease. This is the first indication that BSE may arise as with a genetic rather than feedborne disease in cattle. National Program 103 - Animal Health Action Plan: Component 8: Countermeasures to Prevent and Control Transmissible Spongiform Encephalopathies; Problem Statement 8C: Bovine Spongiform Encephalopathy (BSE).

4. E211K is heritable trait in cattle. With the identification of the E211K polymorphism in cattle it was important to ascertain the heritability of this trait. It was determined that the only known living offspring of this animal has the same polymorphism. Not only is it known that this trait is a heritable trait in cattle it opens many avenues of research on BSE. National Program 103 - Animal Health Action Plan: Component 8: Countermeasures to Prevent and Control Transmissible Spongiform Encephalopathies; Problem Statement 8C: Bovine Spongiform Encephalopathy (BSE).


5.Significant Activities that Support Special Target Populations
None.


6.Technology Transfer

Number of Active CRADAs2

Review Publications
Hamir, A.N., Kunkle, R.A., Miller, J.M., Cutlip, R.C., Richt, J.A., Kehrli, Jr., M.E., Williams, E.S. 2007. Age-related lesions in laboratory-confined raccoons (Procyon lotor) inoculated with the agent of chronic wasting disease of mule deer. Journal of Veterinary Diagnostic Investigation. 19(6):680-686.

Hamir, A.N., Kunkle, R.A., Bulgin, M.S., Rohwer, R.G., Gregori, L., Richt, J.A. 2008. Experimental transmission of scrapie agent to susceptible sheep by intralingual and intracerebral inoculation. Canadian Journal of Veterinary Research. 72:63-67.

Hamir, A.N., Kunkle, R.A., Nicholson, E.M., Miller, J.M., Hall, S.M., Schonenbrucher, H., Brunelle, B.W., Richt, J.A. 2008. Preliminary observations on the experimental transmission of chronic wasting disease (CWD) from elk and white-tailed deer to fallow deer. Journal of Comparative Pathology. 138(2-3):121-130.

Hamir, A.N., Kunkle, R.A., Richt, J.A., Miller, J.M., Greenlee, J.J. 2008. Experimental transmission of US scrapie agent by nasal, peritoneal, and conjunctival routes to genetically susceptible sheep. Veterinary Pathology. 45(1):7-11.

Hamir, A.N., Richt, J., Miller, J.M., Kunkle, R.A., Hall, S., Nicholson, E.M., Orourke, K.I., Greenlee, J.J., Williams, E.S. 2008. Experimental transmission of chronic wasting disease (CWD) of elk (Cervus elaphus nelsoni), white-tailed deer (Odocoileus virginianus), and mule deer (Odocoileus hemionus hemionus) to white-tailed deer by intracerebral route. Veterinary Pathology. 45(3):297-306.

Brunelle, B.W., Hamir, A.N., Baron, T., Biacabe, A.G., Richt, J.A., Kunkle, R.A., Cutlip, R.C., Miller, J.M., Nicholson, E.M. 2007. Polymorphisms of the prion gene promoter region that influence classical bovine spongiform encephalopathy susceptibility are not applicable to other transmissible spongiform encephalopathies in cattle. Journal of Animal Science. 85(12):3142-3147.

Nicholson, E.M., Kunkle, R.A., Hamir, A.N., Lebepe Mazur, S., Orcutt, D. 2007. Detection of the disease-associated isoform of the prion protein in formalin-fixed tissues by Western blot. Journal of Veterinary Diagnostic Investigation. 19:548-552.

O'Rourke, K.I., Spraker, T.R., Zhuang, D., Greenlee, J.J., Gidlewski, T.E., Hamir, A.N. 2007. Elk with a long incubation prion disease phenotype have a unique PrP-d profile. NeuroReport. 18(18):1935-1938.

Schonenbrucher, H., Adhikary, R., Mukherjee, P., Casey, T.A., Rasmussen, M.A., Maistrovich, F.D., Hamir, A.N., Kehrli, Jr., M.E., Richt, J.A., Petrich, J.W. 2008. Fluorescence-Based Method, Exploiting Lipofuscin, for Real-Time Detection of Central Nervous System Tissues on Bovine Carcasses. Journal of Agricultural and Food Chemistry. 56(15):6220-6226.

Kunkle, R.A., Nicholson, E.M., Lebepe-Mazur, S., Orcutt, D.L., Srinivas, M.L., Greenlee, J.J., Alt, D.P., Hamir, A.N. 2008. Western blot detection of PrP**sc in archived paraffin-embedded brainstem from scrapie-affected sheep. Journal of Veterinary Diagnostic Investigation. 20(4):522-526.

Gavier-Widen, D., Noremark, M., Langeveld, J.P., Stack, M., Biacabe, A.B., Vulin, J., Chaplin, M., Richt, J.A., Jacobs, J., Acin, C., Monleon, E., Renstrom, L., Klingeborn, B., Baron, T.G. 2008. Bovine spongiform encephalopathy in Sweden: an H-type variant. Journal of Veterinary Diagnostic Investigation. 20(1):2-10.

Brunelle, B.W., Kehrli, Jr., M.E., Stabel, J.R., Moody Spurlock, D., Hansen, L.B., Nicholson, E.M. 2007. Allele, genotype, and haplotype data for bovine spongiform encephalopathy-resistance polymorphisms from healthy US holstein cattle. Journal of Dairy Science. 91:338–342.

Smith, J.D., Greenlee, J.J., Hamir, A.N., West Greenlee, M.H. 2008. Retinal cell types are differentially affected in sheep with scrapie. Journal of Comparative Pathology. 138(1):12-22.

Last Modified: 10/31/2014
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