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From: TSS ()
Subject: BSE Minimal-Risk Regions and Importation of Live Animals and Commodities from Canada March 4, 2005 To: Importers, Brokers and Other Interested Parties
Date: March 6, 2005 at 4:53 pm PST

-------- Original Message --------
Subject: BSE Minimal-Risk Regions and Importation of Live Animals and Commodities from Canada March 4, 2005 To: Importers, Brokers and Other Interested Parties
Date: Sun, 06 Mar 2005 18:48:51 -0600
From: "Terry S. Singeltary Sr."
To: Bovine Spongiform Encephalopathy
CC: Gary Burkholder


United States
Department of
Agriculture
Animal and Plant
Health Inspection
Service
Veterinary Services
National Animal
Health Policy and
Programs
4700 River Road
Unit 33
Riverdale, MD 20737
March 4, 2005
Subject: BSE Minimal-Risk Regions and Importation of Live Animals and
Commodities from Canada
Delay of Effective Date
To: Importers, Brokers and Other Interested Parties
(301)734-8093
FAX (301) 734-8818
On January 4, 2005, the USDA, Animal and Plant Health Inspection Service
(APHIS)
published a final rule which amended the regulations to provide for the
importation of
certain ruminants, ruminant products and byproducts from regions that
pose a minimal
risk of introducing bovine spongiform encephalopathy (BSE) into the
United States,
and designated Canada as the first minimal-risk region (70 FR 460-553,
Docket
No. 03-080-3). The effective date of the final rule was to be March 7, 2005.
However, on March 2, 2005 the U.S. District Court for the District of
Montana granted
a preliminary injunction to prevent the implementation of the minimal
risk rule until the
R-CALF lawsuit is considered on the merits by the court.
Therefore, until further notice, the current import requirements for
ruminant and
ruminant commodities from Canada will remain unchanged. Only those
commodities
that were listed in the August 15, 2003 notice (republished May 6, 2004)
will be
eligible for importation from Canada, under the risk-mitigation measures
specified in
that notice.
W^-
Jere L. Dick
Associate Deputy Administrator
National Animal Health Policy and Programs

http://www.aphis.usda.gov/lpa/issues/bse/bserisk.pdf

Importation of Processed Canadian Beef Products
Regulatory Timeline
On May 20, 2003, USDA announces Canada’s detection of bovine spongiform
encephalopathy
(BSE) in an animal in that country. USDA places Canada under its BSE
restriction guidelines
and begins prohibiting the entry of all ruminants or ruminant products
from Canada, pending
further investigation into the detection. USDA also dispatches a team to
assist Canadian officials
with the epidemiological investigation and help provide USDA with
necessary scientific
information regarding the situation.
On August 8, 2003, following the completion of Canada’s epidemiological
investigation, USDA
announces it will no longer prohibit the importation of hunter-harvested
wild ruminant products
intended for personal use. In addition, USDA announces that it will
accept applications for
import permits for certain low-risk products. These products include:
• boneless sheep or goat meat from animals under 12 months of age
• boneless bovine meat from cattle under 30 months of age
• veal meat from calves that were 36 weeks of age or younger at slaughter
• fresh or frozen bovine liver
• vaccines for veterinary medicine for non-ruminant use
• and pet products and feed ingredients that contain processed animal
protein and tallow of
non-ruminant sources when produced in facilities with dedicated
manufacturing lines.
Subsequent permit requests were examined thoroughly by USDA’s Animal and
Plant Health
Inspection Service (APHIS), taking into account risk mitigations that
were in place (such as
facility dedication, specified risk material (SRM) removal, and age of
cattle). Permits were
issued, as authorized by existing regulatory authority, for products
that were deemed to be low
risk. The import permits included specific risk mitigation requirements
that must be followed in
order for the permit to be valid.
On August 15, 2003, USDA posts an amended list of allowable products on
its website as a
clarification of the August 8 announcement. The list includes “trim”
from boneless beef from
cattle under 30 months of age and veal (including carcasses) from calves
36 weeks of age or
under.
Permit applications were subsequently submitted to APHIS for processed
product made from
allowable product. APHIS determined that processed product from trim and
boneless beef from
cattle under 30 months of age would be allowed, since processing would
not increase the risk
associated with the products.
On August 27, 2003, APHIS issues the first permit for approved ground
product. Subsequent
permits allow the entry of other processed meat from cattle under 30
months of age, such as hot
dogs, pepperoni pizza toppings, hamburger patties, smoked briskets, dry
cured beef cuts, and
soups/TV dinners containing beef.
From August 27, 2003, to April 26, 2004, a total of 5,611,580 million
pounds of processed
product and ground beef are imported into the United States. More than
half of this product is
not Canadian in origin, but rather originates in the United States (or
another recognized BSE-free
country such as Australia or New Zealand) and simply goes to Canada for
processing. The
product is imported under conditions designed to ensure that there was
no commingling with
Canadian product.
The remaining 2,232,459 pounds of product is imported on permits
allowing for importation of
product that either originated in the United States (or another BSE free
country) or that
originated in Canada, provided that the product was processed strictly
from animals under 30
months of age, and in accordance with a number of processing
requirements designed to further
mitigate any risk.
On October 21, 2003, USDA issues an alert on the policy that allows
transit through the United
States of certain meat and meat products from Canada. These shipments
must be accompanied
by an import permit, and all permit conditions must be met.
On October 22, 2003, USDA issues an amendment adding several new low
risk Canadian
products to the "Low Risk Canadian Products" list. These products are
edible bovine tongues,
edible bovine hearts or kidneys, and edible bovine lips. These products
are enterable if
accompanied by an APHIS permit and other necessary documentation.
On November 4, 2003, USDA publishes in the Federal Register a proposed
rule to allow the
importation of certain live ruminants and ruminant products and
byproducts from minimal risk
regions under specified conditions. The proposed rule would also add
Canada to this list of
minimal risk regions.
Following the completion of USDA’s epidemiological investigation into
the December, 2003,
detection of BSE in Washington State, USDA republishes the proposed rule
in the Federal
Register for public comment. The comment period closed in April 2004,
and the Department is
currently reviewing all of the comments received.
On April 19, 2004, APHIS posts information on its website regarding the
Agency’s decision to
issue import permits for bone-in beef for animals under 30 months of
age. This posting leads the
Ranchers-Cattlemen Action Legal Fund, United Stockgrowers of America
(R-CALF USA) to
file suit against USDA in U.S. District Court for the District of Montana.
On April 26, 2004, a U.S. District Court judge issues a Temporary
Restraining Order that
prohibits APHIS from issuing import permits for products other than
those included as part of
the August 15, 2003, list of low-risk Canadian products. USDA reaches a
subsequent settlement
agreement with R-CALF representatives in which USDA reinstates the
August 15, 2003,
permitting and risk mitigation measures pending completion of the
rulemaking initiated on
November 4, 2003.
June 10, 2004

http://www.aphis.usda.gov/lpa/issues/bse/bsechronjune10.pdf

USDA says Canadian beef imported despite ban was safe

Robert Roos * News Editor

May 21, 2004 (CIDRAP News) – The US Department of Agriculture (USDA)
acknowledged today that it allowed the importation of about 7.3 million
pounds of Canadian beef that was officially banned under rules intended
to keep bovine spongiform encephalopathy (BSE) out of the United States.

But the USDA said the meat, imported since last summer, did not
represent any risk to consumers because it came from cattle under 30
months of age. BSE has very rarely been found in animals younger than 30
months.

"We're talking about products that are safe products," Undersecretary
for Food Safety Elsa Murano said at a news briefing. "All these products
came from animals that were younger than 30 months of age. Therefore,
we're not talking about a food safety issue here."

USDA officials discussed the issue in response to news reports published
yesterday. A Washington Post report, quoting figures from a cattlemen's
organization, said 33 million pounds of "processed" beef was imported
since last summer despite the official ban. The Ranchers-Cattlemen
Action Legal Fund USA (R-CALF USA), based in Billings, Mont., calculated
that amount from statistics from the Census Bureau and the USDA's
Foreign Agricultural Service, the story said.

Also yesterday, a Reuters report said the USDA disclosed that it had
allowed Canadian plants to send into the United States about 10 million
pounds of hamburger and other cuts of beef that were officially banned.

The USDA banned all imports of Canadian beef and live cattle after a BSE
case was discovered in a cow from an Alberta farm in May 2003. In August
the agency reopened the border to boneless meat (but not ground meat)
from cattle younger than 30 months old, boneless veal from calves
younger than 36 weeks, and fresh or frozen beef liver. Live cattle and
other categories of beef are still banned.

Murano said she didn't know how R-CALF USA arrived at the figure of 33
million pounds of processed beef imported. She said the USDA Food Safety
and Inspection service's import records showed that the questionable
imports included 5.6 million pounds of processed products such as hot
dogs, sausages, and deli meats; about 1.5 million pounds of organs, such
as tongue, hearts, and kidneys; 139,298 pounds of bone-in cuts admitted
after Apr 19; and 475 pounds of hamburger.

Ron DeHaven, administrator of the USDA Animal and Plant Health
Inspection Service (APHIS), said all of the processed beef that was
imported was made from beef that would not have been subject to the ban
in its unprocessed form. "All of those items were included in our list
of [permitted] products that we put on the website August 8 as enterable
products," he said. "The only thing that changed was our allowing those
products . . . to be processed before they entered the United States."

While insisting that the products posed no risk to consumers, USDA
officials admitted mistakes in handling the matter. "Clearly the process
and our failure to announce some of these actions was flawed," DeHaven said.

Agriculture Secretary Ann Veneman didn't know about the "additional
process product" coming into the United States, said Alisa Harrison, a
spokeswoman for Veneman. Harrison also said that APHIS followed proper
food safety procedures but "did fail to obtain the approval for their
actions from the appropriate USDA policy and legal representative."

DeHaven said the Canadian Food Inspection Agency certified that the
imported products were not mixed during processing with any other meat
products that could have posed a risk. He also said most of the organ
meats allowed into the country were bound for Mexico.

