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CFIA Completes BSE Investigation

Tam

Well-known member
Oldtimer said:
Tam said:
But all of Canada's cases were younger than the Texas cow so who really had BSE first. :wink:

Tam-- Don't you keep up with the USDA and the sound scientists? They aren't the same strain--SHEESH...Canada has just plain old run of the mill "typical BSE", while the US has something special and rare - "Atypical BSE"..... :wink: :lol:
In this case I'm very glad that Canada has the plain old run of the mill BSE. At least we do have a few answers of where ours comes from and how we can control it. Unlike the "SPECIAL AND RARE" strain you have that there are few to no answers about. Which means it can strike any and all cattle even organic at any time. :wink I still have to ask though who had BSE first, if your atypical special strain has reseachers coming forward with these kinds of press releases.
The small scientific world of prion researchers -- the scientists who investigate "transmissible spongiform encephalopathies" (TSE) such as mad cow disease in cattle and Creutzfeldt-Jakob Disease (CJD) in humans -- is abuzz. That's because the two confirmed cases of US mad cow disease in Texas and Alabama are an "atypical" strain different from the British strain. This really should not be surprising. Sheldon Rampton and I reported in 1997 that very strong evidence of an "atypical" TSE disease in US cattle goes back to the 1985 work of Dr. Richard Marsh, the researcher to whom we dedicated our book Mad Cow USA. Even before Britain confirmed its first case of mad cow, Marsh was investigating a similar disease he traced to Wisconsin dairy cattle, confirming suspicions among US scientists since the 1960s that a deadly TSE disease in mink -- transmissible mink encephalopathy or TME -- resulted from their eating dairy cattle.
If this is the same strain as these researchers were talking about then WHO HAD BSE FIRST? Not having the plain old run of the mill kind just may not be something to laugh about if it can be linked back to the mink and Wisconsin dairy cattle in the 1960"s :roll:
 

Tam

Well-known member
reader (the Second) said:
Tam said:
reader (the Second) said:
If I remember correctly all the U.S. are born after the ban and all but ONE of the Canadian are born after the ban. 10 - 13 in most cases. One 6 or 7 year old.

Let's get this correct the Two US cases were born BEFORE the bans as in the Alabama cow was claimed to be over ten and the Texas cow almost fifteen years of age. And the Canadian cattle, half were born before and the other half were born after, 3 and 3. But all of Canada's cases were younger than the Texas cow so who really had BSE first. :wink:

Give it a break. If you think those are the only North America cows, then you're smoking something we should know about.
Reader are you drinking wine again? :roll: You were wrong on the confirmed cases!!!! Confirmed North American cases of BSE has 5 born BEFORE the bans 2 in the US and 3 in Canada and 3 born AFTER the bans all in Canada . Not 1 before and 6 after. And if you have information about the ages and whereabouts of UNCONFIRMED CASES then by all means tell us.
 

Tam

Well-known member
reader (the Second) said:
[

This post is BS. There are different strains of these diseases and it is no honor to have one strain or the other. Unbelievable :roll: :roll:

Even having a strain similar to UK BSE does not mean the source is UK BSE. Not enough is known about the strains and what they mean.

You guys talk and talk but don't read and learn. All knowledge is just to attack on one narrow issue, not to expand your understanding relative to something that I would think would concern you as a rancher.

First of all who said it was an honor, to have one over the other :roll: Maybe if you would stop and compare the two strains you would see why I'm glad we don't have the same strain. :roll:
Let us compare the two Reader.
We have evidence that has proved to most that the strain that the UK has is the same strain Canada has as we had one case of imported BSE in 1993 that came from guess where Reader the UK. Could another UK cow have slipped by undetected and hit our feed system pre feed ban? Looks like it since we have that same strain showing up years later. :shock: :roll:
We also have evidence from all the research done in the UK and the EU that it spreads by feeding infected ruminant by products back to health ruminant at a young age. The result of this evidence was our FEED BANS in 1997 and updates in 1998 . We no longer feed ruminant by products to our cattle.
If this evidence is true as we believe it is, then organic beef is safe as they have never come into contact with ruminant by product feed.
We seem to be using the correct test to detect and confirm our strain of BSE. As it is the same test the UK and the EU uses to detect their strain which is the same strain we have.
We also know some other things but the ones I listed should be enough to show why I'm glad, if we had to have a strain of BSE, we don't have what the US has.
First the US cases can't be blamed on cattle and feed imported from Canada.
Second, and most important the US strain is classified as Spontaneous as in NO KNOWN SOURCE. or by some, spontaneous means IT JUST HAPPENS. If you don't know the source or reason it happened Reader how do you stop the spread???? Can you tell us that organic beef cattle will not contract Atypical BSE if it is something the JUST HAPPENS?
And I have read that Atypical symtoms are not the same as plain old run of the mill BSE, so animals could be missed if looking and testing is not done for your correct strain of the disease. And since the US is NOW using the same test that the rest of the world is using, will it detect your strain on first try or will it take retest after forced retest to detect it like in the TEXAS COW?
I don't in any way feel honored by have one strain over the other BUT What we know about the Canadian strain over what we know about the US Atypical strain is why I'm glad we in Canada have the strain we have. We have ways of controlling ours DO YOU?
 

flounder

Well-known member
TAM WROTE ;


>>>Second, and most important the US strain is classified as Spontaneous as in NO KNOWN SOURCE. or by some, spontaneous means IT JUST HAPPENS. If you don't know the source or reason it happened Reader how do you stop the spread???? Can you tell us that organic beef cattle will not contract Atypical BSE if it is something the JUST HAPPENS? <<<


:lol2: :lol2: :lol2:


there is no scientific evidence for a spontaneous TSE.
please show us this study? the study by soto and prusiner produced NO typical TSE i.e. natural field TSE of any kind. IF anyone believes that all the sporadic CJD cases in the USA and all the documented and especially the ones that were and are not documented are of a spontaneous source i.e. just happens naturally, then you are smoking some goood stuff, dream on folks. bottom line ;

> I am curious and trying to understand all of this. Why did you say
> "Prusiner's dream come true" when speaking of mandatory TSE testing for
> all species?



his test$$

spontaneous TSE i.e. USA MAD COW would be unstoppable then. no feed ban, no
triple firewalls or anything would stop it then, but 100% testing. USDA does
not even believe in the spontaneous theory, but will spin it as long as
possible. just my opinion. ...

Stanley Prusiner - Discoverer of Prions


http://maddeer.org/video/embedded/prusiner.html


* Prusiner holds more than 35 issued or allowed United States patents all of
which are assigned to the University of California and many of which are
licensed to InPro Biotechnology. ...

http://www.bu.edu/amyloid/events/prusiner-bio.html


CACHING $$$


IF BSE arises spontaneously out of every so many cattle, then why do some
countries have it and some don't?



CHAIRMAN BROWN: Thank you, Dr. Asher.

Questions for Dr. Asher?

All right. Then -- yes.

DR. PRUSINER: There's a page in here, and I'm worried that we come away with the wrong conclusion. I thought it was a very nice presentation. There was one point. Let me see if I can find it now. Here is it.

DR. ASHER: Which page, Stan?

DR. PRUSINER: It's on these slides that say uncertainties concerning theoretical -- twenty-eight. Thank you.

DR. ASHER: Yes.

DR. PRUSINER: I can't see that with my glasses.

DR. ASHER: Uncertainties concerning.

DR. PRUSINER: Right. So it says sources of infection and sporadic CJD are unknown. I mean I would argue all of these epidemiologic studies, I think, clearly argue that sporadic CJD are -- what people are now calling classical CJD -- don't come from infection. Would you agree with that?

I don't understand the Point 6.

DR. ASHER: Don't come from infection? No, I think that the whole issue is still open. They certainly are associated with an infectious agent, and when subsequent subjects are exposed to them, they become infected. I don't believe that the issue is settled at all.

I mentioned the possibility, which is certainly possible, but certainly not demonstrated, that the infection is of endogenous origin, but more than that I wouldn't be prepared to say.

I believe that rigorously the cause of sporadic CJD has not been -- the source of sporadic CJD has not been determined.

DR. PRUSINER: Okay. I just want to make it very clear from my point of view that this is not a scientifically defensible point of view at this point. That's my --

DR. ASHER: I don't think any point of view at the moment is scientifically defensible. I think it's simply not known.

(Laughter.)

CHAIRMAN BROWN: Can we resolve the issue by noting that the source of infection can be the brain itself?

DR. PRUSINER: It's not going to be resolved. I just want to make the point --

CHAIRMAN BROWN: No, but I mean is that -- is that -- if we accept the fact that source of infection does not necessarily imply an external source --

DR. PRUSINER: That's fine.

CHAIRMAN BROWN: -- then, you know, then I think we're talking the same language.

