2003 Annual Report 1.What major problem or issue is being resolved and how are you resolving it? The economic losses to the sheep industry due to ovine scrapie can be reduced by a coordinated program of live animal testing, replacement with sheep of lower susceptibility, and reducing transmission within the flock. Scrapie is a transmissible spongiform encephalopathy associated with deposition of an abnormal isoform of a mammalian glycoprotein, the prion protein, in tissues throughout the sheep. The highest level of accumulation is in the brain, although detectable levels are found in lymphoid tissues and placenta/fetal tissues. Detection of prions in peripheral lymphoid tissue can be used to identify and cull infected animals early in infection. Further, the susceptibility of sheep to clinical scrapie and to accumulation of prions is under genetic control. Quantitative determination of the level of protection associated with commonly occurring genotypes could enable producers and regulatory programs to integrate protective genetics with elimination of infected stock to reduce the amount of disease in U.S. flocks. Control of all domestic prion diseases is important in reducing trade barriers for U.S. sheep and germplasm and for assuring the present and future global markets for cattle. Ovine progressive pneumonia virus causes persistent viral infection and development of multi-organ inflammatory disease in some animals. The infection rates in US sheep flocks range from 9 to 49%. A variable and unpredictable percentage of infected animals will progress to clinical disease with high viral titers and increased risk of transmission to flockmates. This project has produced a serologic test for the disease. The current project includes development of a vaccine to reduce infected animals and identification of genes associated with decreased transmission in infected sheep. 2.How serious is the problem? Why does it matter? Sheep scrapie is a member of a group of diseases that includes bovine spongiform encephalopathy (BSE). Sheep scrapie is associated with minor direct losses to the industry. However, loss of international markets for sheep and sheep germplasm and loss of the domestic access to rendering facilities are major economic losses to the industry. Control programs based on epidemiology alone have failed to control the spread of scrapie throughout the U.S. since the disease was introduced in 1947. A control program based on a live animal diagnostic test supplemented by introduction of replacement stock of lower genetic susceptibility is urgently needed. Transmission barriers of these diseases are not understood. Therefore, the presence of any of these diseases within the U.S. represents a continuous threat for emergence in animals not yet found to have been infected in the U.S. The occurrence of BSE in cattle in the U.S. would have devastating impact on the U.S. access to global markets. Animals with clinical signs of ovine progressive pneumonia virus represent the main risk of virus transmission because disease progression is associated with much higher virus loads than is nonprogressive persistent infection. Therefore, the immunogenetic basis of disease pathogenesis is an important consideration in SRLV research. The most significant problem associated with SRLV infection is the inability to determine which animals will progress to severe clinical disease and death. Our objectives are to reduce the clinical disease rates and viremia levels through a vaccination program targeting the type 1 immune responses and to determine the immunogenetics associated with this response. SRLV research will lead to methods for the induction of immunologic control and genetic predictors of animals with a low potential for transmission if infected. 3.How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? Our research concerning the transmissible encephalopathies and small ruminant lentiviruses address the following elements of our National Program in Animal Production, Product Value and Safety. 103 Animal Health 100%. 