In November 2003 the USDA proposed to open the border to more Canadian
beef products and to live Canadian cattle younger than 30 months, but
that proposal was put on hold after the first US case of BSE was found
last December. In March the agency invited more public comments on the
proposal, and the comments are still being evaluated, according to
Andrea McNally, an APHIS spokeswoman. The proposal would allow
importation of meat from older cattle, but excluding high-risk tissues
such as the skull, brain, eyes, vertebral column, and spinal cord.

Yesterday's news reports of the USDA's actions triggered sharp criticism
from Congress. "This is a breach of process and the public trust," said
Rep. Earl Pomeroy, D-N.D. "For USDA to allow over 30 million pounds of
Canadian beef to be imported without notifying Congress or the public is
completely unacceptable."

At today's briefing, USDA officials also discussed their plan to greatly
increase BSE testing starting in June. DeHaven said the agency hopes to
test "somewhere in excess of 200,000 animals" over the next 12 to 18
months in the effort to determine whether more cases of BSE exist in the
United States.

He said officials expect that the rapid tests used in the surveillance
program will sometimes yield inconclusive results. When that happens,
samples will be sent to a national USDA laboratory in Ames, Iowa, for
confirmatory testing, which will take 4 to 8 days, he said. The USDA
will hold the carcasses involved until the confirmatory test results are
available.

See also:

Transcript of USDA briefing
http://www.usda.gov/Newsroom/0204.04.html

http://www.cidrap.umn.edu/cidrap/content/other/bse/news/may2104beef.html


Docket No. 03-080-1 -- USDA ISSUES PROPOSED RULE TO ALLOW LIVE ANIMAL
IMPORTS FROM CANADA

"Terry S. Singeltary Sr."

11/03/2003 01:19 PM


To:

regulations@aphis.usda.gov

cc:


bcc:


Subject:

Docket No. 03-080-1 -- USDA ISSUES PROPOSED RULE TO ALLOW LIVE ANIMAL
IMPORTS FROM CANADA

I would like to kindly comment on Docket No. 03-080-1 USDA ISSUES
PROPOSED RULE TO ALLOW LIVE ANIMAL IMPORTS FROM CANADA ; >Under this
proposal, ruminant and ruminant products eligible for entry into >the
United States from a BSE minimal risk region would include: > >1) bovine
>animals less than 30 months of age for immediate slaughter; > >2)
bovine >animals for feeding to be moved to a designated feedlot and then
to >slaughter at less than 30 months of age; > snip... >6) fresh
(chilled or frozen) >meat from bovines less than 30 months of age; 7)
fresh (chilled or frozen) >whole or half carcasses of bovines less than
30 months of age; 8) fresh >(chilled or frozen) bovine liver; 9) fresh
(chilled or frozen) bovine >tongues; the myth that cattle under 30
months of age are free from BSE/TSE is just that, a myth, and it's a
false myth ! the youngest age of BSE case to date is 20 months old; As
at: 31 May 2003 Year of onset Age youngest case (mnths) Age 2nd youngest
case (mnths) Age 2nd oldest case (yrs.mnths) Age oldest case (yrs.mnths)
1986 30 33 5.03 5.07 1987 30 31 9.09 10.00 1988 24 27 10.02 11.01(2)
1989 21 24(4) 12.00(2) 15.04 1990 24(2) 26 13.03 14.00 1991 24 26(3)
14.02 17.05 1992 20 26 15.02 16.02 1993 29 30(3) 14.10 18.10 1994 30(2)
31(2) 14.05 16.07 1995 24 32 14.09 15.05 1996 29 30 15.07 17.02 1997
37(7) 38(3) 14.09 15.01 1998 34 36 14.07 15.05 1999 39(2) 41 13.07 13.10
2000 40 42 17.08 19.09 2001 48(2) 56 14.10 14.11 2002 51 52 15.08
15.09(2) 2003 50 62 11.11 14.11
http://www.defra.gov.uk/animalh/bse/bse-statistics/bse/yng-old.html
http://www.defra.gov.uk/animalh/bse/index.html The implications of the
Swiss result for Britain, which has had the most BSE, are complex. Only
cattle aged 30 months or younger are eaten in Britain, on the
assumption, based on feeding trials, that cattle of that age, even if
they were infected as calves, have not yet accumulated enough prions to
be infectious. But the youngest cow to develop BSE on record in Britain
was 20 months old, showing some are fast incubators. Models predict that
200-300 cattle under 30 months per year are infected with BSE and enter
the food chain currently in Britain. Of these 3-5 could be fast
incubators and carrying detectable quantities of prion.
http://www.sare.org/htdocs/hypermail/html-home/28-html/0359.html > 3)
sheep and goats less than 12 >months of age for immediate slaughter; 4)
sheep and goats for feeding to be >moved to a designated feedlot and
then to slaughter at less than 12 months >of age; > even if one believes
that scrapie does not transmit to humans (without scientific proof and
realizing scrapie transmits to primates) what about the potential for
BSE in sheep/goats and what about the many different tissues that are
infectious ? Research into sheep TSEs - audit reports & IAH's response

http://www.defra.gov.uk/animalh/bse/bse-publications/bse-publications-index.html#audit
>5) cervids for immediate slaughter; > are you going to test all
cervids coming into the USA from Canada for CWD/TSEs ? (this should be
mandatory). Commentary by European Microbiologist Roland Heynkes August
26, 2003 Posted to BSE-L@UNI-KARLSRUHE.DE > SECRETARY VENEMAN: "Well,
thank you, Tony, for your question. As > you know, we've spent a
considerable amount of time on this issue > of Canada and the single
case of BSE. The announcement we made on > the 8th had several aspects.
One was we were going to use a permit > process to open the border with
respect to boxed beef from animals > under 30 months. As you know,
animals under 30 months are generally > thought to be of virtually no
risk of having BSE. Now, we will also > begin a regulatory process to
look at the lowest risk animals, > those under 30 months. That
regulation is in process at this point, > but it will take some time to
actually do the regulation. That will > include a risk assessment and so
forth. > in my opinion this is a statement with intent to deceive and it
is not correct. There have been several cases of clinical BSE in British
cattle under 30 months and it is therefore hardly possible to think that
cattle under 30 months have virtually no risk of having BSE. In 1988 the
youngest British BSE case was 24, the second youngest 27 months old. In
1989 the youngest British BSE case was 21 and there were 4 cases only 24
months old. In 1990 there were two cases only 24 and one 26 months old.
In 1991 the youngest British BSE case was 24 and there were 3 cases only
26 months old. In 1992 the youngest British BSE case was 20!, the second
youngest 26 months old. In 1993 there was was a 29 months old case, in
1995 the UK had a 24 months old case and in 1996 one British BSE case
was 29 months old.
http://www.defra.gov.uk/animalh/bse/bse-statistics/bse/yng-old.html But
mainly this wrong statement is misleading, because not the clinically
sick cows are the problem for consumers. The real problem are those
animals that became infected as calves and are still incubating the
infectivity during the incubation time of 5-6 years. For consumers it is
therefore totally irrelevant that cattle are at low risk to reach the
clinical stage before being 30 months old. Important for consumers is
the fact that most British BSE cases became infected as calves
(http://www.heynkes.de/peaks.htm) and that infected calves are already
amplifying the infectivity. The advantage of young calves for consumers
is that the infectivity in infected animals is low and still
concentrated around the gastro- intestinal tract. But this is not
necessarily true for bulls, which are usually slaughtered when they are
19-22 months old. They are too young to give positive results in the
actual BSE tests, but they might be infective for consumers. For US
consumers it is of no importance whether a BSE-infected Canadian cow
will show the first symptoms before or after it becomes 30 months old.
Interesting for the consumers is only 1) if cattle are infected or not,
2) where in the animal is how much of the infectivity and 3) what
happens to the infectivity during slaughtering? If the US government is
really interested to reduce consumers risk, it has to 1) stop
cannibalism among farm animals (no farm animal protein and fat in
feeding stuff for farm animals, no possibility of cross contamination of
concentrate feed in mills and no lambing on pastures where scrapie might
be a problem) 2) test slaughter cattle above 24 months for BSE, 3) avoid
contamination of the beef with prions from CNS by changing slaughter
methods (electrical stunning instead of captive bolt, no immobilisation
with a pithing rod, no spreading of infectivity by sawing through the
spinal cord), 4) destroy the high risk materials (brain, eyes, spinal
cord, dorsal root ganglia and other peripheral ganglia, nervous and
lymphatic tissue associated with intestine) 5) commit the whole chain
from abattoir to counter in shop and restaurant to label products from
cattle and sheep, because it is only a myth that scrapie is less
infective than BSE. In addition the US government should test all cattle
and sheep which died or had to be killed because of illness. This
measure should be hold out for at least one year in order to see the
real BSE- and scrapie-incidence in the USA.... Microbiologist Roland
Heynkes http://www.heynkes.de/default.htm Furthermore, for the USA to
continue to flagrantly ignore the findings from Collinge/Asante et al
that BSE transmission to the 129-methionine genotype can lead to an
alternate phenotype that is indistinguishable from type 2 PrPSc, the
commonest _sporadic_ CJD. These findings could have major implications
for the medical and surgical arena and human health. this type sporadic
CJD is very prevalent in the USA ;
http://www.fda.gov/ohrms/dockets/ac/03/slides/3923s1_OPH.htm > HARVARD
RISK REASSESSMENT > > USDA also released the findings of a second
assessment conducted by the > Harvard Center for Risk Analysis (HCRA)
that confirms the findings of the > initial study released in 2001...
THESE FINDINGS WERE FLAWED FROM THE BEGINNING and the GAO proved this;
Reanalysis of Mad Cow Disease Confirms Risk is Low in the U.S. Policies
put in place in 1997 would reverse any possible disease spread For
immediate release: Friday, October 31, 2003 Boston, MA A study by the
Harvard Center for Risk Analysis (HCRA) at the Harvard School of Public
Health, assessing the likelihood of mad cow disease spreading in the
United States cattle population, confirms the findings of the initial
HCRA analysis done in 2001; that even if infected animals or
contaminated ruminant feed material entered the American animal
agriculture system from Canada, the risk of mad cow spreading
extensively within the American herd would be low, and that any possible
spread would by now have been reversed by controls put in place in the
late 1990s. The new study was initiated at the request of the United
States Department of Agriculture following discovery in the summer of
2003 of a Canadian cow infected with bovine spongiform encephalopathy
(BSE). The reanalysis also finds that any disease that might have been
introduced would eventually be eliminated from the United States. The
reanalysis, done by George Gray, executive director and Joshua Cohen,
senior researcher, both at HCRA, specifically examined scenarios for the
likely introductions of BSE from Canada into the U.S. These hypothetical
introductions included both infected animals (the study assumed 5
infected animals imported even though Canada has only identified one
case to date) and contaminated animal feed. These scenarios were
evaluated using the HCRA computer model that simulates conditions in the
American agricultural system. The analysis found that if BSE infected
animals had been introduced as early as 1990, up to 500-600 cattle in
the U.S. might have become infected, and approximately 20-25 percent
would have demonstrated signs of BSE. Such an outbreak was never
detected, though it would have been below the prevalence level that
surveillance systems in place at that time would likely have found. If
the introduction took place later, the total number of animals infected
in the U.S. would have been smaller. The HCRA study found that the 1997
U.S. imposition of a ban on feeding rendered ruminant protein back to
other ruminants essentially chokes off and then reverses any possible
spread of the disease. Even accounting for incomplete compliance with
that feed ban, the HCRA analysis found that had infected animals or
contaminated feed come in from Canada or elsewhere, the spread of BSE in
the American cattle population would have been reversed by now and that
human exposure to contaminated animal tissue would have been very low.
HCRA has also delivered to the USDA the revised version of the November,
2001 BSE report following extensive peer review by both American and
European experts. The revised document is available on the HCRA website
at http://www.hcra.harvard.edu/publications.html#Evaluation and the HCRA
BSE computer model is available by contacting Joshua Cohen at HCRA
(cohenj@hsph.harvard.edu ). For further
information, please contact: George Gray Executive Director Harvard
Center for Risk Analysis 617-432-4341 ggray@hsph.harvard.edu Kevin C.
Myron Office of Communications Harvard School of Public Health
617-432-3952 kmyron@hsph.harvard.edu
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Contact HSPH

