USDA Licensed Test USDA Licensed Test Kits and Enhanced Kits and Enhanced Surveillance for BSESurveillance for BSE

Larry LudemannLarry Ludemann11 and and Lawrence ElskenLawrence Elsken22

Policy, Evaluation, and LicensingPolicy, Evaluation, and Licensing11

Inspection and ComplianceInspection and Compliance22

Center for Veterinary BiologicsCenter for Veterinary BiologicsVeterinary ServicesVeterinary ServicesAnimal and Plant Health Inspection Animal and Plant Health Inspection ServiceServiceUnited States Department of AgricultureUnited States Department of Agriculture510 S. 17510 S. 17thth Street, Suite 104 Street, Suite 104Ames, Iowa 50010 USAAmes, Iowa 50010 USA

The Causative Agent of TSEsThe Causative Agent of TSEs

PrionsPrions• Abnormally folded proteinAbnormally folded protein

Not a virus. Not a bacteria. No DNA or RNANot a virus. Not a bacteria. No DNA or RNA No known immune responseNo known immune response

• Two forms of prion proteinTwo forms of prion protein Normal prion protein (PrPc) – on most cellsNormal prion protein (PrPc) – on most cells Infectious Prion (PrPSc)Infectious Prion (PrPSc)

• Relatively resistant to disinfectants, sterilizations, Relatively resistant to disinfectants, sterilizations, proteinasesproteinases

• Accumulates and kills the cellsAccumulates and kills the cells

BSE: DiagnosticsBSE: Diagnostics

No effective live animal test for BSENo effective live animal test for BSE Need brain tissueNeed brain tissue Histopathology of brainHistopathology of brain IHC, WB, ELISA – PrPIHC, WB, ELISA – PrPresres

““Negatives” do not guarantee Negatives” do not guarantee absence of infectivity - tests are NOT absence of infectivity - tests are NOT intended as “Food Safety Tests”intended as “Food Safety Tests”

BSE Diagnostic TestsBSE Diagnostic Tests Histology ImmunoHistochemistry ImmunoHistochemistry

(IHC)-(IHC)-an ELISA performed on a tissue section mounted on a slide

“Rapid tests”- ELISA, EIA, Western Blot

““Rapid Tests”Rapid Tests”

As name implies, generally more rapid As name implies, generally more rapid than IHCthan IHC

Associated with occasional “false positive” Associated with occasional “false positive” initial reactions (especially ELISA)initial reactions (especially ELISA)

Western Blot Test lower throughput, Western Blot Test lower throughput, slower, and more involved than ELISAslower, and more involved than ELISA

All approved tests have very good All approved tests have very good sensitivity and specificitysensitivity and specificity

BUT - only serve as screening testsBUT - only serve as screening tests

IMMUNOHISTOCHEMISTRYIMMUNOHISTOCHEMISTRY

Adds immunological confirmation to Adds immunological confirmation to observation of lesionsobservation of lesions

Stain observed in specific (definitive) Stain observed in specific (definitive) anatomic areas associated with BSEanatomic areas associated with BSE

No “false positives”No “false positives” Generally requires several days to Generally requires several days to

complete testcomplete test

January 8, 2004January 8, 2004

CENTER FOR VETERINARY BIOLOGICS NOTICE NO. 04-02CENTER FOR VETERINARY BIOLOGICS NOTICE NO. 04-02

Subject: Diagnostic Test Kits for Bovine Spongiform EncephalopathySubject: Diagnostic Test Kits for Bovine Spongiform Encephalopathy

To: To: Biologics Licensees, Permittees, and ApplicantsBiologics Licensees, Permittees, and ApplicantsVeterinary Services Management TeamVeterinary Services Management TeamDirectors, Center for Veterinary BiologicsDirectors, Center for Veterinary BiologicsArea Veterinarians in Charge, VSArea Veterinarians in Charge, VSState VeterinariansState Veterinarians

I. PURPOSEI. PURPOSE

The purpose of this notice is to inform interested parties that the Center for Veterinary Biologics (CVB)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 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 (including “rapid tests”) intended as an aid in the diagnosis of bovine spongiform encephalopathy (BSE).(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 inthe 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.

Authorities, Regulations, & GuidanceAuthorities, Regulations, & Guidance Virus-Serum-Toxin Act: 21 USC Virus-Serum-Toxin Act: 21 USC

• Authorizes Secretary of Agriculture to Authorizes Secretary of Agriculture to regulate manufacture and distribution of regulate manufacture and distribution of biologics including diagnostics (test kits) biologics including diagnostics (test kits) intended for use in diagnosis of disease in intended for use in diagnosis of disease in animals.animals.

Code of Federal Regulations: Title 9 Code of Federal Regulations: Title 9 VS Memoranda VS Memoranda

• 800.73 (Immunodiagnostic Test Kits), 800.73 (Immunodiagnostic Test Kits), othersothers

What is a regulated test kit?What is a regulated test kit?