DR. PRUSINER: That's fine.

DR. ASHER: I don't believe that an external source -- that the state of knowledge today permits an external source to be excluded.

CHAIRMAN BROWN: Okay.

DR. ASHER: I don't think this is the place to have this kind of discussion, but it is important that these differences, I suppose, that these differences be aired.

DR. ROHWER: Paul.

CHAIRMAN BROWN: Bob.

DR. ROHWER: I don't want Dr. Asher to have to stand alone on this either, and I agree with him fully.

CHAIRMAN BROWN: Maybe we should have what, a seminar, two, three hours?

(Laughter.)

CHAIRMAN BROWN: We have time, don't we?

DR. ROHWER: No, we could have a vote.

(Laughter.)

CHAIRMAN BROWN: That'll take too much time.

Thanks, Dave.

We'll now proceed to the next speaker, who is Professor Almond from Pasteur-Marieux Connaught in France, whose title is "The Potential Risk of Introducing BSE Agent into Sheep and Goats in Europe."

DR. ALMOND: Ladies and gentlemen, it looks like my computer has crashed. All of my slides are on my computer. So I'm going to have to ask you to bear with me for a few moments until I reboot it. I'm sorry about that.

I'm sorry. Everything was set up so I could just come up here and touch the buttons, and it's obviously crashed.

CHAIRMAN BROWN: Jeff, this is not the first time, nor will it be the last time that computer programs have disappointed the speaker. Do you think we're talking, you know, a minute or two or a more extended rebooting?

DR. ALMOND: I hope we're talking about two minutes.

CHAIRMAN BROWN: Okay. We'll just wait then.

(Whereupon, the foregoing matter went off the record at 9:37 a.m. and went back on the record at 9:43 a.m.)


snip...



This comes back to my little spat with David Asher about --
(Laughter.)

DR. PRUSINER: No, I'm serious. I'm coming back to this because it's a very important point.

You can believe, as David believes, that all these diseases happen by exogenous infection or you can believe as I believe that there are these sporadic cases of CJD that we see in the United States, represent the spontaneous conversion of PrP-c into PRP-scrapie or a somatic mutation, and I would argue that happens in sheep all the time.

And I would argue that for whatever reasons, whatever the culture is in New Zealand among sheep farmers than it is in Australia, we're not seeing cases of scrapie as they appear.

And so I'm much more comfortable having well monitored flocks of a limited size and determining that these animals to the best of our methods that are available at any given point in time are free of scrapie than I am with believing that just because the stuff comes from New Zealand or Australia that it's better.

CHAIRMAN BROWN: Yes, to introduce a slight modification from the chair, I think that that's a decent point, to be more comfortable with a heavily surveyed flock. I would think that if scrapie were existing endemically strictly as spontaneous conversion disease, that it would not be expected for flocks which are scrapie free within a year or two suddenly to come down with scrapie affected sheep after the introduction of a scrapie infected sheep into the flock.

That smells like horizontal transmission to me.

DR. PRUSINER: No, I don't mean to say that there isn't horizontal transmission. I believe in horizontal transmission once a case starts, but I'm just saying that there are spontaneous cases that begin that way, and then the infectious mode takes over, and for reasons we don't understand at all, scrapie is a much more infectious disease, a much more infectious prion disease than CJD is among humans.

CHAIRMAN BROWN: Okay.

DR. PRUSINER: I'm in agreement with you.

CHAIRMAN BROWN: Yes. How would we explain the fact that there just isn't any recognized reported clinical scrapie in Australia? A monstrous conspiracy?


snip...






http://www.fda.gov/ohrms/dockets/ac/99/transcpt/3518t2.rtf



http://72.14.209.104/search?q=cache:pKJPlLI2R44J:www.fda.gov/ohrms/dockets/ac/99/transcpt/3518t2.rtf+scrapie+strains+breed+east+friesian&hl=en&gl=us&ct=clnk&cd=23





another thing to ponder. prusiner has had this cdi test for years now.
one problem, it is so sensitive, no one will validate it in the USA.
SO, round and round we go. why do you think they picked the least likely
rapid test to find BSE at first? same reason they chose not to use WB, when
Dr. Detwiler told them in 2003 they would be missing cases if they only use
IHC,
the put her out to pasture. she got the last laugh. and now is at the big
mac as one
of the top TSE advisers now, and now even the big mac is after USDA et al.
money talks and BSe walks though, we have to wait and see from here, but
this
spontaneous excuse is a hoot. there final straw so to speak. ...


14 February 2005

Diagnosis of prions in patients should utilize novel strategy, team says

A technique for detecting prions in tissue, developed in recent years by
UCSF scientists, is significantly more sensitive than the diagnostic
procedures currently used to detect the lethal particles in samples of brain
tissue from patients, according to a study performed by a UCSF team.

The finding indicates that the diagnostic technique, known as the
conformation-dependent immunoassay (CDI), should be established as the
standard approach for brain biopsies of patients suspected of having the
disease, they say. The team is exploring whether the CDI might be adapted to
detect prions in blood and muscle.

The finding suggests that reliance on the current methods for detecting
prions in human brain tissue -- microscopic examination of tissue for the
telltale vacuoles that form in brain cells and immunohistochemistry (IHC),
which involves detecting prions in brain sections using prion
protein-specific antibodies -- may have led to an under diagnosis of the
disease in patients in recent years, they say. (A definitive diagnosis of
the disease in humans is made only on autopsy, when a neuropathologist can
analyze multiple brain regions for vacuoles and evidence of prions by IHC,
and it is estimated that only 50 percent of human cases are autopsied, in
part because many pathologists do not want to risk infection during the
autopsy.)

In the study, the team compared the ability of the CDI and the two
traditional diagnostic techniques to detect prions in various brain samples
from 28 patients diagnosed on autopsy as having one of several human forms
of the disease -- sporadic, familial or iatrogenic Creutzfeldt-Jakob disease
(CJD). While the CDI detected the biochemical signal for prions in 100
percent of the samples studied, the traditional tests failed to detect the
prion in a high proportion of cases. For example, in an experiment that
focused on 18 brain regions from eight patients with sporadic CJD, the CDI
detected prions in 100 percent of the samples, while IHC detected them in 22
percent and routine tissue examination in 17 percent.

"In about 80 percent of the different brain regions examined, prions were
not consistently detected by either IHC or routine histology that measure
vacuolation. In contrast, the CDI was always positive in all regions of the
brain," says the lead author of the study, Jiri Safar, MD, associate adjunct
professor of neurology and a member of the UCSF Institute for
Neurodegenerative Diseases, which is directed by senior author Stanley B.
Prusiner, MD, UCSF professor of neurology and biochemistry.

"These findings indicate that histology and immunohistochemistry should no
longer be used to rule out prion disease in single-site biopsy samples,"
says Safar. "The superior performance of the CDI in diagnosing prion disease
suggests that the CDI be used in future diagnostic evaluations of prion
disease, particularly for single-site brain biopsies during life"

"If the traditional techniques are used at autopsy, they must be applied to
many cortical and subcortical samples," says co-author Stephen J. DeArmond,
MD, PhD, UCSF professor of neuropathology.

Moreover, while the study examined the efficacy of the CDI in comparison to
the two techniques routinely used by neuropathologists to detect prions in
human brain tissue, previous studies at UCSF indicate that the CDI is also
significantly more sensitive than Western blot analysis, the technology used
with IHC to detect prions in brain tissue from cattle suspected of having
bovine spongiform encephalopathy (BSE). That IHC and Western blot analysis
are relatively insensitive methods, the researchers say, supports their
ongoing assertion that the CDI should also be used to evaluate the brain
tissue of cattle.

"The studies reported here are likely to change profoundly the approach to
the diagnosis of prion disease in both humans and livestock," says Safar.

More broadly, the scientists say, the high sensitivity of the CDI suggests
that CDI-like tests could also prove useful for diagnosing other
neurodegenerative diseases, such as Alzheimer's disease, Parkinsons's
disease and fronto-temporal dementias, all of which, like prion diseases,
involve various forms of protein misprocessing. These diseases currently are
diagnosed by neuropathological analysis and immunohistochemistry.

"Whether immunohistochemistry underestimates the incidence of one or more of
these common neurodegenerative diseases is unknown, but the CDI could shed
light on these diseases," says co-author Bruce Miller, MD, UCSF A.W. and
Mary Margaret Clausen Distinguished Professor of Neurology and director of
the UCSF Memory and Aging Center.

The finding will be printed on-line and in print on March 1, 2005 in
Proceedings of the National Academy of Sciences.

The study brings into high relief the different detection strategies of
immunohistochemistry and the CDI, both of which involve revealing the
presence of prions, known as PrPsc, by applying antibodies to brain tissue.

