DOMESTIC AND INTERNATIONAL EFFECTS OF EXOTIC ANIMAL DISEASES AND THEIR ERADICATION

ACCESSION NO: 0182811 SUBFILE: CRIS
PROJ NO: CA-D*-ARE-6585-H AGENCY: CSREES CALB
PROJ TYPE: HATCH PROJ STATUS: NEW
START: 01 OCT 1999 TERM: 30 SEP 2004 FY: 1999

INVESTIGATOR: Jarvis, L. S.

PERFORMING INSTITUTION:
AGRICULTURAL AND RESOURCE ECONOMICS
UNIV OF CALIFORNIA
DAVIS, CALIFORNIA 95616

OBJECTIVES: 1) Examine the potential economic effects of an outbreak of exotic animal diseases like Foot and Mouth Disease (FMD) and Bovine Spongiform Encelphalopathy (BSE) on the US (California) economy and on international markets if an outbreak of s uch a disease were to occur in the United States, and to analyze how changes in US policy can mitigate the expected economic damage from a potential outbreak. 2) Examine the expected domestic and international effects of eradicating exotic animal diseases like FMD and BSE in countries where they exist of have recently existed.

APPROACH: 1) Build and/or extend currently existing models to analyze the potential effects of exotic animal diseases on US and especially California livestock production, consumption and international trade, focusing on the economic welfare effect of changes in important domestic policies that will affect the magnitude and duration of such effects. 2) Carry out industry studies and build and/or extend currently existing models to analyze the potential domestic and international effects from eliminatin g FMD (or BSE) where it exists. For example, analyze the expected effect on domestic beef prices in Argentina from gaining access to the FMD-free international market, the effect that higher prices and increased demand for high quality meat cuts will have on the structure on Argentine beef production, and the effect on international markets from expanded Argentine and shift in Argentine production from the FMD-endemic to the FMD-free market.

PROGRESS: 2000/01 TO 2000/12
Two papers were written that presented estimates of the costs of outbreaks in California of 1) Bovine spongiform encephalopathy (BSE), and 2) Food and Mouth Disease (FMD). An outbreak of each disease would impose huge costs. Each disease would impact dome stic demand and trade, raise long run supply costs, and impose costs to control and/or eradicate the diseases. Strategies to address the threat of BSE for the California should focus on preventing entry, improving surveillance to identify BSE early if it enters, and preventing the spread of BSE in the US herd and human population. Policy issues relate to reviewing compliance of feed processing procedures and developing an individual animal identification system. Strategies to address the threat of FMD in California should focus on improving surveillance to identify FMD early if an outbreak occurs and to impose an effective quarantine and a stamping out or vaccination program.

IMPACT: 2000/01 TO 2000/12
BSE spread from the United Kingdom into Western Europe despite slaughter of infected animals, control of live animal movements, and mandated restrictions on animal feeding practices. Changes are needed in US animal production practices to reduce the proba bility that BSE spreads to the US and becomes diffused throughout the US cattle herd.

PUBLICATIONS: 2000/01 TO 2000/12
1. Sumner, D. A. 1999. [Remarks on economic and public policy aspects of exotic pest control]. Exotic pests and diseases : biology, economics, public policy / Raymond H. Coppock and Marcia Kreith, editors. Davis : University of California, Agricultural Is sues Center, pp. 7-8, 22-30, 31.
2. Jarvis, L. S. 1999. [Remarks on foot-and-mouth disease]. Exotic pests and diseases : biology, economics, public policy / Raymond H. Coppock and Marcia Kreith, editors. Davis : University of California, Agricultural Issues Center, pp. 167-172, 181-182.< BR> 3. Jarvis, L. S. 1999. [Remarks on bovine spongiform encephalopathy]. Exotic pests and diseases : biology, economics, public policy / Raymond H. Coppock and Marcia Kreith, editors. Davis : University of California, Agricultural Issues Center, pp. 206-209, 212, 213.
4. Jarvis, L. S. and Bervejillo, J. E. 2000. Re-emergence of South American beef exporters : new competition for U.S. producers? Agricultural and Resource Economics Update 4(1):1-4.

PROJ CONTACT:

Name: JARVIS, L. S.
Phone: 530-752-7221
Fax: 530-752-5614
Email: jarvis@primal.ucdavis.edu

STUDY OF TRANSMISSION AND EARLY DETECTION OF SCRAPIE

ACCESSION NO: 0178039 SUBFILE: CRIS
PROJ NO: IDA01145 AGENCY: CSREES IDA
PROJ TYPE: HATCH PROJ STATUS: NEW
START: 01 JUL 1998 TERM: 30 JUN 2003 FY: 2000

INVESTIGATOR: Bulgin, M. S.

PERFORMING INSTITUTION:
ANIMAL & VETERINARY SCIENCE
UNIV OF IDAHO
MOSCOW, IDAHO 83843

OBJECTIVES: 1) Obtain and maintain a genotyped scrapie research flock by obtaining the high-risk scrapie-exposed animals designated by State and Federal Animal Health Regulatory Personnel in Idaho, 2) Aid in the validation of a practical ante-mortem te st by providing appropriate researchers with samples of blood, spinal fluid, lymph tissue at appropriate times on appropriate animals for immonohistochemical, western blot examination, capillary electrophoresis immunoassay or any other newly developed tes t for the identification of the scrapie agent, 3) Determine whether scrapie is transmitted to adults, 4) Follow these animals for no less than 8 years or until death. Develop futher protocols as new information is made available from current research.

APPROACH: Sheep identified as high risk for infection with Scrapie will be followed for a minimum of 5 years. Known Scrapie negative animals will be used as both nose-to-nose contact controls and within flock control and followed also. Monitoring will be done initially with state-of-the-present art of immunohistochemistry and western blot tests on biopsy samples of various lymphoid tissues such as mandibular and prescapular lymph node. As improved or different tests are made available, these will also be incoorporated into testing this flock. Sheep with positive test will be followed until death or 5 years as an attempt to correlate test results with disease.

PROGRESS: 2000/01 TO 2000/12
The Scrapie research flock year started with 91 high exposure sheep, 20 low exposures and 14 control sheep. Twelve animals were diagnosed scrapie positive giving a 13% incidence. Eleven were bled out and blood was sent to Baltimore Research and Education Foundation for prion isolation. One animal died having lambs and was also found to be positive. All were of the QQ genotype. Several subclinical, known positive ewes and their lambs were bled semimontly for capillary electrophoresis testing and the lambs showed a positive test at approximately 60 days.

IMPACT: 2000/01 TO 2000/12
No QR genotype animals in the high exposure group have shown clinical Scrapie lending credence to the theory that this genotype is resistant to clinical disease.

PUBLICATIONS: 2000/01 TO 2000/12
No publications reported this period

PROJ CONTACT:

Name: Bulgin, M. S.
Phone: 208-454-8657
Fax: 208-454-8659
Email: cvtc@uidaho.edu

BIOLOGY AND CONTROL OF EMERGING DISEASES SHARED BY LIVESTOCK AND WILDLIFE

ACCESSION NO: 0182439 SUBFILE: CRIS
PROJ NO: IDA01179 AGENCY: CSREES IDA
PROJ TYPE: HATCH PROJ STATUS: NEW
START: 01 JUL 1999 TERM: 30 JUN 2004 FY: 2000

INVESTIGATOR: Zaugg, J. L.; Bulgin, M. S.; Anderson, B. C.; England, J. J.

PERFORMING INSTITUTION:
ANIMAL & VETERINARY SCIENCE
UNIV OF IDAHO
MOSCOW, IDAHO 83843

OBJECTIVES: 1. Determine if mule deer are experimentally susceptible to Scrapie infection. 2. Evaluate the utility of the palpebral site to test for tuberculosis in cervids. 3. Determine of Brucella abortus RB51 vaccine, delivered orally, will stimulat e an immune response in bison and elk.

APPROACH: 1. Intracerebrally inoculate mule deer fawns and domestic lambs with Scrapie-infected brain tissue. Montior for illness. Screen palpebral lymph nodes for prions at 18 and 24 months, and full necropsy at 36 months post inoculation. 2. Inoculat e elk and mule deer with Mycobacterium avium organisms. Compare hypersensitive reactions to tuberculin intradermal inoculation made in the upper eyelids, to those standard test sites in the cervical region. 3. Elk will be fed Brucella RB51 vaccine as a to p dressing in four, five-day series. Blood samples will be taken and evaluations conducted to determine if, and when an immune response is achieved.

NON-TECHNICAL SUMMARY: Diseases (brucellosis, tuberculosis and scrapie) of profound importance to the livestock industry have emerged as having actual, or potential interaction with wildlife. The purpose of this study is to investigate key aspects of t he biology and control of bovine brucellosis, tuberculosis and scrapie emerging diseases shared by livestock and wildlife.

