The Quest for New Prion Tests The next step in curbing the spread of transmissible spongi-
T
he furor over the 1986–1993 outbreak in the United Kingdom of bovine spongiform encephalopathy (BSE) and variant Creutzfeldt–Jakob disease (vCJD), which have devastated the cattle industry and killed ~110 people, is behind us. But it appears that these and related transmissible spongiform encephalopathies (TSEs), or prion protein (PrP) diseases, are here to stay. The first death from vCJD outside the United Kingdom was reported in Canada in 2002. Recent news headlines from Reuters question whether McDonald’s and Wendy’s hamburgers in Canada and Japan contain BSE-infected meat. And chronic wasting disease (CWD) has forced the quarantine and destruction of thousands of North American elk and deer. Many countries are developing policies to limit the spread of TSE-infected products—such as meat, dietary supplements that use antler velvet, and human blood—as
well as testing programs to monitor the incidence of TSEs. For example, surveillance for BSE is now shifting from passive case reporting to active case finding, says Paolo Pergami of the World Health Organization (WHO). Similarly, the U.S. Department of Agriculture (USDA) and the Department of Health and Human Services doubled the number of BSE tests in February 2002. One year later, the USDA plans to add 5 labs to the current list of 10 state and university laboratories contracted for CWD testing, says the department’s Lisa Ferguson. The development of quick, easy, standardized analytical tests, using technologies such as antibodies, capillary zone electrophoresis (CZE), and aptamers, is the key for aggressive TSE prevention. But these tests still pose technical challenges, and so far, no clear winner has emerged.
form encephalopathies is a test
that can be used on live animals, but there are still many challenges to overcome.
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Spreading TSEs The four most common TSEs are “mad cow disease” or BSE; vCJD, which infects humans; “mad elk disease” or CWD, which infects deer and elk; and scrapie, which infects sheep. And there are still other, less prominent TSE diseases, such as feline spongiform encephalopathy and transmissible mink encephalopathy. Although TSEs have been considered regional diseases, new cases in new places, such as Canada, indicate spreading. All European Union (EU) member countries, except Sweden and Austria, now report occurrences of BSE. In 2002, 15 European countries reported 1086 suspect animals; the most cases occurred in the United Kingdom (781) and France (91). The first case of BSE in Finland was reported in December 2001. Outside of the EU, Japan reported five cases of BSE as of September 2001. Likewise, although CWD has been localized in the western United States and Canada since 1967, recent spreading to the eastern United States threatens large numbers of white-tailed deer, says Michael Miller of the Colorado Department of Natural Resources. Previously, only ~2 dozen animals had been identified with CWD at farms in Colorado, Montana, Nebraska, Oklahoma, and South Dakota. In 2002, Wisconsin reported the first bucks infected with CWD, and then killed 25,000 deer to prevent it from spreading farther east. Similarly, the Canadian province Saskatchewan has slaughtered ~7800 animals in recent years to limit spreading.
Policies of containment Isolating and preventing TSE occurrences are global concerns. In Europe, the incidence of BSE and vCJD can be considered a burning house, says Martin Atterby of Altegen, Inc. “The development of TSE isolation and testing policies globally is like putting up fire extinguishers to prevent fires, but also trying to put out [existing] fires.” To prevent BSE and vCJD from reaching the United States, the U.S. Food and Drug Administration (FDA) initiated a deferral policy in May 2002 to prohibit people who have lived in France or the United Kingdom for a certain amount of time from donating blood. “Erring on the side of caution and keeping in mind what happened with the AIDS/HIV epidemic, the FDA’s May 2002 deferral policy will expand in October 2002 to include people who have lived any34 A
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where in Europe,” says Scott Caswell of the Americas Blood Organization (ABO), referring to the public’s loss of confidence in the FDA and the blood industry because of HIV-tainted transfusions in the 1980s. Similarly, the Colorado Department of Agriculture quarantined nine ranches to prevent CWD from spreading among wild populations of deer and elk. In April 2002, the USDA compensated Colorado farmers to kill elk that tested positive for, had been exposed to, or were suspected of CWD infection. USDA also reports that Korea has temporarily suspended the importation of U.S. and Canadian deer and elk and their products.
