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From: TSS ()
Subject: Transmission, Differentiation, and Pathobiology of Transmissible Spongiform Encephalopathies 2005 Annual Report
Date: December 5, 2006 at 8:00 am PST


Sent: Monday, December 04, 2006 10:23 PM
Subject: Transmission, Differentiation, and Pathobiology of Transmissible Spongiform Encephalopathies 2005 Annual Report


Research Project: Transmission, Differentiation, and Pathobiology of Transmissible Spongiform Encephalopathies
Location: Virus and Prion Diseases of Livestock

2005 Annual Report

1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter?
This project deals with Transmissible Spongiform Encephalopathies (TSE), which are fatal degenerative diseases of the central nervous system that can affect several animal species, including humans. The causal agent is believed to be a cellular protein, the prion protein (PrP) that has assumed an unnatural form. Because the altered protein is resistant to protease degradation, it accumulates in nervous tissue and the resulting dysfunction ultimately leads to death. The specific TSEs being investigated in this project are scrapie in sheep, transmissible mink encephalopathy (TME), bovine spongiform encephalopathy (BSE) in cattle, and chronic wasting disease (CWD) in deer and elk. The major concern about these diseases is that BSE has been shown to cross the species barrier to cause a unique TSE in human beings. Although it has not been demonstrated that scrapie, TME, or CWD present any risk to human health, the BSE experience has raised many questions about the potential hazard these TSEs present for transmission to other animal species, especially domesticated livestock and wildlife. The current research is focused on direct experimental challenge of the species barrier effect by animal inoculation. These results are then compared to results obtained with a variety of laboratory procedures to determine if they can be used as predictive models for future risk assessments. These studies also provide information about the clinical and pathological disease characteristics that can be expected if a TSE crosses the species barrier, thus enabling animal health specialists to recognize such situations should they occur. Additional transmission studies in the natural host will focus on determining the modes of transmission and disease development in scrapie and CWD so that appropriate intervention strategies can be devised that will control the spread of these diseases.

Because of the risk for BSE transmission to human beings, the presence of scrapie, TME, CWD, and BSE in the United States presents a potential liability to the U.S. livestock and hunting industries, because the safety of animals and animal products intended for domestic consumption and international trade may be questioned. Efforts are being made by both federal and state regulatory agencies to eradicate scrapie and CWD and to determine the prevalence of BSE. The effectiveness of these programs will depend heavily on having accurate information about the nature of these diseases, not only in the original hosts, but also in other species that may be affected.

The impact of this research project on U.S. agriculture, especially the cattle industry, is significant. The discovery of one BSE positive animal in December 2003 in Washington State resulted in significant losses to the U.S. beef industry. More than 50 countries (including major markets such as Japan and South Korea) banned import of U.S. cattle and beef products within days of the December 2003 announcement. Estimated losses arising from these bans during 2004 range from $3.2 billion to $4.7 billion. Many of these bans are still in effect in 2005 and with the discovery of a second, indigenous case of BSE in June 2005, significant losses from export bans of U.S. beef will most likely be also experienced in 2005. In addition, the regulations introduced in 2004 led to changes in the beef industry with a net economic cost of approximately $200 million in 2004.


2.List the milestones (indicators of progress) from your Project Plan.
Animal experiments: Because of the long-term nature of most TSE experiments in animals, especially those involving cross-species transmission, most of the studies outlined in this project will not be completed within 30 months. Therefore, the following time line primarily presents expectations for when experiments will be initiated.

FY 2003 Cattle inoculated with white-tailed deer CWD. Fallow and white-tailed deer inoculated with CWD. Cattle inoculated with TME. Sheep inoculated with AV136QR171 and Idaho scrapie isolates.

FY 2004 Cattle inoculated with elk CWD. Raccoons inoculated with TME, scrapie, and CWD isolates. Mice inoculated for strain typing of 10 TSE isolates. Swine inoculated with scrapie and CWD. Reindeer inoculated with CWD. White-tailed deer inoculated with scrapie.

FY 2005 Study to assess scrapie and CWD amplification in market age swine completed. Mice inoculated for strain typing of 15 TSE isolates.

Laboratory studies:

FY 2003 Validation of method for genotyping from paraffin sections. Methods developed for biochemical strain typing studies.

FY 2004 Genotyping of archived scrapie tissues from the National Veterinary Services Laboratories (NVSL)/APHIS/VS/USDA. Evaluation of biochemical methods for strain typing of scrapie isolates. Development of mass spectrometry methods for characterization of protein expression in normal sheep brain.

FY 2005 Evaluation of biochemical methods for differentiation of TSE agents after cross-species transmission. Comparison of protein expression (mass spectrometry) in scrapie brain to that in normal sheep brain.


3a.List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why.