PROGRESS: 2000/01 TO 2000/12
Studies will be initiated to assess if mule deer are susceptible to scrapie. Special deer holding facilities have been constructed. Deer fawns were unavailable last spring. Enhanced efforts will be made to ensure adequate deer available to start project i n the spring of 2001. Frozen scrapie-infected sheep brain tissues are on hand for inoculation into deer and sheep hosts in June 2001.

IMPACT: 2000/01 TO 2000/12
The study will determine if mule deer are susceptible to the disease, scrapie.

PUBLICATIONS: 2000/01 TO 2000/12
No publications reported this period

PROJ CONTACT:

Name: Zaugg, J. L.
Phone: 208-454-8657
Fax: 208-454-8659
Email: jzaugg@uidaho.edu

TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES OF ANIMALS AND RETROVIRAL DISEASES

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

PERFORMING INSTITUTION:
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).

USE OF NEW EXTRACTION PROCEDURE FOR PRION PROTEIN TO DEVELOP A DIAGNOSTIC KIT FOR TSE

ACCESSION NO: 0400649 SUBFILE: CRIS
PROJ NO: 3625-32000-025-01T AGENCY: ARS 3625
PROJ TYPE: USDA INHOUSE PROJ STATUS: NEW
START: 30 JAN 1997 TERM: 30 JAN 2000

INVESTIGATOR: SCHMERR M; CUTLIP R C

PERFORMING INSTITUTION:
NATIONAL ANIMAL DISEASE CTR
AMES, IOWA 50010

OBJECTIVES: Development of an extraction procedure for prion protein that is based on a high performance liquid chromatography method. Development of a diagnostic kit for Transmissible spongiform encephalopathies based on the titration procedure and de tection by a micro analytical method.

APPROACH: Organic solvents will be used to extract prion protein and a high performance liquid chromatographic column that is selective for its binding of hydrophobic compounds will be used to purify and to concentrate the prion protein. Conventional t ests such as Western blot will be used to detect the prion protein. Capillary electrophoresis using fluorescent labelled peptides will be used as a sensitive test to detect minute amounts of the prion protein. CRADA funded by Fort Dodge Laboratories, FY-9 7, $160,816. Agreement Number 58-3K95-7-524; Award date 01/23/97.

PROGRESS: 1999/01 TO 1999/09
1. What major problem or issue is being resolved and how are you resolving it? Transmissible spongiform encephalopathies of present in domestic animals and game animals has caused considerable concern both economic and for human health. The outbreak of bo vine spongiform encephalopathy in the United Kingdom resulted in great economic loss to the farmers of that country as well as public concern for human health after it was shown that this disease can be transmitted to humans. The putative agent that cause s this disease an abnormal protein is an insoluble protein that forms large aggregates. It is present in a rather low concentration in naturally infected animals. Development of a method that would efficiently extract this protein and increase the concent ration would help in the development of a test for this protein. This serious problem has been approached by using analytical methods. Different organic solvents were used to extract this protein. A high performance liquid chromatography method was develo ped using a specialized column matrix to purify the protein. 2. How serious is the problem? Why does it matter? The actual incidence of scrapie in sheep and in game animals ranges from 1-10%. The tests that are presently used are postmortem tests and diag nosis animals that have progressed to clinical disease. This may not help in the control of the disease if animals are infectious in early stages. A pre-clinical test using n accessible tissue or fluid would address this problem. The ability to test blood in animals and humans for the presence of abnormal prion protein would make it possible to eliminate this disease from the animal population and to improve safety for human products. 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 were the most significant accomplishments this past year? The ability to extract and concentrate the small amount of abnormal prion protein abnormal prion protein in the b lood of animals infected with a TSE has aided in the development of an assay. Prior to this extraction method, the only way to for abnormal prion protein in blood was by using rodent adapted strains and injecting the material into the brains of these anim als. 5. Describe the major accomplishments over the life of the project including their predicted or actual impact. Over the past few years, a new extraction method was developed that made it possible to develop a test for the abnormal prion protein in th e blood of animals. 6. What do you expect to accomplish, year by year, over the next 3 years? Experiments to improve protocols and methods that are used for extracting the abnormal prion protein. Experiments to study the time at which abnormal prion prote in can be in the blood of TSE exposed 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 const raints if known, to the adoption & durability of the technology product? The CRADA partners for the CRADA No. 58-3K95-7-524 Extraction of the Prion Protein and Its Use in a Diagnostic Test have been trained in the techniques using this technology. Thi s included numerous discussions and has included safety training for protocols using TSE infected material. Provisional Patent granted February 2-2-99 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).

PUBLICATIONS: 1999/01 TO 1999/09
1. SCHMERR, M.J. and JENNY, A.L. 1998. A diagnostic test for scrapie infected sheep ... electrophoresis immunoassay with fluorescent labeled peptides. Electrophoresis 19:409-414.
2. SCHMERR, M.J. 1998. Diagnostic test kit for transmissible spongiform encephalopathies using a new extraction method for the abnormal prion protein. U.S. Patent Application S/N 0046-98.

ADVANCED DIAGNOSTIC METHODS FOR INFECTIOUS DISEASE

ACCESSION NO: 0149998 SUBFILE: CRIS
PROJ NO: 3625-32000-027-00D AGENCY: ARS 3625
PROJ TYPE: USDA INHOUSE PROJ STATUS: NEW
START: 01 OCT 1995 TERM: 30 SEP 2000 FY: 1999

INVESTIGATOR: MILLER J M; VACANT; VACANT

PERFORMING INSTITUTION:
NATIONAL ANIMAL DISEASE CTR
AMES, IOWA 50010

OBJECTIVES: Develop practical methods for rapid detection and speciation of myco- bacteria in formalin-fixed tissues. Evaluate the usefulness of immuno- histochemistry for diagnosis of transmissible spongiform encephalopathies in various animal species . Develop immunohistochemical methods for detection of bovine herpesvirus-4 and bovine adenoviruses in formalin- fixed tissues.

