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Q & A of test methods

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Well-known member
Feb 10, 2005
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Montgomery, Al
Q&A Dr. Jean-Philippe Deslys

1. What is the standard regime for testing of suspect animals in the EU?

The regime is an initial screening by a high-output test, the Bio-Rad
test. If a result raises suspicion, a confirmatory test is conducted
with the Western blot test.

2. How long has this been the case?

Its a fairly recent development. Only recently has the Western blot
test become sensitive enough, with the addition of phospohtungstic acid
precipitation step. The Bio-Rad test (which Deslys helped develop) is
extremely sensitive, and the standard Western blot is extremely reliable
with high-signal test results. However, it had to be made more sensitive
for low-signal (samples with low density of malformed prions) samples.
It has been made more sensitive.

Reproducibility is the problem with the IHC test. It is not
standardized; depending on the lab and its protocols, or even on the
technician involved in the test, one can get conflicting results.

3. Is there a way to measure the three tests in sensitivity, accuracy
and objectivity?

Historically, yes. The IHC was the gold standard at one point, but we
have shifted to the Western blot. It requires less work, it is more
sensitive and its results are reproducible. IHC relies on localization.
If you have a weak signal case, you may get lucky and test a spot with a
high concentration of prions. But the opposite it true too; you can miss
an infection by testing a sample with low concentrations. Western blot
is much better for low signal situations.

4. The USDA in 2003 used the Western blot to confirm the BSE case in
Washington state, and it sent samples to the U.K. for independent
testing. In the case this November, which it announced was negative, it
instead used the IHC test and did not send samples to the U.K. Is this
good science?

Its not logical. If you have two consecutive questionable screenings,
you do another test. I can only advise, its managements duty at USDA
to make the decisions. But when you have a discrepancy between the rapid
test and the IHC, it is only logical to confirm it with another test.

5. We are hearing now about a new strain of BSE, atypical BSE or aBSE.
Or BaSE. We have heard that IHC, the so-called gold standard, cannot
detect the variant. Is this true?

Yes. There have been a few cases, one in Italy, one in Belgium, one here
in France. It seems to only affect very old animals. The distribution in
the brain is very different than we see with BSE, it looks very
different. The IHC test will come back negative.

This his a very recent phenomenon. I have no opinion on its virulence.
We do not know where it comes from. It could be a version of sporadic
infection. Western blot caught them, but we would not even know it
existed if we werent running systematic testing in the EU.

BSE was around for a long time before we caught it and by then, it was
everywhere. It had become highly infectious. It probably amplified due
to low-temperature rendering. The disease was recycled through the food
chain, and was given time to amplify. By the time it was identified,
even good cooking couldnt eliminate it.

I cant stress enough that systematic testing is necessary. Withdrawing
all positives from the food chain is the best way to break the cycle.

What can happen with testing of only cattle that are clearly at risk is
that several can remain undetected. Canada has tested about 30,000 head
of cattle and has three positives. That would indicate that there are
probably undiscovered cases. And what happens then is that the disease
is allowed to amplify. You have to maintain testing.

When people choose to protect their economic interests over public
health, it can have a boomerang effect. It happened all through Europe.
They always deny; its not OUR problem, it is our neighbors problem.
And then a single case is discovered and the public reacts. The economic
results are devastating. It would be better to just assume BSE is
present and use systematic testing as protection. That way, the public
is reassured that it is not entering the food supply.

By systematic testing, I mean doing as we do in the EU, which is to test
every animal over 30 months of age when it is slaughtered. In Europe,
three times as many cases of BSE have been caught by systematic testing
as by clinical testing (of clearly sick animals). In 2004, eight
clinical cases were discovered, 29 were discovered at rendering plants,
and 17 at slaughter. We should be using these tests as a weapon to
protect the public and to give them assurance that the food supply is
being protected.

6. USDAs list of specified risk materials excludes some products, like
blood and bone meal, that are banned in the EU and UK. Is our feed
supply safe?

With SRMs, where do you stop? Tests have found prions in meat, nerves
travel through meat, and so on. The main infectivity is in the brain and
the spinal cord. A blood and bone meal ban in animal feed is not really
necessary, because except in cases of highly infective animals, it is
unlikely that they are dangerous in themselves. If you combine
systematic testing and targeted SRM removal, the brain and the spinal
column in cattle over 30 months, you can have a compromise that is both
safer and less costly than expanded feed bans.

Certainly, you can stop the spread of BSE with a total ban on offal. But
it has to be a total ban. It cant be given to sheep or swine or
poultry. It would be very expensive and virtually impossible to
accomplish. You can have farmers using the wrong feed or transportation

Systematic testing makes far more sense. I think of it as a thermometer.
It not only allows us to catch the disease, it also allows us to monitor
its progress. We can watch the levels of infectivity and if they start
going up instead of down, we can take measures.

To an extent, our environment is contaminated. About 10 percent of wild
animals test positive for TSEs. If you recycle these agents, they can
evolve and get more dangerous. This is probably what happened with BSE.
It wasnt very dangerous until it evolved to the disease we know today.