Diagnostic test kits contain: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 Used to diagnose existence of (usually) or susceptibility to disease agents of animalssusceptibility to disease agents of animals

Reagents are not regulatedReagents are not regulated

Prelicense Assessment of KitsPrelicense Assessment of Kits

Accuracy and precisionAccuracy and precision Diagnostic sensitivityDiagnostic sensitivity

• Dynamic rangeDynamic range• Analytical sensitivityAnalytical sensitivity

Diagnostic specificityDiagnostic specificity Ruggedness, repeatability, suitabilityRuggedness, repeatability, suitability Predictive values for specific disease Predictive values for specific disease

prevalence (especially for Program Diseases)prevalence (especially for Program Diseases)

Prelicense ValidationPrelicense Validation Test large number of known positive and Test large number of known positive and

known negative animal samples covering a known negative animal samples covering a range of reactions and all sample typesrange of reactions and all sample types

Determine onset of detection of diseaseDetermine onset of detection of disease Evaluate (in at least 3 laboratory settings)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 Authorization from USDA per 9 CFR 103.3 (experimental biological products)(experimental biological products)

Manufacturing controlsManufacturing controls Aim is to minimize within-serial and serial-to-Aim is to minimize within-serial and serial-to-

serial variation by ensuring consistency of serial variation by ensuring consistency of production – production – Outline of ProductionOutline of Production

Master Seed/Master Cell conceptMaster Seed/Master Cell concept Validation of new lots of antibody/antiseraValidation of new lots of antibody/antisera Serial release testing of serum or antigen Serial release testing of serum or antigen

panel with range of reactivities and cross-panel with range of reactivities and cross-reactivitiesreactivities• Serial release panel is critical to minimize variation Serial release panel is critical to minimize variation

in test resultsin test results• Panel includes range of reactivityPanel includes range of reactivity

Other RequirementsOther Requirements Inspection of manufacturing facilities Inspection of manufacturing facilities

and other licensed sites (e.g., and other licensed sites (e.g., Permittee quarantine sites)Permittee quarantine sites)

Prelicense serial testingPrelicense serial testing Label approvalLabel approval

USDA Approved Bovine Spongiform Encephalopathy Antigen Test KitsUSDA Approved Bovine Spongiform Encephalopathy Antigen Test Kits(Permits, as of September 30, 2004)(Permits, as of September 30, 2004)

Manufacturer Manufacturer Kit Format Kit Format Trade Name Trade Name Date Approved Date Approved

Bio-Rad Bio-Rad Laboratories,Laboratories,

(permittee: Bio-Rad, (permittee: Bio-Rad, France) France)

ELISAELISA TeSeE® TeSeE® Permit issued:Permit issued:

March 17, 2004 March 17, 2004

Abbott LaboratoriesAbbott Laboratories

(permittee: Enfer, (permittee: Enfer, Ireland) Ireland)

ELISAELISA Enfer BSE Enfer BSE Permit issued:Permit issued:

May 28, 2004 May 28, 2004

Roche Diagnostics Roche Diagnostics Corp.,Corp.,

(permittee Prionics, (permittee Prionics,

Switzerland) Switzerland)

Western BlotWestern Blot Prionics®- Prionics®- Check Check Western Western

Permit issued:Permit issued:

May 28, 2004 May 28, 2004

USDA Approved Bovine Spongiform Encephalopathy Antigen Test KitsUSDA Approved Bovine Spongiform Encephalopathy Antigen Test KitsLicenses, as of (September 30, 2004)Licenses, as of (September 30, 2004)

Manufacturer Manufacturer Kit Format Kit Format Trade Name Trade Name Date Approved Date Approved

IDEXX Laboratories, IDEXX Laboratories, Inc. Inc.

EIA (Enzyme EIA (Enzyme ImmunoassaImmunoassay) y)

HerdChek- HerdChek- BSE BSE

License issued:License issued:

March 17, 2004 March 17, 2004

Pierce Pierce Biotechnology, Inc., Biotechnology, Inc.,

(Produces kit for (Produces kit for Prionics) Prionics)

ELISAELISA Prionics®- Prionics®- Check LIA Check LIA

Licensed:Licensed:

September 1, September 1, 2004 2004

Pierce Pierce Biotechnology, Inc., Biotechnology, Inc.,

For Export OnlyFor Export Only

ELISAELISA

BSE-ScrapieBSE-Scrapie

Ag Test KitAg Test Kit

Prionics®- Prionics®- Check LIA Check LIA

Licensed:Licensed:

September 1, September 1, 2004 2004

VMRD, Inc. VMRD, Inc.