http://www.hsph.harvard.edu/press/releases/press10312003.html BOUGHT AND
PAID FOR (in partial or whole) by your local cattle dealler ; In
addition, USDA cannot rely on the Food and Drug Administrations (FDAs)
1997 BSE feed rule being rigorously enforced. Because of serious lapses,
increased surveillance is needed. The USDA-sponsored Harvard risk
assessment of the risk of BSE in the U.S. noted that compliance with
FDAs 1997 BSE feed rule is the most important factor in preventing a
BSE outbreak. Yet a pair of reports by GAOone published in September
2000 and the other published in January 2002have shown how lax FDA has
been in ensuring compliance with the feed rule. The first report,
published some three years after the BSE feed rule went into effect
found fairly widespread non-compliance: inspection results of the 2,481
firms that were identified as handling prohibited materials . . . 699,
or 28 percent, did not label their products with the required cautionary
statements that the feed should not be fed to cattle or other ruminants.
. . . In addition, of the 1,771 firms that manufacture both prohibited
and non-prohibited material, 361, or 20 percent, did not have a system
in place to prevent commingling and cross contamination, as required
by the regulation (pp. 11-12 in
http://www.gao.gov/new.items/rc00255.pdf). The 2002 GAO report found
that, (C)oncerning the feed ban, FDA has not acted promptly to compel
firms to keep prohibited proteins out of cattle feed and to label animal
feed that cannot be fed to cattle. . . . Moreover, FDAs data on
inspections are severely flawed and, as a result, FDA does not know the
full extent of industry compliance. FDA acknowledges that it has not yet
identified and inspected all firms subject to the ban (pg. 3 in
http://www.gao.gov/new.items/d02183.pdf). The report concludes that
federal actions do not sufficiently ensure that all BSE-infected
animals or products are kept out or that if BSE were found it would be
detected promptly and not spread to other cattle through animal feed or
enter the human food chain italics added (pg. 3 in
http://www.gao.gov/new.items/d02183.pdf). The failure of FDA to fully
implement the 1997 BSE feed ban should spur USDA to exercise greater
vigilance to ensure that if BSE occurred in the US that it would be
found quickly. USDA should therefore dramatically expand the testing of
cattle to ensure, at a minimum, that all downer cows (e.g. all emergency
slaughter and all fallen stock) are tested for BSE using one of the
rapid tests, preferably the one found to be the most accurate (e.g. with
the lowest rate of false positives and false negatives). We also believe
that USDA should act to ensure that no CNS tissue is found in meat
destined for human consumption. We note that the results of the Food
Safety Inspection Services 2002 AMR survey found that about 74 percent
(25 of 34) of the establishments tested in the AMR Survey of 2002 had
positive laboratory results for CNS tissue in their final beef AMR
products; the other 26 percent had negative laboratory results (see pg.
2 of http://www.fsis.usda.gov/OA/topics/AMRSurvey.pdf). The USDA should
take appropriate action to ensure that there is zero CNS contamination
of meat destined for human consumption... Gerald Wells: Report of the
Visit to USA, April-May 1989 snip... The general opinion of those
present was that BSE, as an overt disease phenomenon, _could exist in
the USA, but if it did, it was very rare. The need for improved and
specific surveillance methods to detect it as recognised... snip... It
is clear that USDA have little information and _no_ regulatory
responsibility for rendering plants in the US... snip... 3. Prof. A.
Robertson gave a brief account of BSE. The US approach was to accord it
a _very low profile indeed_. Dr. A Thiermann showed the picture in the
''Independent'' with cattle being incinerated and thought this was a
fanatical incident to be _avoided_ in the US _at all costs_... snip...
http://www.bseinquiry.gov.uk/files/mb/m11b/tab01.pdf

FULL TEXT ;

https://web01.aphis.usda.gov/BSEcom.nsf/0/b78ba677e2b0c12185256dd300649f9d?OpenDocument&AutoFramed


Docket No, 04-047-l Regulatory Identification No. (RIN) 091O-AF46 NEW
BSE SAFEGUARDS


https://web01.aphis.usda.gov/regpublic.nsf/0/eff9eff1f7c5cf2b87256ecf000df08d?OpenDocument


Working Group Report on
the Assessment of the Geographical BSE-Risk (GBR) of
CANADA

snip...

Annex to the EFSA Scientific Report (2004) 2, 1-15 on the Assessment of the
Geographical BSE Risk of Canada
- 11 -

snip...

- 2 -
2. EXTERNAL CHALLENGES
2.1 Import of cattle from BSE-Risk2 countries
An overview of the data on live cattle imports is presented in table 1 and is based on
data as provided in the country dossier (CD) and corresponding data on relevant exports
as available from BSE risk countries that exported to Canada. Only data from risk
periods are indicated, i.e. those periods when exports from a BSE risk country already
represented an external challenge, according to the SSC opinion on the GBR (SSC July
2000 and updated January 2002).
• According to the CD, 231 cattle were imported from UK during the years 1980 to
1990 and no cattle imports from UK were recorded after 1990.
• According to Eurostat, altogether 198 cattle have been imported from the UK during
the years 1980 to 1990, Additionally 500 were recorded in 1993; this import is
1 For the purpose of the GBR assessment the abbreviation “MBM” refers to rendering products, in particular
the commodities Meat and Bone Meal as such; Meat Meal; Bone Meal; and Greaves. With regard to imports
it refers to the customs code 230110 “flours, meals and pellets, made from meat or offal, not fit for human
2 BSE-Risk countries are all countries already assessed as GBR III or IV or with at least one confirmed
Annex to the EFSA Scientific Report (2004) 2, 1-14 on the Assessment of the
Geographical BSE Risk of Canada
- 3 -
mentioned in Eurostat and the updated UK export statistic as male calves, but not
mentioned in the original UK export statistics. According to the CD, detailed
investigations were carried out and it is very unlikely that the 500 calves have been
imported. Therefore, they were not taken into account.
• According to the CD, in 1990 all cattle imported from UK and Ireland since 1982
were placed in a monitoring program.
• Following the occurrence of the BSE index case in 1993 (imported from UK in 1987
at the age of 6 months), an attempt was made to trace all other cattle imported from
UK between 1982 and 1990.
• Of the 231 cattle imported from the UK between 1980 and 1990, 108 animals had
been slaughtered and 9 had died. From the remaining, 37 were exported, 76 were
sent to incineration and one was buried; these were not entering the rendering system
and therefore not taken into account.
• According to the CD, 16 cattle were imported from Ireland (according to Eurostat
20), of which 9 were slaughtered, 3 died. The remaining 4 were incinerated and did
therefore not enter the rendering system. According to the CD, the 6 animals which
were imported in 1990 according to Eurostat, were never imported.
• Moreover 22 cattle have been imported from Japan (through USA), of which 4 were
exported (excluded from the table) and 14 were destroyed and therefore not entering
the rendering system, 4 were slaughtered.
• Of 28 imported bovines from Denmark, 1 was destroyed and 1 was exported. Of the
19 buffalos imported in 2000, 1 was incinerated and the others were ordered to be
destroyed.
• Additionally in total 264 cattle according to the CD (276 according to other sources)
were imported from Austria, France, Germany, Hungary, Italy, The Netherlands and
Switzerland.
• The numbers imported according to the CD and Eurostat are very similar. Some
discrepancies in the year of import can be explained by an extended quarantine;
therefore it is likely that imports according to Eurostat in 1980 and imports
according to the CD in 1981 are referring to the same animals.
• Additionally, between 16.000 and 340.000 bovines have annually been imported
from US, almost all are steers and heifers. In total, between 1981 and 2003,
according to the CD more than 2.3 million, according to other sources 1.5 million
cattle have been imported.
• According to the CD, feeder/slaughter cattle represent typically more than 90% of
the imported cattle from the USA; therefore, only 10% of the imported cattle have
been taken into account.

snip...


