Advanced Diagnostic Methods for Infectious Disease


PROJ NO: 3625-32000-027-00D AGENCY: ARS 3625
START: 01 OCT 1995 TERM: 30 SEP 2000 FY: 1999



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.

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