People complain that testing is very expensive. It is much more
expensive to kill and test whole herds.

7. In your opinion, is infected feed the sole method of transmission of
BSE, apart from the very rare maternal transmission?

Feed is the main problem. However, we are seeing some other
possibilities, including through fat and greases. Calves are fed milk
extracts, with the cream removed. To make it nutritious, they are using
fat and grease from cattle.

(FOLLOW QUESTION: Would that allow BSE to develop into an infective
level in cattle younger than 30 months, assuming they might be getting
infected at a younger age?)

8. You were involved in a study that tested two primates who were fed
infected brain tissue. One eventually died of TSE; the other survived.
The press reported that the main finding was that it would take
something on the order of 1.5 kilograms of infected matter to create an
infection, but that seems to be an oversimplification. Could you explain
it further?

The findings suggest that as little as five grams is enough to infect.
The 1.5 kilo figure is the amount of infected tissue that would have to
be ingested from an animal that would be below the threshold of
infection, and would test negative. In other words, even though a
younger animal may be developing the disease, it would take a
considerable amount of tissue to transmit the disease.

An animal could be just below the testing level, and not be particularly
dangerous. But that is why you have to keep testing. Once it reaches the
threshold, it can become highly infective.

9. BSE is a pretty horrifying disease, but overall, it has killed less
than 200 humans, and only a handful in recent years. Listeria, by
comparison, kills thousands every year. Overall, how do you rate the
threat from BSE?

The overall risk is not particularly high. Over two million infected
animals went into the food chain in Europe, 400,000 of them before the
SRMs, the brains and spinal column, were removed from the carcass. Less
than 200 died, and less than 4,000 are at risk of developing the
disease. What we know now is that one particle is not going to kill you.
There has to be condensation of the prions to be truly dangerous.

This is not a sterile world. But the danger is that now that the crisis
appears to be over, attention will turn elsewhere and that will allow
the disease to amplify again. Just as we stopped paying attention to
AIDS when medication seemed to control it, then were surprised when a
new and more infectious and aggressive strain appeared, we could be
surprised by a more serious strain of BSE. That is why I support
systematic testing for the long term. The object is to keep levels of
BSE low, and to recognize the danger if it suddenly pops back up. ...END
Stanley Prusiner has a patent pending using phosphotungstic acid coating "magnetic beads". Where else have we heard about magnetic energy and prions??? Mark Purdey's hypothesis!

United States Patent Application 20030180706
Kind Code A1
Prusiner, Stanley B. ; et al. September 25, 2003

Removal of prions from blood, plasma and other liquids

Devices such as flow through columns, substrates such as spherical polymer beads, and methods of using such to remove prions from any liquid sample are disclosed. A surface of a substrate is coated with a prion complexing agent, such as a salt of phosphotungstic acid. Blood or plasma passing through a column containing beads coated with prion complexing agent are rendered prion free.

That which is claimed is:

1. A method of removing prions from a sample, comprising the steps of: contacting a sample in a flowable liquid state with a solid substrate comprised of a prion complexing agent; and allowing the sample to remain in contact with the substrate for time such that prions in the sample bind to the substrate.

2. The method of claim 1, wherein the complexing agent is a heteropoly acid or salt thereof.

3. The method of claim 2, wherein the complexing agent is a metal salt of phosphotungstic acid.

4. The method of claim 1, wherein the complexing agent is a peptide.

5. The method of claim 1, wherein the complexing agent is an antibody.

6. The method of claim 5, wherein the antibody is an antibody that selectively binds to PrP.sup.Sc.

7. The method of claim 1, wherein the sample comprises a human body fluid.

8. The method of claim 2, wherein the sample comprises human blood.

9. The method of claim 1, wherein the substrate is further comprised of a polymer.

10. The method of claim 9, wherein the substrate is further comprised of a metal having the polymer coated thereon.

11. The method of claim 9, wherein the substrate is in the shape of spherical beads.

12. The method of claim 10, wherein the metal is a ferromagnetic metal.

13. The method of claim 12, further comprising: removing the substrate from contact with the sample by application of magnetic energy.

14. A substrate, comprising: a water insoluble polymer; and a prion complexing agent.

15. The substrate of claim 14, further comprising: a metal wherein the polymer is coated on the metal and salt of phosphotungstic acid is coated on the polymer.

16. The substrate of claim 14, wherein the prion complexing agent is comprised of an antibody which binds PrP.sup.Sc bound to a surface of the polymer.

17. A prion free liquid, characterized by being subjected to the method of claim 1.

18. The prion free liquid of claim 17, wherein the liquid is further characterized by being derived from a human.

19. The prion free liquid of claim 18, wherein the liquid is human blood.

20. The prion free liquid of claim 18, wherein the liquid is human plasma.

21. A device for removing prions from a liquid sample, comprising: a housing through which a liquid sample flows; and substrate surfaces comprised of prion complexing agent.

22. The device of claim 19, wherein the housing is cylindrical and the substrate is polymer beads having sodium phosphotungstate coated on their surface.

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