Annex to the EFSA Scientific Report (2004) 2, 1-15 on the Assessment of the
Geographical BSE Risk of Canada
- 5 -
2.2 Import of MBM or MBM-containing feedstuffs from BSE-Risk
countries
An overview of the data on MBM imports is presented in table 2 and is based on data
provided in the country dossier (CD) and corresponding data on relevant exports as
available from BSE risk countries that exported to Canada. Only data from risk periods
are indicated, i.e. those periods when exports from a BSE risk country already
represented an external challenge, according to the SSC opinion on the GBR (SSC, July
2000 and updated January 2002).
According to the CD, no imports of MBM took place from UK since 1978 (initially
because of FMD regulations).
• According to Eurostat data, Canada imported 149 tons MBM from the UK in the
period of 1993 to 2001. According to up-dated MBM statistics from UK (August
2001) no mammalian MBM was exported to Canada from 1993 – 1996. As it was
illegal to export mammalian meat meal, bone meal and MBM from UK since
27/03/1996, exports indicated after that date should only have included nonmammalian
MBM. Therefore, these imports were not taken into account.
• According to the CD, imports of MBM have taken place from Denmark, Germany,
France, Japan and US.
• According to Eurostat Canada imported MBM from Denmark, Belgium, France and
Ireland.
• According to the CD further investigations concluded that all imported MBM from
Denmark consisted of pork and poultry origin and was directly imported for
aquaculture, the imported MBM from France was feather meal, the imported MBM
from Germany was poultry meal for aquaculture and the imported MBM from
Belgium was haemoglobin; therefore these imports were not taken into account.
• The main imports of MBM were of US origin, according to the CD around 250.000
tons, according to other sources around 310.000 tons between 1988 and 2003.

snip...

Annex to the EFSA Scientific Report (2004) 2, 1-15 on the Assessment of the
Geographical BSE Risk of Canada
- 7 -
2.3 Overall assessment of the external challenge
The level of the external challenge that has to be met by the BSE/cattle system is
estimated according to the guidance given by the SSC in its final opinion on the GBR of
July 2000 (as updated in January 2002).
Live cattle imports:
In total the country imported according to the CD more than 2.3 million, according to
other data 1.5 million live cattle from BSE risk countries, of which 231 (CD)
respectively 698 (other sources) came from the UK. The numbers shown in table 1 are
the raw import figures and are not reflecting the adjusted imports for the assessment of
the external challenge. Broken down to 5 year periods the resulting external challenge is
as given in table 3. This assessment takes into account the different aspects discussed
above that allow to assume that certain imported cattle did not enter the domestic
BSE/cattle system, i.e. were not rendered into feed. In the case of Canada, the 500 cattle
imported from UK according to Eurostat were not taken into account and it is assumed
that all incinerated, buried, exported animals and the animals still alive did not enter the
rendering system and were therefore excluded from the external challenge.
MBM imports:
In total the country imported according to the CD around 300.000 tons, according to
other sources nearly 360.000 tons of MBM from BSE risk countries, of which 149 tons
came from the UK. The majority consisted of MBM imported from the US. The
numbers shown in table 2 are the raw import figures and are not reflecting the adjusted
imports for the assessment of the external challenge. Broken down to 5 year periods the
resulting external challenge is as given in table 3. This assessment takes into account
the different aspects discussed above that allow to assume that certain imported MBM
did not enter the domestic BSE/cattle system or did not represent an external challenge
for other reasons. As it was illegal to export mammalian meat meal, bone meal and
MBM from UK since 27/03/1996, exports indicated after that date should only have
included non-mammalian MBM. In the case of Canada all imported MBM from UK,
Germany, Belgium, Denmark and France was not taken into account.
On the basis of the available information, the overall assessment of the external
challenge is as given in table 3 below.
Annex to the EFSA Scientific Report (2004) 2, 1-15 on the Assessment of the
Geographical BSE Risk of Canada
- 8 -
External Challenge experienced by CANADA
External challenge Reason for this external challenge
Period Overall Level Cattle
imports
MBM
imports
Comment
1980 to 1990 Low Low Negligible
1991 to 1995 High Moderate High
1996 to 2000 Extremely
high High Extremely
high
2001 to 2003 Very high High Very high
Table 3: External challenge resulting from live cattle and/or MBM imports from the UK and other BSE risk
countries. The challenge level is determined according to the SSC-opinion on the GBR of July 2000 (as
updated in January 2002).
3. STABILITY
3.1 Overall appreciation of the ability to avoid recycling of BSE
infectivity, should it enter processing
Feeding
The annual Canadian production of MBM is approximately 575,000 tons of which
approx. 40,000 tons are exported each year, mainly to USA.
Use of MBM in cattle feed
• Before the feed ban, dairy cattle received supplementary feed containing MBM
during their productive life (maximum 200-400 g MBM per day). Beef cattle in the
western part of the country do not usually receive complementary feed. Beef cattle
in the eastern part receive normally no supplement protein but the calves could have
access to creep feeds containing MBM, after weaning the ratios may have contained
supplemental protein containing MBM (100-400 g per day).
• According to the CD, MBM is mainly fed to pigs and poultry and included in pet
food.
• According to the CD, only a proportion of dairy cattle may have received MBM.
Feed bans
• Before 1997, there was no legal restriction to include MBM into cattle feed.
• An MBM-ban was introduced in August 1997; it is forbidden since to feed
mammalian MBM to ruminants except if of pure porcine, equine and non
mammalian origin, i.e. in practice a ruminant-to-ruminant ban (RMBM-ban).
Annex to the EFSA Scientific Report (2004) 2, 1-15 on the Assessment of the
Geographical BSE Risk of Canada
- 9 -
Potential for cross-contamination and measures taken against
• Cross-contamination in the about 600 feed mills is assumed to be possible as long as
cattle and pig feed is produced in the same production lines, and premises.
• Cross-contamination during transport is possible, particularly if the same trucks are
used for transporting ruminant MBM (RMBM) and non-ruminant MBM (porcine or
poultry MBM which still might be included into cattle feed) or for transporting
pig/poultry feed and cattle feed.
• On-farm cross-contamination is regarded to be possible.
• Cross-contamination of cattle feed with RMBM can not be excluded. Hence, as
reasonable worst case scenario, it has to be assumed that cattle, in particular dairy
cattle, can still be exposed to RMBM and hence to BSE-infectivity, should it enter
the feed chain.
Control of Feed bans and cross-contamination
• With the introduction of the RMBM ban (1997) the feed mills (approximately 600)
were checked for compliance with the ban, including good manufacturing practices
(GMP) and record keeping, i.e. the separation in production of MBM containing
ruminant material (RMBM) from non-ruminant MBM.
• The feed mills had previously – since 1983 – been regularly checked in relation to
production of medicated feed.
• No examinations are performed to assess cross-contamination with RMBM of the
protein (e.g. non ruminant MBM) that enters cattle feed. Differentiation would
anyway be difficult.
Rendering
Raw material used for rendering
• Ruminant material is rendered together with material from other species, but
according to the CD only in the production of MBM prohibited for use in ruminant
feeds.
• Slaughter by-products, including specified risk material (SRM) and fallen stock are
rendered.
• The country expert estimated that 20% of the rendering plants, processing 20% of
the total amount of raw material, are connected to slaughterhouses. Their raw
material is more than 98 % animal waste from these slaughterhouses while less than
2 % is fallen stock. No estimation was given for the remaining 80% of the rendering
capacity.
• There are 32 rendering plants of which 3 are processing blood exclusively.
Rendering processes
• The rendering systems (parameters) were specified for 6 plants producing mixed
MBM, none of these fulfilled the 133/20/3 standard. Of these, 5 have dedicated
facilities to produce products for use in ruminant feed and products not permitted for
use in ruminant feed.
• The remaining plants process porcine or poultry material exclusively.
SRM and fallen stock
• There is an SRM ban for human food in place since 2003.
Annex to the EFSA Scientific Report (2004) 2, 1-15 on the Assessment of the
Geographical BSE Risk of Canada
• However, SRM are rendered together with other slaughter waste and fallen stock.
However, according to the CD, MBM with SRM is not permitted to be fed to
ruminants.
Conclusion on the ability to avoid recycling
• Between 1980 and 1997 the Canadian system would not have been able to avoid
recycling of the BSE-agent to any measurable extent. If the BSE-agent was
introduced into the feed chain, it could have reached cattle.
• Since 1997 this ability gradually improved with the introduction of the ruminant
MBM ban and its implementation.
• Since cross-contamination cannot be excluded, and as SRM is still rendered by
processes unable to significantly reduce BSE-infectivity, the system is still unable to
avoid recycling of BSE-infectivity already present in the system or incoming.
3.2
BSE surveillance
laboratory tests).
i.e. formalin fixation.

snip...


