Medical Device Link.

Originally Published IVD Technology June 2001

Ahead of the flock?

USDA's Mary Jo Schmerr struggles on to refine a promising technology

  IVD Technology: What is the scope of the problem of disease transmission from animals to humans?

Mary Jo Schmerr, PhD, is a respiratory and neurologic disease research chemist at the National Animal Disease Center (Ames, IA), a branch of the U.S. Department of Agriculture's agricultural research service. She can be reached via mschmerr@nadc.ars.usda.gov.

Mary Jo Schmerr: Animal diseases can be transmitted to humans, and there are some that are very old that we all know about. One is brucellosis, which causes undulant fever. Other classic examples include bovine tuberculosis and E. coli O157:H7, both of which can be transmitted from animal populations to humans.

There are also a whole host of minor-type diseases that you don't hear very much about. Cowpox, of course, provided the original basis of a vaccine against human smallpox.

Some animal-borne bacterial infections can be quite serious—leptospiral infections, for instance—but they are rarely transmitted to humans. Although it's pretty rare, in the case of hoof-and-mouth disease some infections can cross over into the human species from the animal diseases.

Then of course there are the transmissible spongiform encephalopathies (TSEs) that we're just beginning to learn about. Unlike TB or the other diseases I've mentioned, TSEs are fairly uncommon. In humans the infection rate is about one or two individuals per million people. In animal populations where the disease is present, the infection rate is about 1% of the animals in a large herd, or perhaps a slightly higher percentage in a smaller herd. So even in animal populations TSEs are not as highly infectious as hoof-and-mouth disease.

TSEs are a relatively recent discovery. What makes them of unique concern?

The biggest thing about a TSE is that it's a fatal disease; there's no treatment for it. People who contract a TSE—whether from nonhuman contact or from their own genetic makeup—simply die. And it's a very unpleasant death. The same must also be true for animals. Although we don't perceive them as having a lot of pain, they do suffer tremors and muscle spasms associated with the disease, and the progress of the disease must be somewhat painful.

The bovine form of TSE is commonly known as mad cow disease. When was the first case diagnosed?

BSE was identified in 1985, but it was written up for publication somewhat later. A new form of the human Creutzfeldt-Jakob disease (CJD) was identified in 1996. Following this time, researchers demonstrated that this variant of CJD was the same as the agent that causes BSE.

The theory behind the transmission of TSEs is that they are prion infections. Could you explain how that mechanism works?

Prions are actually normal proteins that are located on the surfaces of many, many cells in the body. There are more on the neurons and glial cells of the brain than elsewhere. We're not exactly sure what role that normal prion protein plays, but it's probably related to the regulation of copper metabolism in the brain. That's an especially crucial issue, because the brain requires copper to be at a certain level. So the normal prion protein binds copper, and I think the abnormal one does as well. The normal protein has a shape that is about 30–40% alpha helix, which is a type of secondary structure for proteins. This protein is processed normally and is commonly found on the surfaces of cells.

Abnormal prion proteins have the same primary structure as normal ones, but they're folded differently. In addition to the 30% alpha helix found in the normal form, about 30% of an abnormal protein is folded into a beta sheet form. The normal proteins have hardly any beta sheet form. So it's this abnormal beta-sheet folding, which looks a bit like a pleated curtain, that gives the abnormal protein its properties.

The normal prion protein is soluble, can be digested by proteases, and does not aggregate. The abnormal form is insoluble, resistant to proteases, and aggregates into long fibrils. Those are the major differences in the properties of the two proteins. When a susceptible animal is infected with the abnormal form of the protein, it generally comes down with the related disease.

And TSEs are the result?

That's right. TSEs are the family of diseases that we think are caused by infection with abnormal prion proteins. Most of the data support that theory, and it's starting to be less of a theory and more of an established fact.

The tests that are currently being used to detect prion infection are all postmortem tests, is that correct?

That's correct. The researchers usually need to have a piece of the brain, brain stem, or spinal cord; in some animals they can use lymph nodes. And with these samples there are several technologies that can be used to make a diagnosis.

In one method, the researcher sections the tissue and uses immunohistochemistry to stain the prions with an antibody specific to the prion protein.

Another method uses the Western blot test, in which the researcher prepares the brain material and runs it out on an SDS gel, and then blots that onto a special paper. A specific antibody is used again to detect the prion proteins. Proteinase K is used to digest away the normal prion protein. The abnormal one is not digested by this enzyme. Three distinct bands are observed in positive samples. These represent the glycosylation states of the protein.

A third method uses an enzyme-linked immunosorbent assay (ELISA) technology. In this method, the researcher disrupts the prion using a denaturant such as guanidine hydrochloride, which is then washed off. Then the researcher can either use a capture antibody to isolate the prion proteins, or can bind the prion directly to the plates, apply specific antibodies to isolate the proteins, and then amplify the signal.

Working at the limits of detection

I understand that you're working on a method to test for prion proteins in blood, which can be used while the animal is still alive.

Yes, the technology we've developed is completely different from those now in use. We realized that if you are trying to detect prion proteins in blood samples, there are problems related to low amounts, solubility, and insolubility—especially in biological buffers. The challenge is to find a way to overcome these parameters and to detect the prion protein.

So we devised a new process that uses organic solvents to extract the abnormal prion proteins and capillary electrophoresis technology to detect them. Because we extract with organic solvents, we can dry down and concentrate the material. Then we use that sample for a test in a capillary electrophoresis immunoassay, which is based on direct fluorescence. We measure the first event—of the antibody binding to the fluorescent-labeled peptide—and then we look for displacement by the abnormal prion protein. Since that displacement changes the binding of the peptide to the antibody, we can calculate how much abnormal prion protein is present.