APPROACH: Two approaches will be used to develop methods for detection and speciation of mycobacteria in formalin-fixed tissues. The first will focus on development of an immunohistochemical technique; the second will utilize the polymerase chain react ion technique to amplify specific bacterial genomic sequences. Immunohistochemical diagnosis of transmissible spongiform encephalopathies will be evaluated using affected brains from several different animal species. A variety of monoclonal and polyclonal antisera to both normal and abnormal isoforms of the prion protein will be tested. If necessary, new antisera will be developed using monoclonal or baculovirus production technologies. These methods also will be used to develop antibodies for immunohisto chemical detection of bovine adenovirus and herpesvirus 4 infections in formalin-fixed tissues. NADC, Ames, IA. Bldg. 2, B-18: BL-2; 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? Some animal diseases have been determined to be of such significance that a national effort has been initiated to establish programs for their control or eradication. One of th e most important requirements for successful application of these programs is availability of accurate, economical, and practical diagnostic tests. We identify situations in which new or emerging technologies might be used to improve the diagnostic capabi lities of federal and state animal health agencies. The most appropriate methodologies are selected for investigation and when suitable procedures have been developed the new tests are evaluated to determine their applicability to specific problems presen ted by the disease under consideration. 2. How serious is the problem? Why does it matter? Animal diseases targeted for control or eradication are usually considered potential human health risks. The diseases currently being addressed in this project are tuberculosis and transmissible spongiform encephalopathie (TSEs). Besides their relevance to public health concerns, another important reason to eradicate these diseases relates to international trade issues. Our ability to export animals or animal produc ts can be seriously impeded by the continued presence of these diseases in the U.S. 3. How does it relate to the National Program(s) and National Component(s) to which it has been assigned? National Program 103, Animal Diseases, (100%) Current research ob jectives are focused on development of more rapid tests for diagnosis of tuberculosis in domestic ruminants and wildlife, and for more specific and sensitive tests to diagnose TSEs in all species. 4. What were the most significant accomplishments this pas t year? Sheep from several scrapie-infected flocks were examined to determine if their tissues contained prion protein (PrP), which is a diagnostic marker for scrapie infection. The animals had been previously tested with a newly described live-animal tes t that involves microscopic examination of lymphoid tissue taken from the third eyelid. Only eyelid-negative animals were examined immediately because eyelid-positive animals are being held to observe whether they will eventually develop clinical scrapie (several such cases have occurred already). This work is on-going but to date PrP has been found in brains from only 2 eyelid-negative sheep, suggesting the eyelid test has high specificity. The overall project is a collaborative project under the directi on of Dr. Katherine O'Rourke at the ARS laboratory in Pullman, WA. 5. Describe the major accomplishments over the life of the project including their predicted or actual impact. A polymerase chain reaction (PCR) technique was developed for identification of Mycobacterium bovis, the cause of bovine tuberculosis, in formalin-fixed tissues. The method was shown to successfully detect the organism in 93% of culture-positive tissues and it is now being used by USDA for the rapid diagnosis of tuberculosis in ti ssues submitted as a part of the eradication program for this disease. Because other mycobacteria can cause lesions similar to those produced by M. bovis, the PCR method was later adapted to detect 2 subspecies of M. avium, i.e., paratuberculosis and aviu m. Sensitivity of the method in culture-positive tissues was 100% for paratuberculosis but only 57% for avium. Even though the effectiveness of PCR for diagnosis of these non-tuberculous diseases is less efficient than for tuberculosis, application of the technique has been added to the USDA testing protocol because identification of these cases allows regulatory personnel to avoid necessary epidemiologic investigations, herd quarantines, etc. Although the primary purpose of developing a PCR test for myco bacteria was to provide a more rapid diagnosis (2-3 days as compared to the 6-8 weeks required for bacterial culture), a recent study has shown that the test also can sometimes detect mycobacteria in culture-negative tissues. A panel of 102 such tissues t hat had demonstrable organisms by light microscopy were examined by the PCR test and in 63% of the cases a specific mycobacterium was identified (44 M. bovis, 20 M. avium). A second focus of work in this project is improvement and evaluation of an immunoh istochemical (IHC) test for diagnosis of TSEs in various animal species. The primary emphasis has been on adaptation of the method for use in diagnosing scrapie in sheep and chronic wasting disease (CWD) in deer and elk. The IHC technique was found to be much more sensitive and reliable than the traditional method, which required histopathologic examination of brain, and the procedure is now the official test used by USDA for diagnosis of TSEs. The technique has also been used for a variety of ARS scrapie and CWD projects at NADC and Pullman, Washington, and for collaborative research projects with scientists at other institutions. These projects have included evaluation of reagents, diagnostic evaluation of animals on transmission experiments, and studie s to determine prevalence of these diseases. 6. What do you expect to accomplish, year by year, over the next 3 years? We have recently begun investigations to explore the possibility of using a new technology, immuno-PCR, for development of serological t ests that can detect exposure to an infectious agent. This technique uses a format like that of a commonly used diagnostic method, enzyme-linked immunoassay (ELISA), but the detection system is based on a nucleic acid, rather than an enzyme, tag which is then amplified by PCR for enhanced sensitivity. To evaluate the potential usefulness of this technology, we intend to compare the immuno-PCR test to previously developed ELISA procedures for a bovine herpesvirus (infectious bovine rhinotracheitis virus) a nd a retrovirus (bovine leukemia virus). Should these model systems provide good results we plan to apply the technique to other diseases that are characterized by an unusually weak, or delayed antibody response to the etiological agent. In these situatio ns the availability of more sensitive serological tests might allow development of more effective control or eradication programs. The diseases which will be of primary interest to us initially are tuberculosis and paratuberculosis. 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 p roduct? Because the PCR test for mycobacterial identification and the IHC test for TSE diagnosis were designed to meet the needs of animal disease control or eradication programs, the primary user will be the Pathobiology Laboratory, National Veterinary S ervices Laboratories (NVSL), Animal and Plant Health Inspection Service, USDA. We have already transferred the technology for IHC diagnosis of TSEs to that laboratory. A few state diagnostic laboratories have also expressed interest in using the procedure but because test results have regulatory ramifications it will be necessary for NVSL to develop training and certification standards before the technology can be made available to them. With regard to the PCR test for mycobacterial identification, we hav e recently been successful in transferring that technology to NVSL. The Pathobiology Laboratory is currently evaluating its proficiency for the technique by testing a set of 106 unknowns that we prepared for them. Because tuberculosis is almost eradicated from the U.S., it is unlikely that any state laboratories will be interested in trying to provide this service. The primary application will be at the federal laboratory for examination of tissues submitted through the tuberculsosis surveillance program in slaughter animals. After eradication of the disease has been declared, the procedure will be part of the monitoring program necessary to assure that the disease does not recur in the U.S. 8. List your most important non-peer reviewed publications and p resentations to non-scientific organizations, and articles written about your work(NOTE: this does not replace your peer reviewed publications which are listed below). Prion diseases - new species, new concerns. Presented at the Conference on Emerging Dis eases, University of Georgia College of Veterinary Medicine and Georgia Center for Continuing Education, Athens, GA, August, 1999.

PUBLICATIONS: 1999/01 TO 1999/09
1. MILLER, J.M., JENNY, A.L. and ELLINGSON, J.L. 1999. Polymerase chain reaction identification of Mycobacterium avium in formalin-fixed, paraffin-embedded animal tissues. J. Vet. Diagn. Invest. 11:434-438.
2. ELLINGSON, J.L.E., STABEL, J.R., BISHAI, W.R., FROTHINGHAM, R. and MILLER, J.M. 1999. Evaluation of the accuracy and ... Mycobacterium avium subspecies. Molec. Cell. Probes 13: Accepted February 2, 1999.
3. MILLER, J.M. 1999. Prion diseases - new species, new concerns. Conference on Emerging Diseases. Abstr. p. 4.
4. PETERS, J., JENNY, A.L., PETERSON, T.L. and MILLER, J.M. 1999. Immunohistochemical diagnosis of chronic wasting disease in ... elk from a captive herd. 48th Ann. Wildlife Dis. Assoc. Conf. Abstr. p. 50.
5. WILLIAMS, E.S., MILLER, M.W., SPRAKER, T.R., SIGURDSON, C., JENNY, A. and MILLER, J.M. 1999. Diagnosis of chronic wasting disease of deer and elk. 48th Ann. Wildlife Dis. Assoc. Conf. Abstr p. 68.

SPECIFIC EFFECT OF PRP SCRAPIE ON TYPES OF BRAIN CELLS

ACCESSION NO: 0177178 SUBFILE: CRIS
PROJ NO: IOWV-109-05-9856 AGENCY: CSVM IOWV
PROJ TYPE: STATE PROJ STATUS: TERMINATED
START: 01 JUL 1997 TERM: 30 JUN 2000 FY: 1999

INVESTIGATOR: Jeftinija, S.

PERFORMING INSTITUTION:
VETERINARY MEDICINE
IOWA STATE UNIVERSITY
S. AND 16TH ELWOOD
AMES, IOWA 50011

OBJECTIVES: Prion diseases are transmissible neurodegenerative disorders that cause typical reactive astrocytosis. The conversion of normal prion protein (PrPc) to PrPsc (scrapie) and the accumulation of PrPsc in the astrocytes precede neuronal death i n the course of the disease. These observations suggest a role for astrocytes in pathogenesis of prion diseases. However, it is still unknown whether scrapie pathology comes about by neurotoxicity of PrPsc, acute depletion of PrPc, or some other mechanism . We propose to focus on the astrocyte's role in the neurotoxicity of PrPsc. Recently, we have demonstrated glial release of glutamate. In this study we propose to determine the mechanism of PrPsc neurotoxicity.

APPROACH: Using astrocyte or neuron enriched cortical cultures of normal animals (rat) in combination with ratiometric imaging with Fura-2 to detect intracellular calcium changes, we will determine whether PrPsc has an early effect on intracellular cal cium levels in cultured brain cells. Fragments of PrPsc consisting of several amino acids will be tested on same cultures (e.g. PrP106-126). Using HPLC we will monitor resting and PrPsc or peptide-induced changes in extracellular glutamate concentration a nd corresponding intracellular calcium changes. Astrocytic cultures will be exposed for determined period of time to FITC labeled PrP106-126 or PrPsc to determine binding sites of PrPsc and intensity of fluorescence will be determined. Following infection period with PrPsc, cultures will be processed for immunocytochemistry using anti- PrPsc antibodies to demonstrate the presence of PrPsc on the cell surface. Total antibody binding will be done on parallel cultures that will be permeabilized. Positive fin dings in latter experiment will support hypothesis that PrPsc is being internalized by infected cells.

PROGRESS: 1997/07 TO 2000/06
The fatal sheep disease scrapie is a transmissible degenerative encephalopathy resulting from infection by prions. Prions are infectious pathogens that differ from bacteria, fungi, parasites, virions, and viruses, both with respect to their structure and with respect to the diseases that they cause. A post-translational process converts the cellular prion protein (PrPc) into an abnormal isoform (PrPsc). The conversion of PrPc to PrPsc and the accumulation of PrPsc in the glial cells precede neuronal death in the course of the disease. These observations suggest a role for astrocytes in pathogenesis of prion diseases. However, it is still unknown whether scrapie pathology comes about by neurotoxicity of PrPsc, acute depletion of PrPc, or some other mechani sm. We focused our study on the astrocyte's role in the neurotoxicity of PrPsc. During the past decade, interest in the neurobiology of glutamate has greatly increased stemming from the recognition that glutamate is excitotoxic. We have demonstrated glial release of glutamate induced by a fragment of PrPsc consisting of amino acids 106-126 (PrP106-126). We studied the effect of PrPsc by measuring factors associated with excitotoxicity, calcium increase in glial cells and increase in glutamate release. The re are two major outcomes of this study: We will be able to determine the role of glia in the mechanism of PrPsc neurotoxicity; and, it may become possible to identify a site of action for drugs to be used in the treatment of prion diseases.

