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From: TSS ()
Date: September 6, 2005 at 9:11 am PST

EFSA Scientific Report on the Evaluation of Rapid post mortem TSE Tests intended for Small Ruminants

Last updated: 22 June 2005
Adopted on 17 May 2005 (Question N° EFSA-Q-2003-084)

Summary of the Report

The European Food Safety Authority (EFSA) and its Scientific Expert Working Group on Transmissible Spongiform Encephalopathy (TSE) Testing were asked by the European Commission (EC) to take over the mandate of the former Scientific Steering Committee (SSC) for the scientific evaluation of rapid TSE/BSE (Bovine Spongiform Encephalopathy) tests.

Until 2004 no evaluation of rapid TSE tests on material from small ruminants was conducted by the European Commission. In the absence of such data, 5 rapid post mortem TSE tests performing satisfactorily on bovine tissue were provisionally approved by the EC for the TSE monitoring of small ruminants in accordance with the TSE Regulation (EC) No 999/2001. Following an EC call for expression of interest in the Official Journal of the European Union (No C15) on 22 January 2003, several parties indicated their interest in participating in this third European evaluation exercise for newly developed rapid post mortem and live animal TSE/BSE tests.

EFSA was asked to take over the scientific aspects of this evaluation and to assess the outcome of the IRMM evaluation of rapid TSE post mortem tests, taking also into account an opinion of the French Food Safety Agency (AFSSA), and to give recommendations on the approval of the tests.

During January to June 2004, the European Commission’s Institute of Reference Materials and Measurements (IRMM) carried out an evaluation of the diagnostic and analytical sensitivities, of the diagnostic specificity and repeatability of six rapid post mortem tests on samples from natural scrapie cases. Additionally the ability of these tests and their analytical sensitivity for the detection of “atypical” scrapie strain (Nor 98) in sheep tissue were evaluated. During August 2004, further brain samples from three clinical cases of sheep orally challenged with BSE-affected cattle brain homogenate were screened using each of the six rapid tests. In March 2005, in response to concerns of the EFSA WG on TSE Testing following confirmation of a case of BSE in a French goat, the six rapid tests were re-evaluated against dilutions of brain homogenates from experimentally BSE infected sheep to provide analytical sensitivity for this material comparable to that previously obtained for scrapie.

The assessment comprised the assessment of the application dossiers, of the results obtained from the laboratory evaluation, organised, carried out and analysed by the IRMM, and of the package inserts. Based on an overall assessment on the application dossiers, the laboratory evaluation and the approval of the package inserts, the experts of EFSA´s Working Group on TSE Testing express their opinion on 6 rapid post mortem TSE tests for small ruminants.

Keywords: BSE, Bovine Spongiform Encephalopathy, TSE, Transmissible Spongiform Encephalopathy, post mortem, rapid BSE test, small ruminants, evaluation, Regulation (EC) No 999/2001, TSE monitoring.

Publication date: 22 June 2005



Research Project: Bse and Other Tses Ruminant Herbivors: Diagnosis, Transmission and Genetics
Location: Animal Diseases Research

2004 Annual Report

1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter?
Bovine spongiform encephalopathy (BSE) is a fatal disease of cattle, first reported in the United Kingdom and now found in many European countries and Japan, with isolated cases in Canada and the United States. The disorder is related to consumption of feed contaminated with the carcasses of BSE-infected cattle. Bans on recycling ruminant material in ruminant feed are effective in reducing the number of affected animals. However, identification of even a single case results in economically devastating restriction in international trade, limits the availability of some byproducts derived through rendering carcasses, and increases the need for costly surveillance. Chronic wasting disease (CWD) is another member of the TSE family of disorders, with a more limited host range and no evidence to date of a zoonotic potential. CWD is reported in free ranging and farm raised deer and elk in the U.S. and Canada. CWD is a relatively uncharacterized TSE with novel patterns of transmission and agent distribution, as well as providing the considerable challenge of a controlling a persistent disease in free ranging wildlife.

The objectives of the project are to develop inexpensive, high capacity live animal and postmortem diagnostic tests, prion detection methods for monitoring environmental contamination, and pathogenesis/transmission experiments that provide direction to intervention techniques and reagents. Control of all domestic prion diseases is important in reducing trade barriers for U.S. sheep and germplasm and for assuring the present and future global markets for cattle.

The goals of the project are (i) to develop diagnostic strategies that contribute to disease control in captive and free ranging animals, (ii) to identify genetic factors involved in resistance to disease and reduction of transmission from infected animals, and (iii) to determine routes of transmission and disease persistence in the population and the environment.

