It’s a Mad, Mad, Mad, Mad Cow Test

The title and subject matter for this month’s column were inspired by a symposium presented at PittCon 2001, the analytical laboratory instruments show that was held in New Orleans, La., March 4-9. “It’s a Mad, Mad, Mad, Mad Cow: Detection and Quantification of Prions” was arranged by Brian K. Nunnally of Eli Lilly and Co. and presented some of the latest information on testing for bovine spongiform encephalopathy (BSE).

BSE is known as “mad cow disease.” The chronic, degenerative disease affects the central nervous system of cattle. Worldwide there have been more than 178,000 cases since the disease was first diagnosed in 1986 in Great Britain. The disease has also been confirmed in native-born cattle in other European countries. However, over 95% of all BSE cases have occurred in the United Kingdom. BSE is not known to exist in the United States, although the similar chronic wasting disease is found in wild elk and deer in the U.S.

BSE belongs to the family of diseases known as the transmissible spongiform encephalopathies. These diseases are caused by a transmissible agent which is yet to be fully characterized. The diseases share the following characteristics: (1) a prolonged incubation period of months or years; (2) a progressive debilitating neurological illness which is always fatal; (3) examination of extracts of affected brain tissue by electron microscopy reveal the presence of scrapie associated fibrils; (4) pathological changes such as vacuolization appear in the central nervous system; and (5) the transmissible agent elicits no detectable specific immune response in the host.

A proteinase-resistant form of the proteins called prions have been proposed by Stanley Prusiner, the Institute for Neurodegenerative Diseases, University of Calif., as the transmissible agent that causes BSE and other similar neurodegenerative diseases. Prusiner, who made a presentation at PittCon 2001, was the recipient of the 1997 Nobel Laureate in Medicine for his discovery of prions (PrP)–a new biological principle of infection.

According to Prusiner, prions are the cause of Creutzfeldt-Jakob disease of humans, scrapie of sheep, and BSE of cattle. He outlined four new concepts that have emerged from studies of prions. First, prions are the only known example of infectious pathogens that are devoid of nucleic acid. All other infectious agents possess genomes composed of either RNA or DNA that direct the synthesis of their progeny. Second, prion diseases may be manifested as infectious, genetic, and sporadic disorders. This means that prion diseases have a wide range of clinical manifestations. Third, prion diseases result from the accumulation of PrP Sc, the conformation of which differs substantially from that of its precursor PrP C. Fourth, PrP Sc can exist in a variety of different conformations, each of which seems to manifest in a specific disease phenotype.

One of the difficulties encountered when testing for BSE is that the prions elicit no detectable specific immune response in the host. This has hindered the development of a live animal diagnostic test. Currently, there is no test to detect the disease in a live animal. The only means of diagnosis in live animals at present is by clinical signs by veterinarians. After death, microscopic examination of brain tissue is the primary laboratory method used to confirm a diagnosis of BSE. In addition to microscopic examination, there are several techniques used to detect the partially proteinase-resistant form of the prion protein, PrP Sc. These techniques are the Western blot test and immunocytochemistry.

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These diagnostic tests are based on the interaction of specific antibodies with the disease-causing protein, PrP Sc. The abnormal form of the protein, PrP Sc is more resistant to degradation by enzymes and can be differentiated from normal form, PrP C.

The Western blot test was developed by a Swiss company, called Prionics, (www.prionics.ch). The test is used to detect PrP Sc protein purified from treated brain material (obtained post mortem) by its molecular weight and reaction with specific antibodies. The company is developing the technique to increase the sensitivity and allow its application to tissues other than brain tissue, which may allow it to be used on live animals. Currently, only brain material testing has been fully validated.