Study to assess scrapie and CWD amplification in market age swine completed.
Milestone Fully Met Milestone Substantially Met

Mice inoculated for strain typing of 15 TSE isolates.
Milestone Not Met

Evaluation of biochemical methods for differentiation of TSE agents after cross-species transmission.
Milestone Substantially Met

Comparison of protein expression (mass spectrometry) in scrapie brain to that in normal sheep brain.
Milestone Not Met

3b.List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone?
FY 2006 Mouse bioassay with various TSE isolates to determine if this strain typing procedure is useful for identification of unique TSE strains in the U.S.

Cattle inoculation with both U.S. BSE isolates in order to amplify BSE material for subsequent pathogenesis studies.

Inoculation of reindeer with CWD agent.

FY 2007 and FY 2008 Because the incubation period for TSE transmission is typically 2 years or longer, it is anticipated that the major activity during these years will involve termination of many animal inoculation experiments that began in FY03. The work will include necropsies and tissue analysis to confirm TSE transmissions, followed by laboratory analysis using strain characterization methods developed and evaluated in FY 04 and FY05.


4a.What was the single most significant accomplishment this past year?
Transmissible Mink Encephalopathy (TME) transmission to cattle: Intracerebral inoculation of cattle with brain material from mink with TME and TME from cattle (i.e. from 1st cattle passage) resulted in lesions of spongiform encephalopathy and distribution of the abnormal form of the prion protein, PrPres (as determined by immunohistochemistry and Western blot) in the central nervous system (CNS) which resembles those found in cattle infected with Bovine Spongiform Encephalopathy (BSE). This indicates that TME is able to induce a neurological disease and pathological lesions in cattle which are similar to BSE. Comparison of cattle- passaged TME with both U.S. BSE cases by Western Blot analysis revealed differences in molecular weight. This would allow us to differentiate between cattle infected with BSE and TME. This is a significant finding that might enable us to distinguish BSE in cattle from the other animal TSEs should they ever appear in cattle.


4b.List other significant accomplishments, if any.
Intracerebral transmission of Chronic Wasting Disease (CWD) to white-tailed deer: Under free-ranging conditions, CWD has been observed in elk, mule deer and white-tailed deer. Since there is no information to indicate that CWD from these 3 cervid species is the same (or if these are 3 different strains of cervid CWDs), we inoculated CWD from these 3 sources to 3 groups of white-tailed deer fawns. White-tailed deer were susceptible to all three sources of CWD with similar incubation times. This finding is important in that it indicates that there are no differences in clinical susceptibility of white-tailed deer to CWD from different cervid sources.

Oral transmission of CWD to elk: To compare genetic susceptibility of elk to CWD, eight 8-month-old elk calves of 3 different genotypes (MM, LM and LL) for the prion protein were orally dosed with CWD infected brain material from elk. Elk with the MM and LM genotype developed CWD within 23 and 40 months, respectively, whereas LL elk are still alive and clinically normal at 4 years post inoculation. The finding that elk with the LL genotype have a reduced susceptibility to oral infection with the chronic wasting disease agent is important to elk farmers. This study will continue for additional 2 years.

Scrapie transmission to swine: Swine were inoculated with scrapie-affected sheep brain homogenate via the oral and intracerebral route and necropsied at 6 months post inoculation. No evidence of prion disease was found in these market aged swine. Monitoring of scrapie-inoculated littermates will continue until the termination of the study in approximately 5 years. This work is important for pork producers.

Retinal pathology in sheep with scrapie: A study of retinal pathology in scrapie-affected sheep has been completed. It was demonstrated that cells of the retina in scrapie-affected sheep with prion accumulation in the retina express proteins that indicate retinal degeneration, whereas cells of the normal sheep retina do not express these proteins. The implication of this report is that scrapie-affected sheep may have deficits of the visual system which are detectable by various methods. Laboratory verification of second U.S. BSE case: Studies were conducted which confirmed the BSE diagnosis of an inconclusive bovine brain sample. The PrPres profile from the second BSE case diagnosed in the United States showed different molecular properties when compared to the PrPres pattern described for the 2003 U.S. isolate. A germline mutation in the bovine PrP gene was not evident. This work benefited the APHIS-National Veterinary Services Laboratories.

B cells in sheep scrapie: In scrapie-infected sheep, an over-representation of the B-1 subset of B cells was detected in the peripheral blood. In addition, a significant reduction in the expression of the normal prion protein, PrPc, on B cells, was found. This correlated with the progression of scrapie in these animals. The implication of these findings is that scrapie-affected sheep may have detectable changes in the peripheral blood that may lead to a live animal test.


4c.List any significant activities that support special target populations.
None.