These goals will be met through the following specific objectives: (1) development of higher throughput, very inexpensive PrPd detection methods, to be used diagnostically for postmortem and live animal testing and in transmission and environmental contamination studies (2) an efficient biological assay model using a panel of species-specific transgenic mice, in which the murine gene is replaced by the appropriate cervid PrP gene for genetic resistance and transmission studies; and (3) characterization of the cervid PrP genes for identification of alleles associated with genetic resistance

Our research concerning the transmissible encephalopathies addresses the elements of our National Program in Animal Production, Product Value and Safety. 103 Animal Health 100%, Pathogen Detection and Diagnostics, Genetic Resistance to Disease, Epidemiology of Disease, and Strategies to Control Infectious and Non-infectious diseases.. It also relates to National Program 108, Food composition and Intake Food Safety.

The TSEs present a serious problem to US agriculture. Identification of a single imported cow with BSE in December 2003 resulted in devastating direct and indirect losses to the cattle, rendering, and pharmaceutical industries with long term changes in handling of meat and byproducts implemented by USDA, the Food and Drug Administration, and the Food Safety Inspection Service. Although CWD affects a far smaller segment of the agricultural community, the losses of domestic and foreign markets, restrictions on trade and movement of live animals, and the substantial costs associated with surveillance of hunter-killed animals represent major economic losses for the cervid farming and hunting industries. Agriculture and public health will benefit from reduced disease prevalence, cost-effective diagnostic and surveillance methods, prevention measures, and appropriate handling of facilities to reduce the possibility of re-infection.

2. List the milestones (indicators of progress) from your Project Plan.
Year 1 (FY2003)

Develop monoclonal antibody and target binding molecules for PrP-d detection.

Transgenic mouse (elk gene) development: construct preparation and embryonic cell development.

Define the PRNP alleles in white tailed deer.

Identify a potentially contaminated cervid holding area, provide appropriate biosecurity to insure that no wild ruminant animals enter or leave the facility from the surrounding area, and restock with susceptible species

Year 2 (FY2004)

Develop suitable assays for PrP-d detection.

Transgenic mouse (elk gene) development: characterization of homozygous founder stock.

Describe the organization of the mule deer PRNP gene.

Prepare construct for white tailed deer transgenic mouse.

Monitor livestock in potentially contaminated facility.

Initiate oral exposure of mink to the CWD agent from elk.

Year 3 (FY2005)

Develop applications for PrP-d detection assays in animal tissues and environmental samples.

Transgenic mouse development (elk gene): assay selected mouse lines.

Prepare construct for mule deer transgenic mouse.

Continue to monitor animals in potentially contaminated facility.

3. Milestones:
A. List the milestones that were scheduled to be addressed in FY 2004. Which were fully or substantially met?

The milestones below were scheduled for FY2004 and have been partially met.

Develop suitable assays for PrP-d detection. This work is in progress. We have developed assays using immunohistochemistry and enzyme-linked immunosorbent assay. These techniques have not been shown to be sensitive or specific enough to detect a marker in the blood of naturally infected sheep. Additional work with more sensitive techniques including mass spectroscopy and infrared spectroscopy will be performed in year 2.

Transgenic mouse (elk gene) development: characterization of homozygous founder stock. Prepare construct for white tailed deer transgenic mouse

Transgenic mouse development is progressing but is still in stage 1 (development of defined constructs and embryic stem cells.

The following milestones were described for FY 2004 and have been fully met.

Describe the organization of the mule deer PRNP gene.

Monitor livestock in potentially contaminated facility.

Initiate oral exposure of mink to the CWD agent from elk.

B. List the milestones that you expect to address over the next three years. What do you expect to accomplish year by year?

Development and application of assays for diagnostic testing (FY2005) and field testing of candidate assays (FY2006 and FY2007).

Development of assays for environmental contamination (FY2005 and 2006) with remedial measures evaluated (FY2007).

Transgenic mouse development (elk and gene) with founder stock developed in FY2005 and bioassays performed in FY2006.

Elk in potentially contaminated facility monitored in every year of the project.

The potential for transmission of BSE through the placenta of experimentally infected sheep will be evaluated (FY2005).

4. What were the most significant accomplishments this past year?
A. Genetics can be an important component of TSE control programs if some polymorphisms in the prion gene are associated with reduced disease incidence. In conjunction with Colorado State University, private landowners (the Richard Edwards family), the Canadian Food Inspection Agency, and the Nebraska Department of Game and Parks, the Animal Disease Research Unit identified susceptible prion genotypes in elk, white tailed deer and mule deer, characterized the first reported prion pseudogene in placental mammals, and developed protocols for differentiating between the functional gene and the pseudogene. We demonstrated that all reported genotypes of mule deer are susceptible, identified the single genotype in white tailed deer associated with a reduced susceptibility to disease and demonstrated that elk of the most resistant genotype can be affected by disease under some farm conditions. This accomplishment supports the state and federal regulatory agencies' control programs, and allows research laboratories to correctly genotype cervid livestock.

