1. USDA Licensed Test Kits and Enhanced Surveillance for BSE Larry Ludemann 1 and Lawrence Elsken 2 Policy, Evaluation, and Licensing 1 Inspection and Compliance 2 Center for Veterinary Biologics Veterinary Services Animal and Plant Health Inspection Service United States Department of Agriculture 510 S. 17 th Street, Suite 104 Ames, Iowa 50010 USA

2. The Causative Agent of TSEs  Prions Abnormally folded proteinAbnormally folded protein  Not a virus. Not a bacteria. No DNA or RNA  No known immune response Two forms of prion proteinTwo forms of prion protein  Normal prion protein (PrPc) – on most cells  Infectious Prion (PrPSc) Relatively resistant to disinfectants, sterilizations, proteinasesRelatively resistant to disinfectants, sterilizations, proteinases Accumulates and kills the cellsAccumulates and kills the cells

3. BSE: Diagnostics  No effective live animal test for BSE  Need brain tissue  Histopathology of brain  IHC, WB, ELISA – PrP res  “Negatives” do not guarantee absence of infectivity - tests are NOT intended as “Food Safety Tests”

4. BSE Diagnostic Tests   Histology  ImmunoHistochemistry (IHC)-  ImmunoHistochemistry (IHC)-an ELISA performed on a tissue section mounted on a slide   “Rapid tests”- ELISA, EIA, Western Blot

5. “Rapid Tests”  As name implies, generally more rapid than IHC  Associated with occasional “false positive” initial reactions (especially ELISA)  Western Blot Test lower throughput, slower, and more involved than ELISA  All approved tests have very good sensitivity and specificity  BUT - only serve as screening tests

6. IMMUNOHISTOCHEMISTRY  Adds immunological confirmation to observation of lesions  Stain observed in specific (definitive) anatomic areas associated with BSE  No “false positives”  Generally requires several days to complete test

8. January 8, 2004 CENTER FOR VETERINARY BIOLOGICS NOTICE NO. 04-02 Subject: Diagnostic Test Kits for Bovine Spongiform Encephalopathy To: Biologics Licensees, Permittees, and Applicants Veterinary Services Management Team Directors, Center for Veterinary Biologics Area Veterinarians in Charge, VS State Veterinarians I. PURPOSE The purpose of this notice is to inform interested parties that the Center for Veterinary Biologics (CVB) is accepting veterinary biologics product license and permit applications for diagnostic test kits (including “rapid tests”) intended as an aid in the diagnosis of bovine spongiform encephalopathy (BSE). II. Background Numerous test methods (e.g., immunohistochemistry, ELISA, Western blot) and commercial test kits have been developed to aid in the diagnosis of BSE. Some of these kits have been approved by foreign regulatory officials (e.g., European Union, Japan) for use in their BSE testing programs. Due to recent confirmed cases of BSE in North America and the anticipated increase in surveillance for BSE in the U.S., the CVB will accept and consider license and permit applications for diagnostic test kits intended to aid in the diagnosis of BSE. The CVB will proceed with the review and testing processes of each license or permit application up to the point of issuing the actual U.S. Veterinary Biological Product License or Permit. In the future, the CVB will issue additional guidance on the final U.S. Veterinary Biological Product License or Permit restrictions required for distribution and sale of these diagnostic test kits in the U.S.

9. Authorities, Regulations, & Guidance  Virus-Serum-Toxin Act: 21 USC Authorizes Secretary of Agriculture to regulate manufacture and distribution of biologics including diagnostics (test kits) intended for use in diagnosis of disease in animals.Authorizes Secretary of Agriculture to regulate manufacture and distribution of biologics including diagnostics (test kits) intended for use in diagnosis of disease in animals.  Code of Federal Regulations: Title 9  VS Memoranda 800.73 (Immunodiagnostic Test Kits), others800.73 (Immunodiagnostic Test Kits), others

10. What is a regulated test kit?  Diagnostic test kits contain: All unique reagents required to conduct testAll unique reagents required to conduct test Complete instructions for the testComplete instructions for the test Instructions for interpretation of test resultsInstructions for interpretation of test results Claims, uses, and limitationsClaims, uses, and limitations  Used to diagnose existence of (usually) or susceptibility to disease agents of animals  Reagents are not regulated

11. Prelicense Assessment of Kits  Accuracy and precision  Diagnostic sensitivity Dynamic rangeDynamic range Analytical sensitivityAnalytical sensitivity  Diagnostic specificity  Ruggedness, repeatability, suitability  Predictive values for specific disease prevalence (especially for Program Diseases)

12. Prelicense Validation  Test large number of known positive and known negative animal samples covering a range of reactions and all sample types  Determine onset of detection of disease  Evaluate (in at least 3 laboratory settings) Serum or antigen panel (ruggedness)Serum or antigen panel (ruggedness) Well-characterized patient samplesWell-characterized patient samples Compared to “Gold” standard assayCompared to “Gold” standard assay Suitability of test kitSuitability of test kit Adequacy of instructionsAdequacy of instructions  Authorization from USDA per 9 CFR 103.3 (experimental biological products)

13. Manufacturing controls  Aim is to minimize within-serial and serial- to-serial variation by ensuring consistency of production – Outline of Production  Master Seed/Master Cell concept  Validation of new lots of antibody/antisera  Serial release testing of serum or antigen panel with range of reactivities and cross- reactivities Serial release panel is critical to minimize variation in test resultsSerial release panel is critical to minimize variation in test results Panel includes range of reactivityPanel includes range of reactivity

