Diagnostic Tests and Surveillance

There is no approved laboratory test able to detect the disease in live animals.
Veterinary pathologists confirm BSE by microscopic examination of brain tissue.

• thus this is a postmortem test
• strain isotyping is determined by the time consuming injection of PrP^Sc from infected animal into a test species
• strain isotyping is analyzed using PrP^Sc collected from brain tissue of the test species by gel electrophoresis

Extensive testing of bovine materials using a mouse bioassay have failed to detect BSE in muscle, meat or milk; BSE infectivity has been detected only in brain, spinal cord and retina. As a test for BSE infectivity, intracerebral inoculation is 1000 times more sensitive than the standard mouse assay (SEAC, Nov. 18, 1996). No infectivity has been detected in lymph tissue or spleen using this more sensitive test. Lymph and spleen are suspect tissues because they are infective in sheep. Results using more sensitive tests on meat, milk, tallow and gelatin have not yet been reported.

Despite recent publicity about a possible detection method the test is not specific for BSE. The presence of 14-3-3 brain protein in cerebrosplinal fluid indicates nerve cell death and may result from inflammatory brain disease (BSE is not inflammatory), herpes, encephalitis or coronary infarction.

van Keulen et al. developed antibodies to selected synthetic PrP based peptides in 1995 . Schreuder et al. used these antibodies to detect PrP^sc in the tonsils of a group (n=55) of sheep naturally infected with scrapie (clinically positive for scrapie based on immunohistochemistry). This antibody was then used to detect scrapie in sheep during the preclinical phase of infection in susceptible sheep. Susceptible sheep (n=6) were homozygous for the PrP^VQ allele (valine at position 16, glutamine at 171). More resistant sheep were heterozygous possessing one PrP^VQ allele and one PrP^AR (alanine at position 136 and arginine at position 171). This form of heterozygosity has been shown to confer scrapie resistence in several breeds . Schreuder et al. were able to detect infection in less than half (10 mo) the expected incubation period (25 mo) using tonsil biopsy. The results of the biopsies were subsequently confirmed with immunostaining.

List of current tests available for diagnosis of TSE diseases are:

• bioassay - the long tedious and expensive exposure of animals to PrP^Sc and wait for expression of the disease
• analysis of tissues from affected animals (and of animals used for the bioassay) by:
  • histopathological examination of brain
  • SAF detection by electron microscopy
  • immunohistochemical demonstration of PrP^res
  • western blotting for PrP^res
  • immunoassay for PrP^res

Tests for diagnosis of TSE diseases must be validated for:

• feasibility
• standardization
• assay performance
• monitoring of performance
• maintenance and extension of validation criteria
• specificity - the assay must be specific for a given analyte or isoform of the analyte
• predictibility - the assay must be able to distinguish between normal and diseases tissue
  • this is not always the same as specificity
  • results that correlate with a disease state are not always predictive

Tests for TSE may be affected by:

• sampling
  • selection
    • which tissue is collected
    • sampling location within tissue
  • quality of the tissue sample collected
    • storage after collection and before analysis
    • time of collection relative to death of the affected animal
    • method of collection and tissue dissection

The development of sensitive, reliable tests for BSE infected animals, especially for those with preclinical condition, is a top priority as is the detection of PrP's in food.

Three diagnostic kits have been approved for use in Europe for the detection of BSE and a fourth has been submitted ot the European Commission (EC) for validation. All are antibody based.

Prionics AG (Zurich) has developed the Prionics Check Assay, will distribute through Roche Diagnostics (Basel, Switzerland)

• 100% accurate (no false positive or negatives)
  
• uses brain tissue (thus this is a postmortem test)
  
• Western blot - i.e. samples are digested with proteinase K whereby normal PrP^C is degraded but most of the PrP^Sc is only partially degraded - remaining proteins are separated by size using electrophoresis (recommend Novex Nu-PAGE gels), transferred to a membrane and then detected with prion-specific antisera linked to an enzyme for a chemiluminescent reaction
• each gel tests ~ a dozen samples
  
• 24 hr turnaround
  
• used since 1999
  
• detects BSE in preclinical animals
• use approved by the EC

working on blood tests for both BSE and CJD

CEA (Commissariat à l'Energie Atomique; the French Atomic Energy Commission) is distributing its test named Platelia™ through Bio-Rad

• 100% accurate (no false positive or negatives)
• uses brain tissue (thus this is a postmortem test)
  
• Sandwich DAS-ELISA Immunometric assay (EIA) - i.e. prions in bovine samples are digested with proteinase K whereby normal PrP^C is degraded but most of the PrP^Sc is only partially degraded -specific antibodies coat multiwell plates before the sample is applied and the amount of PrP^Sc bound by the antibodies is quantified with a second antibody linked to an enzyme to form a detectible product
  • ELISA = enzyme-linked immunosorbent assay
• each plate can test numerous samples (high throughput; several hundred per plate possible in this assay format)
  
• 4 hr turnaround
  
• potential for detecting BSE in preclinical animals
• use approved by the EC

Protherics has licensed its assay to Enfer Scientific (Tipperary, U.K.)