What is the current status of your work on this test?

Right now we're working on the reproducibility of the assay. We are attempting to get to the point where a test run on the same sample taken from the same animal at the same time produces a variation of 5–10%.

Achieving that level of reproducibility is important, however, because it will give us some room for error when we actually call the results of an animal's test positive or negative. Those results are not based on a subjective reading, but are actually based on numbers provided by the test. The instrumentation calculates the values for the respective peaks which represent the amount of binding and of the free peptide. We are now determining our cutoff values—to decide where positive and where negative should be called. Once that's done and the process is reproducible, biological validation of the test shouldn't present any problem.

Licensing for development

Your research on this test is being conducted as part of your work at the U.S. Department of Agriculture (USDA). Once the research phase is complete, how will the test actually be commercialized?

When we were developing the extraction process we had a cooperative research agreement with the company Fort Dodge Animal Health (Overland Park, KS), which is a subsidiary of American Home Products. The licensing of the patent is now being negotiated and the company will develop a kit to test animals using this technology.

So by virtue of the company's work during the early phases of development, the licenses on this technique are already spoken for?

Only the license for applications in animal diagnostics has been spoken for. But now there are more applications, other things that this method could be used for. For instance, drug companies and others wanting to test for TSEs could find this technique useful. So USDA or my coinventor, Andew Alpert of PolyLC Inc., will grant other licenses for companies that want to develop tests for use with human blood. Human applications are not included in the original license, and a couple of companies have already expressed interest in human testing applications.

Political disease

In Europe, the transmission of BSE to humans has created a politically volatile atmosphere. How are the Europeans handling TSE testing now, and what are they doing to develop new test methods?

First, I would like to commend the European scientists on their quick response to the need for massive testing. All together, the Europeans have conducted well over a million and a half tests on their cattle since the beginning of the year. That is impressive mobilization of laboratory resources.

They are using a couple of approved validated tests. There's a Western-blot test by Prionics, and an ELISA-based test marketed by Bio-Rad. Some government agencies are also performing tests using their own in-house assays. There are five other tests that are currently being validated for use in the EU. They will probably come out this summer.

All of these are still postmortem tests, and most use brain samples. One test developed by Enfer (Tipperary, Ireland), uses spinal cord samples.

But none of them are using blood?

No. And I wish there were more researchers who could and would do so, because I think that would drive development forward considerably faster.

BSE is decreasing in Europe and will eventually go away. But there will probably still be a need to test and remove animals with preclinical infections. Because if you remove and test an animal's brain at slaughter—and the animal is then found to be positive for a TSE—it will already have contaminated all the slaughter equipment in the plant.

But if animals could be tested on the farm where they grew up, or where they're being housed for breeding or finishing, that would eliminate some of the transmission that may occur in holding pens.

We probably can't get rid of TSEs completely. In the Western United States there is a residual wildlife population that carries a form of TSE called chronic wasting disease, and that would be very difficult to control. But we probably can eliminate most of these animal diseases if we can just develop some really good tests to detect preclinical infection.

When will the test you're developing first be available, and for what exact purposes will it be labeled? How long will it take to test it further for BSE?

That's always the hardest question. The company that we're working with has a timeline of about two years, at the end of which it expects to release a kit that can be used for diagnosis of sheep scrapie. In the meantime, if we find that prion proteins are also in the blood of BSE-infected cows, the test could be marketed for that as well. The other potential market would be for animals with chronic wasting disease.

Once we get through the analytical validation part of development—getting all our variables nailed down to that 5% level—the testing technique could pretty quickly be turned over to a company that's set up to automate it. That would be very good, because then we could test lots of samples and more quickly get a sense for the biological variability of the tests. Right now, we can't process that many samples.

Progress under pressure

The atmosphere surrounding your work has become politically charged. How did this occur and what has been the effect on your work?

Politics and science do not make a very good mix. Unfortunately, prion diseases are political because of all of the ramifications. TSEs in general have had their share of controversy among the researchers. This happened long before I started my research. In the recent past, I have noted that there is much more of a cooperative effort. I also believe that the expertises within TSEs varies considerably. This does cause some friction. Unfortunately, the political influence is not usually a productive one for science.

To what extent do such controversies relate to congressional politics, or politics at the national level?

Congress appropriates the funding for the governmental agency for which I work. There is a broad constituency of people and organizations that would like to have this test. More specifically, the people who are nearest to the animals—veterinarians, food-safety people, those who slaughter cattle, and those who sell cattle products for human use or consumption—are all very interested in having this kind of a test. So I'm sure there is pressure exerted on Congress to fund development of live animal tests.

Are large corporations supportive? Are they a source of funding for your work?

A lot of companies are willing, and ask to fund my research. But because I work for the government, formal agreements are required to obtain such funds.

Actually I find that pharmaceutical houses that use animal products for the production of vaccines and industries that use blood-based products are the most interested in the research and the test development that I am doing.

A researcher in a small city in Iowa holds the most promising technology for detecting abnormal prion proteins—thought to be the infectious agents of transmissible spongiform encephalopathies (TSEs)—in the blood of living animals. Mary Jo Schmerr is a research chemist at the USDA's National Animal Disease Center (Ames, IA) whose achievement was first announced in 1999. Since then, Schmerr's still-developing technique has generated a great deal of excitement in the prion-research community and is a source of some controversy. In this excerpted interview with IVD Technology editor Steve Halasey, Schmerr describes the potential utility of her technique and its current development status. The full interview can be found by visiting the IVD Technology Web site at http://www.devicelink.com/ivdt.

Copyright ©2001 IVD Technology