For Export OnlyFor Export Only

IHCIHC License issued:License issued:

August 5, 2004 August 5, 2004

Licensed BSE Test Kits:Licensed BSE Test Kits:Format or TechniqueFormat or Technique

All use obex tissueAll use obex tissue Purify normal and abnormal PrPPurify normal and abnormal PrP Treatment to “remove” normal PrPTreatment to “remove” normal PrP

• Western Blot – separate protein by Mol WtWestern Blot – separate protein by Mol Wt Antibody binds “abnormal” PrPAntibody binds “abnormal” PrP Indicator enzyme (on antibody) produces Indicator enzyme (on antibody) produces

“visible” reaction: “visible” reaction: • ELISA, EIA, chemiluminescence, solid precipitate ELISA, EIA, chemiluminescence, solid precipitate

(Western Blot, IHC)(Western Blot, IHC)

Diagnostics: Under DevelopmentDiagnostics: Under Development

““Cowside” or on-site testsCowside” or on-site tests Capillary electrophoresis – bloodCapillary electrophoresis – blood Blood – other methodologies - Blood – other methodologies -

CaptureCapture Urine – UPrPscUrine – UPrPsc Increased sensitivity – cyclic Increased sensitivity – cyclic

amplificationamplification Non-PrP markers – “Upregulated” Non-PrP markers – “Upregulated”

MarkersMarkers

What does licensure provide?What does licensure provide? USDA controlled manufacturing of all USDA controlled manufacturing of all

components and kitscomponents and kits• Minimal variation within and between serialsMinimal variation within and between serials

USDA evaluation of test kit before and after USDA evaluation of test kit before and after licensure (serial release)licensure (serial release)

Monitoring of field performance and Monitoring of field performance and investigations investigations • CVB-Inspection and Compliance 800-752-6255CVB-Inspection and Compliance 800-752-6255

Ongoing facilities inspectionsOngoing facilities inspections Questions? Questions? www.aphis.usda.gov/vs/cvb/www.aphis.usda.gov/vs/cvb/

BSE Surveillance ProgramBSE Surveillance Program

Expanded or Enhanced surveillance Expanded or Enhanced surveillance began June 1, 2004. Purpose:began June 1, 2004. Purpose:• Determine if BSE is present in U.S. cattle Determine if BSE is present in U.S. cattle

populationpopulation• Determine if risk-management policies Determine if risk-management policies

adequateadequate For animal health – NOT food safetyFor animal health – NOT food safety

EU testing: Jan-Dec 2001EU testing: Jan-Dec 2001

SamplesSamples PositivePositive % % positivepositive

RiskRisk 776,266 776,266 756756 .097%.097%

Em slaugEm slaug 96,774 96,774 324324 .335%.335%

CNSCNS 27,991 27,991 3434 .121%.121%

FallenFallen 651,501 651,501 397397 .061%.061%

HealthyHealthy 7,677,5767,677,576 279279 .0036%.0036%

SuspectSuspect 3,267 3,267 11031103 33.76%33.76%

BSE Surveillance ProgramBSE 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.

BSE Surveillance ProgramBSE Surveillance Program

US high-risk population estimate: US high-risk population estimate: about 446,000 about 446,000 • 246,000 on-farm deaths with 246,000 on-farm deaths with

unexplained causes or causes consistent unexplained causes or causes consistent with BSEwith BSE

• 200,000 ante-mortem condemnations 200,000 ante-mortem condemnations by FSISby FSIS

• 129 (2004 data) Foreign Animal Disease 129 (2004 data) Foreign Animal Disease investigations for CNS disease investigations for CNS disease

BSE Surveillance ProgramBSE Surveillance Program The majority of samples from the following The majority of samples from the following

categories:categories:• Nonambulatory cattle; Nonambulatory cattle; • Cattle exhibiting signs of a central nervous Cattle exhibiting signs of a central nervous

system disorder; system disorder; • Cattle exhibiting other signs that may be Cattle exhibiting other signs that may be

associated with BSE, such as emaciation or associated with BSE, such as emaciation or injury; and injury; and

• Dead cattle. Dead cattle. USDA personnel will also sample all cattle USDA personnel will also sample all cattle

condemned on ante-mortem inspection by condemned on ante-mortem inspection by USDA’s Food Safety and Inspection Service.)USDA’s Food Safety and Inspection Service.)

BSE Surveillance ProgramBSE 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

BSE Surveillance ProgramBSE Surveillance Program

Enhanced surveillance plan Enhanced surveillance plan background, statistical background, statistical inferences:inferences:• http://cofcs66.aphis.usda.gov/lpa/http://cofcs66.aphis.usda.gov/lpa/

issues/bse/bse.htmlissues/bse/bse.html

BSE Surveillance ProgramBSE Surveillance Program

As of October 10, 2004, no positive As of October 10, 2004, no positive BSE test results in Enhanced BSE test results in Enhanced Surveillance ProgramSurveillance Program• Cumulative “rapid” tests from June 1, Cumulative “rapid” tests from June 1,

2004 – October 10, 2004: 79,7662004 – 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/http://www.aphis.usda.gov/lpa/issues/

bse_testing/test_results.htmlbse_testing/test_results.html