In 1990, when BSE was made notifiable, this awareness was extended to
suspicions of BSE.
" Since 1993 the number of brains examined per year did exceed the number
recommended by OIE (300 - 336 for countries with a cattle population over 24
months of age of 5.0 to 7.0 Million)

PLEASE NOTE BEFORE GOING ANY FURTHER THAT MOST EVERY COUNTRY THAT WENT
BY THOSE SAME OIE BSE GUIDELINES HAVE BSE NOW. THE ONLY REASON IT WAS
NOT DETECTED SOONER
IN THESE COUNTRIES WERE BECAUSE OF THESE SAME OIE GUIDELINES. SIMPLY PUT,
THEY ARE WRONG IN RELATIONS TO TSEs. IT'S NOTHING MORE THAN AN EXCUSE,
ONE THAT FLIES ABOUT LIKE A COW WOULD...TSS


in all years, except in 1995 (table 4).
year 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003
samples 225 645 426 269 454 759 940 895 1´020 1´581 3´377 3´361
Table 4: Number of bovine brains annually examined for CNS diseases,
including BSE.
" According to the CD approx. 98% of the examined cattle were older than
24 months
and approx. 90% exhibited neurological symptoms. Although the identification
system of Canada does not document the birth date or age of the animals,
according
to the CD, examination of the dentition is used to ascertain the
maturity of the
animals.
" The list of neurological differential diagnoses for the 754 brains
examined in 1997
included encephalitis (70 cases), encephalomalacia (19), hemophilus (7),
hemorrhage (2), listeriosis (38), meningoencephalitis (36), rabies (22),
tumors (2),
other conditions (135) and no significant findings (423).
" Compensation is paid for suspect BSE cases as well as for animals
ordered to be
destroyed (90-95% of market value with a maximum of 2,500 Can$ per cow).
" Diagnostic criteria developed in the United Kingdom are followed at ADRI,
Nepean. According to the very detailed protocol for the collection,
fixation and
submission of Bovine Spongiform Encephalopathy (BSE) specimens at abattoirs
under inspection by the Canadian Food Inspection Agency, the specimen
shall be
shipped to National Center for Foreign Animal Disease, Winnipeg, Manitoba.
" In 2003, around 3000 animals from risk populations have been tested.
" According to the CD, it is aimed to test a minimum of 8000 risk
animals (animals
with clinical signs consistent with BSE, downer cows, animals died on
farm animals
diseased or euthanized because of serious illness) in 2004 and then
continue to
progressively increase the level of testing to 30,000.
" In May 2003, Canada reported its first case of domestic BSE. A second
case was
detected in the US on 23 December 2003 and traced back to Canadian
origin. Both
were born before the feed ban and originated from Western Canada.
3.3 Overall assessment of the stability
For the overall assessment of the stability, the impact of the three
main stability factors
(i.e. feeding, rendering and SRM-removal) and of the additional
stability factor,
surveillance, has to be estimated. Again, the guidance provided by the
SSC in its
opinion on the GBR of July 2000 (as updated January 2002) is applied.
Until 1997, it was legally possible to feed ruminant MBM to cattle and a
certain fraction of
cattle feed (for calves and dairy cattle) is assumed to have contained
MBM. Therefore
feeding was Not OK. In August 1997 a ruminant MBM ban was introduced
but feeding
of non-ruminant MBM to cattle remained legal as well as feeding of
ruminant MBM to
non-ruminant animals. This makes control of the feed ban very difficult
because laboratory
differentiation between ruminant and non ruminant MBM is difficult if
not impossible.
Annex to the EFSA Scientific Report (2004) 2, 1-15 on the Assessment of the
Geographical BSE Risk of Canada
- 12 -
Due to the highly specialised production system in Canada, various
mammalian MBM
streams can be separated. Such a feed ban would therefore be assessed as
"reasonably
OK", for all regions where this highly specialised system exists.
However, several areas
in Canada do have mixed farming and mixed feed mills, and in such
regions, an RMBM
ban would not suffice. Additionally, official controls for cattle feeds
to control for the
compliance with the ban were not started until the end of 2003. Thus,
for the whole
country, the assessment of the feeding after 1997 remains "Not OK".
Rendering
The rendering industry is operating with processes that are not known to
reduce infectivity.
It is therefore concluded that the rendering was and is Not OK.
SRM-removal
SRM and fallen stock were and are rendered for feed. Therefore
SRM-removal is assessed
as Not OK
BSE surveillance
Before 1989, the ability of the system to identify (and eliminate)
BSE-cases was limited.
Since 1990 this ability is improved, thanks to a specific (passive) BSE
surveillance.
Today the surveillance should be able to detect clinical BSE-cases
within the limits set
by an essentially passive surveillance system.
" Passive surveillance has been carried out since 1990. In 1993
surveillance was
intensified and has considerably improved with mandatory reporting and basic
compensation ensured, awareness raising measures and education of
veterinarians, and
a specific BSE-surveillance programme targeting cattle showing clinical
signs that
could be compatible with BSE.
" The initiated introduction of active surveillance should improve the
system
significantly.
Stability of the BSE/cattle system in CANADA over time
Stability Reasons
Period Level Feeding Rendering SRM
removal
BSE
surveillance
1980 to 2000 Mainly
passive
2001 to 2003
Extremely
unstable Not OK Not OK Not OK
Improving
with some
testing of
risk groups
Table 5: Stability resulting from the interaction of the three main
stability factors and the BSE
surveillance. The stability level is determined according to the
SSC-opinion on the GBR of July 2000 (as
updated in 2002).
Annex to the EFSA Scientific Report (2004) 2, 1-15 on the Assessment of the
Geographical BSE Risk of Canada
- 13 -
On the basis of the available information, it has to be concluded that
the country's
BSE/cattle system was extremely unstable until today, i.e., it would
have recycled and
amplified BSE-infectivity very fast, should it have entered the system.
The stability of the
BSE/cattle system in Canada overtime is as given in table 5 above.
4. CONCLUSION ON THE RESULTING RISKS
4.1 Interaction of stability and challenges
In conclusion, the stability of the Canada BSE/cattle system in the past
and the external
challenges the system has coped with are summarised in the table 6.
INTERACTION OF STABILITY AND EXTERNAL CHALLENGE IN CANADA
Period Stability External Challenge Internal challenge
1980 to 1990 Low Unlikely but not excluded
1991 to 1995 High
1996 to 2000 Extremely high
Likely and rapidly growing
2001 to 2003
Extremely
unstable
Very high Confirmed at a lower level
Table 6: Internal challenge resulting from the interaction of the
external challenge and stability. The
internal challenge level is determined according to guidance given in
the SSC-opinion on the GBR of
July 2000 (as updated in 2002).

From the interaction of the two parameters stability and external
challenge a

conclusion is drawn on the level of internal challenge that emerged
and had to be met
by the system, in addition to external challenges that occurred.
An external challenge resulting from cattle import could only lead to an
internal
challenge once imported infected cattle were rendered for feed and this
contaminated
feed reached domestic cattle. Cattle imported for slaughter would
normally be
slaughtered at an age too young to harbour plenty of BSE infectivity or
to show signs,
even if infected prior to import. Breeding cattle, however, would
normally live much
longer and only animals having problems would be slaughtered younger. If
being 4-6
years old when slaughtered, they could suffer from early signs of BSE, being
approaching the end of the BSE-incubation period. In that case, they
would harbour,
while being pre-clinical, as much infectivity as a clinical BSE case.
Hence cattle imports
could have led to an internal challenge about 3 years after the import
of breeding cattle
(that are normally imported at 20-24 months of age) that could have been
infected prior
to import. In case of Canada this implies that cattle imported in the
mid eighties could
have been rendered in the late eighties and therefore led to an internal
challenge in the
early 90s.
On the other hand imports of contaminated MBM would lead to an internal
challenge in
the year of import, if fed to cattle. The feeding system is of utmost
importance in this
context. If it could be excluded that imported, potentially contaminated
feed stuffs
reached cattle, such imports might not lead to an internal challenge at
all. In case of
Annex to the EFSA Scientific Report (2004) 2, 1-15 on the Assessment of the
Geographical BSE Risk of Canada
- 14 -
Canada this implies that it was possible that imported MBM reached
domestic cattle and
lead to an internal challenge in the early 90s.
4.2 Risk that BSE infectivity entered processing
A certain risk that BSE-infected cattle entered processing in Canada,
and were at least
partly rendered for feed, occurred in the early 1990s when cattle
imported from UK in
the mid 80s could have been slaughtered. This risk continued to exist,
and grew
significantly in the mid 90s when domestic cattle, infected by imported
MBM, reached
processing. Given the low stability of the system, the risk increased
over the years with
continued imports of cattle and MBM from BSE risk countries.
4.3 Risk that BSE infectivity was recycled and propagated
A risk that BSE-infectivity was recycled and propagated exists since a
processing risk
first appeared; i.e. in the early 90s. Until today this risk persists
and increases fast
because of the extremely unstable BSE/cattle system in Canada.
5. CONCLUSION ON THE GEOGRAPHICAL BSE-RISK
5.1 The current GBR as function of the past stability and challenge
The current geographical BSE-risk (GBR) level is III, i.e. it is
confirmed at a lower level
that domestic cattle are (clinically or pre-clinically) infected with
the BSE-agent.
This assessment deviates from the previous assessment (SSC opinion,
2000) because at
that time several exporting countries were not considered a potential risk.
5.2 The expected development of the GBR as a function of the past and
present stability and challenge
" As long as the system remains unstable, it is expected that the GBR
continues to
grow, even if no additional external challenges occur.
" Since recent improvements in the safety of MBM production in many
countries or
significant recent reductions in the incidence of BSE are not taken into
account for
the assessment of the external challenge, the external challenge
assessed after 2001
could be overestimated and is the worst case assumption. However all
current GBR
conclusions are not dependent on these assumptions in any of the
countries assessed.
For future assessments and when the impact of the production,
surveillance and true
incidence changes has been fully quantified, these developments should
be taken
into account.
5.3 Recommendations for influencing the future GBR
" Enhancing the stability of the system, in particular by ensuring that
cattle have no
access to mammalian MBM in combination with appropriate rendering and
exclusion of
SRM and fallen stock from any feed chain could lead, over time, to a
reduction of the
GBR.
" Improved passive and active surveillance, i.e. sampling of animals not
showing
signs compatible with BSE from at-risk cattle populations, such as
adult cattle in
Annex to the EFSA Scientific Report (2004) 2, 1-15 on the Assessment of the
Geographical BSE Risk of Canada
- 15 -
fallen stock and emergency slaughter, by means of rapid screening, would
allow
monitoring the efficiency of stability enhancing measures.
Documentation provided to EFSA
" Letter with the ref D(2003)KVD/ip/420722 from the European Commission
requesting a geographical risk assessment for the appearance of BSE in a
country.
" Country Dossier as prepared by the country in response to the EC and EFSA
data collection request.
" Other sources of data information i.e. exports from third countries and
Eurostat data.
" SSC, July 2000. Final opinion on the Geographical Risk of Bovine
Spongiform Encephalopathy (GBR).
" SSC, January 2002. Updated opinion on the Geographical Risk of Bovine
Spongiform Encephalopathy (GBR).
Acknowledgment
Members of the EFSA Scientific Expert Working Group on GBR are
acknowledged for
their valuable contribution to this mandate. The members are: Didier
Calavas, Aline De
Koeijer, Michael Gravenor, John Griffin, Dagmar Heim, Matthias Kramer,
Riitta
Maijala, Mo Salman, Vittorio Silano, Emmanuel Vanopdenbosch, and Stig
Widell.

