ACCESSION NO: 0149364 SUBFILE: CRIS
PROJ NO: 3625-32000-025-00D AGENCY: ARS 3625
PROJ TYPE: USDA INHOUSE PROJ STATUS: NEW
START: 01 OCT 1995 TERM: 30 SEP 2000 FY: 1999
INVESTIGATOR: MENGELING W L; HAMIR A N; SCHMERR M; VACANT; MILLER J M
NATIONAL ANIMAL DISEASE CTR
AMES, IOWA 50010
OBJECTIVES: Determine if U.S. agents that cause spongiform encephalopathy in sheep and mule deer will cause a disease in cattle resembling bovine spongiform encephalopathy (BSE) in the United Kingdom; to determine if the deer agent will cause scrapie i n sheep. Develop diagnostic methods that can detect transmissible spongiform encephalopathies in living and dead animals. Determine the structure of carbohydrates attached to surface proteins and relationship to strain of bovine leukemia & opp viruses.
APPROACH: Cattle that were previously fed the scrapie agent as raw or rendered product will be observed for signs, lesions and prion protein that are characteristic of BSE. To evaluate the effect of the agent of deer agent on cattle and sheep, calves a nd lambs will be inoculated intracerebrally and observed for no less than four years for signs of disease, and tissues evaluated histologically, histochemically and chemically for lesions and the agent. Capillary electrophoresis will be used to improve th e diagnosis of the transmissible spongiform encephalopathies by enhancing the sensitivity many times through the use of labeled synthetic peptides in a competitive binding assay. Structure of surface carbohydrates of BLV viruses will be determined using s pecial capillary electrophoresis methods. Ames, IA, NADC, Bldg. 2, Modules B7/B8 & Barns 110, 119, 127 & 136; BL2: required for pathogens. No biotechnology research in this CRIS; no IBC approval is needed.
PROGRESS: 1999/01 TO 1999/09
1. What major problem or issue is being resolved and how are you resolving it? Transmissible spongiform encephalopathies (TSEs) or prion diseases are a group of similar diseases that occur infrequently in some domestic and game animals in the United State s. The TSEs that have occurred in the United States are scrapie of sheep and goats, chronic wasting disease (CWD) of deer and elk, and transmissible mink encephalopathy (TME) of farmed mink. They were considered of minor significance until the outbreak of bovine spongiform encephalopathy (BSE) in the United Kingdom resulted in great economic loss to that country as well as public concern for human health after it was shown that this disease probably is transmitted to humans. BSE does not occur in the Unit ed States. The risk of it occurring is considered low but it could be imported or transferred from another form of TSE such as scrapie of sheep or CWD of deer and elk. Should BSE occur in the United States, the economic loss to the cattle industry and the loss from the human health hazard would be large. To compound this problem there is no rapid and accurate method to diagnose TSE in the living animal, an extremely long incubation period causing difficulty in evaluating host range, pathogenesis and contr ol of the diseases and poor characterization of the abnormal prion protein, the putative cause of the diseases. An improved animal model with a short incubation period would assist greatly in accomplishing these tasks. This serious problem has been approa ched by using analytical methods to improve diagnosis, intracerebral inoculation of different species to determine host range of TSEs in the United States, oral inoculations of sheep to evaluate pathogenesis, and inoculation of raccoons to evaluate them a s an experimental model of the TSEs. The diagnostic tests that are presently used are postmortem tests in animals that have progressed to clinical disease. Analytical procedures are being used to develop a diagnostic test based on competition immunoassay using fluorescein-labeled peptides from prion protein and capillary electrophoresis to detect abnormal prion protein in affected animal tissues and fluids. Five species of animals are under examination to determine the host range of the TSEs. Transmission and pathogenesis studies are very slow and expensive but are the only way to learn if different species of animals are susceptible to various TSEs and characteristics of the disease. 2. How serious is the problem? Why does it matter? As stated above, the incidence of TSEs in domestic and game animals in the United States is low. Scrapie in sheep is the most common, affecting some 50 flocks annually. The importance of these diseases lies in their potential to cross species and cause a devastating disease in other species including humans such as has recently occurred in cattle and humans in Europe, mainly in Great Britain. This epizootic has cost the lives of over 180,000 cattle from disease and approximately 1.5 million cattle have been euthanized as a c ontrol measure. The U.K. National Audit Office on BSE estimates that the total cost for the disease will exceed 5.5 billion dollars. Should BSE occur in the U.S., loss of the beef export market alone would cost about 2.5 billion dollars annually. Because of the potential of transmission of this fatal disease to humans, the potential loss is enormous. 