4.What were the most significant accomplishments this past year? A. Single Most Significant Accomplishment during FY 2003 year: The specific immune cells in lymph nodes involved in PrPSc accumulation remain unknown. Therefore, we analyzed lymph nodes for the presence of PrPSc and macrophage or follicular dendritic cell (FDC) markers using dual immunohistochemistry. Results indicated that lymph node follicular macrophages acquire PrPSc by phagocytosis of CD21+ FDC processes and process full-length PrPSc to N-terminally truncated PrPSc. These data provide the first knowledge in determining the pathogenesis of sheep scrapie in FDC and macrophages. B. Other Significant Accomplishment(s), if any: 1. A new caprine arthritis-encephalitis virus (CAEV) competitive inhibition enzyme-linked immunosorbent assay (cELISA) had previously been developed. We tested 200 goat sera for the presence of CAEV antibodies using cELISA against the standard of comparison, immunoprecipitations (IP) of [S35] methionine-labeled CAEV lysate. The CAEV cELISA validation resulted in 100% sensitivity and 96.4% specificity against the standard of comparison. By annually testing goats for CAEV using cELISA, a CAEV-free herd could be established. 2. There is no vaccine for Babesia bovis. We have collaboration with WSU. We have made significant progress in developing a B. bovis BAC library representing 100X coverage of the babesial genome. This will enhance vaccine design through the identification of parasite genes important in the life cycle, which will be targets for vaccination or gene knockout strategies. 3. Standardized, validated diagnostic tests are needed for eradication of scrapie. In FY2001-FY2003, a cooperative ARS-APHIS-state test validation program resulted in submission of samples from more than 2,000 sheep. These samples are being used to validate the third eyelid live animal test, the postmortem immunohistochemistry test, and to develop novel rapid, high throughput tests suitable for slaughter surveillance. A panel of internationally accepted tests suitable for diagnostic and surveillance purposes will result. C. Significant Accomplishments/Activities that Support Special Target Populations: Control of scrapie and the small ruminant lentiviruses (SRLV) directly benefits small farms that raise sheep and goats for supplementary income. Direct and indirect losses to these producers because of these diseases are significant. Control programs that include identification of infected flocks and animals should reduce the economic consequences of SRLV and prion diseases. D. None 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. A practical live animal test for scrapie and preclinical postmortem tests for scrapie were developed and transferred to the regulatory agencies for use in the US. Monoclonal antibodies useful in assays on routinely formalin fixed tissue from infected sheep, deer, elk, cattle, humans, mink, domestic cats and a wide variety of captive wildlife potentially exposed to prion diseases were developed. Mechanisms for preventing transmission of scrapie through genetic selection of sires were demonstrated. Along with sire testing, genetic testing in bred ewes will ensure less transmission of scrapie from ewe to lamb. We have initiated another research project relating to transmission of OPPV at the Experimental Sheep Station in Dubois, ID. We continue to test a small sheep flock (Future Farmers of America Herd) (<30) for OPPV in Sumner, WA, associated with Sumner High School. 6.What do you expect to accomplish, year by year, over the next 3 years? FY2004: The last phase of scrapie diagnostic test validation for the third eyelid test will be completed. Preclinical test methodology for slaughter sheep will be completed, with identification of the optimal sites in the lymphoreticular system and reproductive tract examined and testing protocols developed for slaughter surveillance. Inexpensive genotyping test formats will be developed and validated. PrPc characterization in goat trophoblast cells will be investigated. The cELISA serologic test for CAEV infection will be commercially available once the larger validation studies for licensing have been completed in 6-12 months. We will determine the immunodominant OPPV protein and correlate serum antibody titers to viral load in sheep naturally infected with OPPV. We will continue to evaluate serum antibody titers and viral load over a 1-3 year period in a natural transmission experiment. We will determine if sheep naturally infected with OPPV maintain low viral loads and Th1 type immune responses associate with specific MHC Class II DRB1 exon 2 haplotypes. FY2005: Placental, uterine, fetal tissues, and blood will be analyzed using cell culture