Standard immunohistochemistry, developed in the DeArmond lab 20 years ago,
involves using an enzyme known as a protease, or a combination of harsh acid
and high temperature treatment, to destroy normal prion protein (PrPC),
which is ubiquitous in brain tissue. Once this occurs, scientists apply
fluorescently lit antibodies that react with residues of the relatively
resistant abnormal prion protein (PrPSc), thereby highlighting it.

The limitation of this technique is that scientists have since learned that
there is a large part of the abnormal prion protein that is protease
sensitive, and that portion escapes detection by the standard technique.
Thus, this traditional method underestimates the level of PrPSc in tissue.

The CDI addresses this limitation by revealing the region of PrPSc that is
exposed in the normal PrPC but is buried in infectious PrPSc, using high
affinity, newly generated antibodies that identify PrPSc through the
distinct shape of the molecule, independent of proteolytic treatments. This
makes it possible to detect potentially large concentrations of protease
sensitive PrPSc molecules.

Detractors would say that it is not necessary to detect the minute level of
infectious agent that the CDI is capable of revealing, as it would be
unlikely to be lethal, says Safar. But Prusiner and his colleagues maintain
that any risk is too great when it comes to having prions in the food
supply. In addition, because even low levels of prions are extremely
resistant to inactivation, they may contaminate the environment for many
years.

Prusiner won the 1997 Nobel Prize in Physiology or Medicine for discovering
that a class of neurodegenerative diseases known as spongiform
encephalopathies was caused by prions. Prion diseases develop in humans,
cattle, sheep, deer, elk and mink.

The CDI was developed by members of the Prusiner lab. The CDI methodology
has been licensed to InPro Biotechnology, Inc.

Prusiner, Safar, DeArmond and other members of the Institute for
Neurodegenerative Diseases are scientific advisors to, or own stock in,
InPro.

Other co-authors of the study were Michael D. Geschwind, Camille Deering,
Svetlana Didorenko, Mamta Sattavat, Henry Sanchesz, Ana Serban, Kurt Giles,
of UCSF, and Martin Vey, of Behring, Marburg, Germany, and Henry Baron, of
Behring, Paris.

The study was funded by the National Institutes of Health, the John Douglas
French Foundation for Alzheimer's research, the McBean Foundation, the State
of California, Alzheimer's Disease Research Center of California and the
RR00079 General Clinical Research Center.

The UCSF Institute for Neurodegenerative Diseases:
http://ind.medschool.ucsf.edu/.

FURTHER COMPARISON OF THE CDI TO THE STANDARD DIAGNOSTIC PROCEDURES,
PROVIDED BY STEPHEN J. DEARMOND, MD, PHD, UCSF PROFESSORS OF NEUROPATHOLOGY:

Explanation as to why the CDI is more sensitive than Western blot analysis:
Studies at UCSF during development of the CDI showed that CDI could detect
prions in brain homogenates at levels that fail to produce disease in
animals (bioassay for prions). Therefore, the CDI is more sensitive than the
bioassay method, which was considered to be the most sensitive technique for
detecting prions. In contrast, Western blot analysis for prions is
significantly less sensitive than the bioassay and is, therefore,
significantly less sensitive than the CDI. Currently, the USDA uses a
combination of Western blot analysis of brainstem homogenates and
immunohistochemistry of the medulla to test cattle suspected of having
bovine spongiform encephalopathy ("mad cow disease"). The relative
insensitivity of IHC and Western blot analysis, says DeArmond, supports the
UCSF scientists' ongoing assertion that the CDI should also be used to
evaluate the brain tissue of cattle.

DeArmond cites additional evidence about Western blot analysis from a World
Health Organization (WHO) study group, which compared the CDI method with
Western blots for detection of prions in sporadic and variant CJD brains.
Based on the smallest amount of prions that could detected by the two
techniques, they found that the CDI was from 1000- to 100,000-fold more
sensitive than Western blot analysis performed in six different research
laboratories (Minor et al. Standards for the assay of Creutzfeldt-Jakob
disease specimens. J. Gen. Virol. 85: 1777-1784, 2004).

Explanation as to why IHC for prions is less sensitive than the CDI: IHC is
routinely performed on formalin-fixed, paraffin-embedded samples of brain.
Formalin fixation markedly decreases the ability of antibodies to bind to
proteins in general, which greatly weakens the IHC signal for prions
(PrPSc). In contrast, homogenates for the CDI are not treated with reagents
that decrease prion antigenicity. Moreover, to concentrate the PrPSc for
measurement by the CDI, the homogenates are exposed to phosphotungstic acid,
which selectively precipitates both protease-sensitive and
protease-resistant PrPSc that comprise prions, but not the normal prion
protein conformer found in uninfected animals, PrPC. This step results in a
higher concentration of PrPSc for detection by the CDI. Because the PrPSc
was not exposed to proteases, the CDI measures all forms of abnormally
folded PrPSc molecules. Protease-sensitive PrPSc can account for 50 percent
of the total PrPSc. For Western analysis, homogenates of brain are treated
with protease to eliminate PrPC; however, this step also eliminates
protease-sensitive PrPSc leaving only protease-resistant PrPSc for Western
blot detection and decreasing the PrPSc signal at least in half.

###

Nature Biotechnology 20, 1147 - 1150 (2002)
Published online: 21 October 2002; | doi:10.1038/nbt748
Measuring prions causing bovine spongiform encephalopathy or chronic wasting
disease by immunoassays and transgenic mice
Jiri G. Safar1, 2, Michael Scott1, 2, Jeff Monaghan1, 5, Camille Deering1,
Svetlana Didorenko1, Julie Vergara1, Haydn Ball1, Giuseppe Legname1, 2,
Estelle Leclerc4, Laura Solforosi4, Hana Serban1, Darlene Groth1, Dennis R.
Burton4, Stanley B. Prusiner1, 2, 3 & R. Anthony Williamson4

1 Institute for Neurodegenerative Diseases, University of California, San
Francisco, CA 94143-0518.

2 Department of Neurology, University of California, San Francisco, CA
94143-0518.

3 Department of Biochemistry and Biophysics, University of California, San
Francisco, CA 94143-0518.

4 Departments of Immunology and Molecular Biology, The Scripps Research
Institute, La Jolla, CA 92037.

5 Current address: InPro Biotechnology, 870 Dubuque Avenue, South San
Francisco, CA 94080.
Correspondence should be addressed to Stanley B. Prusiner
[email protected] or Dennis R. Burton [email protected]
There is increasing concern over the extent to which bovine spongiform
encephalopathy (BSE) prions have been transmitted to humans, as a result of
the rising number of variant Creutzfeldt−Jakob disease (vCJD) cases. Toward
preventing new transmissions, diagnostic tests for prions in livestock have
been developed using the conformation-dependent immunoassay (CDI), which
simultaneously measures specific antibody binding to denatured and native
forms of the prion protein (PrP). We employed high-affinity recombinant
antibody fragments (recFab) reacting with residues 95−105 of bovine (Bo) PrP
for detection and another recFab that recognizes residues 132−156 for
capture in the CDI. We report that the CDI is capable of measuring the
disease-causing PrP isoform (PrPSc) in bovine brainstems with a sensitivity
similar to that of end-point titrations in transgenic (Tg) mice expressing
BoPrP. Prion titers were approx107 ID50 units per gram of bovine brainstem
when measured in Tg(BoPrP) mice, a figure approx10 times greater than that
determined by bioassay in cattle and approx10,000times greater than in
wild-type mice. We also report substantial differences in BoPrPSc levels in
different areas of the obex region, where neuropathology has been
consistently observed in cattle with BSE. The CDI was able to discriminate
between PrPSc from BSE-infected cattle and Tg(BoPrP) mice as well as from
chronic wasting disease (CWD)-infected deer and elk. Our findings argue that
applying the CDI to livestock should considerably reduce human exposure to
animal prions.
To apply the CDI for immunodetection of BSE and CWD prions, we generated a
panel of recFabs binding to residues 90−115 of ungulate (hoofed mammals,
including cattle, sheep, deer, and elk) PrP. Three IgG1kappa antibody Fab
libraries were displayed on the surface of filamentous phage and
individually selected against a panel of PrP antigens, yielding >28 distinct
recombinant antibody clones. The heavy-chain-complementary-determining
region 3 (HCDR3) sequence of P Fab selected against recBoPrP(23−231) and
chimeric mouse−bovine (MBo2M) PrP and displaying the most robust ELISA
signals was: GAYYIKEDF.