14. Other Requirements  Inspection of manufacturing facilities and other licensed sites (e.g., Permittee quarantine sites)  Prelicense serial testing  Label approval

15. USDA Approved Bovine Spongiform Encephalopathy Antigen Test Kits (Permits, as of September 30, 2004) Manufacturer Kit Format Trade Name Date Approved Bio-Rad Laboratories, (permittee: Bio-Rad, France) ELISATeSeE® Permit issued: March 17, 2004 Abbott Laboratories (permittee: Enfer, Ireland) ELISA Enfer BSE Permit issued: May 28, 2004 Roche Diagnostics Corp., (permittee Prionics, (permittee Prionics, Switzerland) Switzerland) Western Blot Prionics®- Check Western Permit issued: May 28, 2004

16. USDA Approved Bovine Spongiform Encephalopathy Antigen Test Kits Licenses, as of (September 30, 2004) Manufacturer Kit Format Trade Name Date Approved IDEXX Laboratories, Inc. EIA (Enzyme Immunoassa y) HerdChek- BSE License issued: March 17, 2004 Pierce Biotechnology, Inc., (Produces kit for Prionics) ELISA Prionics®- Check LIA Licensed: September 1, 2004 Pierce Biotechnology, Inc., For Export Only ELISABSE-Scrapie Ag Test Kit Prionics®- Check LIA Licensed: September 1, 2004 VMRD, Inc. For Export Only IHC License issued: August 5, 2004

17. Licensed BSE Test Kits: Format or Technique  All use obex tissue  Purify normal and abnormal PrP  Treatment to “remove” normal PrP Western Blot – separate protein by Mol WtWestern Blot – separate protein by Mol Wt  Antibody binds “abnormal” PrP  Indicator enzyme (on antibody) produces “visible” reaction: ELISA, EIA, chemiluminescence, solid precipitate (Western Blot, IHC)ELISA, EIA, chemiluminescence, solid precipitate (Western Blot, IHC)

18. Diagnostics: Under Development  “Cowside” or on-site tests  Capillary electrophoresis – blood  Blood – other methodologies - Capture  Urine – UPrPsc  Increased sensitivity – cyclic amplification  Non-PrP markers – “Upregulated” Markers

19. What does licensure provide?  USDA controlled manufacturing of all components and kits Minimal variation within and between serialsMinimal variation within and between serials  USDA evaluation of test kit before and after licensure (serial release)  Monitoring of field performance and investigations CVB-Inspection and Compliance 800-752-6255CVB-Inspection and Compliance 800-752-6255  Ongoing facilities inspections  Questions? www.aphis.usda.gov/vs/cvb/

20. BSE Surveillance Program  Expanded or Enhanced surveillance began June 1, 2004. Purpose: Determine if BSE is present in U.S. cattle populationDetermine if BSE is present in U.S. cattle population Determine if risk-management policies adequateDetermine if risk-management policies adequate  For animal health – NOT food safety

21. EU testing: Jan-Dec 2001 SamplesPositive% positive Risk 776,266756.097% Em slaug 96,774324.335% CNS 27,99134.121% Fallen 651,501397.061% Healthy7,677,576279.0036% Suspect 3,267110333.76%

22. BSE Surveillance Program   Experience in Europe has demonstrated that targeting surveillance efforts at certain high risk populations is the most effective way to identify BSE, if it is present.

23. BSE Surveillance Program  US high-risk population estimate: about 446,000 246,000 on-farm deaths with unexplained causes or causes consistent with BSE246,000 on-farm deaths with unexplained causes or causes consistent with BSE 200,000 ante-mortem condemnations by FSIS200,000 ante-mortem condemnations by FSIS 129 (2004 data) Foreign Animal Disease investigations for CNS disease129 (2004 data) Foreign Animal Disease investigations for CNS disease

24. BSE Surveillance Program  The majority of samples from the following categories: Nonambulatory cattle;Nonambulatory cattle; Cattle exhibiting signs of a central nervous system disorder;Cattle exhibiting signs of a central nervous system disorder; Cattle exhibiting other signs that may be associated with BSE, such as emaciation or injury; andCattle exhibiting other signs that may be associated with BSE, such as emaciation or injury; and Dead cattle.Dead cattle.  USDA personnel will also sample all cattle condemned on ante-mortem inspection by USDA’s Food Safety and Inspection Service.)

25. BSE Surveillance Program   If 268,500 samples are obtained from a target population of 446,000, and if no positives were found after completion of the testing plan, then we can state that we are 99 percent confident that there were less than 5 positive animals in the target population

26. BSE Surveillance Program  Enhanced surveillance plan background, statistical inferences: http://cofcs66.aphis.usda.gov/lpa/ issues/bse/bse.htmlhttp://cofcs66.aphis.usda.gov/lpa/ issues/bse/bse.html

27. BSE Surveillance Program  As of October 10, 2004, no positive BSE test results in Enhanced Surveillance Program Cumulative “rapid” tests from June 1, 2004 – October 10, 2004: 79,766Cumulative “rapid” tests from June 1, 2004 – October 10, 2004: 79,766 Also 1,583 IHN negative testsAlso 1,583 IHN negative tests Currently: More than 5,000/weekCurrently: More than 5,000/week http://www.aphis.usda.gov/lpa/issues/b se_testing/test_results.htmlhttp://www.aphis.usda.gov/lpa/issues/b se_testing/test_results.html