B. Diagnostic testing is performed on a limited number of tissues and correct identification of the most appropriate target tissue for each species is critical for accurate surveillance. In collaboration with Colorado State University, the Canadian Food Inspection Agency, and the Nebraska Division of Game and Parks, the Animal Disease Research Unit described the distribution of PrP-Sc in large populations of captive elk and free ranging white tailed deer. This study demonstrated that the most accurate testing of elk requires examination of the both the brain and the retropharyngeal lymph node but testing of white tailed deer can be accomplished by examination of the retropharyngeal lymph node alone. This work provides the scientific basis for tissue selection for diagnostic testing of elk and white tailed deer, complementing the previous study on mule deer testing.

2) Although the transmission routes of CWD are unknown, placentas shed by infected deer or elk are potential sources of environmental contamination. Testing of reproductive tissues for assessing prion contamination is performed in collaboration with Colorado State University and the University of Idaho. These studies have shown no evidence that CWD is transmitted by contact with the placenta of infected females, as is seen in sheep scrapie.

3) Large and small carnivores feed on dead deer and elk in the CWD endemic area. If these animals are susceptible to a TSE following oral exposure to the agent in the tissues of dead deer or elk, they represent another reservoir of disease. Under a cooperative agreement with the University of Washington, mink were exposed to tissues from CWD-positive elk. The incubation period is expected to exceed 18 months and results are expected in FY2005.

C. Significant activities that support special target populations.


D. Progress Report opportunity to submit additional programmatic information to your Area Office and NPS (optional for all in-house (¿D¿) projects and the projects listed in Appendix A; mandatory for all other subordinate projects).


5. Describe the major accomplishments over the life of the project, including their predicted or actual impact.
Diagnostic test development: Postmortem diagnostic test methodology was developed and transferred to the regulatory agencies for use in the U.S. The test was modified for use in live deer, although the test requires general anesthesia and re-trapping of deer found to be positive. Monoclonal antibodies useful in assays on routinely formalin fixed tissue from infected sheep, deer, elk, cattle, humans, mink, domestic cats and a wide variety of captive wildlife potentially exposed to prion diseases were developed. The technology was transferred to USDA, Animal Plant Health Inspection Service, which contracts with a network of 20 veterinary diagnostic laboratories to provide surveillance for CWD in the US. Gold standard testing of cattle for BSE is conducted only at the National Veterinary Services Laboratory in Ames, IA. The technology and antibodies are also used by the Canadian Food Inspection Agency in Nepean, Ontario, and the Foreign Animal Disease Diagnostic Laboratory in Winnipeg.

CWD genetics: We provided the first report of a processed prion pseudogene in livestock, allowing research laboratories and wildlife managers to correctly identify the prion functional gene in wildlife. Using the appropriate testing technology, we demonstrated that none of the reported genotypes of cervids are associated with disease resistance and genetic measures cannot be a component of the federal CWD control program. However, one genotype in elk is associated with very low disease prevalence and it is possible that selection for this genotype in captive elk with very minor potential exposure to CWD would reduce the disease incidence.

6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products?
The antiprion monoclonal antibodies have been licensed to private companies for development of immunohistochemistry and enzyme linked immunosorbent assays. An IHC test for BSE using one of the monoclonal antibodies is licensed and marketed internationally. In the US, TSE testing is conducted by the federal government or by federally approved laboratories. All the IHC testing performed in the US for livestock TSEs is performed with the technology and antibody reagents developed by this project.

7. List your most important publications in the popular press and presentations to organizations and articles written about your work.
1. BSE diagnostic testing in the US. Invited presentation to the 12th Annual Food Safety Farm to Table Conference, May 2004, Moscow, ID

2. BSE Testing Technology. Invited presentation to the Beef Industry Food Safety Council Industry Summit on Bovine Spongiform Encephalopathy, Fort Worth, TX, April 26, 2004

3. Is BSE in sheep at threat to the US sheep industry? Invited presentation to the Sheep and Goat Health Committee Seminar, National Institute for Animal Agriculture, April 4, 2004, Salt Lake City, UT

4. Prion susceptibility genetics in three cervid species, invited presentation to the Annual meeting of the American College of Veterinary Pathologists, Banff, Canada, November 15, 2003

Review Publications
Hamir, A.N., Miller, J.M., O'Rourke, K.I., Bartz, J.C., Stack, M.J., Chaplin, M.J. 2004. Transmission of transmissible mink encephalopathy (TME) to raccoons (Procyon lotor) by intracerebral inoculation. Journal of Veterinary Diagnostic Investigation. 16(1):57-63.

Brayton, K.A., O'Rourke, K.I., Lyda, A.K., Miller, M.W., Knowles, D.P. A processed pseudogene contributes to apparent mule deer prion gene heterogeneity. Gene. 2003. v. 326. p. 167-173.

O'Rourke, K.I., Spraker, T.R., Hamburg, L.K., Besser, T.E., Brayton, K.A., Knowles, D.P. 2004. Polymorphisms in the prion precursor functional gene but not the pseudogene are associated with susceptibilty to chronic wasting disease in white-tailed deer. Journal of General Virology. 85:1339-1346.


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