Epitope mapping of O, P, and S Fabs demonstrated a single linear epitope
lying between residues 96 and 105 of BoPrP. However, P Fab also displayed
strong reactivity with PrP of other species (see Supplementary Fig. 1
online), which share the common epitope motif: HG(S,N)QWNKPSKPKTN. This
epitope is present in human PrP as well as in all ungulate PrP sequences,
including cattle, deer, elk, kudu, goat, and sheep. Using surface plasmon
resonance to measure binding kinetics, we found that both the P and S
antibody clones bound more tightly when expressed as chimeric human−mouse
(HuM) proteins than when expressed as mouse Fabs (see Supplementary Table 1
online). Binding of the HuM-P Fab to BoPrP was particularly tight, with Kd
values of 0.3 nM and 0.5 nM measured against MBo2MPrP(23−231) and
BoPrP(90−145) antigens, respectively.

The CDI incorporating Eu-labeled HuM-P Fab was initially calibrated with
recombinant MBo2M PrP that was refolded into a beta-sheet conformation and
designated rec beta-MBo2M PrP (ref. 1). Rec beta-MBo2M PrP was detected to a
concentration as low as 20 ng/ml, and after sodium phosphotungstate (NaPTA)
precipitation2, to a concentration as low as 1 ng/ml, with <7% interassay
variation (see Supplementary Fig. 2 online).

Using Eu-labeled HuM-P Fab in a manual CDI protocol2, we determined the
ratios of time-resolved fluorescence (TRF) signals recorded for native (N)
and denatured (D) samples of normal bovine brain homogenate containing only
normal, cellular PrP (PrPC) to be = 2.37. In contrast, D/N ratios obtained
for a BSE-infected brain homogenate containing a mixture of PrPC and PrPSc
were consistently >2.37. Similarly, the presence of BoPrPSc could also be
deduced if the difference between fluorescence signals measured in native
and denatured aliquots (D−N) of the same sample2 exceeded a calculated
threshold value.

To evaluate the sensitivity of the CDI in detecting BSE and CWD prions,
homogenates from pooled BSE-infected Tg(BoPrP)Prnp0/0 mice or pooled
CWD-infected deer brains were serially diluted into homogenates of normal
mice or normal deer brain, respectively (Fig. 1A). These results demonstrate
a robust quantitative response over a dynamic range of several orders of
magnitude (Fig. 1A). The sensitivity of the CDI in detecting CWD prions was
equal to or greater than that for the detection of BSE prions in
Tg(BoPrP)Prnp0/0 mice (Fig. 1A).

Figure 1. Similar sensitivity of the CDI and bioassays in Tg(BoPrP)Prnp0/0
mice for BSE prions.
Figure 1 thumbnail

(A) Dynamic range and analytical sensitivity of the manual direct CDI in
detecting PrPSc in BSE- and CWD-infected brains. Brain homogenates from
either pooled BSE-infected Tg(BoPrP)Prnp0/0 mice or CWD-infected deer were
serially diluted into homologous normal brain homogenate and tested by the
CDI. The (D−N) value measured in counts per minute (c.p.m.) is directly
proportional to the concentration of PrPSc (ref. 2). Data points and bars
represent average plusminus s.d. obtained from three or four independent
measurements. Manual CDI cutoff values were calculated by (mean + 3(s.d.))
using samples from uninoculated Tg(BoPrP) mice (solid horizontal line) and
normal deer (broken horizontal line). (B) Direct and sandwich CDI protocols
for the detection of BoPrPSc were compared using pooled BSE-infected
brainstem homogenates, serially diluted into normal brain homogenate
prepared from the same brainstem area. The automated CDI was used for these
studies. Cutoff values for the direct aCDI (solid horizontal line) and
sandwich aCDI (broken horizontal line) were calculated by (mean + 3(s.d.))
and determined from 782 tests on 432 normal bovine brainstems. (C) Inverse
exponential relationship between titers of BSE prions and incubation times
in Tg(BoPrP+/+)4092/Prnp0/0 mice. The data points are the average plusminus
s.e.m. calculated from three independent end-point titrations. (D) Direct
relationship between BoPrPSc detected by CDI and BSE prions measured in
Tg(BoPrP)Prnp0/0 mice. The (D−N) value is directly proportional to the
concentration of PrPSc (ref. 2). The percentage of ill mice at each BSE
sample dilution used in the bioassay was calculated from three independent
end-point titrations.


Full FigureFull Figure and legend (71K)
To determine the sensitivity of the automated CDI (aCDI) in detecting BSE
prions, brainstem homogenates from BSE-infected cattle (Veterinary
Laboratory Agency (VLA), New Haw, UK), were serially diluted into
homogenates of normal bovine brain. We found a robust quantitative response
and sensitivity limit at 10-3.7 dilution of BSE-infected brain (Fig. 1B).
Introduction of the sandwich protocol using Fab D18 to capture BoPrP
increased the sensitivity of the aCDI up to 20-fold (Fig. 1B).

Using a homogenate prepared from the medulla of a Hereford bull with BSE
(case PG31/90), we did three separate titration series in parallel in
Tg(BoPrP+/+)4092/Prnp0/0 mice, giving an average end-point titer of 106.9
ID50 units/g of brain tissue (Fig. 1C). This finding compares with 103.1
ID50 units/g of BSE-infected brain tissue titrated intracerebrally (i.c.) or
intraperitoneally (i.p.) in RIII mice3, 4, 5, 6, and with 106 ID50 units/g
reported for end-point titration in cattle7. These data indicate that
Tg(BoPrP+/+)4092/Prnp0/0 mice are approx10 times more sensitive than cattle
and >1,000 times more sensitive than RIII mice to infection with BSE prions.
Any possibility that the BSE isolate from PG31/90 contains an unusually high
titer of prions seems highly remote, because previous comparison of PG31/90
with other BSE brain samples from the VLA gave similar or shorter incubation
periods in Tg(BoPrP)4125/Prnp0/0 mice8.

The sensitivity of the aCDI applied to brain tissues of BSE-infected
Tg(BoPrP)Prnp0/0 mice approaches that of bioassay in cattle and
Tg(BoPrP)Prnp0/0 mice (Fig. 1A). Some variability in both CDI data and
end-point titration experiments observed at high dilutions (from 10-4 to
10-6) of prion-infected brain homogenates may be attributed to the
stochastic distribution of prions in small aliquots used for bioassay (30
mul/mouse) and CDI (100 mul/well). One BSE infectious unit, which was
calculated as the dilution point of BSE prions titrated with an average 50%
survival rate in Tg(BoPrP)Prnp0/0 mice, is equal to approx10-4 dilution of a
10% brain homogenate. Applying this, the 10-4 dilution that results in a 50%
infection rate in the most sensitive bioassay in Tg mice correlates with a
10-4.6 BSE brain dilution that produces a positive result in 50% of aCDI
tests (Fig. 1D). To examine the diagnostic performance of the aCDI, 100
confirmed BSE cases and 432 normal cows were tested. The distribution of
(D−N) values in the normal group was Gaussian with a median value of -75
c.p.m. (Fig. 2A). A percentile plot of all measurements obtained from
BSE-infected tissues expressed as the differences of (D−N) indicates a
median value of approx66,000 c.p.m., and a ratio of approx880 for the median
of positive to median of negative samples (Fig. 2B). Introduction of the
sandwich aCDI increased the detection sensitivity for BSE prions
approx5-fold for median values and up to 20-fold for samples with a low
concentration of BoPrPSc (Fig. 2). After 1,729 tests had been done, the aCDI
identified all BSE cases with 100% accuracy, and no false positives occurred
in the control group.

Figure 2. Statistical evaluations of the direct and sandwich automated CDI.
Figure 2 thumbnail

Data for a group of (A) normal and (B) BSE-infected cattle. The obex area of
each normal (n = 432) and BSE-infected brainstem (n = 100) was tested. Some
samples were tested multiple times. Results are expressed as (D−N)
differences in c.p.m.


Full FigureFull Figure and legend (46K)
To understand the relatively broad distribution of (D−N) values within the
BSE group, three BSE-infected brainstems were studied in greater detail. In
each case, two adjacent, approx5-mm-thick cross sections were cut at the
level of the obex. Transverse slices were cut from the midline to the
periphery, yielding an average of 11 samples per brainstem (Fig. 3).
Greatest (D−N) differences were seen in samples taken from the midline of
the brainstem, but these values progressively decreased in samples collected
more laterally (Fig. 3). Importantly, we found up to an eightfold variation
in (D−N) values within one brainstem, indicating the importance of
consistent sampling for accurate diagnostic performance.