5.Describe the major accomplishments over the life of the project, including their predicted or actual impact.
This project was initiated January 28, 2002, as a result of the FY 2002 Appropriations Bill passed by Congress and signed by the President for research on Emerging & Exotic Diseases of Pests. The major objectives are to assess transmissibility of the TSEs that affect livestock and wildlife species, to develop methods for differentiation of TSE strains, and to determine the pathobiology of these diseases in the natural host and after cross-species transmission. Experiments are in progress to determine the transmissibility of different CWDs into cattle, sheep and swine. Based on results of our studies, it may be concluded that under natural conditions cattle exposed to mule deer CWD would require a rather large dose of inoculum, and an extremely long incubation time to develop a TSE-associated disease. In contrast, intracerebral inoculation of cattle with brain material from mink with TME or cattle-passaged TME resulted in incubation times, lesions of spongiform encephalopathy and PrP**res distribution which resemble those found in cattle infected with BSE. We were able to compare the first U.S. BSE isolate (2003) with other typical BSE isolates (Canadian, European) and found them to be indistinguishable. In contrast, the molecular phenotype of the second U.S. BSE isolate (2004) was different from the 2003 U.S. BSE isolate as determined by Western blot analysis. Completed work has determined that sheep scrapie can be transmitted to elk and that the resulting disease is indistinguishable from CWD in that species. When elk CWD was transmitted orally to elk of different genotypes, one genotype (LL) seems to be less susceptible to CWD. White-tailed deer have been inoculated with CWD from elk, mule deer and white-tailed deer in order to determine if strain differences in the CWD agent exist that depend on the host of origin. The cross-species transmission experiments will provide information and tissues that can be used to evaluate the effectiveness of current diagnostic protocols for the TSEs. An important contribution of this project has been the demonstration of the utility of raccoons as an animal model to distinguish scrapie, CWD and TME based on attack rate and time to clinical disease. Raccoons inoculated with TME develop disease within 6 months time and as such represent one of the fastest available, non-transgenic models of TSE disease. We developed a method to extract DNA from formalin-fixed paraffin-embedded brainstem tissue to determine prion gene polymorphisms in scrapie-infected sheep. This method has been successfully transferred to APHIS and might have important implications for future scrapie surveillance efforts.


6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products?
Preliminary inactivation studies with prions in different materials were initiated with 2 independent companies with interest in commercial feed manufacturing. Discussions in establishing CRADAs are underway.


7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below).
ARS Agricultural Research Magazine: Article on research activities in December 2004 edition (Title: TSEs Touch Off ARS Research). Invited lecture presented at USAHA meeting on "Characterization of the recent U.S. BSE case and methods for surveillance." Invited lecture presented at AAVLD meeting on "Molecular characterization of prion isolates from livestock and cervids."” Invited lecture presented at the USDA's Agricultural Outlook Forum on the "BSE and strategies for testing." Invited lecture presented at APHIS-ARS Annual Conference on "Review of key accomplishments in TSE Research." Invited lecture presented at the ARS BSE Industry Consultation Meeting on "Critical gaps in our knowledge of Bovine Spongiform Encephalopathy." Invited lecture presentated at the AOAC International Midwest Section Annual Meeting on "Biochemistry of Prion Diseases." Invited lecture presented at the 39th US-Japan Cooperative Program in Natural Resources on "TSE inactivation in feed production."


Review Publications
Greenlee, J.J., Nicholson, E.M., Hamir, A.N., Richt, J.A., Kunkle, R.A., Noyes, G., Hotzapple, M.T., Kehrli Jr., M.E. 2005. Loss of prion protein immunoreactivity after boiling in saturated calcium hydroxide solution [abstract]. Keystone Symposia: Molecular Mechanisms of Transmissible Spongiform Encephalopathies. Poster No. 125.

Nicholson, E.M., Greenlee, J.J., Holtzapple, M., Noyes, G., Hamir, A.N., Kunkle, R.A., Richt, J., Kehrli, Jr., M.E. 2004. TSE inactivation in feed production [abstract]. 39th United States-Japan Cooperative Program in Natural Resources. USDA:APHIS:NVSL, Session 4, Abstract no. 6.

Hamir, A.N., Kunkle, R.A., Cutlip, R.C., Miller, J.M., Orourke, K.I., Williams, E.S., Miller, M.W., Stack, M.J., Chaplin, M.J., Richt, J. 2005. Experimental transmission of chronic wasting disease agent to cattle by intracerebral route. Journal of Veterinary Diagnostic Investigation. 17:276-281.


Hamir, A.N., Kunkle, R.A., Richt, J.A., Miller, J.M., Cutlip, R.C., Jenny, A.L. 2005. Experimental transmission of sheep scrapie by intracerebral and oral routes to genetically susceptible Suffolk sheep in the United States. Journal of Veterinary Diagnostic Investigation. 17(1):3-9.