IMPACT: 1997/07 TO 2000/06
The importance of understanding the mechanism of scrapie has been heightened by the possibilities that bovine prions have been transmitted to teenagers and young adults who developed variant Creutzfeldt-Jakob disease. More than 160,000 cattle have died of BSE since the disease was first reported. It is thought that BSE originated from supplementary feed containing meat and bone meal contaminated by a scrapie-like agent derived from sheep or cattle. Changes in the preparation of meat and bone meal in the p ractice of industrial cannibalism resulted in new prion diseases with big economic impacts.

PUBLICATIONS: 1997/07 TO 2000/06
No publications reported this period

PROJ CONTACT:

Name: Jeftinija, S.
Phone: 515-294-3243
Fax: 515-294-2315
Email: sjeftini@iastate.edu

THIOFLAVINE T DETECTION OF THE PRION PROTEIN

ACCESSION NO: 0185958 SUBFILE: CRIS
PROJ NO: NEBR-2000-02031 AGENCY: CSREES NEBR
PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: NEW
CONTRACT/GRANT/AGREEMENT NO: 00-35204-9228 PROPOSAL NO: 2000-02031
START: 01 OCT 2000 TERM: 30 SEP 2002 GRANT YR: 2000

INVESTIGATOR: Bartz, J. C.

PERFORMING INSTITUTION:
MEDICAL MICROBIOLOGY AND IMMUNOLOGY
CREIGHTON UNIV
2500 CALIFORNIA PLAZA
OMAHA, NEBRASKA 68178

OBJECTIVES: The goal of this proposal is to develop a spectrofluorometric assay using the amyloid binding dye thioflavine T (ThT) to distinguish different conformations of PrPSc in a strain-specific manner. This will provide a rapid method for identifi cation of transmissible spongiform encephalopathy strains in livestock.

APPROACH: A thioflavine T (ThT) based fluorometric assay will be developed to identify TSE strains based on the conformation of the disease-specific amyloid isoform of the prion protein, PrPSc. ThT fluorometry is a conformational assay used to detect a myloid proteins that is based on the binding of ThT to amyloid B-sheet structure. ThT is a benzothiazole dye that undergoes a spectral shift in excitation and emission maxima in the presence of amyloid protein but not in the presence of precursor amyloid, amorphous aggregates, and non-amyloid proteins with high B-sheet content. Since the molecular basis of TSE strain diversity is throught to be based on PrPSc conformation ThT fluorometry will be used to distinguish different conformations of PrPSc. First, ThT fluorometry will be optimized for identification of TME strains based on ThT binding to the PrPSc conformations (Aim 1). In Aim 2, ThT fluorometry will be used to determine i) the number of distinct strains (i.e. PrPSc conformations) that can be iden tified in a single host species, and ii) in how many distinct host species (i.e. different prion protein genotypes) can the same TME strain be identified. In aim 3 isolates of scrapie and CWD from U.S. outbreaks will be examined by ThT fluorometry in orde r to investigate TSE strain diversity in livestock and cervids.

NON-TECHNICAL SUMMARY: Identification of transmissible spongiform encephalopathy (TSE) strains has taken on increased importance with the emergence of bovine spongiform encephalopathy in the United Kingdom and its subsequent transmission to other speci es including humans. Chronic wasting disease is an emerging TSE of deer and elk in the United States that has an unknown host range and may pose a risk to sheep and cattle since they share pasturelands. Currently, no method exists to monitor for the preva lent or emerging TSE strains of livestock and wild animals. The objective of this proposal is to develop a rapid method for surveillance of TSE strains in livestock. An assay will be developed to identify TSE strains based on the ability of a fluorescent dye to interact with a TSE-specific protein in a strain-specific manner. First, reaction conditions will be optimized. Second, the number of distinct strains that can be identified in a single host species and in how many host species can the same TSE str ain can be identified. Finally, isolates of naturally occurring TSEs from the U.S. will be examined in order to investigate TSE strain diversity in livestock. The ability to monitor endemic and emerging TSE strains in domestic animals can facilitate contr ol measures to reduce the spread of TSEs and to assess the risk that emerging strains pose to animal and human health.

PROJ CONTACT:

Name: BARTZ, J. C.
Phone: 402-280-1811
Fax: 402-280-1875
Email: jbartz@creighton.edu

MECHANISMS OF STRAIN DIVERSITY IN TRANSMISSIBLE MINK ENCH-ALO

ACCESSION NO: 0180541 SUBFILE: CRIS
PROJ NO: NEBR-9802070 AGENCY: CSREES NEBR
PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: NEW
CONTRACT/GRANT/AGREEMENT NO: 98-35204-6409
START: 01 DEC 1998 TERM: 30 NOV 2001 FY: 1999 GRANT YR: 1998

INVESTIGATOR: Bessen, R. A.

PERFORMING INSTITUTION:
MICROBIOLOGY & IMMUNOLOGY
CREIGHTON UNIV
OMAHA, NEBRASKA 68131

OBJECTIVES: 9802070. The specific aims of the research are 1) to investigate the biosynthesis of strain-specific PrP-res molecules using two strains of the transmissible mink encephalopathy (TME) agent; 2) to examine the rate of PrP-res formation betwe en TME strains in a cell-free PrP conversion reaction; 3) To investigate the mechanisms of inhibition of PrP-res formation between TME strains.

APPROACH: This study will use spleen organotypic slice cultures infected with the HY and DY strains of TME to examine the rate of formation and turnover of PrP-res from radiolabeled PrPc precursor. Several biochemical parameters used to distinguish the Prp-res strains will be analyzed and related to the in vivo biological and chemical properties of the TME strains. The in situ PrP conversion reaction will be performed on tissue slices from TME-infected spleens in order to measure the rates of strain-sp ecific PrP-res formation. Anti-TSE drugs that prolong the length of the TSE incubation period will be tested for their effects on both PrP-res biosynthesis in spleen organ cultures and PrP-res conversion in the in situ PrP conversion reaction. The mechani sm of action of these drugs will be investigated as well as their effectiveness between TME strains.

PROGRESS: 1999/10 TO 2000/09
The specific aims of this proposal are to study the strain-specific properties of prion protein (PrP-res) formation in lymphoreticular tissues from the HY and DY strains of transmissible mink encephalopathy (TME) passaged in hamsters. As indicated in the previous Progress Report, the DY TME strain appeared weakly pathogenic in lymphoreticular tissues which made it difficult to effectively pursue specific aims 2 and 3. Therefore, the scientific approaches were modified and now focus on 1) the development o f a fluorometric assay to rapidly identify TSE strains, and 2) the route(s) of agent spread to the central nervous system. 1. Thioflavine T binding assay for identification of HY and DY TME. The current methods used for the identification of prion strains are expensive and time consuming, and therefore, routine identification of prion strains isolated from natural outbreaks is not performed. An assay is being developed which can distinguish TSE strains based on the interaction of thioflavine T (ThT), an a myloid binding dye, with the abnormal, amyloidogenic isoform of the prion protein, PrP res. A comparison of the ThT binding properties of HY and DY PrP-res revealed strain-specific differences and indicate that ThT fluorometry may provide a new method to identify prion strains based on PrP-res amyloid conformation. 2. Establishing the sites of DY TME replication in peripheral tissues. DY TME peripheral infection can interfere with a subsequent HY TME infection in the same host, but our data suggests that DY TME replication does not occur in lymphoreticular tissues or peripheral nerves. This is a very atypical finding among the TSE agents since the dogma is that the TSE agents initially replicate in the lymphoreticular system and then spread to peripheral nerves where axonal transport results in infection of the central nervous system. Our data suggests DY TME must reside or replicate in tissues not previously demonstrated to harbor the TSE agent. Currently, additional peripheral organs are under examinati on for the presence of DY TME PrP-res. A manuscript describing these studies will be submitted in the fall of 2001. 3. Rapid disease onset following peripheral nerve routes of TME infection. Direct inoculation of the sciatic nerve with HY TME resulted in a shorter incubation period than that found with other peripheral routes of inoculation. We hypothesized that direct infection of nerves is an efficient route for TME to gain access to the spinal cord and brain. Additional studies demonstrated that HY TME inoculation of the tongue resulted in spread of the TME agent along axons and led to a rapid disease onset. The HY TME agent could also be detected in axons within the tongue following peripheral inoculation. These findings indicate that TME can travel a long cranial nerves to the tongue, and possibly other oral cavity tissues. These findings demonstrate that 1) prion exposure to nerve endings in the tongue (e.g., lesions, sores) could serve as a route of infection, and 2) prion infection of tongue, which is not banned from human consumption in countries with BSE, is a potential source of prion infection for humans.

IMPACT: 1999/10 TO 2000/09
The transmissible spongiform encephalopathies (TSEs) are neurodegenerative diseases of domestic sheep and cattle, and wild deer and elk that are transmitted by a novel type of pathogen. The focus of this research is to identify existing and emerging TSE s trains in order to prevent TSE spread and TSE contamination of feed and biological products of animal origin that are used for both animal and human application. These studies can safeguard animal and human health through improved TSE surveillance.