Figure 3. Transverse distribution of PrPSc in bovine brainstems at the level
of the obex as determined by CDI analysis correlates with known BSE
pathology.
Figure 3 thumbnail

The averaged distribution of (D−N) values measured by CDI using six slices
from three brainstems taken from BSE-infected cattle is shown in the upper
panel. The numbered position of each slice corresponds to the anatomical
location and structures in the lower panel drawing. Values (average
plusminus s.e.m., n = 6) are directly proportional to the concentration of
PrPSc (ref. 2). The data in the lower panel diagram were compiled from
histoblots20 and standard pathological techniques21; the light-to-dark
shading indicates, from mild to intense, respectively, the severity of
vacuolation and degree of PrPSc staining.


Full FigureFull Figure and legend (88K)
Previously, we plotted the D/N ratio against the PrPSc concentration to
distinguish different prion strains passaged in hamsters2. Because the PrPSc
concentration is directly proportional to (D−N) value, we plotted the D/N
ratio against the (D−N) difference (Fig. 4). The D/N ratio depends on the
antibody binding affinity to PrPSc conformers, in which D is considered a
reference point and N depends on the antibody binding affinity and the
epitope availability in undenatured PrPSc. Thus, the ratio gives simple
quantitative information about the native conformation of PrPSc, which is
prion strain-specific2, 9. Using this approach, we found that three
individual cases of BSE- or vCJD-infected Tg(BoPrP) mice showed D/N ratios
within the range of BSE-infected cattle, indicating similar conformational
characteristics with a somewhat higher concentration of BoPrPSc accumulating
in the brains of Tg mice.

Figure 4. Different conformational characteristics of ungulate prion strains
revealed by direct CDI.
Figure 4 thumbnail

CDI data were plotted as D/N ratios against (D−N) values recorded for
BSE-infected British cattle (n = 100; magenta square), first passage of
pooled brain homogenates from BSE- and vCJD-infected Tg(BoPrP)Prnp0/0 mice
(cyan square and orange square, respectively), CWD-infected mule deer (n =
10; open square), white-tailed deer (n = 6; open triangle), and elk (n = 19;
open circle). The arcs link cutoff values for differences and ratios in
normal US cattle (green; n = 432) and in normal deer and elk (orange; n =
60).


Full FigureFull Figure and legend (94K)
We applied the same plot analysis with CWD samples. However, CWD may not be
compared directly with BSE because of the Gright arrowS polymorphism at
position 97 within the P Fab epitope. When comparing results from different
CWD hosts sharing the same sequence, samples from white-tailed and mule deer
grouped in the same area, whereas samples from elk grouped separately (Fig.
4). This finding suggests a different PrPSc conformation in elk from that in
white-tailed and mule deer. Whether this observation indicates prion strain
differences between elk and deer remains to be established.

For years, detection of protease-resistant (r) PrPSc by immunoblot or
immunohistochemistry represented the only means of identifying prions in
tissue in vitro. Reliance on the complete enzymatic hydrolysis of
ubiquitous, immunoreactive PrPC, however, has limited both the specificity
and sensitivity of these assays6, 10, 11. In our studies, recFabs
recognizing ungulate PrP with high affinity and specificity have been
incorporated into a high-throughput aCDI, creating a rapid and sensitive
methodology for detecting BSE and CWD prions. The CDI, directly identifying
PrPSc through its distinct conformation, may be conducted independently of
proteolytic treatments. Consequently, the CDI can detect potentially large
in vivo concentrations of protease-sensitive (s) PrPSc molecules2. In
hamster brains, sPrPSc is found much earlier than rPrPSc (unpublished data);
whether a similar situation occurs in cattle or other ungulates remains to
be determined.

The performance characteristics and advantages of the aCDI for detecting
PrPSc in BSE- and CWD-infected brainstems include (i) detection of both
sPrPSc and rPrPSc; (ii) a readily scalable, quantitative diagnostic system
using robotic protocols to maintain specificity and sensitivity; (iii)
automated quality control and data processing; (iv) extremely accurate
diagnostic for detection of PrPSc in brainstems collected postmortem; (v)
prion positive:control ratio as high as 1,000 for BSE- and 4,000 for
CWD-infected samples; (vi) interassay variation <7%; (vii) detection limit
for BSE prions similar to bioassays in Tg(BoPrP)Prnp0/0 mice; and (viii)
potential for rapid prion strain typing.

To insure public safety, prion assays should detect one infectious unit.
However, prion titers can be greatly altered by the host in which
measurements are made. For example, BSE infectivity measured in cattle
showed that earlier studies using an RIII mouse bioassay3 underestimated BSE
prion titers by a factor of approx1,000 (refs. 5, 7). We report here that
Tg(BoPrP)Prnp0/0 mice, expressing multiple copies of the bovine PRNP gene,
are approx10 times more sensitive to infection with BSE prions than cattle.
This finding indicates that previous attempts to quantify BSE and scrapie
prions in milk or nonneural tissues, such as muscle, may have underestimated
infectious titers by up to a factor of 104, raising the possibility that
prions could be present in these products in sufficient quantities to pose
some risk to humans12. Additionally, in the studies reported here, we found
that PrPSc levels can vary by site within a brainstem (Fig. 3) by a factor
of 8. This suggests that multiple samples from each animal are required to
minimize the number of false negatives.

We demonstrate that the CDI is capable of measuring PrPSc in bovine
brainstems with a sensitivity similar to the infectivity levels determined
by end-point titrations in Tg(BoPrP) mice (Fig. 1). Although the brain is
known to contain the highest levels of PrPSc in all prion diseases, the
inaccessibility of this tissue will continue to hinder epidemiologic
investigations of these diseases. It will be important to apply the CDI to
the development of an antemortem test for prions. Using the CDI, we have
detected sPrPSc consistently in the blood of rodents infected with prions13.
Using western blots and ELISAs, we have detected rPrPSc in the hindlimbs of
mice12. Whether blood or muscle will be a suitable matrix for the
development of an antemortem test for prions remains to be determined.

Experimental protocol
Immunization and recovery of BoPrP-specific recFabs.
Six-week-old Prnp-ablated (Prnp0/0) mice were injected i.p. with 100 mug of
a synthetic BoPrP(96−115) peptide crosslinked to keyhole limpet hemocyanin
and fully emulsified in RIBI adjuvant. Three mice with the most robust and
specific anti-PrP serum IgG titers received a final boost with 25 mug of
unconjugated BoPrP(96−115) peptide and were sacrificed six days later. Three
IgG Fab libraries displayed on the surface of filamentous phage were
independently prepared from spleen and bone marrow RNA derived from each of
these animals14.

Phage antibody libraries were individually panned against a panel of
different recombinant and synthetic PrP antigens immobilized onto ELISA
wells14. The recovery and epitope recognition characteristics of Fab D18
were described15.

Expression and purification of HuM Fabs.
Fabs P, S, and D18 were expressed in bacteria as both mouse and HuM Fabs, in
which the murine heavy- and light-chain variable genes were fused to genes
encoding human CHgamma1 and CK sequences16. The HuM recFabs were expressed
and fermented in Escherichia coli 33B6 competent cells and purified as
described16. Selected antibody clones were also inserted sequentially into a
eukaryotic expression vector containing human IgG1 constant region and human
kappa constant region genes and expressed in Chinese hamster ovary (CHO)
cells17.

Surface plasmon resonance.
Recombinant BoPrP(23−231), SHaPrP(29−231), or synthetic antigens were
immobilized to carboxy-methyl groups present on the surface of CM5 sensor
chips using N-hydroxysuccinimide and
N-ethyl-N'-((dimethylamino)propyl)-carbodiimidehydrochloride18. Kinetic
constants for the binding of PrP-specific recFabs or IgG for a series of
concentrations were analyzed globally using BiaEval 3.1 software (Biacore).

Preparation of brain homogenates.
Slices from the obex area of the brainstem were homogenized to a final 15%
(wt/vol) in 4% (wt/vol) Sarkosyl in PBS, pH 7.4. The homogenate was diluted
to 5% (wt/vol) using PBS containing 5 mug/ml of proteinase K and incubated
for 60 min at 37°C. After clarification, samples were precipitated with
0.32% NaPTA. After a 60 min incubation at 37°C, samples were centrifuged at
14,000 g in a Jouan MR23i centrifuge for 15−30 min, and resuspended pellets
were assayed by the CDI.

