News of the first known case of bovine spongiform encephalopathy (BSE, or “mad cow disease”) in the United States on December 23 shook the country's beef industry. But if ranchers were caught unaware, it wasn't because of a lack of available diagnostic procedures.
All testing for BSE now occurs postmortem, using tissue from the obex region of the brain stem. The most sensitive test, which is also the lengthiest, is the mouse bioassay, in which suspect brain tissue is injected into the brain of a mouse, and 6 months to 1 year later, the mouse is killed and its brain examined to see if it has developed disease. “Very conclusive ultimately, but not very rapid to say the least,” Brad Crutchfield, vice president of the life sciences group at Bio-Rad Laboratories, told
At the National Veterinary Services Laboratory, a US Department of Agriculture (USDA) testing facility in Ames, Iowa, researchers screen samples for BSE using immunohistochemistry, which is the gold standard, according to a USDA spokesperson. In this case, brains are sectioned, stained for structure and with an antibody to highlight the prion protein, and examined under a microscope for the telltale spongiform morphology. The benefit of this approach, Crutchfield said, is that the tissue retains its shape, so a pathologist can make a definitive determination.
Though faster by far than the bioassay, immunohistochemistry is still lengthier and more tedious than most common diagnostic tests. That's because prion diseases like BSE present particular challenges to diagnostics developers. The usual indirect methods—looking for antibodies to the infectious agent or evidence of a viral or bacterial genome by polymerase chain reaction—won't work because the infectious agent is a conformational isomer of a “self” protein. As a result, prion tests are direct—they look for the presence of abnormal prion protein using antibodies.
The European Commission has approved five tests from four vendors for rapid, postmortem diagnostic testing for BSE in its member states. The United States, in contrast, currently has no licensed rapid tests. All of the European Commission–licensed tests employ antibodies against the prion protein, and four of them rely on the fact that the abnormal protein is resistant to digestion by proteinase K, while the normal isoform is not. The four are Bio-Rad Laboratories' TeSeE, Prionics' Check Western, Abbott/Enfer TSE, and the Prionics Check LIA. The Bio-Rad, Prionics Check LIA, and Enfer tests are all enzyme-linked immunosorbent assay based; they differ in the types of antibodies and the detection method used. The Prionics Check Western uses Western blotting to identify proteinase-resistant prion protein.
The fifth test, the InPro Conformation-Dependent Immunoassay (CDI-5), utilizes a “conformation dependent immunoassay” to quantify abnormal prion protein without proteinase treatment. This test compares the antigen/antibody binding affinities of prions in their native and unfolded states.
Experts say that the future of BSE testing lies in two related areas: developing a live animal diagnostic test and boosting sensitivity. The availability of a live animal test—one that could detect infectious particles in blood or urine—could prevent the wholesale slaughter of a herd simply because of one infected animal. And if such a test was similarly effective for humans, it could be used to screen blood banks and corneal donors.
But existing methods simply are not sensitive enough to detect infectious agents in these samples, Richard Johnson, a professor of neurology, microbiology, and neuroscience at Johns Hopkins, told
And Johnson cites other unresolved questions, such as the detailed structure of the prion protein, the method of conversion from normal protein to infectious particle, and even the definition of an “infectious unit.” All of these issues present complicated problems in diagnostics development, said Johnson, who chaired a committee on transmissible spongiform encephalopathies at the National Academy of Sciences that recently issued a report on prion research.
Nevertheless, at least 50 companies are in the hunt for a better test, according to a recent New York Times report. One of those companies, GeneThera, announced on January 6 that it has “developed a commercially viable system for the collection of blood and the testing of live cattle for mad cow disease.” But with no published reports attesting to the validity of the company's claims, experts like Johnson remain skeptical.
Developing the required sensitivity will not come from incremental improvements in existing methods, he said, but from some entirely new, conceptual approach. And there's no guarantee researchers will ever find one. “I'm not sure this is a doable project,” Johnson said. “We have to stand up and say this is not as simple as landing a man on Mars.”