PUBLICATIONS: 1999/10 TO 2000/09
1. Bessen, R.A., Heystek, L. and J.C. Bartz. Transmissible mink encephalopathy strain identification by thioflavine T binding to prion amyloid. (Submitted 2000)
2. Bessen, R. A. and J.C. Bartz. Variant CJD: Role of the tongue. (Submitted 2000)

BIOLOGICAL ENGINEERING APPLICATIONS OF THE ELECTRONIC NOSE

ACCESSION NO: 0165758 SUBFILE: CRIS
PROJ NO: NC06379 AGENCY: CSREES NC.
PROJ TYPE: HATCH PROJ STATUS: REVISED
START: 01 OCT 1999 TERM: 01 OCT 2004 FY: 1999

INVESTIGATOR: Blanchard, S. M.; Hale, A. S.; Bottcher, R. W.; Larick, D. K.; Nagle, H. T.; Schiffman, S. S.

PERFORMING INSTITUTION:
BIOLOGICAL & AGR ENGINEERING
NORTH CAROLINA STATE UNIV
RALEIGH, NORTH CAROLINA 27695

OBJECTIVES: General Objective: This research will use electronic nose technology to study problems involving environmental odors and food safety, freshness, and quality and will improve the sensitivity and specificity of the E-nose for making appropria te measurements by improving the device itself. Specific Objectives: 1. Determine quantitative information on emission of odors from livestock operations and relate the odors to the processes which produce them so that corrective measures can be evaluated . 2. Determine quantitative information on odors emanating from food and relate the odors to product safety and freshness so that corrective measures can be applied to improve food processing techniques. 3. Determine quantitative information on odors asso ciated with food products so that adulterants can be discovered and, if possible, identified. 4. Develop new signal processing methods to improve the sensitivity and specificity of E-nose measurements.

APPROACH: Odors from Livestock Operations: The electronic nose (E-nose) will be trained on fifteen different dilutions in factors of two of synthetic swine manure slurry. The E-nose will then be used to test samples from the NC State swine operation on Lake Wheeler Road. Other studies will look at odors from ozonated versus non-ozonated livestock buildings, from buildings with and without windbreak walls, and at the effects of dust mass levels and dust particle size on odor detection. Another experimen t will involve development and evaluation of a dust trap to filter dust from air samples and to volatize odorants from the trapped dust to enable quantification of gaseous and dust-borne odors. Results from E-nose studies will be compared to results from gas chromatography to quantify odorant levels and to results from human panels. These experiments will address the first objective of the project. Food Quality: The E-nose will be used to test for product decomposition in processed crab by analyzing diffe rences at several shelf ages. The ability of the E-nose to sort the samples by age will be determined. The experiment will be repeated 3 times, and the results will be compared to information previously obtained with spectral analysis. North Carolina has many important fresh and saltwater fish and shellfish that will be tested in a similar manner since spoilage profiles differ depending upon the food product. These experiments will address the second objective of the project. Product Adulteration: The E-n ose will be trained to recognize a variety meat products, e.g. pork, beef, sheep, and chicken, and then will be used to test mixtures. When the nose is fully trained, it will be tested with unknown mixtures to determine whether meat products contain other adulterants. The same process will be used to differentiate between fish varieties, e.g. flounder, catfish, Tilapia, and to test marketed products. Other tests might include using the E-nose to detect bovine-derived materials in bovine feed or sheep-deri ved materials in sheep feed since this practice can lead to infections such as BSE, bovine spongiform encephalopathy (mad cow disease). The actual odor libraries that will be built, the tests that will be performed, and the timeline for accomplishing the third specific objective will depend upon available funding from commodities groups. E-nose Improvements: Initial work to improve the sensitivity and specificity of the E-nose will focus on improving methods for data analysis. The NC State E-nose currentl y uses principle component analysis (PCA) and/or linear discriminatory analysis (LDA) for compression of data points into a 2- or 3-dimensional array following feature extraction. Three different methods K Nearest Neighbor (KNN), Least-Mean Square (LS), a nd ANNs are used to classify unknown odors. Additional statistical methods, e.g. cluster analysis (CA), canonical discriminant analysis (CDA), feature weighting (FW), radial basis function (RBF) and other methods will be explored over the duration of the project. This work will address the fourth specific objective.

NON-TECHNICAL SUMMARY: Characterizing and documenting livestock waste odors, improving quality evaluation in the seafood industry, and testing for food adulteration are all problems that can be addressed by the electronic nose. This project uses the el ectronic nose to assess livestock waste odors and food freshness and quality.

PROGRESS: 1999/10 TO 2000/09
The NC State Electronic Nose (ENoseII) located in the Department of Biological and Agricultural Engineering is currently being used to build odor libraries so that it can be trained to recognize freshness in and safety of a variety of food products. Two m ain studies have been made to accomplish this goal - one focusing on seafood products (such as tilapia, tuna, and catfish), the other on ground coffee (Ethiopian, Sumatra, Colombia, and Kenya). A third study involving cooking temperature is underway on sk ipjack tuna. In this study, cooked samples of tuna (55 deg-C and 85 deg-C) were compared to raw samples to determine the effect of cooking on tuna aroma. Early results indicate that Discriminate Analysis was able to sort the data into the three different cooking categories. The analysis algorithms will be modified and tested on unknown samples to determine their overall robustness. The building of odor libraries is a time consuming but essential process to correctly train the ENoseII. Similar studies will be performed to expand the odor libraries. In addition, permission has been acquired to import artificial hog dust, which will be used to build an odor library for livestock operations.

PUBLICATIONS: 1999/10 TO 2000/09
Dodd TH, Hale SA, and Blanchard SM. An aroma based method for determining the storage time of fishery products. Paper 00307313. Presented at 2000 ASAE Annual International Meeting, Milwaukee, July 2000.

PROJ CONTACT:

Name: Blanchard, S. M.
Phone: 919-515-6726
Fax: 919-515-7760
Email: susan_blanchard@ncsu.edu

LABORATORY FOR DIAGNOSING DISEASES OF LIVESTOCK

INVESTIGATOR: Reddy, C. C.

PERFORMING INSTITUTION:
VETERINARY SCIENCE
PENNSYLVANIA STATE UNIVERSITY
UNIVERSITY PARK, PENNSYLVANIA 16802

OBJECTIVES: Operate a laboratory to diagnose diseases of livestocks.

APPROACH: Examine specimens from livestock owners; by appropriate methods, in order to diagnose the disease involved.

PROGRESS: 2000/01 TO 2000/12
The livestock diagnostic program provides comprehensive services to detect, diagnose and aid in the control of endemic and emerging disease of livestock. Cases are often submitted as dead or dying animals. During the past year many cases were submitted as samples selected from field necropsies because renovation activities prevented handling of animals over 500 pounds at ADL. Additional renovations to the recently constructed Dunne wing of the Animal Diagnostic Laboratory (ADL) were completed this year. C abinets and an overhead large animal rail system were added to the necropsy/incinerator complex. This permits receipt of animal carcasses over 500 pounds which was not possible for the past year. The administrative/client reception area should be done soo n and complete the transition to the new main entrance on the west side of the new wing. Despite several interruptions in the routine operation of the incinerator facility, over 200,000 pounds of infectious and pathologic waste was incinerated. Major pers onnel changes occurred at ADL during the year. Two professionals were replaced and two additional professionals were hired to join the staff in 2001. Preparations continued for the implementation of a new generation laboratory information management syste m (LIMS) and the process of selecting a consultant to assist in this effort is in progress. Major research initiatives this year focused on the bovine viral diarrhea (BVD) virus and Neospora protozoan infectious agents and evaluation of bovine reproductiv e efficiency. The incidence and prevalence in Pennsylvania cattle, improved diagnostic regimes, development of a polymerase chain reaction (PCR) assay, and improved understanding of the epidemiology of the organism are focal aspects of the Neospora resear ch. Molecular diagnosis and development of a real-time PCR assay are features of the BVD viral research. The bovine reproductive research seeks to describe, evaluate and enhance the efficiency of the entire reproductive cycle of the bovine.

IMPACT: 2000/01 TO 2000/12
The primary goal of the laboratory for diagnosing diseases of livestock, aquatic and avian species is to reduce the production and financial losses in the agricultural enterprises that depend on these species. This was accomplished during the past year us ing the existing services as efficiently as possible. At the same time, research activities were pursued to provide faster and more efficient assays to identify existing and new infectious agents and production related diseases. Increased emphasis was pla ced on identification of zoonatic infectious agents transmissible from animals to people, such as Salmonella enteritidis and 0157 E. coli bacteria. Added awareness of foreign animal agents was stressed so that the impact of diseases such as bovine spongif orm encephalopathy and classical swine fever would be minimized in the event of introduction to the United States.

PUBLICATIONS: 2000/01 TO 2000/12
Zhang, S. and Maddox, C.W. 2000. Cytotoxic activity of coagulase-negative staphylococci in bovine mastitis. Infect. Immun. 68(3):1102-8.