Direct and sandwich CDI for ungulate PrPSc.
The principle, development, calibration, and calculation of PrPSc
concentration from CDI data have been described2. The CDI data described in
this paper were generated with recFab HuM-P labeled with Eu-chelate. Each
sample was divided into two aliquots: (i) untreated (designated native) and
(ii) mixed to a final concentration of 4 M GdnHCl and heated for 5 min at
80°C (designated denatured). Both samples were diluted with H2O containing
protease inhibitors, and aliquots were loaded on a 96-well black polystyrene
plate (Packard, Meriden, CT) that had been activated with 0.2%
glutaraldehyde for direct CDI or coated overnight with 5 mug/ml recFab D18
in sodium bicarbonate buffer, pH 8.6 for sandwich CDI. The plates were
incubated for 2 h and then blocked with Tris-buffered saline (TBS), pH 7.8,
containing 0.5% BSA (wt/vol) and 6% sorbitol (wt/vol) for 1 h at room
temperature. The plates were washed with TBS containing 0.05% (vol/vol)
Tween-20, incubated with Eu-labeled recFab HuM-P for 2 h, washed, then
developed in enhancement solution (Wallac Inc., Turku, Finland). Signals
were counted on a Discovery dual-wavelength, time-resolved fluorometer
(Packard).

aCDI with sample tracking and data processing.
The scaled-up aCDI protocol was developed for the Genesis Robotic Sample
Processor 150 liquid-handling station (Tecan, Research Triangle Park, NC).
All data were analyzed using a custom-built MS SQL interface to distinguish
between positive and negative samples by comparing with internal standards
and threshold values.

End-point titration of BSE prions in Tg(BoPrP)Prnp0/0 mice.
We established Tg(BoPrP+/+)4092/Prnp0/0 mice that are homozygous for the
bovine transgene array. The inoculum was a 10% (wt/vol) homogenate prepared
from the medulla of a Hereford bull with BSE (case PG31/90). Three separate
dilution series were done in parallel, and 30 mul from each dilution were
inoculated i.c. into groups of five mice. Triplicate end-point titrations of
BSE prions were done; titers were calculated according to the methods of
Karber as well as Reed and Munch19, and the average from these three
titrations was determined. The results are expressed as the median infective
dose (ID50) per gram of tissue.

Note: Supplementary information is available on the Nature Biotechnology
website.

Top
Received 21 March 2002; Accepted 20 August 2002; Published online: 21
October 2002.

REFERENCES

1. Mehlhorn, I. et al. High-level expression and characterization of a
purified 142-residue polypeptide of the prion protein. Biochemistry 35,
5528–5537 (1996). | Article | PubMed | ISI | ChemPort |
2. Safar, J. et al. Eight prion strains have PrPSc molecules with
different conformations. Nat. Med. 4, 1157–1165 (1998). | Article | PubMed
| ISI | ChemPort |
3. Bruce, M. et al. Transmission of bovine spongiform encephalopathy and
scrapie to mice: strain variation and the species barrier. Phil. Trans. R.
Soc. Lond. B 343, 405–411 (1994). | ISI | ChemPort |
4. Fraser, H., Bruce, M.E., Chree, A., McConnell, I. & Wells, G.A.H.
Transmission of bovine spongiform encephalopathy and scrapie to mice. J.
Gen. Virol. 73, 1891–1897 (1992). | PubMed | ISI |
5. Moynagh, J., Schimmel, H. & Kramer, G.N. The evaluation of tests for
the diagnosis of transmissible spongiform encephalopathy in bovines. 1–36
(European Commission, 1999).
6. Deslys, J.P. et al. Screening slaughtered cattle for BSE. Nature 409,
476–478 (2001). | Article | PubMed | ISI | ChemPort |
7. Wells, G.A.H. et al. Preliminary observations on the pathogenesis of
experimental bovine spongiform encephalopathy (BSE): an update. Vet. Rec.
142, 103–106 (1998). | PubMed | ISI | ChemPort |
8. Scott, M.R. et al. Compelling transgenetic evidence for transmission
of bovine spongiform encephalopathy prions to humans. Proc. Natl. Acad. Sci.
USA 96, 15137–15142 (1999). | Article | PubMed | ChemPort |
9. Peretz, D. et al. Strain-specified relative conformational stability
of the scrapie prion protein. Protein Sci. 10, 854–863 (2001). | Article |
PubMed | ISI | ChemPort |
10. Schaller, O. et al. Validation of a western immunoblotting procedure
for bovine PrPSc detection and its use as a rapid surveillance method for
the diagnosis of bovine spongiform encephalopathy (BSE). Acta Neuropathol.
98, 437–443 (1999). | Article | PubMed | ISI | ChemPort |
11. Biffiger, K. et al. Validation of a luminescence immunoassay for the
detection of PrPSc in brain homogenate. J. Virol. Methods 101, 79–84 (2002).
| Article | PubMed | ISI | ChemPort |
12. Bosque, P.J. et al. Prions in skeletal muscle. Proc. Natl. Acad. Sci.
USA 99, 3812–3817 (2002). | Article | PubMed | ChemPort |
13. Prusiner, S.B. & Safar, J.G. Method of concentrating prion proteins in
blood samples. US 6,166,187 (2000).
14. Williamson, R.A. et al. Circumventing tolerance to generate autologous
monoclonal antibodies to the prion protein. Proc. Natl. Acad. Sci. USA 93,
7279–7282 (1996). | Article | PubMed | ChemPort |
15. Peretz, D. et al. A conformational transition at the N terminus of the
prion protein features in formation of the scrapie isoform. J. Mol. Biol.
273, 614–622 (1997). | Article | PubMed | ISI | ChemPort |
16. Peretz, D. et al. Antibodies inhibit prion propagation and clear cell
cultures of prion infectivity. Nature 412, 739–743 (2001). | Article |
PubMed | ISI | ChemPort |
17. Burton, D.R. & Barbas, C.F. Human antibodies from combinatorial
libraries. Adv. Immunol. 57, 191–280 (1994). | PubMed | ISI | ChemPort |
18. Johnsson, B., Lofas, S. & Lindquist, G. Immobilization of proteins to
a carboxymethyldextran-modified gold surface for biospecific interaction
analysis in surface plasmon resonance sensors. Anal. Biochem. 198, 268–277
(1991). | PubMed | ISI | ChemPort |
19. Dougherty, R. in Techniques in Experimental Virology (ed. Harris,
R.J.C.) 169–224 (Academic Press, New York, 1964).
20. Prusiner, S.B. et al. Immunologic and molecular biological studies of
prion proteins in bovine spongiform encephalopathy. J. Infect. Dis. 167,
602–613 (1993). | PubMed | ISI | ChemPort |
21. Wells, G.A.H. & Wilesmith, J.W. The neuropathology and epidemiology of
bovine spongiform encephalopathy. Brain Pathol. 5, 91–103 (1995). | PubMed
| ISI | ChemPort |



============================

BIO-RAD

> > -------- Original Message --------
> > Subject: USA BIO-RADs INCONCLUSIVEs
> > Date: Fri, 17 Dec 2004 15:37:28 -0600
> > From: "Terry S. Singeltary Sr."
> > To: [email protected]
> >
> >
> >
> > Hello Susan and Bio-Rad,
> >
> > Happy Holidays!
> >
> > I wish to ask a question about Bio-Rad and USDA BSE/TSE testing
> > and there inconclusive. IS the Bio-Rad test for BSE/TSE that
complicated,
> > or is there most likely some human error we are seeing here?
> >
> > HOW can Japan have 2 positive cows with
> > No clinical signs WB+, IHC-, HP- ,
> > BUT in the USA, these cows are considered 'negative'?
> >
> > IS there more politics working here than science in the USA?
> >
> > What am I missing?
> >
> >
> >
> > -------- Original Message --------
> > Subject: Re: USDA: More mad cow testing will demonstrate beef's safety
> > Date: Fri, 17 Dec 2004 09:26:19 -0600
> > From: "Terry S. Singeltary Sr."
> > snip...end
> >
> >
> > Experts doubt USDA's mad cow results
>
>
>
> snip...END
>
> WELL, someone did call me from Bio-Rad about this,
> however it was not Susan Berg.
> but i had to just about take a blood oath not to reveal
> there name. IN fact they did not want me to even mention
> this, but i feel it is much much to important. I have omitted
> any I.D. of this person, but thought I must document this ;
>
> Bio-Rad, TSS phone conversation 12/28/04
>
> Finally spoke with ;
>
>
> Bio-Rad Laboratories
> 2000 Alfred Nobel Drive
> Hercules, CA 94547
> Ph: 510-741-6720
> Fax: 510-741-5630
> Email: XXXXXXXXXXXXXXXXXX
>
> at approx. 14:00 hours 12/28/04, I had a very pleasant
> phone conversation with XXXX XXXXX about the USDA
> and the inconclusive BSE testing problems they seem
> to keep having. X was very very cautious as to speak
> directly about USDA and it's policy of not using WB.
> X was very concerned as a Bio-Rad official of retaliation
> of some sort. X would only speak of what other countries
> do, and that i should take that as an answer. I told X
> I understood that it was a very loaded question and X
> agreed several times over and even said a political one.
>
> my question;
>
> Does Bio-Rad believe USDA's final determination of False positive,
> without WB, and considering the new
> atypical TSEs not showing positive with -IHC and -HP ???
>
> ask if i was a reporter. i said no, i was with CJD Watch
> and that i had lost my mother to hvCJD. X did not
> want any of this recorded or repeated.
>
> again, very nervous, will not answer directly about USDA for fear of
> retaliation, but again said X tell
> me what other countries are doing and finding, and that
> i should take it from there.
> "very difficult to answer"
>
> "very political"
>
> "very loaded question"
>
> outside USA and Canada, they use many different confirmatory tech. in
> house WB, SAF, along with
> IHC, HP, several times etc. you should see at several
> talks meetings (TSE) of late Paris Dec 2, that IHC- DOES NOT MEAN IT IS
> NEGATIVE. again, look what
> the rest of the world is doing.
> said something about Dr. Houston stating;
> any screening assay, always a chance for human
> error. but with so many errors (i am assuming
> X meant inconclusive), why are there no investigations, just false
> positives?
> said something about ''just look at the sheep that tested IHC- but were
> positive''. ...
>
>
> TSS
>
> -------- Original Message --------
> Subject: Your questions
> Date: Mon, 27 Dec 2004 15:58:11 -0800
> From: To: [email protected]net
>
>
>
> Hi Terry:
>
> ............................................snip Let me know your phone
> number so I can talk to you about the Bio-Rad BSE test.
> Thank you
>
> Regards
>
>
>
> Bio-Rad Laboratories
> 2000 Alfred Nobel Drive
> Hercules, CA 94547
> Ph: 510-741-6720
> Fax: 510-741-5630
> Email: =================================
>
>
> END...TSS
>
>
> ######### https://listserv.kaliv.uni-karlsruhe.de/warc/bse-l.html
##########