Pre- and postmortem tests Pre- and postmortem analytical TSE tests are crucial for new and expanding testing programs that complement the isolation policies. While the United States is doubling BSE and CWD surveillance testing, WHO plans to expand the monitoring of the incidence of BSE and causally linked vCJD beyond the existing programs in 22 countries. In August 2002, Saskatchewan declared mandatory CWD testing for farmed elk and deer in response to a Canadian’s death from vCJD. How many choices of tests there will be is still unknown. Currently, three EU-approved, antibody-based postmortem tests are available, whereas the U.S. gold standard is the immunohistochemistry (IHC) test, says Ferguson. Some tests apparently have reached a dead end; after years of research, Boehringer Ingelheim discontinued development of a live cattle BSE test that measures PrP on white blood cells. Nevertheless, several organizations are developing premortem (or liveanimal) tests, which are expected to prevent unnecessary slaughtering of healthy animals or aid in the diagnosis of human brain diseases, such as dementia. Although some of these tests are for symptomatic animals, others are meant for animals that do not yet exhibit symptoms. “The presymptomatic live-animal tests [are designed to help] keep herds clean, which saves . . . money,” says Atterby. One challenge is developing premortem tests that can be used on various samples, such as the urine or blood of sheep, cattle, deer, or humans. For example, the 14-3-3 cerebrospinal fluid immunoassay—named for the two proteins that it uses as surrogate markers for TSE—is used to diagnose symptomatic humans. How-
ever, recently published research from Jeffrey Tyler at the University of Missouri indicates that this assay does not have any value in the clinical diagnosis of scrapie in sheep. “It is not surprising that the 14-33 assay is less than optimal in sheep,” says Tyler, because the 14-3-3 proteins are not specific indicators of TSE infection, nor are they structural components of the prion. For sheep, the test of choice relies on IHC staining of third-eyelid-associated lymphoid tissue, Tyler says. Unfortunately, this test does not work for deer, nor does it work well for elk. Instead, CWD testing for symptomatic deer involves an IHC tonsil biopsy, says Elizabeth Williams of the University of Wyoming. But the tonsil biopsy, too, is limited. “PrP does not deposit in the tonsils or other peripheral lymphoid tissues of cattle with BSE, and the sensitivity [of the technique] in elk is much less than deer,” she explains. In addition, the tonsil test is not practical for widespread use because the animals have to be anesthetized. Although much of the TSE diagnostic research field is focused on live-animal tests, Robert Petersen of Prion Developmental Laboratories (PDL) says, “there will always be a requirement for postmortem tests.” Detecting disease PrP in dilute blood or urine samples is a challenge for the sensitivity of premortem tests. Therefore, he explains, a postmortem test using a brain sample, which has the highest titer of disease PrP, would be done after a premortem test on a blood or urine sample. Petersen also questions how animal husbandry will affect use of live animal tests: “How frequently are you testing? At what point are you testing—just before slaughter or at feeding? Running around in the mud with a cup for urine could look like the Three Stooges.” A need for easier postmortem tests is driving the technology beyond the current EU-validated tests, which must be performed in the laboratory. One new approach is immunochromatographic point-of-care (POC) postmortem TSE tests, which are being developed by PDL. These tests are designed to be used at a slaughterhouse or a hunter’s checkpoint, “as easy as a pregnancy test,” says Petersen. However, studies to evaluate three CWD postmortem antibody tests may not be finished until the end of 2002, according to Sandy Hayes of the USDA’s Agricultural Research Service (ARS). “I hope [this approach] works,” Williams comments, “but until we see some data, I’m not quite ready to hold my breath.”
New TSE tests The newer live-animal tests under development use a wide range of techniques, such as antibodies, CZE, and MS. Mary Jo Schmerr
of ARS–Iowa and her colleagues have been working on a CZE technique to separate a complex of abnormal PrP bound to fluorescent peptides from free PrP molecules in blood. CZE is sensitive, rapid, and, unlike HPLC, can be cleaned after each use to prevent carry-over contamination, according to Schmerr. “Several of the sheep [that tested] positive in the blood assay have developed clinical scrapie at 2–3 years of age, and there is excellent correlation with [the] tonsil biopsy assay,” she says. The Veterinary Laboratories Agency (VLA; United Kingdom) is further evaluating the technique for scrapie testing, and the University of Göttingen (Germany) is evaluating the technique for vCJD testing. The test also works for CWD. However, the researchers have not been able to test for BSE because it is not present in U.S. cattle, and they cannot import blood from infected animals, Schmerr notes. There is a lot of interest in protein-binding assays for TSE because such tests are used routinely in diagnostics. The binding of plasminogen factor to PrP in blood is the approach cho-
When it comes to validating new live-animal tests, one problem is the lack of a standard infectious blood sample to use as a benchmark.
sen by Adriano Aguzzi and colleagues from the University of Zurich (Switzerland) in collaboration with Abbott Laboratories. An enzyme-linked immunosorbent assay (ELISA) or striptest for urine samples may be possible from the work between scientists from Hadassah University (Israel), PrioSense (Israel), and Prionics (Switzerland). Still other tests, such as bloodbased antibody tests for vCJD and BSE, are being pursued by companies such as Caprion Pharmaceuticals, Inc., in collaboration with Ortho-Clinical Diagnostics and Idexx. However, experts still don’t know which test will have the best sensitivity and accuracy. One of the better-developed new tests is a blood-based, presymptomatic BSE assay that uses antibodies to detect a “piggyback gene” product, prionin-1 protein, instead of detecting PrP directly. The prionin-1 protein is expressed very J A N U A R Y 1 , 2 0 0 3 / A N A LY T I C A L C H E M I S T R Y
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One notable advantage of postmortem early in the prion disease pathway and is dormant until infection occurs. Piggyback genes are also found in other diseases, such as Alzheimer’s and breast cancer. Because a prionin-1 test would detect the presence or absence of disease potential—as opposed to indicating whether an animal will get sick—“to be confirmed negative is the asset of the test,” states Atterby. Farmers in Europe are voluntarily using the diagnostic assay to screen live cattle on a trial basis, in addition to the required postmortem tests. Regulatory authorities in a number of countries are currently evaluating two assays for BSE from Altegen that are already in use. The ELISA-based test is performed in a volume as small as 0.1 µL. The assay can be run in