3. How does it relate to the National Program(s) and National Component(s) to which it has been assigned? National Program 103, Animal Health, 100% 4. What w ere the most significant accomplishments this past year? During the past year, the major accomplishments were termination of a project that demonstrated that cattle are not susceptible to scrapie by the oral route and improvements in the capillary electro phoresis immunoassay for detecting abnormal prion protein in the blood of animals affected with a TSE. Capillary electrophoresis was also used to determine characteristics of the prion protein from sheep that may lead to a method of differentiating strain s or types of the abnormal form of prion protein. A new method to extract the prion protein has been developed and in now used to prepare blood samples. Sheep were inoculated intracerebrally with CWD and raccoons were inoculated with CWD and with scrapie to determine their susceptibility to the respective agents and to evaluate any disease induced in raccoons as a model for the TSEs. 5. Describe the major accomplishments over the life of the project including their predicted or actual impact. Over the pas t years, a test has been under development for detecting abnormal prion protein in blood of living animals infected with TSE. Detection of the protein in the living animal is very important because it will provide a means to identify and destroy affected animals for eradication of the disease and improve safety for human products. Tests that are presently used are based on postmortem examination of animals that have progressed to clinical disease. Sheep scrapie has been demonstrated to transmit to cattle by the intracerebral route but not by the oral route after eight years of incubation. 6. What do you expect to accomplish, year by year, over the next 3 years? For the next three years, experiments will continue to improve protocols and methods for extrac ting and detecting the abnormal prion protein, to correlate analytical data with biological data from TSE infected animals and to determine the time at which abnormal prion protein appears in the blood of TSE exposed animals. Over the next three to five y ears, animals (sheep, cattle, elk and raccoons) that were inoculated with scrapie or CWD in the last two years, will be monitored for disease and the tissues of any diseased animals evaluated and the results reported. It is anticipated that a new scientis t will join in a pathogenesis studies of scrapie by adding sophisticated technical experience in evaluation of tissues for prion protein and by starting studies with transgenic animals. 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 & durability of the technology product? Provisional Patent Application "Method and Kit for Extracting Prion Protein" 8. List your most important non-peer reviewed publications and presentations to non-scientific organizations, and articles written about your work(NOTE: this does not replace your peer reviewed publications which are listed below). Detecting the Presence of Abnormal Prion Protein from Blood: Clearing a Major Analytical Hurdle PACE Setter Vol 3, Issue 2 June 1999
PUBLICATIONS: 1999/01 TO 1999/09
1. DUBEY, J.P., MATTSON, D.E. SPEER, C.A., BAKER, R.J., MULROONEY, D., TORNQUIST, S.J., HAMIR, A.N. and GERROS, T.C. 1999. Characterization of a ... infected case from Oregon. J. Eukaryotic Microbiol. 46:500-506.
2. HAMIR, A.N. 1999. Neuronal vacuolation of raccoons. 1999. IXth Symp. World Assoc. Vet. Lab. Diagn. Abst. p. 116.
3. HAMIR, A.N. 1999. Pulmonary adiaspiromycosis in raccoons (Procyon lotor) from Oregon. J. Vet. Diagn. Invest. 11:545-567.
4. HAMIR, A.N., DUBEY, J.P. and RUPPRECHT, C.E. 1999. Prevalence of Sarcocystis kirkpatricki ... nervous system and striated muscles of raccoons from eastern United States. J. Parasitol. 85(4):748-750.
5. HAMIR, A.N. and FISCHER, K.A. 1999. Neuronal vacuolation in raccoons from Oregon. J. Vet. Diagn. Invest. 11(4):303-307.
6. HAMIR, A.N., SNYDER, D.E., LICHENFELS, J.R. 1999. Cerebral larva migrans in a raccoon (Procyon lotor). Vet. Pathol. 36: Accepted March 15, 1999.
7. SCHMERR, M.J.,ALPERT, A. and JENNY, A. L. 1999. Purification of the prion protein from sheep brain using hydrophilic interaction chromatography. J. Chromatog. B. Accepted March 3, 1999.
8. SCHMERR, M.J. and JENNY, A. L. 1999. The use of capillary electrophoresis to detect the binding of prion peptides ... isolated from scrapie infected sheep brain. J. Biochem. Biophys. Methods. Accepted January 22, 1999.
9. SCHMERR, M.J., JENNY, A.L., BULGIN, M.S., MILLER, J.M., HAMIR, A.N., CUTLIP, R.C. and GOODWIN, K.R. 1999. The use of capillary electrophoresis and fluorescent ... encephalopathy. J. Chromatog. A 853:207-214 (1999).