CANADA

http://www.efsa.eu.int/science/efsa_scientific_reports/gbr_assessments/scr_annexes/563/sr02_biohaz02_canada_report_annex_en1.pdf


Geographical BSE Risk of USA
European Food Safety Authority
Scientific Expert Working Group on GBR
Working Group Report on
the Assessment of the Geographical BSE-Risk (GBR) of
UNITED STATES OF AMERICA
2004

snip...

- 12 -
3.3 Overall assessment of the stability
For the overall assessment of the stability, the impact of the three
main stability
factors, (i.e. feeding, rendering and SRM-removal) and of the additional
stability
factor surveillance has to be estimated. Again, the guidance provided by
the SSC in its
opinion on the GBR of July 2000 (as updated in 2002) is applied.
Annex to the EFSA Scientific Report (2004) 3, 1-17 on the Assessment of the
Geographical BSE Risk of USA
- 13 -
Feeding
Until August 1997, RMBM was legally fed to cattle. Feeding was therefore
"not
OK". In August 1997 an RMBM-ban was introduced but feeding of non-ruminant
MBM to cattle remained legal as well as feeding of RMBM to non-ruminant
animals
(farm animals and pets). An RMBM ban is difficult to maintain, as only
labels can
distinguish the various MMBMs. This makes control of the feed ban very
difficult
because analytical differentiation between ruminant and non-ruminant MBM is
difficult if not impossible.
Due to the highly specialised production system in the USA, various
mammalian
MBM streams can be separated. Such a feed ban would therefore be assessed as
"reasonably OK", for all regions where this highly specialised system
exists.
However, several areas in the USA do have mixed farming and mixed feed
mills, and
in such regions an RMBM ban would not suffice. Additionally, official
controls for
cattle feeds to control for compliance with the ban started in 2002.
Thus, for the
whole country, the assessment of the feeding after 1997 remains "not
OK", but
improving.
Rendering
The rendering industry is operating with processes that are not known to
reduce
infectivity. It is therefore concluded that rendering was and is "not OK".
SRM-removal
SRM were and are still rendered for feed, as are (parts of) the fallen
stock. SRMremoval
is therefore regarded as "not OK".
BSE-surveillance
Before 1989, the ability of the system to identify (and eliminate)
BSE-cases was
limited. Since 1990 this ability is improved, thanks to a specific
(passive) BSE
surveillance. The initiated introduction of active surveillance in risk
populations
should improve the system significantly.
Stability of the BSE/cattle system in the USA over time
Stability Reasons
Period Level Feeding Rendering SRM removal BSE
surveillance
1980 to
2003
Extremely
unstable Not OK Not OK Not OK
Passive but
improving
with some
testing of risk
groups
Table 4: Stability resulting from the interaction of the three main
stability factors and the BSE
surveillance. The stability level is determined according to the
SSC-opinion on the GBR of July
2000 (as updated in 2002).
Annex to the EFSA Scientific Report (2004) 3, 1-17 on the Assessment of the
Geographical BSE Risk of USA
- 14 -
On the basis of the available information, it has to be concluded that
the country's
BSE/cattle system was extremely unstable until today, i.e., it would
have recycled and
amplified BSE-infectivity very fast, should it have entered the system.
The stability of
the BSE/cattle system in the USA overtime is as given in table 4.
The present assessment modifies the stability assessment of the previous
GBR report
in 2000 mainly due to a different perception of the impact of BSE
surveillance on
stability and of the efficiency of the RMBM feed ban.
4. CONCLUSION ON THE RESULTING RISKS
4.1 Interaction of stability and challenges
In conclusion, the stability of the USA BSE/cattle system in the past
and the external
challenge the system has coped with, are summarised in table 5 below.
From the interaction of the two parameters “stability” and “external
challenge” a
conclusion is drawn on the level of “internal challenge” that emerged
and had to be
met by the system, in addition to external challenges that occurred.
Interaction of stability and external challenge in the USA
Period Stability External Challenge Internal challenge
1980 to
1985
1986 to
1990
Moderate Possibly present
1991 to
1995 Very high
1996 to
2000
2001 to
2003
Extremely
unstable
Extremely high
Likely to be present and
growing
Table 5: Internal challenge resulting from the interaction of the
external challenge and stability.
The internal challenge level is determined according to guidance given
in the SSC-opinion on
the GBR of July 2000 (as updated in 2002).
An external challenge resulting from cattle import could only lead to an
internal
challenge once imported infected cattle were rendered for feed and this
contaminated
feed reached domestic cattle. Cattle imported for slaughter would
normally be
slaughtered at an age too young to harbour plenty of BSE infectivity or
to show signs,
even if infected prior to import. Breeding cattle, however, would
normally live much
longer and only animals having problems would be slaughtered younger. If
being 4-6
years old when slaughtered, they could suffer from early signs of BSE, being
approaching the end of the BSE-incubation period. In that case, they
would harbour,
while being pre-clinical, as much infectivity as a clinical BSE case.
Hence cattle
imports could have led to an internal challenge about 3 years after the
import of
Annex to the EFSA Scientific Report (2004) 3, 1-17 on the Assessment of the
Geographical BSE Risk of USA
- 15 -
breeding cattle (that are normally imported at 20-24 months of age) that
could have
been infected prior to import.
In the case of the USA a few potentially infected cattle were imported
from the UK
and more from other BSE-risk countries. Furthermore, large numbers of
imported
animals came from Canada. This implies that cattle imported in the mid
eighties could
have been rendered in the late eighties and therefore led to an internal
challenge in the
early 90s.
On the other hand imports of contaminated MBM would lead to an internal
challenge
in the year of import, if fed to cattle. The feeding system is of utmost
importance in
this context. If it could be excluded that imported, potentially
contaminated feed stuffs
reached cattle, such imports might not lead to an internal challenge at all.
In case of the USA this implies that it was possible that imported MBM
reached
domestic cattle and lead to an internal challenge in the early 90s.
If Canadian imports would be excluded from this assessment, we find that
the USA
receives a moderate challenge for all 5-year intervals since 1980, a
high challenge
between 1985 and 2000 and a low challenge thereafter. If combining these
moderate
to high challenges due to imports with the extremely unstable system,
the conclusion
would still be that the occurrence of an internal challenge is possible
during the early
80s and likely in the late 80s.
4.2 Risk that BSE infectivity entered processing
A processing risk developed in the late 80s/early 90s when cattle
imports from BSE
risk countries were slaughtered or died and were processed (partly) into
feed, together
with some imports of MBM. This risk continued to exist, and grew
significantly in the
mid 90s when domestic cattle, infected by imported MBM, reached
processing. Given
the low stability of the system, the risk increased over the years with
continued
imports of cattle and MBM from BSE risk countries.
4.3 Risk that BSE infectivity was recycled and propagated
A risk that BSE-infectivity was recycled and propagated exists since a
processing risk
first appeared, i.e. in the early 90s. Until today this risk persists
and increases fast
because of the extremely/very unstable BSE/cattle system in the USA.
5. CONCLUSION ON THE GEOGRAPHICAL BSE-RISK
5.1 The current GBR as function of the past stability and challenge
• The current geographical BSE risk (GBR) level is III, i.e. it is
likely but not
confirmed that domestic cattle are (clinically or pre-clinically)
infected with the
BSE-agent.
Note1: It is also worth noting that the current GBR conclusions are not
dependent on
the large exchange of imports between USA and Canada. External challenge
due to
exports to the USA from European countries varied from moderate to high.
These
Annex to the EFSA Scientific Report (2004) 3, 1-17 on the Assessment of the
Geographical BSE Risk of USA
challenges indicate that it was likely that BSE infectivity was
introduced into the
North American continent.
Note2: This assessment deviates from the previous assessment (SSC
opinion, 2000)
because at that time several exporting countries were not considered a
potential risk.
5.2 The expected development of the GBR as a function of the past
and present stability and challenge
• As long as there are no significant changes in rendering or feeding,
the stability
remains extremely/very unstable. Thus, the probability of cattle to be
(preclinically
or clinically) infected with the BSE-agent persistently increases.
• Since recent improvements in the safety of MBM production in many
countries
or significant recent reductions in the incidence of BSE are not taken into
account for the assessment of the external challenge, the external challenge
assessed after 2001 could be overestimated and is the worst case assumption.
However all current GBR conclusions are not dependent on these assumptions
in any of the countries assessed. For future assessments and when the
impact of
the production, surveillance and true incidence changes have been fully
quantified, these developments should be taken into account.
5.3 Recommendations for influencing the future GBR
• Measures that improve the stability of the system, will, over time,
reduce the
probability that cattle could get infected with the BSE-agent. Possible
actions
include
- removal of SRM and/or fallen stock from rendering of animal
by-products into
feed,
- high pressure standards in rendering processes,
- significant improvement of ban on use of ruminant MBM in cattle feed,
supported by regular sampling of feed for the occurrence of such MBM.
• Improved passive and active surveillance, i.e. sampling of animals not
showing
signs compatible with BSE from “at-risk” cattle populations, such as
adult cattle
in fallen stock and emergency slaughter, by means of rapid screening, would
allow monitoring the efficiency of stability enhancing measures.
Documentation...

snip...

http://www.efsa.eu.int/science/efsa_scientific_reports/gbr_assessments/scr_annexes/574/sr03_biohaz02_usa_report_annex_en1.pdf


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FULL
TEXT....TSS

PNAS | March 1, 2005 | vol. 102 | no. 9 | 3501-3506

NEUROSCIENCE

Diagnosis of human prion disease

Jiri G. Safar *, , Michael D. Geschwind , , Camille Deering
*, Svetlana Didorenko *, Mamta Sattavat ¶, Henry Sanchez ¶,
Ana Serban * , Martin Vey ||, Henry Baron **, Kurt Giles *,
, Bruce L. Miller , , Stephen J. DeArmond * , ¶ and Stanley
B. Prusiner *, , ,