ACQUISITION OF AN ABI PRISM 7700 SEQUENCE DETECTION SYSTEM

ACCESSION NO: 0180635 SUBFILE: CRIS
PROJ NO: SDR-9803756 AGENCY: CSREES SD.R
PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: TERMINATED
CONTRACT/GRANT/AGREEMENT NO: 98-35208-6657
START: 01 SEP 1998 TERM: 31 AUG 1999 FY: 1999 GRANT YR: 1998

INVESTIGATOR: Christopher-hennings, J.; Nelson, E. A.; Benfield, D. A.

PERFORMING INSTITUTION:
VETERINARY SCIENCE
SOUTH DAKOTA STATE UNIVERSITY
BROOKINGS, SOUTH DAKOTA 57007

OBJECTIVES: 9803756. Our goal is to use the Perkin-Elmer 7700 Sequence Detection System to aid in investigations of the pathogenesis and diagnosis, as well as host genetic resistance or susceptibility to infectious disease of livestock. Porcine Reprodu ctive and Respiratory Syndrome Virus (PRRSV), Johne's disease in cattle, Scrapie disease in sheep, bovine herpes virus 1 (BHV-1) and bovine viral diarrhea (BVD) are the primary infectious diseases currently under investigation. With this equipment we will be able to detect and quantitate polymerase chain reaction (PCR) products of viral, bacterial and animal host RNA or DNA.

APPROACH: Our approach is to develop primers and probes for use in a polymerase chain reaction (PCR) which will be utilized to detect infectious agents or host gene sequences on the instrumentation made available through this grant. Probes called "mole cular beacons" will be synthesized for each infectious agent or host gene sequence. These probes are single-stranded oligonucleotide probes that become fluorescent when they bind to perfectly complementary nucleic acids. The Sequence Detection System will detect the fluorescence using an argon laser and software which is used for detection and quantitation in a high throughput manner. Using this system we will be able to quantitate the amount of PRRSV in boar semen which would be useful in correlating imm une factors with reduction or elimination of PRRSV in semen: genetically type pigs based on their major histocompatibility antigens which could be related to the persistence of PRRSV; sample tissues to determine if there is a predominant site for harborin g Mycobacterium paratuberculosis organisms; measure viral shedding after various treatments for BVD and BHV-1; and determine if susceptibility to natural scrapie in sheep correlates with polymorphisms of the prion protein.

PROGRESS: 1998/09 TO 1999/08
The 7700 Perkin-Elmer Sequence Detection System is used within the Animal Disease Research and Diagnostic Laboratory at South Dakota State University for automated polymerase chain reaction (PCR) assays. These assays are for the detection, quantitation an d/or genotyping identification of infectious agents including Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Mycobacterium paratuberculosis (Johne's Disease in cattle) and Bovine Virus Diarrhea (BVD) along with other Pestiviruses. By using t his equipment, we are able to conduct experiments involving the pathogenesis of these diseases as well as provide field diagnosis of these agents. To date, we have been able to use the "molecular beacon" fluorescent probe technology on this equipment, to identify and quantitate Mycobacterium paratuberculosis, A plasmid prep has been obtained for quantitative standard curve measurement.

PUBLICATIONS: 1998/09 TO 1999/08
No publications reported this period.

MODES AND MECHANISM OF TRANSMISSION AND INFECTION OF SCRAPIE

ACCESSION NO: 0080372 SUBFILE: CRIS
PROJ NO: UTA00102 AGENCY: SAES UTA
PROJ TYPE: STATE PROJ STATUS: TERMINATED
START: 01 JUL 1997 TERM: 30 JUN 2002 FY: 1999

INVESTIGATOR: Holyoak, G. R.

PERFORMING INSTITUTION:
ANIMAL DAIRY & VET SCIENCE
UTAH STATE UNIVERSITY
LOGAN, UTAH 84322

OBJECTIVES: The purpose of this study is to determine if scrapie is transmitted by the parents through the embryo and/or the uterus, and at which point the embryos are at risk of exposure and infection. This information will aid in control and eradicat ion of this disease. It will assist in the development of procedures (models), and identification of genotype of sheep for import.

APPROACH: Embryo transfers conducted between natural scrapie infected embryo donor ewes and scrapie-free embryo recipient ewes.

PROGRESS: 1997/07 TO 2002/06
Ovine embryos were collected from scrapie-exposed donors at Mission, Texas, and transferred to non-exposed recipients at Logan, Utah. Offspring obtained from embryo transfer were kept in a quarentined area for 60 months to observe for incidence of scrapie infection. The scrapie agent was not transmitted to either the recipients or their offspring. The results imply that embryos handled and transfered following protocols prescribed by the International Embryo Transfer Society is a reasonably safe method to introduce new genetic resources or salvage valuable genetics from scapie infected flocks. The results of the study are as follows: Embryos from 38 scrapie exposed (natural) donors were transferred to 58 recipients resulting in 94 lambs. Ten of the donors were histopathologically (HIS) positive, producing 32 lambs with 20 surviving to 60 months. Eight of the donors were immunohistopathologically (IHC) positive, producing 21 lambs of which 13 survived to 60 months. Five donors were both histopathologically and histopathologically positive, producing 16 lambs and 11 survived to 60 months. Fifty-eight recipients lambed, of which 27 received embryos from histopathologically scrapie-positive donors. Ninety-four offspring were obtained from embryo transfer. Of these, 27 lambs originated as embryos from histopathological positive donors and 21 lambs from immunohistochemical positive donors. Within these two catagories were 16 lambs born from embryos that orginated from both histopathologically and immunohistoche mically positive donors (total of 5 donors). Sixty-four of the 94 lambs were tested with HIS. The 30 not tested were stillbirths or died as premature lambs. Sixty-three were tested with IHC. Thirty-one did not test for IHC because the lambs were stillborn or died prematurely and one sample could not be located. The molecular genetic analyses is still underway to determine whether resistance to scrapie challenge or through natural exposure to the infectious agent is due to the host's genomic constitution. Codon substitution polymorphisms in the ORF region of the PrP gene cluster are being analyzed. DNA from a group of scrapie artificially challenged goats will be extracted and sequencied. Artifical exposure to the scrapie agent in goats results in a high i ncidence of clinical scrapie.

IMPACT: 1997/07 TO 2002/06
This study infers that embryos collected from clinical scrapie or scrapie exposed donors and then transferred to scrapie free recipients is a reasonably safe method for salvaging valuable genetics or circumventing the route of transmission of scrapie.

DIAGNOSIS, PATHOGENESIS AND ANTIGENIC AND GENETIC INTERACTIONS OF TRANSMISSIBLE ENCEPHALOPATHIES

ACCESSION NO: 0149400 SUBFILE: CRIS
PROJ NO: 5348-32000-011-00D AGENCY: ARS 5348
PROJ TYPE: USDA INHOUSE PROJ STATUS: NEW
START: 01 JUN 1995 TERM: 31 MAY 2000 FY: 1999

INVESTIGATOR: OROURKE K I; HERRMANN L M; SUAREZ C E; KNOWLES JR D P; VALDEZ R A

PERFORMING INSTITUTION:
AGRICULTURAL RESEARCH SERVICE
PULLMAN, WASHINGTON 99164

OBJECTIVES: Develop control measures for sheep scrapie & cervid chronic wasting disease through improved diagnostic tests, identification of genes associated with lower susceptibility to disease, & definition of transmission routes thru cells & amp; secreted molecules. Through interaction w/APHIS and regional & national sheep associations, provide for the validation of the nictating membrane biopsy technique for the preclinical diagnosis of scrapie. REPL: -32000-007-00D. FY Prgm Inc.($270,00 0) for Prion diseases, 1 new SY.

APPROACH: Validate recently developed live animal test for scrapie in U.S. Suffolk sheep. Determine live animal test modifications necessary for testing other breeds of sheep in U.S. & abroad, and for testing mule deer, white tailed deer & elk for chronic wasting disease. Determine whether sheep with prion genetic polymorphisms associated with low susceptibility are resistant to disease or subclinical carriers. Identify the prion gene polymorphisms associated with chronic wasting disease in mul e deer, white tailed deer and elk. Identify tissues and cell types of the reproductive tract & digestive system (including salivary tissues) which produce normal cellular prions, those which accumulate the scrapie-associated isoform of prion, & th ose involved in transmission of infectious prions to offspring & flockmates. Develop fluid-based assays for detecting normal & abnormal forms of prion protein in secretions. Develop and characterize reagents useful for screening ruminant-derived p roducts entering the pharmaceutical chain. Pullman, WA, Bustad 336 & vivarium, USDA bldgs-airport Rd, BL-1: 11/23/98.