Hamir, A.N., Kunkle, R.A., Richt, J.A., Cutlip, R.C., Miller, J.M., Orourke, K.I., Williams, E.S., Miller, M.W., Stack, M.J., Chaplin, M.J. 2004. Experimental transmission of chronic wasting disease agent to cattle by intracerebral route: final outcome of the study [abstract]. Animal Prion Diseases and the Americas. p. 78.


Hamir, A., Miller, J., Cutlip, R., Stack, M., Chaplin, M., Bartz, J., Jenny, A., Williams, E. 2004. Experimental inoculation of TME, scrapie, and CWD to raccoons: an update [abstract]. Animal Prion Diseases and the Americas. p. 79.


Hamir, A.N. 2004. Experimental cross-species transmission of chronic wasting disease (CWD) at the National Animal Disease Center (NADC), Ames, Iowa: an update [abstract]. Animal Prion Diseases and the Americas. p. 42.


Nicholson, E.M., Young, A.J., Richt, J. 2004. Establishment of a real time-PCR based assay for the determination of ovine PrP mRNA [abstract]. Animal Prion Diseases and the Americas. p. 90.


Richt, J.A., Kluge, J.P., Alt, D.P., Kunkle, R.A., Hamir, A.N., Czub, S., Davis, A.J., Hall, S.M. 2004. Identification and characterization of the U.S. bovine spongiform encephalopathy case [abstract]. Animal Prion Diseases and the Americas. p. 38.


Nicholson, E.M., Greenlee, J.J., Richt, J., Hamir, A.N., Kunkle, R.A., Kehrli, Jr., M.E. 2005. Biochemistry of prion diseases. Association of Analytical Communities International, Midwest Section [abstract]. Abstract No. 601. p. 38.


Hamir, A.N., Kunkle, R.A., Richt, J.A., Cutlip, R.C., Miller, J.M., O'Rourke, K.I., Williams, E.S., Miller, M.W., Stack, M.J., Chaplin, M.J. 2004. Experimental transmission of chronic wasting disease agent to cattle by intracerebral route: final outcome of the study [abstract]. Annual Conference of the American College of Veterinary Pathologists. 41:588.


Bessen, R.A., Dejoia, C., Dlakic, W., Sorg, R., O'Connell, K., Tucker, T., Hamir, A.N., Richt, J.A., Rocke, T. 2005. Prion infection of mucosal tissue [abstract]. 2nd International Chronic Wasting Disease Symposium. p. 38.


Hamir, A.N., Cutlip, R.C., Miller, J.M., Kunkle, R.A., Greenlee, J.J., Richt, J.A., Bartz, J. 2005. Experimental transmission of transmissible spongiform encephalopathies (TSEs) at the National Animal Disease Center (NADC), Ames, Iowa, USA: An update [abstract]. Annual Wildlife Disease Association Conference. p. 263.


Richt, J.A., Kluge, J.P., Alt, D.P., Kunkle, R.A., Hamir, A.N., Czub, S., Davis, A.J., Hall, S.M. 2004. Characterization of the recent U.S. BSE case and methods for surveillance. In: Proceedings of the 108th Annual Meeting of the United States Animal Health Association, October 22-27, 2004, Greensboro, North Carolina. p. 91-92.


Young, A.J., Elmubark, G., Nthale, J., Hamir, A., Richt, J. 2004. Scrapie-associated alterations in the composition and prion protein expression of the peripheral blood B cell pool [abstract]. Conference of Research Workers in Animal Diseases. p. 130.


Young, A.J., Elmubark, G., Nthale, J., Hamir, A.N., Richt, J. 2005. Genetic and scrapie-associated differences in PrPc expression of peripheral blood B cell subsets [abstract]. Molecular Mechanisms of TSEs (Prion Diseases), Keystone Symposia. p. 61.


Anantharam, V., Vorberg, I., Choi, C., Kanthasamy, A., Richt, J., Priola, S.A., Kanthasamy, A.G. 2005. Differential role of prion protein in oxidative stress- and ER stress-induced apoptotic signaling in neural cells [abstract]. Molecular Mechanisms of TSEs (Prion Diseases), Keystone Symposia. p. 39.


Anantharam, V., Vorberg, I., Choi, C., Kanthasamy, A., Richt, J.A., Priola, S.A., Kanthasamy, A.G. 2004. Proteolytic activation of PKC-delta contributes to oxidative- and ER-stress induced apoptotic cell death in PrP*sen-overexpressing mouse neuronal cells [abstract]. Animal Prion Diseases and the Americas. p. 48.


http://www.ars.usda.gov/research/projects/projects.htm?ACCN_NO=408808&showpars=true&fy=2005

TSS




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