*Institute for Neurodegenerative Diseases, Memory and Aging
Center, and Departments of Neurology, ¶Pathology, and
Biochemistry and Biophysics, University of California, San
Francisco, CA 94143; ||ZLB Behring, 35041 Marburg, Germany;
and **ZLB Behring, 75601 Paris, France

Contributed by Stanley B. Prusiner, December 22, 2004

Abstract

With the discovery of the prion protein (PrP),
immunodiagnostic procedures were applied to diagnose
Creutzfeldt–Jakob disease (CJD). Before development of the
conformation-dependent immunoassay (CDI), all immunoassays
for the disease-causing PrP isoform (PrPSc) used limited
proteolysis to digest the precursor cellular PrP (PrPC).
Because the CDI is the only immunoassay that measures both
the protease-resistant and protease-sensitive forms of
PrPSc, we used the CDI to diagnose human prion disease. The
CDI gave a positive signal for PrPSc in all 10–24 brain
regions (100%) examined from 28 CJD patients. A subset of 18
brain regions from 8 patients with sporadic CJD (sCJD) was
examined by histology, immunohistochemistry (IHC), and the
CDI. Three of the 18 regions (17%) were consistently
positive by histology and 4 of 18 (22%) by IHC for the 8
sCJD patients. In contrast, the CDI was positive in all 18
regions (100%) for all 8 sCJD patients. In both gray and
white matter, 90% of the total PrPSc was protease-sensitive
and, thus, would have been degraded by procedures using
proteases to eliminate PrPC. Our findings argue that the CDI
should be used to establish or rule out the diagnosis of
prion disease when a small number of samples is available as
is the case with brain biopsy. Moreover, IHC should not be
used as the standard against which all other
immunodiagnostic techniques are compared because an
immunoassay, such as the CDI, is substantially more
sensitive.

------------------------------------------------------------
--------------------
Human prion diseases include Creutzfeldt–Jakob disease
(CJD), kuru, and Gerstmann–Sträussler–Scheinker disease.
Sporadic CJD (sCJD) accounts for 85% of all cases of human
prion disease, familial CJD (fCJD) for 10–15%, and infection
from exogenous, frequently iatrogenic CJD (iCJD) prions, for
<1% (1). Prions consist solely of a disease-causing prion
protein (PrPSc) that is derived from the cellular isoform
(PrPC) (2). During prion replication, PrPSc stimulates
conversion of PrPC into nascent PrPSc.
Human prions from many cases of sCJD, fCJD, and iCJD were
transmitted to apes and monkeys, but few titrations were
performed, so there is little quantitative data on the
levels of prions from these investigations (3). The
development of mice expressing human prion protein (HuPrP)
and chimeric mouse–human transgenes (MHu2M) (4–7) allowed us
to measure the levels of prions in human brains as reported
here. The incubation times of these mice were sufficiently
abbreviated to allow endpoint titrations. Based on these
endpoint titrations, we surmise that each of three cases of
sCJD harbors a different strain of prion. We also used the
titrations to calibrate PrPSc measurements that were
determined by the conformation-dependent immunoassay (CDI).
Full-length, protease-resistant PrPSc (rPrPSc) and
previously unrecognized protease-sensitive forms of PrPSc
(sPrPSc) can be detected by the CDI (8). Most PrPSc
accumulating in the frontal cortex and white matter of sCJD
cases was sPrPSc. Other immunoassays for PrPSc detect the
N-terminally truncated protein PrP 27–30 derived from PrPSc;
these include Western blotting, ELISA, and histoblotting. It
is unclear what forms of PrPSc are detected by
immunohistochemistry (IHC) after hydrolytic autoclaving in
the presence of formic acid.

Because the CDI can readily detect PrPSc molecules
comprising one ID50 unit, we examined the diagnostic
sensitivity of the test by measuring PrPSc in many different
brain regions. We performed these measurements on brains of
28 people who died of either sCJD, fCJD(E200K), or iCJD.
Whereas the CDI registered a positive signal in every brain
region examined in all of the cases, standard histopathology
and IHC were much less effective in diagnosing CJD. Indeed,
the poor performance of these histological techniques
indicates that they should no longer be used to rule out
prion disease in a brain biopsy from a single cortical site
and must be applied to multiple cortical and subcortical
brain samples at autopsy.

Materials and Methods

Preparation of Brain Homogenates. For biochemical analysis
only, slices from 24 different anatomical areas of human
brains weighing 250–350 mg were homogenized to a final 15%
(wt/vol) in 4% (wt/vol) Sarkosyl in PBS, pH 7.4, by three
75-s cycles in a reciprocal homogenizer MiniBeadBeater-8
(BioSpec Products, Bartlesville, OH) as described in refs.
8–10. The resulting homogenate was diluted to a final 5%
(wt/vol) by using PBS containing 4% (wt/vol) Sarkosyl. The
diluted samples were either treated with a proteinase
inhibitor mixture for measurements of PrPSc or digested with
2.5 or 10 µg/ml proteinase K (PK) for 60 min at 37°C on the
shaker. After a clarification spin at 500 x g for 5 min at
room temperature in a drum rotor (Jouan, Winchester, VA),
the samples were mixed with stock solution containing 10%
sodium phosphotungstate (NaPTA) and 85 mM MgCl2, pH 7.4, to
obtain a final concentration of 0.32% NaPTA. After a 1-h
incubation at 37°C on a rocking platform, the samples were
centrifuged at 14,000 x g in a Jouan MR23i centrifuge for 30
min at room temperature. The resulting pellets were
resuspended in H2O containing protease inhibitors (0.5 mM
phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, and 2
µg/ml leupeptin) and assayed by the CDI.

Sandwich CDI for PrPSc. For capture of HuPrP, the mAb MAR1
was used (11), and detection was accomplished with mAb 3F4
(12) labeled with Eu-chelate of
N-(p-isothiocyanatobenzyl)-diethylenetriamine-N1,N2,N3,N3-te
traacetic acid at pH 9.6 for 16 h at room temperature
according to the manufacturer's protocols (Wallac, Turku,
Finland), as described in ref. 8. The principle,
development, calibration, and calculation of PrPSc
concentration from CDI data have been described in refs.
8–10. The results were expressed as the difference in
Ab-binding between native and denatured samples [(D - N)] of
the time-resolved fluorescence of aliquots measured in cpm.
In some cases, the concentration of PrPSc is directly
proportional to (D - N) value and was calculated from the
formula described in refs. 8–10.

Histopathologic Procedures. Autopsies were performed shortly
after death, and brain tissue was either immediately frozen
or immersion-fixed in 10% buffered formalin for embedding in
paraffin. We stained 8-µm-thick sections with hematoxylin
and eosin (H&E) to evaluate vacuolation. Vacuolation scores,
visual estimates of the percentage of gray matter area in a
slide occupied by vacuoles, were determined by a single
observer (S.J.D.). Reactive astrocytic gliosis was evaluated
by glial fibrillary acidic protein immunostaining by using a
rabbit antiserum (DAKO). Hydrolytic autoclaving pretreatment
of the formalin-fixed tissue sections was used to detect
PrPSc, as described in ref. 13. The Bielschowsky silver
stain and IHC for -synuclein, tau, and ubiquitin were used
as needed to test for Alzheimer's disease,
synucleinopathies, tauopathies, and other neurodegenerative
processes.

Additional methods describing the diagnosis of prion
disease, human samples acquisition, construction of
transgenic (Tg) mice, endpoint titrations in Tg mice, sample
tracking and data processing, and sandwich CDI for PrPSc are
described in Supporting Text, which is published as
supporting information on the PNAS web site.

Results

Patient Groups, Clinical Diagnosis, and Codon 129 Polymorphi
sm. Human brain specimens were obtained from 46 patients who
underwent pathologic evaluation. Of the 28 cases in the
prion disease group, 24 were diagnosed with sCJD, three with
fCJD(E200K), and one with iCJD (see Table 3, which is
published as supporting information on the PNAS web site).
The PrP polymorphism at codon 129 [methionine (M) or valine
(V)] was determined by DNA sequencing for all 28 patients in
the prion disease group. As shown for sCJD, 13 patients were
MM, 7 were MV, and 4 were VV (Table 3).

Analytical Sensitivity of the CDI for Detection of Human
Prions. We used Eu-labeled 3F4 mAb (12) for detection and
MAR1 mAb (11) to capture HuPrPSc in a sandwich CDI format
(10). For a normal human brain homogenate that contains only
PrPC, the (D - N) was 1,789 cpm.

Brain homogenates from three cases of sCJD and one case of
fCJD were serially diluted in 3-fold increments into normal
human plasma and assayed by the CDI (Fig. 1). The
sensitivity of the CDI in detecting both sCJD and fCJD
prions was equal to or greater than that for the detection
of human prions by bioassay in Tg(MHu2M)5378/Prnp0/0 mice
(Fig. 1; see also Fig. 5 and Table 4, which are published as
supporting information on the PNAS web site). Within the
linear range, there was a good correlation between PrPSc
concentration measured by CDI and prion titer measured by
bioassay.

Fig. 1. Correlation between CDI and bioassay in Tg mice.
Sandwich CDI protocol for the detection of PrPSc was
compared to titration bioassays in Tg(MHu2M)5378/Prnp0/0
mice for three sCJD brains (a) and one fCJD brain (b).
Samples were precipitated with PTA and digested with 2.5
µg/ml PK for 1 h at 37°C. The MAR1 mAb was used (11) for
capture, and Eu-labeled 3F4 mAb was used for detection. The
(D - N) values measured in cpm are directly proportional to
the concentration of PrPSc (8, 10). Data points and bars
represent the average ± SD obtained from three to four
independent measurements. The cutoff (D - N) value of 1,789
cpm for this sandwich CDI protocol was calculated by [mean +
3(SD)] and determined from 100 brain samples obtained from
patients who died from nonneurologic disease (n = 6),
Alzheimer's disease (n = 7), and other neurologic diseases
(n = 5).