The other technique is immunocytochemistry. It also is based on the detection of PrP Sc using specific antibodies. As in the Western blot test, post-mortem brain material is required. This test is performed directly on the tissue section and does not involve any protein purification steps. Immunocytochemistry can also work on other tissues, such as the third eyelid or tonsil of sheep. Research in Europe is trying to determine exactly how useful it would be to take biopsies of these tissues while the animal is still alive as a means of screening flocks. They are not perfect because it looks as if the genetic makeup of the livestock will influence how soon after infection one of these tissues becomes positive for abnormal PrP Sc.

Besides the tests provided by Prionics, the European Union has evaluated others. These include tests manufactured by CEA, Enfer Scientific, and the Veterinary Laboratories Agency. All the tests have only been evaluated on adult cattle showing clinical signs of BSE and confirmed to have the disease. They have not yet been proved to be effective in detecting infected animals during the long incubation period, except perhaps in the last few months before disease becomes apparent.

CEA, a research group in France, has produced a test based on the sandwich immunoassay technique, which is an adapted ELISA test. It also tests brain tissue collected post-mortem. Because it appears to be more sensitive than other tests, it has been used to see if it can detect abnormal protein in the wall of the small intestine of experimentally infected calves. It has also been used to see if it can detect abnormal protein in the brain and spinal cord of experimentally infected animals at an earlier stage than other tests. Stefan Weiss of CEA related at PittCon that the company has selected an RNA aptamer against purified PrP Sc preparations which interacts specifically with PrP Sc. The aptamer recognizes abnormal prions but not the normal type, PrP C. Weiss suggested that the aptamer could act as a powerful tool in diagnosis of BSE.

Enfer Scientific, Tipperary, Ireland, has developed an enzyme linked immunosorbent assay or ELISA test for BSE. The antibody used by the company was manufactured by another British company called Proteus. The assay, like many ELISAs, is very sensitive, and allows a rapid turnaround time with results usually available within 24 hr. The test can be done in high volumes. The company has been commercially testing spinal cord from cattle slaughtered for human consumption in Ireland for a couple of years, and has also tested cattle from BSE affected herds that have been killed by the Irish Authorities. The labs in Ireland test 2,500 samples per day with overnight turnaround. Abbott Laboratories in the U.S. has entered into an agreement with Enfer Scientific Ltd. for the marketing and distribution of two diagnostic tests used to detect BSE in cattle.

An assay, developed by U.S. Dept. of Agriculture/Agricultural Research Service chemist Mary Jo Schmerr, can detect the presence of abnormal prions in the blood of animals and humans. Schmerr, who works at ARS’ National Animal Disease Center in Ames, Iowa, and Andrew Alpert of PolyLC, Inc. in Columbia, Md., are co-inventors of the assay. ARS and Fort Dodge Animal Health of Fort Dodge, Iowa, have signed a Cooperative Research and Development Agreement (CRADA) to develop a test kit for use in diagnosing TSEs in animals.

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Another area of research interest are urine tests. These tests in live animals involve the detection of disease-specific physiological or metabolic markers. These are secondary substances produced as a result of infection. Such markers, apparently related, have been identified in serum and in urine. Whether or not they are specific to BSE or scrapie remains to be proven. Changes have been detected in the constituents of urine of CJD patients, sheep with scrapie and cattle with BSE. Research is being done to determine when the changes occur during the long incubation period of BSE.

None of the tests currently being developed have the prospect of providing reassurance that an animal or carcass is BSE free, only that evidence of infection can or cannot be detected at the time of sampling. Most of the claims for diagnostic tests for BSE have been made on the basis of checks on cattle that are at the end of their incubation period (clinically affected) or on cattle that may never have been infected. According to the European Union testing commission, it is essential that BSE tests are validated on animals that are known to be infected, but which are only part way through their incubation period, before they can legitimately claim to be of value for testing of cattle while alive on farm or dead after slaughter. Another factor in the use of the tests is which tissue should be tested. Most of the immunological tests that can detect abnormal PrP work best on brain tissue. A test which works on the brain of a clinically affected cow may not work as well when used with other body tissue.

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by JAMES GIESE
Internet Editor