----- Original Message -----
From: Amanda Kolling
To: Terry S. Singeltary Sr.
Sent: Thursday, May 26, 2005 8:11 AM
Subject: RE: Prion biology relevant to bovine spongiform encephalopathy (ANIMALSCI Feedback Form)


Dear Mr. Singeltary,

Thank you for your comments. Contrary to a previous e-mail sent to you by an employee of HighWire, the Journal of Animal Science does accept Letters to the Editor. If you are interested in submitting this as a letter to the editor, I urge you to contact our editor-in-chief, Dr. Michael Gaylean at [email protected]

Best regards,

Amanda Kolling
Technical Editor,
Journal of Animal Science


At 09:11 AM 5/17/2005 -0700, Terry S. Singeltary Sr. wrote:
>------------------------------------------------------------
>Comments sent via JAS Feedback Page
>------------------------------------------------------------
> NAME: Terry S. Singeltary Sr.
> EMAIL: [email protected]
> IP ADDRESS: 216.119.139.23
> HOSTNAME: pool139-23.dial-p1.hou.wt.net
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>------------------------------------------------------------
>COMMENTS:
>J. Anim. Sci. 2005. 83:1455-1476
>© 2005 American Society of Animal Science
>SPECIAL TOPICS
>Prion biology relevant to bovine spongiform encephalopathy1
>J. Novakofski*,2, M. S. Brewer{dagger}, N.
>Mateus-Pinilla{ddagger}, J. Killefer* and R. H. McCusker*
>
>* Departments of Animal Sciences and {dagger} Food
>Science and Human Nutrition, University of Illinois at
>Urbana­Champaign 61801-4737; and {ddagger} Illinois
>Natural History Survey, Center for Wildlife and Plant
>Ecology, Champaign, IL 61820
>
>2 Correspondence: 1503 South Maryland Dr. (phone:
>217-333-6181; e-mail: [email protected]).
>
>Bovine spongiform encephalopathy (BSE) and chronic
>wasting disease (CWD) of deer and elk are a threat to
>agriculture and natural resources, as well as a human
>health concern. Both diseases are transmissible
>spongiform encephalopathies (TSE), or prion diseases,
>caused by autocatalytic conversion of endogenously
>encoded prion protein (PrP) to an abnormal, neurotoxic
>conformation designated PrPsc. Most mammalian species
>are susceptible to TSE, which, despite a range of
>species-linked names, is caused by a single highly
>conserved protein, with no apparent normal function. In
>the simplest sense, TSE transmission can occur because
>PrPsc is resistant to both endogenous and environmental
>proteinases, although many details remain unclear.
>Questions about the transmission of TSE are central to
>practical issues such as livestock testing, access to
>international livestock markets, and wildlife
>management strategies, as well as intangible issues
>such as consumer confidence in the safety of the meat
>supply. The majority of BSE cases seem to have been
>transmitted by feed containing meat and bone meal from
>infected animals. In the United Kingdom, there was a
>dramatic decrease in BSE cases after neural tissue and,
>later, all ruminant tissues were banned from ruminant
>feed. However, probably because of heightened awareness
>and widespread testing, there is growing evidence that
>new variants of BSE are arising "spontaneously,"
>suggesting ongoing surveillance will continue to find
>infected animals. Interspecies transmission is
>inefficient and depends on exposure, sequence homology,
>TSE donor strain, genetic polymorphism of the host, and
>architecture of the visceral nerves if exposure is by
>an oral route. Considering the low probability of
>interspecies transmission, the low efficiency of oral
>transmission, and the low prion levels in nonnervous
>tissues, consumption of conventional animal products
>represents minimal risk. However, detection of rare
>events is challenging, and TSE literature is
>characterized by subsequently unsupported claims of
>species barriers or absolute tissue safety. This review
>presents an overview of TSE and summarizes recent
>research on pathogenesis and transmission.
>
>Key Words: Bovine Spongiform Encephalopathy . Chronic
>Wasting Disease . Prion
>
>http://jas.fass.org/cgi/content/abstract/83/6/1455
>
> >there is growing evidence that new variants of BSE are
>arising "spontaneously,"
>
>
>THERE is NO evidence of a 'spontaneous' TSE anywhere that
>is infectious and shows the pathology of any natural TSE.
>if i have missed something, could someone please site this
>science to me please.
>
>
> >Considering the low probability of interspecies
>transmission, the low efficiency of oral transmission,
>and the low prion levels in nonnervous tissues,
>consumption of conventional animal products represents
>minimal risk.
>
>
>I DISAGREE with all of the above. all one has to do is
>read transmission
>studies. scrapie infected sheep and goats, CWD infected
>deer and
>elk (who knows how many strains) and undocumented TSEs
>in the
>USA bovine have been rendered and fed to animals for
>humna/animal
>consumption for decades. it's only a pipe dream that
>none of this
>was infectious. to think of a 'spontaneous' TSE as just
>popping
>up from nowhere, is like believing in Santa Claus. remember
>the USA scrapie research in Mission, Texas. IT did NOT look
>like BSE...
>
>
>1: J Infect Dis 1980 Aug;142(2):205-8
>
> Oral transmission of kuru, Creutzfeldt-Jakob
>disease, and scrapie to nonhuman primates.
>
> Gibbs CJ Jr, Amyx HL, Bacote A, Masters CL,
>Gajdusek DC.
>
> Kuru and Creutzfeldt-Jakob disease of humans and
>scrapie disease of sheep and goats were transmitted to
>squirrel monkeys (Saimiri sciureus) that were exposed
>to the infectious agents only by their nonforced
>consumption of known infectious tissues. The
>asymptomatic incubation period in the one monkey
>exposed to the virus of kuru was 36 months; that in the
>two monkeys exposed to the virus of Creutzfeldt-Jakob
>disease was 23 and 27 months, respectively; and that in
>the two monkeys exposed to the virus of scrapie was 25
>and 32 months, respectively. Careful physical
>examination of the buccal cavities of all of the
>monkeys failed to reveal signs or oral lesions. One
>additional monkey similarly exposed to kuru has
>remained asymptomatic during the 39 months that it has
>been under observation.
>
>PMID: 6997404
>http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6997404&dopt=Abstract
>
>
>3. You will recall that the advice provided by
>Professor Smith in
>1993 and by Dr. Gore this month used the sub-population
>of dairy
>farm workers who had had a case of BSE on their farms -
>63,000, which is approximately half the number of dairy
>farm
>workers - as a denominator. If the above sums are
>repeated using
>this denominator population, taking an annual incidence
>in the general
>population of 1 per million the observed rate in this
>sub-population
>is 10 TIMES, and taking an annual incidence of 0.7 per
>million,
>IT IS 15 TIMES (THE ''WORST CASE'' SCENARIO) than
>that in the general population...
>
>http://www.bseinquiry.gov.uk/files/yb/1995/01/31004001.pdf
>
>
>It was, however, performed in the USA in 1979, when it
>was shown that
>cattle inoculated with the scrapie agent endemic in the
>flock of Suffolk
>sheep at the United States Department of Agriculture in
>Mission, Texas,
>developed a TSE quite unlike BSE. 32 <
>
>http://www.bseinquiry.gov.uk/report/volume2/chaptea3.htm#820543
>
>The findings of the initial transmission, though not of
>the clinical or
>neurohistological examination, were communicated in
>October 1988 to Dr
>Watson, Director of the CVL, following a visit by Dr
>Wrathall, one of
>the project leaders in the Pathology Department of the
>CVL, to the
>United States Department of Agriculture. 33
>
>
>http://www.bseinquiry.gov.uk/report/volume2/chaptea3.htm#820546
>
>
>The results were not published at this point, since the
>attempted
>transmission to mice from the experimental cow brain
>had been
>inconclusive. The results of the clinical and
>histological differences
>between scrapie-affected sheep and cattle were
>published in 1995.
>Similar studies in which cattle were inoculated
>intracerebrally with
>scrapie inocula derived from a number of
>scrapie-affected sheep of
>different breeds and from different States, were
>carried out at the US
>National Animal Disease Centre. 34
>
>
>http://www.bseinquiry.gov.uk/report/volume2/chaptea3.htm#820549
>
>
>The results, published in 1994, showed that this source
>of scrapie
>agent, though pathogenic for cattle, did not produce
>the same clinical
>signs of brain lesions characteristic of BSE.
>
>
>http://www.bseinquiry.gov.uk/report/volume2/chaptea3.htm
>
>
>Visit to USA ... info on BSE and Scrapie
>
>
>http://www.bseinquiry.gov.uk/files/yb/1988/10/00001001.pdf
>
>
>HOUND STUDY
>
>AS implied in the Inset 25 we must not _ASSUME_ that
>transmission of BSE to other species will invariably
>present pathology typical of a scrapie-like disease.
>
>snip...
>
>http://www.bseinquiry.gov.uk/files/yb/1991/01/04004001.pdf
>
>
>In Confidence - Perceptions of unconventional slow
>virus diseases
>of animals in the USA - APRIL-MAY 1989 - G A H Wells
>
>
>http://www.bseinquiry.gov.uk/files/mb/m11b/tab01.pdf
>
>WHY is USA insisting _now_ not to use WB, when on the
>1st _confirmed_
>case Dec. 23, 2003
>USA mad cow, WB was used ???
>
>maybe this is the reason ;
>
>JAPAN BSE # 8 & 9 cow
>
>8. 6/10/2003 Holstein Steer 13/10/2001 23 mths
>No clinical signs WB+, IHC-, HP-
>
>
>9. 4/11/2003 Holstein Steer 13/1/2002
>21 mths No clinical signs WB+, IHC-, HP-
>
>===========
>
>More information on the first 11 Japanese BSE-cases can
>be found on the

