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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

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Usda licensed test kits and enhanced surveillance for bse

USDA Licensed Test Kits and Enhanced Surveillance for BSE

Larry Ludemann1 and

Lawrence Elsken2

Policy, Evaluation, and Licensing1

Inspection and Compliance2

Center for Veterinary Biologics

Veterinary Services

Animal and Plant Health Inspection Service

United States Department of Agriculture

510 S. 17th Street, Suite 104

Ames, Iowa 50010 USA

The causative agent of tses
The Causative Agent of TSEs

  • Prions

    • Abnormally folded protein

      • Not a virus. Not a bacteria. No DNA or RNA

      • No known immune response

    • Two forms of prion protein

      • Normal prion protein (PrPc) – on most cells

      • Infectious Prion (PrPSc)

        • Relatively resistant to disinfectants, sterilizations, proteinases

        • Accumulates and kills the cells

Bse diagnostics
BSE: Diagnostics

  • No effective live animal test for BSE

  • Need brain tissue

  • Histopathology of brain

  • IHC, WB, ELISA – PrPres

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

Bse diagnostic tests
BSE Diagnostic Tests

  • Histology

  • ImmunoHistochemistry (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 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


  • 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

Usda licensed test kits and enhanced surveillance for bse

January 8, 2004


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


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


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.

Authorities regulations guidance
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.

  • Code of Federal Regulations: Title 9

  • VS Memoranda

    • 800.73 (Immunodiagnostic Test Kits), others

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

  • Diagnostic test kits contain:

    • All unique reagents required to conduct test

    • Complete instructions for the test

    • Instructions for interpretation of test results

    • Claims, uses, and limitations

  • Used to diagnose existence of (usually) or susceptibility to disease agents of animals

  • Reagents are not regulated

Prelicense assessment of kits
Prelicense Assessment of Kits

  • Accuracy and precision

  • Diagnostic sensitivity

    • Dynamic range

    • Analytical sensitivity

  • Diagnostic specificity

  • Ruggedness, repeatability, suitability

  • Predictive values for specific disease prevalence (especially for Program Diseases)

Prelicense validation
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)

    • Well-characterized patient samples

    • Compared to “Gold” standard assay

    • Suitability of test kit

    • Adequacy of instructions

  • Authorization from USDA per 9 CFR 103.3 (experimental biological products)

Manufacturing controls
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 results

    • Panel includes range of reactivity

Other requirements
Other Requirements

  • Inspection of manufacturing facilities and other licensed sites (e.g., Permittee quarantine sites)

  • Prelicense serial testing

  • Label approval

Usda approved bovine spongiform encephalopathy antigen test kits permits as of september 30 2004
USDA Approved Bovine Spongiform Encephalopathy Antigen Test Kits(Permits, as of September 30, 2004)

Usda approved bovine spongiform encephalopathy antigen test kits licenses as of september 30 2004
USDA Approved Bovine Spongiform Encephalopathy Antigen Test KitsLicenses, as of (September 30, 2004)

Licensed bse test kits format or technique
Licensed BSE Test Kits: KitsFormat or Technique

  • All use obex tissue

  • Purify normal and abnormal PrP

  • Treatment to “remove” normal PrP

    • Western 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)

Diagnostics under development
Diagnostics: Under Development Kits

  • “Cowside” or on-site tests

  • Capillary electrophoresis – blood

  • Blood – other methodologies - Capture

  • Urine – UPrPsc

  • Increased sensitivity – cyclic amplification

  • Non-PrP markers – “Upregulated” Markers

What does licensure provide
What does licensure provide? Kits

  • USDA controlled manufacturing of all components and kits

    • Minimal 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 800-752-6255

  • Ongoing facilities inspections

  • Questions?

Bse surveillance program
BSE Surveillance Program Kits

  • Expanded or Enhanced surveillance began June 1, 2004. Purpose:

    • Determine if BSE is present in U.S. cattle population

    • Determine if risk-management policies adequate

  • For animal health – NOT food safety

Eu testing jan dec 2001
EU testing: Jan-Dec 2001 Kits

Samples Positive % positive

Risk 776,266 756 .097%

Em slaug 96,774 324 .335%

CNS 27,991 34 .121%

Fallen 651,501 397 .061%

Healthy 7,677,576 279 .0036%

Suspect 3,267 1103 33.76%

Bse surveillance program1
BSE Surveillance Program Kits

  • 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 program2
BSE Surveillance Program Kits

  • US high-risk population estimate: about 446,000

    • 246,000 on-farm deaths with unexplained causes or causes consistent with BSE

    • 200,000 ante-mortem condemnations by FSIS

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

Bse surveillance program3
BSE Surveillance Program Kits

  • The majority of samples from the following categories:

    • Nonambulatory cattle;

    • Cattle exhibiting signs of a central nervous system disorder;

    • Cattle exhibiting other signs that may be associated with BSE, such as emaciation or injury; and

    • Dead cattle.

  • USDA personnel will also sample all cattle condemned on ante-mortem inspection by USDA’s Food Safety and Inspection Service.)

Bse surveillance program4
BSE Surveillance Program Kits

  • 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 program5
BSE Surveillance Program Kits

  • Enhanced surveillance plan background, statistical inferences:


Bse surveillance program6
BSE Surveillance Program Kits

  • As of October 10, 2004, no positive BSE test results in Enhanced Surveillance Program

    • Cumulative “rapid” tests from June 1, 2004 – October 10, 2004: 79,766

    • Also 1,583 IHN negative tests

    • Currently: More than 5,000/week