PROGRESS: 1999/01 TO 1999/09
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, a nd 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 accumula tion is in the brain, although detectable levels are found in lymphoid tissues and 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 US flocks. Free-ranging and farm raised deer and elk in the US develop a different prion disorder, chronic wasting disease. Control of all domestic prion diseases is important in reduci ng trade barriers for US sheep and germplasm. 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 undetermined direct lo sses 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 epidemilogy alone have failed to contro l the spread of scrapie throughout the US since the disease was introduced in 1947. A control program based on a live animal diagnostic test is urgently needed. 3. How does it relate to the National Program(s) and National Component(s) to which it has bee n assigned? Our research concerning the transmissible encephalopathies addresses the following National Program in Animal Production, Product Value and Safety: 103 Animal Health 100%. 4. What were the most significant accomplishments this past year? Due t o the impact of scrapie on the industry, we in collaboration with NADC and WSU scientists validated the non-invasive live animal test for scrapie and transferred the third-eyelid test to APHIS-VS- NVSL. This is significant because it allows regulatory age ncies to identify infected animals before clinical signs appear. It also allows scientists to identify naturally infected sheep for use in transmission and pathogenesis experiments. The potential outcome is eradication of U. S. scrapie and the opening of important international markets. 5. Describe the major accomplishments over the life of the project including their predicted or actual impact. Over the past two years, a live animal test for scrapie was developed. Further, 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 have been developed. These accomplishments are extensions of work p erformed throughout the life of this project in which genetically susceptible sheep were identified and peripheral lymphoid tissues from sheep were examined for prions. 6. What do you expect to accomplish, year by year, over the next 3 years? During the n ext year (2000), additional validation data showing the accuracy (sensitivity, specificity, reproducibility, and suitability for use under a variety of laboratory conditions) of the live animal scrapie test will be published. Validation of the test in she ep in Canada will be conducted. During the next two years (2000-2001) genetic data on sheep with clinical scrapie will be expanded to include data on clinically normal sheep with prion accumulation in nodes. Also the infection status of sheep with the low - susceptibility genotypes born to infected ewes will be determined, and entire flock surveys conducted to determine statistically the role of genetics in scrapie. Our ongoing goal, reachable by 2002 is that placental, uterine, and fetal tissues will be a nalyzed to determine the role of the normal cellular PrP in accumulation of the abnormal PrP-Sc and to determine the cell types associated with spread of scrapie from the ewe at the time of lambing. This information will be useful to proposing specific mo des of transmission and in developing husbandry plans to reduce the spread of the disease. 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, f armer, other scientists)? What are the constraints if known, to the adoption & durability of the technology product? Monoclonal antibodies and the live animal test have been made available for technology transfer. One antibody was licensed in this fis cal year and the companion antibody will be made available shortly. 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 co llaborative 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 through transfer of two monoclonal antibody-secreting hybridoma cell lines, evaluation of automated equipment for immunostaining, and on site training by an ARS scientist. A check panel of tissues will be provided as the last step in the transfer. Scrapie is a program disease and use of the eyelid test will be restricted to authorized laboratories with results reported to the regulatory agencies. 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 you r peer reviewed publications which are listed below). 1999 ARS Technology Transfer Award for outstanding effort in technology transfer for the successful development and transfer of diagnostic methodologies for the important animals diseases of scrapie, b ovine anaplasmosis, and equine piroplasmosis, which have a major impact domestically and on U.S trade awarded to K. ORourke and D. Knowles. Scrapie epidemiology and program management course handbook: we prepared a training video, wrote a handbook chapter , and presented a training course for federal veterinarians on collection of tissue from sheep for the live animal scrapie test.

PUBLICATIONS: 1999/01 TO 1999/09
1. OROURKE, K.I. 1999. Monoclonal antibodies and antibody cocktail for detection of prion protein as an indication of transmissible spongiform ... U.S. Patent App. docket PC 0124.99, serial No. 09/353,348.
2. TYLER, J.W., WEAVER, D.M., TURK, J.R., OROURKE, K.I., HARRINGTON, M.G., TAYLOR, W., and JENNY, A. 1999. Naturally occurring scrapie in Southdown sheep. J. of Veterinary Internal Medicine 13: 213-216.

DIAGNOSIS, PATHOGENESIS AND ANTIGENIC AND GENETIC INTERACTIONS OF TRANSMISSIBLE ENCEPHALOPATHIES

ACCESSION NO: 0403351 SUBFILE: CRIS
PROJ NO: 5348-32000-015-00D AGENCY: ARS 5348
PROJ TYPE: USDA INHOUSE PROJ STATUS: NEW
START: 12 APR 2000 TERM: 30 JUN 2002

INVESTIGATOR: OROURKE K I; HERRMANN L M; SUAREZ C E; KNOWLES JR D P; VALDEZ R A

PERFORMING INSTITUTION:
AGRICULTURAL RESEARCH SERVICE
PULLMAN, WASHINGTON 99164

OBJECTIVES: Develop control measures for sheep scrapie & cervid chronic wasting disease through improved diagnostic tests, defining genetics (prion) involved and definition of transmission routes through cells & secreted molecules. Provide for validation of the nictating membrane biopsy technique for the preclinical diagnosis of scrapie. Determine the role of immunogenetics in ovine progressive pneumonia virus and develop diagnostic assay. Formerly 5348-32000-011-00D (3/00).

APPROACH: Validate recently developed live animal test for scrapie in U.S. Suffolk sheep. Determine live animal test modifications necessary for testing other breeds of sheep in U.S. & abroad, and for testing mule deer, white tailed deer & elk for chronic wasting disease. Determine whether sheep with prion genetic polymorphisms associated with low susceptibility are resistant to infection or are subclinical carriers. Identify the prion gene polymorphism associated with chronic wasting disease i n mule deer, white tailed deer and elk. Identify tissues and cell types of the reproductive tract & digestive system (including salivary tissues) which produce normal cellular prions, those which accumulate the scrapie-associated isoform of prion, &am p; those involved in transmission of infectious prions to offspring & flockmates. Develop fluid-based assays for detecting normal & abnormal forms of prion protein in secretions. Develop and characterize reagents useful for screening ruminant-deri ved products entering the pharmaceutical chain. Pullman, WA, Bustad 336 & vivarium, USDA bldgs-airport Rd, BL-1: 11/23/99.

EARLY PRECLINICAL DISTRIBUTION OF PRPSC IN SHEEP WITH NATURAL SCRAPIE

ACCESSION NO: 0182084 SUBFILE: CRIS
PROJ NO: WNV-00143 AGENCY: CSREES WN.V
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: TERMINATED
START: 01 JUL 1999 TERM: 30 JUN 2000

INVESTIGATOR: Baszler, T. V.; O'Rourke, K. I.

PERFORMING INSTITUTION:
ANIMAL HEALTH RESEARCH CENTER
WASHINGTON STATE UNIVERSITY
PULLMAN, WASHINGTON 99164

OBJECTIVES: This proposal will focus on investigation of detecting preclinical infection in sheep with natural scrapie. Recently, researchers at WSU developed and validated a preclinical diagnostic test for sheep scrapie based upon detection of PrPSc i n third eyelid lymphoid tissue biopsies using monoclonal antibody based immunohistochemistry. Prospective validation studies have indicated that identification of third eyelid PrPSc in 18-24 month old sheep reliably predicts future clinical disease and co uld be of great value in removing infected animals from flocks long before significant disease transmission occurs. However, it is not known if PrPSc deposition occurs in lymphoid tissues before 18 months of age (a more practical age for testing), or whet her or not PrPSc accumulates in anatomic sites other than third eyelid or general lymphoid tissues during preclinical infection. For disease surveillance purposes it is critical to determine if a negative third eyelid test in a 1-2 year old sheep truly in dicates lack of scrapie infection. The current proposal will test the hypothesis that PrPSc accumulation during preclinical infection occurs at anatomic sites other than lymphoid tissue. We will use immunohistochemical detection of PrPSc in entire, define d scrapie flocks to address the objectives: 1. Determine spatially the spectrum of tissues with PrPSc accumulation during natural preclinical scrapie 2. Determine the temporal accumulation pattern of PrPSc in effected tissues during natural preclinical sc rapie

APPROACH: Our general strategy for accomplishing objectives 1 and 2 is to implement immunohistochemical detection of PrPSc on entire sheep flocks with naturally-occurring scrapie. Immunohistochemical detection of PrPSc will form the basis for determini ng preclinical scrapie infection status. Both objectives 1 and 2 will be implemented on the same flocks. To be selected for study, flocks must: (1) Have an incidence of scrapie high enough to warrant depopulation by regulatory personnel; (2) Contain 50% s usceptible 171-QQ genotype sheep; (3) Maintain accurate genealogy records; and (4) Be composed of 75% black-faced sheep (90% of scrapie in the US is reported in Suffolks and Hampshires). The high incidence of scrapie in flocks containing over 50% 171-QQ w ith increase the probability of obtaining sufficient numbers of scrapie infected sheep, and also would increase the probability of ongoing scrapie transmission occurring in the flock. Accurate genealogy records will ensure accurate tracking of scrapie exp osed maternal lines in order to identify sheep with the high potential for preclinical infection. The flocks must contain a range of animals from 1-6 years in order to have sufficient populations of sheep in both the preclinical and clinical stages. The t emporal accumulation of PrPSc in objective 2 will be assessed by comparing PrPSc immunoreactivity in sheep of different ages. Assuming that most transmission occurs during the perinatal period (5) and that disease occurs most commonly in 3-4 year old shee p, we anticipate most preclinical infection will occur in 1 and 3 year old sheep. Thus, targeted flock must contain 50% of animals <3 years of age. Investigations will focus on tissues that are proven or potential transmission routes, and will include lym phoid tissue (including the third eyelid, peripheral and internal lymph nodes, tonsil, and spleen), gastrointestinal tissue, mammary tissue, reproductive tissue. hematopoietic tissue and brain. The results of the proposed studies will further validate the use of the "third eyelid test" for preclinical testing in surveillance testing for natural sheep >scrapie, and will provide valuable preliminary data for future experimental studies of disease transmission.

