Quickening the Diagnosis of Mad Cow Disease

Quickening the Diagnosis of Mad Cow Disease
New tests for prions shorten the time frame for detection

June 11, 2001 The Scientist (Volume 15[12]:22) by Laura DeFrancesco

Europeans have destroyed 4.5 million cows since 1996, the height of the epidemic in the United Kingdom, because they were believed to be at risk for mad cow disease (bovine spongiform encephalopathy, or BSE).1 Necropsies, however, showed that only a few hundred thousand of them actually were infected.2 Had a diagnostic test for mad cow disease existed when this epidemic erupted, these numbers might have been different. But no such test did exist. The only available assay was a bioassay in which host animals are inoculated with material from suspected animals, and some number of months to years later, an answer appears.

Now basic research on prions, the infectious proteinaceous particles that cause mad cow disease and other cases of transmissible spongiform encephalopathy (TSEs), has caught up with the disease. The latest batch of assays validated for use by the European Commission cuts the time down from months to hours. And even faster, more sensitive tests, including one that purports to detect single prion aggregates,3 are on the horizon. Millions of tests for the BSE prion have now been sold throughout Europe, and as a result, wholesale slaughter of herds is no longer taking place. Instead, animals en route to slaughter for food use are tested for BSE prior to their entering the food chain--testing up to 100 percent of feed animals in Germany, for example.

Prions are found throughout virtually all species, and for the most part are innocuous. However, certain events, such as a mutation or exposure to infected material, causes a conformational change in the protein that makes it deadly and infective. This change, from an a-helical form (PrPc) to a -flat sheet (PrPsc), is all that distinguishes the normal prion from the abnormal, infective version. The identity of the two forms, in terms of amino acid composition and even post-translational modifications, has made detecting and quantifying abnormal prions a challenge.

The first real breakthrough in prion research came in 1997, when Michael Scott and colleagues from the Institute for Neurodegenerative Diseases (directed by Nobel laureate Stanley Prusiner) at the University of California, San Francisco, created a strain of transgenic (Tg) mice that overexpressed the bovine prion PrP protein.4 The wild mouse strain, without the extra gene, was relatively resistant to BSE. Adding the bovine gene resulted in the mice not only becoming sensitive, but much more so than the cows, the natural host. Tg mice die within a few months of infection, rather than the years it takes for the disease to appear in a cow. This was an important first step in designing experimental tools for studying BSE, and for determining the precise prion concentration in infected cows. According to Prusiner colleague Jiri Safar, the wild mice test underestimates the prion concentration by as much as 1,000-fold, which means the exposure of general population to BSE prions in the United Kingdom was probably much higher than previously thought.

Serious and exhaustive screening efforts, however, require even speedier tests, preferably ones that can test animals by the thousands. Currently, several immunodiagnostics tests, which can distinguish the normal from the abnormal prion, are filling this need. The European Commission has validated three such tests: a western blot assay developed by the Zurich, Switzerland-based Prionics AG; an ELISA assay made by Enfer Scientific Ltd. of Newbridge, Ireland; and a sandwich immunoassay developed by the French agency CEA and marketed by Bio-Rad of Hertfordshire, U.K., the most sensitive one to date.5

A Conformation-Dependent Assay

While western blotting and other immunodetection methods can detect abnormal prion protein, they are laborious, and only work on postmortem material. The hunt is on for assays that are sensitive enough to detect prions in central nervous system fluids and other biomaterials that could potentially be used in clinical diagnosis. To this end, Safar and Prusiner at UCSF are looking for antibodies that can directly distinguish normal from abnormal protein. Capitalizing on the fact that the normal and abnormal proteins, because of differences in conformation, have different epitopes exposed, they have isolated antibodies that recognize epitopes exposed in the normal prion, but are hidden in the abnormal. They found that with such antibodies, they cannot only quantify abnormal protein but also differentiate eight different prion strains, each with a particular and different conformation.6

Mad cow disease, one of a group of degenerative diseases of the brain, has created not just scientific problems, but economic, ecological--think 4.5 million carcasses--and political ones as well. Designing surveillance programs present their own challenges, as farmers stand to lose their livelihood in the process. At the PittCon 2001 meeting in New Orleans this past March, Bruno Oesch of the University of Zurich described how the approaches to surveillance in different European countries has had an impact on disease reporting. In Switzerland, for example, every living thing is slaughtered on farms (including pets) where infected animals are found. Predictably, the number of reported cases is low. In contrast, in Portugal, where farmers are compensated more than 125 percent of the herd's value, BSE reporting was considerably higher.

