# Presentation: 663875

## 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**

## 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

- 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

- Histology
- ImmunoHistochemistry (IHC)-an ELISA performed on a tissue section mounted on a slide
- “Rapid tests”- ELISA, EIA, Western Blot

## “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

## 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

- 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.

## 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

**Notes:**
Don’t regulate:
pregnancy tests (physiologic status)
food safety tests
IgG kits unless claim for Failure of Passive Transfer is made.

## 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

- 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

- 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)

**Notes:**
If kit recommends using serum, plasma, and whole blood, data from all tissue types must be submitted.
At least 2 serials in final format should be evaluated in at least 3 laboratories.
If target is Program Disease, NVSL must participate in prelicense evaluation.
If target is exotic (Foreign Animal Disease), get proper permits for importing samples.
Samples must be randomized and blinded.
Kits must be labeled “For Experimental Use Only” until licensed.

## 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

- 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)

| Manufacturer  | Kit Format  | Trade Name  | Date Approved  |
| --- | --- | --- | --- |
| Bio-Rad Laboratories,
(permittee: Bio-Rad,  France)  | ELISA | TeSeE®  | Permit issued:
March 17, 2004  |
| Abbott Laboratories
(permittee: Enfer,  Ireland)  | ELISA
 | Enfer BSE  | Permit issued:
May 28, 2004  |
| Roche Diagnostics  Corp.,
 (permittee  Prionics, 
 Switzerland)  | Western Blot | Prionics®- Check Western  | Permit issued:
May 28, 2004  |

## 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 Immunoassay)  | 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 
 | ELISA
BSE-Scrapie
Ag Test Kit | Prionics®- Check LIA  | Licensed:
September 1, 2004  |
| VMRD, Inc. 
For Export Only | IHC |  | License issued:
August 5, 2004  |

## 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 Wt
- Antibody binds “abnormal” PrP
- Indicator enzyme (on antibody) produces “visible” reaction:
- ELISA, EIA, chemiluminescence, solid precipitate (Western Blot, IHC)

## 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

## What does licensure provide?

- 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?  www.aphis.usda.gov/vs/cvb/

## BSE Surveillance Program

- 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

- 				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 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 Program

- 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 Program

- 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 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 Program

**Enhanced surveillance plan background, statistical inferences:**
**http://cofcs66.aphis.usda.gov/lpa/issues/bse/bse.html**

## 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: 79,766**
**Also 1,583 IHN negative tests**
**Currently:  More than 5,000/week**
**http://www.aphis.usda.gov/lpa/issues/bse_testing/test_results.html**** **