PROGRESS: 1999/07 TO 2000/06
One flock of 50 sheep and one flock of 28 sheep with flock histories of scrapie were euthanatized and necropsied for examination of PrPSc deposition in targeted tissues. All tissues were examined for PrPSc accumulation by immunohistochemistry (IHC) using anti-prion specific monoclonal antibody previously reported by our laboratory. Double-label IHC, using PrPSc specific monoclonal antibody and cellular markers, was done to unequivocally determine cellular specificity of PrPSc deposition. PrPSc immunoreact ivity was identified in 13/78 sheep ranging from 14 months to 6 years of age. 70% (9/13) of scrapie infected sheep, in these two typical scrapie infected flocks, were detected during the preclinical stage of disease. All 13 PrPSc positive sheep, including all 9 sheep in which PrPSc was detected preclinically, had PrPSc deposition within multiple alimentary tract tissues or their related lymph nodes (jejunum, ileum, colon, or rectum). The findings emphasize the importance of the alimentary tract as a sourc e of scrapie transmission because PrPSc was not detected in cells of other likely natural transmission sources including uterus, mammary gland, and bone marrow. All sheep with clinical signs of scrapie had PrPSc deposition in the brain and multiple extra- neural tissues. Of the nine preclinical animals, five had PrPSc deposits in the CNS as well as other tissues, while four were devoid of PrPSc immunoreactivity in the CNS. Of the four sheep without PrPSc in the brain, three were 2 years of age and one 6 ye ars of age. In the 5 young preclinical scrapie sheep (<2 years of age), PrPSc deposition was widespread in multiple lymphoid tissues and alimentary tract; deposition did not occur early in a particular tissue, lymph node, or lymphoid structure. A famil ial linkage of PrPSc accumulation was not identified. PrPSc deposition in the alimentary tract, whether during preclinical or clinical disease, localized primarily to submucosal lymphoid follicles (GALT) and submucosal or myenteric nerve plexus. In alimen tary tract related peripheral nervous tissue PrPSc accumulations were detected within perikarya and neurites. Immunoreactivity in lymphoid germinal centers was within the cytoplasm of follicular dendritic-like cells (FDC), and within phagocytic tingible b ody macrophages-like cells. Using sequential double-labeling techniques, co-localization of PrPSc and anti-CNA.42 mAb (FDC marker) was evident in germinal center cells. Approximately 25% of PrPSc immunoreactivity co-localized in the same cell with anti-CN A.42 immunoreactivity (FDC). This finding emphasizes the importance of FDCs in the pathogenesis of natural sheep scrapie. All sheep in the study were genotyped at codon 136 and 171 of the prion gene. All sheep in both flocks under study were homozygous fo r alanine at codon 136 (136AA). There was polymorphism at codon 171 associated with both clinical scrapie and PrPSc deposition. All thirteen carriers of PrPsc were 171QQ while none of the 30 sheep with genotype 171QR or 2 sheep with 171RR showed PrPSC dep osition in tissues.

IMPACT: 1999/07 TO 2000/06
The data confirm the importance of the alimentary tract as a route of natural sheep scrapie. Furthermore, specific cell targets for scrapie prion accumulation were identified in which to focus future studies investigating the mechanism of genetic resistan ce to sheep scrapie. Most scrapie positive sheep in the 2 flocks studied harbored scrapie prion during preclinial disease emphasizing the importance of targeting sheep during early infection for effective scrapie eradication and surveillance.

PUBLICATIONS: 1999/07 TO 2000/06
1. Herrmann LM, Baszler TV, Knowles DP: Prion mRNA, but not PrPSc, is found in the salivary glands of scrapie-infected sheep. Biochemica et Biophysica Acta, 1479:147-154, 2000
2. O'Rourke KI, Baszler TV, Miller JM, Cutlip RC, Well GAH, Parish SM, Hamir AN, Cockett NE, Jenny A, Knowles DP: Preclinical diagnosis of scrapie by immunohistochemistry of third eyelid lymphoid tissue. J. Clin. Microbiol. 38: 3254-3259, 2000

ETIOLOGY AND HOST RESTRICTION OF TRANSMISSIBLE MINK ENCEPHALOPATHY

ACCESSION NO: 0170939 SUBFILE: CRIS
PROJ NO: WIS03905 AGENCY: CSREES WIS
PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: TERMINATED
CONTRACT/GRANT/AGREEMENT NO: 95-37204-2238
START: 15 SEP 1995 TERM: 30 SEP 1998 FY: 1998 GRANT YR: 1995

INVESTIGATOR: Aiken, J. M.; Marsh, R. F.; McKenzie, D. I.

PERFORMING INSTITUTION:
ANIMAL HEALTH & BIOMEDICAL SCIENCE
UNIV OF WISCONSIN
MADISON, WISCONSIN 53706

OBJECTIVES: 9502055. To characterize the role of the prion protein in the species barrier effect in scrapie infection to facilitate our abilities to predict a host range for given strains of transmissible spongiform encephalopathies.

APPROACH: Our approaches are: examination of the interaction of the HYPER and DROWSY strains of TME through competition experiments using denatured/renatured HYPER and DROWSY PrP, and characterization of the role of PrP in the species barrier effect be tween mink and ferret using in vitro cell-free conversion experiments.

PROGRESS: 1995/09 TO 1998/09
This proposal addressed the etiology and host restriction of transmissible mink encephalopathy in two ways. First, we investigated the relationship between the two hamster-adapted TME strains, HY and DY, by serially passaging a biological clone of TME int o Syrian hamsters. Following interspecies passage, DY-adapted hamster TME was the predominant strain identified based on Western blot analysis. Additional serial passage in hamsters res ulted in the emergence of the HY strain unless passage was performed with low titer inoculum when DY was the predominant phenotype observed. Experimental competition assays demonstrated that DY was able to block, either partially or completely, depending upon the dose, the HY phenotype. These findings indicate that intersp ecies transmission of biologically cloned TME results in a competition between several strains of the TSE and that a daptation is due to this competition. We also used cell-free conversion experiments to address the role of PrP in the species barrier. We expressed recombinant hamster PrPC in baculovirus and combined it with PrPSc-enriched preparations derived from scrapie- infected hamster brains and TME-infected mink brains. Cell-free conversion reactions can be strain-specific conversion when the source of PrPC is mammalian (i.e., from mouse neuroblastoma cells). To determine whether bacHaPrPC conversion works in an analogous manner, we performed similar cell-free conversion experiments using the HY and DY strains of hamster-adapted TME. Strain-specific conversion was determined by the presence and size of 35S-bacHaPrP-res using autoradiography and quantitated using a Phosphoimager (Molecular Dynamics) and ImageQuant software. PrPHY- and PrPDY-enriched preparations drive the conversion of 35S-bacHaPrPC into two products that differ in size. The observed 1-2 kDa size difference in the converted products is reproducible at 2 different incubation conditions and is the same as observed between PK-treated PrPHY and PrPDY . The cell-free conversion reactions include radiolabelled PrPC and, thus, are readily quantitated. Conversion efficiency is determined by comparing the amount of protease resistant PrP generated during the reactions with the initial amount of 35SPrPC present. We obtain 23% conversion effici ency with PrP purified from HY infected hamsters (PrPHY)and 28% conversion efficiency using PrPDY as a template. These efficiencies are comparable with the efficiencies obtained using mammalian derived PrPC (20-35% efficiency). The specificity of these re actions (in addition to the retention of strain-specific properties of the converted product) is further suggested by the low conversion efficiency of heterologous PrPSc. When PrP purified from TME infected mink was denatured in 2M GdnHCl and incubated in the presence of hamster 35SPrPC, the conversion efficiency was very low (4%).

PUBLICATIONS: 1995/09 TO 1998/09
1. McKenzie, D., J. Bartz and J.M. Aiken. 1998. A molecular basis for transmissible spongiform encephalopathy strain differences. Bulletin de l'Institut Pasteur 96, 35-47.
2. Bartz, J.C., Marsh, R.F., McKenzie, D.M. and Aiken, J.M. 1998. The host range of chronic wasting disease is altered upon passage in ferrets. Virology 251:297-301.