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 2006 and with the discovery of a third case of BSE (second indigenous BSE case in the U.S.) in March 2006, significant losses from export bans of U.S. beef will most likely be also experienced also in 2006. 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 only.

2.List by year the currently approved milestones (indicators of research progress)
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.

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

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

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

2006 Initiate mouse bioassay of various TSE isolates to determine if this strain typing procedure is useful for identification of unique TSE strains in the U.S. Initiate cattle inoculation with both U.S. BSE isolates in order to amplify BSE material for subsequent pathogenesis studies. Inoculation of reindeer with the CWD agents derived from white-tailed deer, mule deer and elk. Initiate oral pathogenesis studies of raccoons with TME.

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

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.

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.

2006 Determination of the feasibility of PrP**Sc detection from formalin fixed tissues. Evaluate methods of biochemical strain typing of TSE isolates.

4a.List the single most significant research accomplishment during FY 2006.
Identification and characterization of third U.S. BSE case: Studies were conducted which confirmed the BSE diagnosis of an inconclusive bovine brain sample. The PrP**res profile from the third BSE case diagnosed in the United States showed different molecular properties when compared to the PrP**res pattern described for the 2003 U.S. isolate, however had similar molecular properties as described for the second U.S. BSE case. This finding and our findings from the two previous cases of BSE support the presence of only atypical BSE in the U.S. indigenous cattle population.

This work is of critical relevance to the National Program Action Plan, Animal Health (103), program component pathogen detection, because it had an important impact on the confirmation of an inconclusive BSE diagnosis (based on a rapid test) and the incidence of BSE in the U.S. cattle population.

4b.List other significant research accomplishment(s), if any.
Intracerebral transmission of CWD from white-tailed deer to cattle: Tissues were collected from cattle inoculated intracerebrally with CWD from white-tailed deer (WTD) at the termination of a 4-year study. The WTD CWD attack rate (7/8) in cattle was higher and the incubation time shorter than that previously seen in cattle challenged intracerebrally with mule deer CWD (3/8). The absence of lesions of spongiform encephalopathy, the prion accumulation as detected by immunohistochemistry and the Western blot profiles were similar to those observed in cattle inoculated with mule deer and elk derived CWD.

Intracerebral transmission of mule deer CWD into sheep: Tissues were collected from sheep at the termination of the 6-year study to assess transmission of mule deer CWD to sheep by intracerebral inoculation. These tissues were analyzed by histopathology, immunohistochemistry and Western blot. Two of eight inoculated sheep had spongiform encephalopathy with prion accumulation detected by immunohistochemistry and Western blot. Results of analyses indicated a prion disease in sheep indistinguishable from sheep scrapie.

Intracerebral transmission of CWD from elk, mule-deer, and white-tailed deer into white-tailed deer: Tissues were collected from white-tailed deer inoculated intracerebrally with mule deer, elk, and white-tailed deer CWD at the termination of a 3-year study. The clinical course, lesions of spongiform encephalopathy, prion accumulation detected by immunohistochemistry and Western blot profiles did not differ between treatment groups. The results suggest that the host-species source of CWD does not affect the outcome of CWD presentation in white-tailed deer after intracerebral inoculation.

Transmission of scrapie and CWD to swine: Tissues from swine inoculated with scrapie and CWD by intracerebral and oral routes were collected at 6 months post-inoculation and were analyzed by histopathology, immunohistochemistry and Western blot. There were no ante mortem clinical signs suggestive of neurologic disease, no histologic lesions suggestive of spongiform encephalopathy, and no indication of prion accumulation by immunohistochemistry and Western blot. Monitoring of a replicate of inoculated littermates will continue until the termination of the study in approximately 5 years.

Retinal Pathology in sheep with scrapie: A study was completed which demonstrated that the retina responds to the accumulation to abnormal prion protein in a way that may affect visual system function. It could be shown that specific cell populations of the retina were affected in scrapie-infected sheep.

Relevance to ARS National Program Action Plan, Animal Health (103): Above described research accomplishments have a critical impact on pathogen detection, epidemiology of disease, host/pathogen interactions, genetic resistance to disease and disease control strategies. In particular, 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. Transmission studies focusing on the mode of TSE transmission and disease development and on the evaluation of changes in the retina of animals infected with TSEs might enable us to design appropriate intervention or ante mortem diagnostic tools that might enable us to devise strategies to control the spread of these fatal diseases.

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

5.Describe the major accomplishments to date and 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. Such studies are of utmost importance to the ARS National Program Action Plan, Animal Health (103), which includes pathogen detection, epidemiology of disease, host/pathogen interactions, genetic resistance to disease, and disease control strategies. Experiments are in progress to determine the transmissibility of different CWDs into white-tailed deer, 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 white-tailed deer results in a higher incidence rate (7/8 animals, 88%) compared to mule deer CWD (3/8 animals, 38%). In addition, 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. Transmission of scrapie and CWD to swine revealed that CWD- or scrapie-infected market-aged swine (6-month-old) showed no ante mortem clinical signs, no histologic lesions suggestive of spongiform encephalopathy, and no indication of prion accumulation by immunohistochemistry and Western blot. 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 and third U.S. BSE isolate (11/2004, diagnosed 06/2005; 03/2006) 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. Biopsy of the gut-associated lymphoid tissue (GALT) present at the rectal-anal junction revealed that 3 of the 4 remaining LL elk contained PrP-Sc positive cells. 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. Upon completion (6 years post infection) of a study with sheep intracerebrally inoculated with mule deer CWD revealed that two of eight inoculated sheep had spongiform encephalopathy with prion accumulation detected by immunohistochemistry and Western blot. The prion disease observed in sheep infected with mule deer CWD was indistinguishable from scrapie. These 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-affected 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?
Two Cooperative Research and Development Agreements (CRADAs) were established with industry.

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).
Invited lecture presented at the NAEBA meeting on "Chronic Wasting Disease Research at the NADC".

Invited lecture presented at the German TSE Platform Meeting titled "Natural and Experimental Prion Diseases of Cattle".

Invited lecture presented at Interagency Working Group (IWG) on Prion Science on "TSE Research at the National Animal Disease Center".

Invited lecture presented at OIE meeting "Prion Diseases of Domestic Livestock" titled "Identification and Characterization of U.S. BSE Cases".

Invited lecture presented at the Third International Rushmore Conference on "Natural and Experimental Prion Diseases of Cattle".

Invited lecture presented at the AAVLD Meeting titled "Identification and Characterization of U.S. BSE cases".

Invited to participate in a workshop held in Saskatoon, Canada, on February 8, 2006, to establish research priorities for PrioNet Canada and the Alberta Prion Research Institute (APRI) with respect to chronic wasting disease (CWD). Presented a seminar at the workshop titled "Transmissible Spongiform Encephalopathy (TSE) studies at the National Animal Disease Center NADC, Agricultural Research Service."