systems to determine the cell types associated with spread of scrapie from the ewe at the time of lambing. The cELISA serologic test for OPPV infection will be commercially available once the CAEV cELISA has been licensed and larger validation studies on sheep sera have been completed. Using an infectious molecular clone, we will determine if there is a correlation of high viral load with a Th2 type immune response to OPPV and low viral load with a Th1 type immune response to OPPV. In addition, specific immune responses will be correlated with specific MHC Class II DRB1 exon 2 haplotypes. FY2006: Tests for prion contamination in soil and water will be developed. A vaccination strategy will be developed to ensure a Th1 type immune response to OPPV. 7.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 monoclonal antibodies to the prion protein and their use in combination as detection reagents for prions have been patented. Both antibodies are commercially available. Only non-exclusive licenses have been offered, to insure the widest possible use of these reagents in diagnostics, industry, and research. The antibodies are in use internationally and collaborative programs to train personnel in Canada, Mexico, and China are in progress. The preclinical test for scrapie has been transferred to the National Veterinary Services Laboratory. APHIS has established a national testing network, through which veterinary and state diagnostic laboratories will apply the technology under contract with APHIS. CAEV cELISA will be licensed and commercially available in the U.S. in 6-12 months. 8.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: This does not replace your peer-reviewed publications listed below). 1. Detection of serum antibodies to small ruminant lentiviruses using a competitive enzyme-linked immunosorbent assay (cELISA): A tool for successful eradication, Conference for Research Workers in Animal Disease, November 2002, St.Louis, MO. 2. CD21 and Follicular Dendritic Cells: A Possible Source of PrPSc in Lymph Node Macrophages of Sc-infected Sheep, Conference for Research Workers in Animal Disease, November 2002, St.Louis, MO. Review Publications Proc. Western Sec. Amer Soc Anim Sci 2003, 54:101 Herrmann, L.M., Cheevers, W.P., Davis, W.C., Knowles, Jr., D.P., O'Rourke, K.I. 2005. CD21 positive follicular dendritic cells: A possible source of PrPSc in lymph node macrophages of scrapie-infected sheep. American Journal of Pathology. 162(4):1075-1081. Herrmann, L.M., Cheevers, W.P., McGuire, T.C., Adams, D.S., Hutton, M.M., Gavin W.G., Knowles D.P. Competitive-inhibition enzyme-linked immunosorbent assay for detection of serum antibodies to caprine arthritis-encephalitis virus: Diagnostic tool for successful eradication. Clinical and Diagnostic Laboratory Immunology. 2003. v. 10(2). p. 267-271. HERRMANN, L.M., CHEEVERS, W.P., MARSHALL, K.L., MC GUIRE, T.C., HUTTON, M.M., LEWIS, G.S., KNOWLES JR, D.P. DETECTION OF SERUM ANTIBODIES TO OVINE PROGRESSIVE PNEUMONIA VIRUS (OPPV) IN SHEEP USING A CAPRINE ARTHRITIS-ENCEPHALITIS VIRUS (CAEV) COMPETITIVE-INHIBITION ENZYME-LINKED IMMUNOSORBENT ASSAY (CELISA). CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY. 2003. v. 10 p. 862-865. Paramithiotis, E., Pinard, M., Lawton, T., Laboissiere, S., Leathers, S., Zou, W., Estey, L., Lamontagne, J., Lehto, M.T., Kondejewski, L.H., Francoeur, G.P., Papadopoulos, M., Haghighat, A., Spatz, S.J., Head, M., Will, R., Ironside, J., Orourke, K.I., Tonelli, Q., Ledebur, H.C., Chakrabartty, A., Cashman, N.R. 2003. A prion protein epitope selective for the pathologically misfolded conformation. Nature Medicine. 9(7):893-899. Valdez, R.A., Rock, M.J., Anderson, A.K., O'Rourke, K.I. 2003. Immunohistochemical detection and distribution of prion protein in a goat with natural scrapie. Journal of Veterinary Diagnostic Investigation. 15(2):157-162. Virology 306 2003 116-125 O'Rourke, K.I., Duncan, J.V., Logan, J.R., Anderson, A.K., Norden, D.K., Williams, E.S., Combs, B.A., Stobart, R.H., Moss, G.E., Sutton, D.L. 2002. Active surveillance for scrapie utilizing third eyelid biopsy and genetic susceptibility testing in flocks of sheep in Wyoming. Clinical and Diagnostic Laboratory Immunology. 9(5):966-971. Van Everbroeck, B., O'Rourke, K.I., Cras, P. 1999. Immunoreactivity of the monoclonal antibody F89/160.1.5 for the human prion protein. Eur. J. Histochem. 43(4):335-338.