>website of the Japanese Embassy in the US:
>
>http://www.us.emb-japan.go.jp/english/html/fafacts/bse/bse.htm
>
>
>IN fact, the new strain of TSE in cattle BaSE, does not
>look like nvCJD in humans, but very similar
>to the sporadic CJD;
>
>
>BASE in cattle in Italy of Identification of a second
>bovine amyloidotic spongiform encephalopathy: Molecular
>similarities with sporadic Creutzfeldt-Jakob disease
>
>http://www.pnas.org/cgi/content/abstract/0305777101v1
>
>
>Adaptation of the bovine spongiform encephalopathy
>agent to primates and comparison with Creutzfeldt-
>Jakob disease: Implications for human health THE
>findings from Corinne Ida Lasmézas*, [dagger] ,
>Jean-Guy Fournier*, Virginie Nouvel*, Hermann Boe*,
>Domíníque Marcé*, François Lamoury*, Nicolas Kopp
>[Dagger ] , Jean-Jacques Hauw§, James Ironside¶, Moira
>Bruce [||] , Dominique Dormont*, and Jean-Philippe
>Deslys* et al, that The agent responsible for French
>iatrogenic growth hormone-linked CJD taken as a control
>is very different from vCJD but is similar to that
>found in one case of sporadic CJD and one sheep scrapie
>isolate;
>
>
>http://www.pnas.org/cgi/content/full/041490898v1
>
>
>Characterization of two distinct prion strains derived
>from bovine spongiform encephalopathy transmissions to
>inbred mice
>
>
> http://vir.sgmjournals.org/cgi/content/abstract/85/8/2471
>
>
>USA BSE GBR III
>
>http://www.efsa.eu.int/science/efsa_scientific_reports/gbr_assessments/scr_annexes/574/sr03_biohaz02_usa_report_annex_en1.pdf
>
>http://www.fda.gov/ohrms/dockets/dockets/03n0312/03N-0312_emc-000001.txt
>
>https://web01.aphis.usda.gov/regpublic.nsf/0/eff9eff1f7c5cf2b87256ecf000df08d?OpenDocument
>
>https://web01.aphis.usda.gov/BSEcom.nsf/0/b78ba677e2b0c12185256dd300649f9d?OpenDocument&AutoFramed
>
>
>Terry S. Singeltary SR.
>P.O. Box 42
>Bacliff, Texas USA 77518

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

La Vonne Gallo
HighWire Press
1454 Page Mill Road
Palo Alto, CA 94304
fax: 650.725.9335
[email protected]

~~~~~~~~~~~
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
 

Tam

Well-known member
reader (the Second) said:
Murgen wrote:
What are the ages of the cows, in the US compared to Canada?


R2: If I remember correctly all the U.S. are born after the ban and all but ONE of the Canadian are born after the ban. 10 - 13 in most cases. One 6 or 7 year old.


Excuse me Ms Tam but it's back to remedial reading for you. First, I caveated my statement by saying "if I remember correctly." Then I said that most of the cattle with BSE, both Canadian and U.S., were 10 - 13 years old and only one was young. YES I MEANT TO SAY MOST WERE BORN BEFORE THE BAN AND MISSPOKE BUT THAT IS OBVIOUS FROM THE REST OF MY POST SINCE I SPEAK OF CATTLE BORN BEFORE 1997 IN MOST CASES.
Does my remedial reading also change the number of cattle actually born after the bans Reader. :wink: You said "all but ONE of the Canadian are born after the ban." meaning of course before the bans. and now you are saying "both Canadian and U.S., were 10 - 13 years old and only one was young." Since Oldtimer has made it his task in life to point out daily that Canada has had THREE born after the bans I just took it upon myself to correct your memory and your NUMBERS But you would rather make snipes like Give it a break. If you think those are the only North America cows, then you're smoking something we should know about. than to admit the information posted by you was wrong. Have another glass of wine Reader. :roll:
 

Murgen

Well-known member
another thing to ponder. prusiner has had this cdi test for years now.
one problem, it is so sensitive, no one will validate it in the USA.
SO, round and round we go. why do you think they picked the least likely
rapid test to find BSE at first? same reason they chose not to use WB, when Dr. Detwiler told them in 2003 they would be missing cases if they only use IHC, the put her out to pasture. she got the last laugh. and now is at the big mac as one
of the top TSE advisers now, and now even the big mac is after USDA et al. money talks and BSe walks though, we have to wait and see from here, but this spontaneous excuse is a hoot. there final straw so to speak. ...

Still think the USDA didn't think there were cases within the US, and so didn't test, all the while exporting MBM to other countries, Sandhusker?

"you won't find them, if you don't test" Should be USDA's motto.

Please start testing everything, but use an independent lab, (UK preferably)
 

Mike

Well-known member
The first approved rapid test used by the U.S. was the Bio-Rad. It was certainly not the least likely to find a positive. In fact, it has had several false positives in Japan because it is extremely sensitive and the samples must be almost perfect.

Did you forget that the Western Blot found the Washington cow?

Go back to bed Murgen. You're conspiracies have gotten the best of you.
 

Bill

Well-known member
Mike said:
The first approved rapid test used by the U.S. was the Bio-Rad. It was certainly not the least likely to find a positive. In fact, it has had several false positives in Japan because it is extremely sensitive and the samples must be almost perfect.

Did you forget that the Western Blot found the Washington cow?

Go back to bed Murgen. You're conspiracies have gotten the best of you.
No offense Mike but Murgens "conspiracy theories" are miniscule compared to Oldtimer, Sandhusker, Econ, et al!!!!!!!!! :lol: :lol: :lol:
 

Murgen

Well-known member
No offense Mike but Murgens "conspiracy theories" are miniscule compared to Oldtimer, Sandhusker, Econ, et al!!!!!!!!!

My conspiracy theories are backed up by RCALF. I'm thinking of becoming a member!
 

Econ101

Well-known member
What "conspiracy" theory are you talking about, Bill? Do you not think that Tyson has enough money to have the best economists and support to think out how best to game the markets while ranchers like you can only sit back, act dumb when it is to your own advantage, and be voracious in any attacks on people who are able to see the bigger picture?
 

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