• 100% accurate (no false positive or negatives)
• uses spinal cord tissue (thus this is a postmortem test)
  
• ELISA - specific antibodies coat multiwell plates before the sample is applied and the amount of PrP^Sc bound by the antibodies is quantified with a second antibody linked to an enzyme to form a detectible product
  • ELISA = enzyme-linked immunosorbent assay
• each plate can test numerous samples (high throughput; 1000 tests/day per system possible)
  
• 4 hr turnaround
  
• potential for detecting BSE in preclinical animals
• use approved by the EC

EG&G Wallac (Gaithersburg, MD) has developed a test based on the Delfia system

• immunoassay
• uses CNS tissue (thus this is a postmortem test)
• each plate can test numerous samples (high throughput)
  
• not yet approved by the EC

Several other promising tests for live-animal screening are being developed.

The USDA has developed several tests for Scrapie and Chronic Wasting Disease. These new, as yet unapproved tests, offer promises towards detection of TSE in live animals.

• uses lymph tissue
• not yet developed for BSE

• uses eyelid tissue
• not yet developed for BSE

• uses blood
• not yet developed for BSE

The discovery that plasminogen can descriminate between PrP^C and PrP^Sc has lead to interest in developing tests for circulating PrP^{Sc based on this observations.} Several companies are actively persuing this market.

Similar to plasminogen binding, synthetic RNA aptamers have been shown to bind PrP^C . More recently, Stefan Weiss (CEA) has apparently selected a RNA aptamer that specifically binds PrP^Sc and not PrP^{C (reported at the PittCon 2001 Symposium, New Orleans, La, March 4-9,2001)}. Development of a diagnostic assay is promising.

A molecular marker (expression of an individual gene that shows a unique change) is depressed in spleens of mice, sheep and cattle with TSE compared to controls . The usefulness of this marker (erythroid differentiation-related factor; EDRF) as a preclinical indicator or TSE infection is being explored.

A novel assay that has the potential to indicate the presence of PrP^Res within tissues and to identify the strain of TSE has been studied. This assay (Protein-misfolding cyclic amplification; PMCA) is based on the in vitro conversion of PrP^C to PrP^Sc after exposure of PrP^C to PrP^Res and published by Saborio in Nature 411:810,2001.Click on the image below to learn more about this type of assay.

The PMCA assay.

A new application of the protease resistance test has shown that PrP^Res is present in the urine of hamsters, cattle and humans with TSEs but not in the urine of healthy individuals . See BSE_Urine page for further details. UPrP^Sc was detectable before clinical signs in experiments with hamsters, indicating a strong potential use of this information to develop an early marker of TSE transmission.

Several tests for PrP's present in food have been developed.

Bayer Corp. (Research Triangle Park, NC) has joined with BioReliance Corp. (Rockville, MD)

• the test is a Western blot assay to quantify the removal (clearance) of protease K resistant PrP^Sc from biological samples

Q-One Biotech (Glasgow, Scotland)

• the test is a Western blot assay to quantify the removal (clearance) of PrP^Sc from biological samples

ABC Research Corporation (Gainesville, FL)

• announced (April 2001) through the PRNewswire Interactive News Release that it will use a new test to detect meats contaminated with brain tissue
• the test was developed at the Colorado State University
• this ELISA tests for the presence of at neural protein: glial fibrillary acidic protein
• the test does not measure BSE infectivity but looks for contamination of meat with tissue from the CNS (brain and spinal cord)
• since CNS tissue is the major culprit in spreading BSE (not meat itself), eliminating meats with CNS contamination will greatly reduce the chance of BSE infection
• this assay will detect meat contaminated with normal CNS or BSE CNS
• the assay works with both raw and cooked meats

R-Biopharm Inc. (Marshall, MI)

• announced a test (RIDASCREEN® Risk Material 10/5) to detect meats contaminated with brain tissue
• the test was developed in cooperation with Dr. Glenn Schmidt at the Colorado State University
• this ELISA tests for the presence of at neural protein: glial fibrillary acidic protein (GFAP)
• detects 0.11% CNS in meat
• the test only takes 15minutes
• the test does not measure BSE infectivity but looks for contamination of meat with tissue from the CNS (brain and spinal cord)
• since CNS tissue is the major culprit in spreading BSE (not meat itself), eliminating meats with CNS contamination will greatly reduce the chance of BSE infection