In this country, the government has taken several measures to keep mad cow disease out; the most recent being the ban on blood donations from people who spent more than six months in the United Kingdom during the height of the epidemic. There are no reported cases of BSE in the United States, nor of new variant Creutzfeldt-Jakob disease (nvCJD), the human prion disease that has been shown conclusively to originate from tainted beef7 and has so far killed roughly 100 Britons.

U.S. Efforts Falling Short?

The European experience shows that wherever people have looked seriously, BSE has been found. But surveillance efforts in the United States seem modest compared to those in the European Union (EU); the U.S. Department of Agriculture tests but a fraction of animals each year--2,700 last year out of some 3.4 million [I assume the author means 34 million (36 million were kiilled in 1999)--BSE coordinator] cattle slaughtered for food during the same time period.8 And while the EU requires all countries to set up mandatory surveillance centers for CJD, so far in this country, it is being done on a voluntary basis.

Since the United States seems to be BSE-free, one might wonder why surveillance is necessary. Says Pierluigi Gambetti, director of the National Prion Disease Pathology Surveillance Center at Case Western Reserve University: "It is extremely important to know whether we have a vCJD or not. Other countries would resent it if the U.S. were careless. With communication and exchange of people and travel, this could put other countries at risk."

Gambetti's surveillance agency also acts as a repository of tissue that is available to researchers. He sees it as "an invaluable resource [without which] the U.S. would be put at a tremendous disadvantage in research on these diseases as compared to EU, which has been banking tissues for years." Furthermore, while BSE has not been found here, other TSEs have. Chronic wasting disease (CWD), which infects deer and elk, is endemic in Colorado, where millions of cattle are being raised. One widely held theory on the origins of the BSE epidemic in the United Kingdom proposes that it arose from scrapie-infected sheep material that got into cattle feed. If true, this would mean that prions can cross species barriers. It's only a small leap to suppose that the CWD prion could find its way into the food chain in the United States.

Although no one is ringing alarm bells yet, whether mad cow disease poses a serious threat to the United States is still largely unknown. Furthermore, while the BSE epidemic appears to be under control in the United Kingdom, where most of the mad cow cases have occurred, it's unclear where it will end in Europe and what the impact on people will be because the incubation period for mad cow is about five years. Some epidemiologists estimated that the cases of nvCJD in the United Kingdom could run into the hundreds of thousands, based on the numbers of people affected so far. Recent work showing that animals with different genetic backgrounds have varying incubation times suggest that an estimate based on the reported cases, all of which had the same genetic background, may be optimistic and that what has appeared to date might only be the tip of the iceberg.9 Laura DeFrancesco can be contacted at defrancesco1@earthlink.net.


1. P. Brown et al., "Bovine spongiform encephalopathy and variant Creutzfeldt-Jakob disease: background, evolution, and current concerns," Emerging Infectious Diseases, 7[1]:6-16, January-February 2001.

2. Ministry of Agriculture, Fisheries, and Food BSE weekly cumulative statistics: www.maff.gov.uk/animalh/bse/bse-statistics/level-4-weekly-stats.html.

3. J. Bieschke et al., "Ultra-sensitive detection of pathological prion protein aggregates by dual-color scanning for intensely fluorescent targets," Proceedings of the National Academy of Sciences (PNAS), 97:5468-73, 2000.

4. M.R. Scott et al., "Identification of a prion protein epitope modulating transmission of bovine spongiform encephalopathy to transgenic mice," PNAS, 94:14279-84, 1997.

5. "The Evaluation of Tests for the Diagnosis of Transmissible Spongiform Encephalopathy in Bovines," European Commission, Directorate General XXIV, Consumer Policy and Consumer Health Protection, July 8, 1999

6. J. Safar et al., "Eight prion strains have PrPSc molecules with different conformations," Nature Medicine, 4:1157-65, 1998.

7. M.E. Bruce et al., "Transmission to mice indicate that nvCJD is caused by the BSE agent," Nature, 389:498-501, 1997.

8. BSE Surveillance, U.S. Department of Agriculture, Animal and Plant Health Inspection Service.

9. S.E. Lloyd et al., "Identification of multiple quantitative trait loci linked to prion disease incubation period in mice," PNAS, 98:6279-83, May 22, 2001. Resources Ministry of Agriculture Fisheries and Food (MAFF)'s BSE site www.maff.gov.uk/animalh/bse/index.html

Office International des Epizooties statistics on BSE in Europe www.oie.int/eng/info/en_esbmonde.htm

The National Prion Disease Pathology Surveillance Center www.cjdsurveillance.com

USDA's BSE site www.aphis.usda.gov/oa/bse

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