Diagnostic Sensitivity of the CDI for Detection of Human
Prions. To evaluate the diagnostic sensitivity of the CDI
for the detection of different CJD prions, we performed
multiple blind tests on brain tissue from 24 sCJD cases, 3
fCJD cases, 1 iCJD case, and 18 controls (Table 3). Data
from the controls exhibited a Gaussian distribution, with
the median (D - N) value oscillating around zero, as
expected for samples containing only residual PrPC after
phosphotungstate (PTA) precipitation (Fig. 2 and Table 5,
which is published as supporting information on the PNAS web
site). In contrast, median (D - N) values for sCJD and fCJD
cases are 106, which is six orders of magnitude higher than
the median of the control group (Fig. 2). The dynamic range
of the CJD data approaches 104, and all values are above
threshold. After performing 493 tests, the CDI identified
all CJD cases with 100% accuracy, and no false positives
occurred in the control group (Table 6, which is published
as supporting information on the PNAS web site).

Fig. 2. Statistical distribution of the CDI data for the
detection of PrPSc. Multiple samples from control (n = 18)
(a) and CJD brains (n = 27) (b) were tested. The control
group included cases of Alzheimer's disease (AD), patients
who died from other neurological diseases (OND), and cases
with no specific brain pathology at autopsy (NSPA). Each
brain sample was tested two to four times. Results are
expressed as (D - N) in cpm.

Codon 129 and the Anatomical Distribution of PrPSc in sCJD
Brains. By using the CDI, we determined the levels of PrPSc
in 24 brain regions for some patients and as few as 10 for
others because frozen samples for all regions were not
available (Fig. 6, which is published as supporting
information on the PNAS web site). Generally, the highest
concentrations of HuPrPSc were detected in the primary
visual cortex, thalamus, and cerebellum. All other areas of
the cortex and subcortical gray matter displayed substantial
accumulation of PrPSc. From these quantitative studies, we
conclude that, although the codon 129 genotype may influence
the levels of PrPSc deposition, the differences among the
three codon 129 genotypes (MM, MV, and VV) are less
prominent than expected from qualitative lesion profiles and
IHC.
Biopsies of Human Brains. A 52-year-old female experienced
memory problems, difficulty with concentration, anxiety,
confabulation, and visual hallucinations. A left parietal
lobe biopsy was performed 3 months after symptoms began to
rule out CJD. The biopsy contained a sample of cortex
extending from the pial surface to the underlying white
matter. No characteristic vacuolation or PrPSc deposits were
identified in sections of the biopsy (Fig. 3 b and f).

Fig. 3. Routine histology and PrPSc IHC on brain sections of
two CJD patients. (a and e) Patient with CJD (MV2 subtype)
in which vacuolation and PrPSc deposits were identified in
all brain regions sampled, and kuru-type plaques were
identified in the cerebellar cortex. (b and f and c and g)
Patient with CJD (MM1 subtype) in which neither vacuolation
nor PrPSc deposits were found in a brain biopsy (b and f) or
at routine autopsy (c and g). (d and h) By using
high-intensity MRI signals as a guide, a second set of brain
sections were prepared from this patient; vacuolation of the
neuropil as well as coarse PrPSc deposits were found. (a–d)
H&E stain. (Scale bar, 50 µm.) (e–h) IHC for PrPSc. (Scale
bar, 30 µm.)

The patient died 3.5 months after the biopsy. Routine
sampling of multiple brain regions again failed to reveal
sufficient degrees of gray matter vacuolation (Fig. 3c) to
make the diagnosis of human prion disease. IHC for PrPSc
show rare punctate deposits in the neocortical regions
sampled (Fig. 3g). At the time, one of us (S.J.D.) believed
such infrequent deposits might be an artifact, and,
therefore, the inconclusive IHC and routine histology
prevented a definitive diagnosis of CJD. By using the MRI
scan performed a week before death as a guide, new samples
were obtained from cortical regions with high signal
intensities. In these regions, clusters of vacuoles
measuring 40–60 µm in diameter were found in cortical layers
2 and 3 (Fig. 3d) and coarse PrPSc deposits were associated
with the clusters of vacuoles (Fig. 3h). Small amounts of
PrPSc deposits were also found away from the vacuoles. The
clusters of vacuolation and deposits of PrPSc tended to be
small and highly focal, occupying <1% of the cortical
cross-sectional area.
Two years later, when unfixed frozen samples from the right
hemisphere corresponding to neuropathologically positive and
negative contralateral brain regions were analyzed by the
CDI, all 13 of the regions examined were strongly positive
for PrPSc. The data from histology with H&E staining, IHC,
and the CDI are summarized in Table 7, which is published as
supporting information on the PNAS web site. (D - N) values
varied from almost 300,000 cpm in the medulla to >3,000,000
cpm in the thalamus, globus pallidus, frontal cortex,
occipital cortex, as well as the parietal cortex, the region
where the initial biopsy was taken on the contralateral
side.

Diagnostic Sensitivity of the CDI and IHC. When brain
sections from 10 CJD cases [8 sCJD and 2 fCJD(E200K)] were
analyzed by H&E staining, IHC with -PrP mAbs, and the CDI
(Tables 1 and 2), we discovered that, like the biopsied
patient reported above, the CDI was vastly superior to both
histologic examination for vacuolation of the neuropil and
IHC for PrP immunostaining. The microscopic studies were
performed on formalin-fixed, paraffin-embedded tissue
sections with knowledge that the patients were clinically
diagnosed with CJD, but without knowledge of the CDI
results.

Table 1. Comparison of the diagnostic sensitivity of the
vacuolation profile, IHC, and sandwich CDI in the detection
of PrPSc in different anatomical areas of sCJD brains

Table 2. Comparison of the diagnostic sensitivity of the
vacuolation profile, IHC, and sandwich CDI for the detection
of PrPSc in different anatomical areas of fCJD brains

> From 18 brain regions of the 8 sCJD cases, we compared the

results of histology, IHC, and CDI. Only the CDI gave
consistently positive (100%) PrPSc signals for all 18 brain
regions in all 8 patients. By routine histology, only 3 of
18 regions were found positive in the 8 sCJD cases (Table
1); this represents a diagnostic sensitivity of 17%. The
entorhinal cortex, temporal lobe cortex, and the caudate
nucleus were 100% positive from the brains of all eight
patients analyzed by histology. The remaining 15 regions
varied from 0% to 87% positive for the 8 sCJD brains
examined.
The results with IHC were similar to those obtained by
histological examination, which was unexpected because IHC
is generally thought to be more sensitive than histology. By
IHC, only 4 of 18 regions were found positive in all 8 sCJD
patients (Table 1); these results represent a diagnostic
sensitivity of 22%. The frontal cortex, parietal cortex,
temporal lobe cortex, and the insula were 100% positive from
the brains of all 8 patients. The remaining 14 regions
varied from 13% to 87% positive for the 8 sCJD brains
examined. Comparing histology, IHC, and the CDI, only the
temporal lobe region gave consistently positive results
(100%) for the 8 sCJD patients (Table 1).

Next, we compared histology, IHC, and CDI analysis on the
brains of two patients who died of fCJD(E200K). Of the 18
regions examined by histology or IHC, 9 were found positive
in the 2 patients (Table 2); these results represent a
diagnostic sensitivity of 50%. In contrast, the CDI was
positive in all 18 regions for both patients.

Asymmetric Lesions and PrPSc Deposits in the Brain. To
address the possibility of sampling bias, we examined the
brain of a 79-year-old female in whom rapidly progressive
motor and language decline were associated with myoclonic
jerks and an electroencephalogram with periodic spikes
characteristic of CJD. The patient died 14 days after a
diffusion-weighted MRI scan showed high intensity signals in
the left cerebral cortex but few or no such signals in the
right.

Histologic analysis showed moderately severe vacuolation
scores in multiple cortical regions of the left cerebral
hemisphere with little or none in analogous regions on the
right (Table 8, which is published as supporting information
on the PNAS web site). Unbiased stereological counts of
neurons in different cerebral cortical layers showed marked
loss from all layers of the left frontal cortex (Brodmann
areas 44 and 45), but no loss was found on the right (data
not shown). Morphometric quantification of IHC for glial
fibrillary acidic protein showed marked astrocytic gliosis
in the left cerebral cortex and only focal, mild gliosis on
the right. We found more PrPSc deposits in the left cortex
than in the right but these were not quantified (Table 8).
PrPSc was found in all locations of analogous right and left
cortical samples by the CDI; levels of PrPSc in the right
cortex were 5–50% lower than those from the left. Much lower
levels of PrPSc in the right cortex might have been expected
based the minimal microscopic changes, reflecting again the
incongruity between microscopic and CDI analyses.

Levels of sPrPSc and rPrPSc. To determine the relationship
between sPrPSc and rPrPSc in the brains of sCJD patients,
samples were either PTA-precipitated to measure the
concentration of total PrPSc or PK-digested then
PTA-precipitated to obtain the concentration of rPrPSc, as
described in refs. 8 and 9. These treated samples were then
subjected to analysis by the CDI. Surprisingly, >80% of
total PrPSc was susceptible to proteolytic degradation (Fig.
4). Despite a 20-fold lower concentration of PrPSc in white
matter, the ratio between sPrPSc and rPrPSc remained
constant. In conclusion, sPrPSc constitutes a major fraction
of total PrPSc in the frontal cortex and white matter of the
sCJD-infected brains.

Fig. 4. Most PrPSc in the frontal cortex and white matter of
sCJD brains is protease-sensitive [gray bars, calculated
from measurements of total PrPSc (white bars) and rPrPSc
(black bars)]. Before measurement by the CDI, undigested
samples were PTA-precipitated to measure total PrPSc or
digested with 50 µg/ml PK at 37°C for 1 h, followed by PTA
precipitation to determine rPrPSc (8, 9). The graph shows
the means ± SEM obtained from duplicate measurements of
samples from the frontal cortex (n = 19) and white matter (n
= 12) of sCJD-infected brains.

It is noteworthy that IHC of formalin-fixed,
paraffin-embedded tissue sections occasionally showed PrPSc
deposits in white matter; however, histoblot analysis, which
is our most sensitive and specific tissue-based method,
routinely failed to identify rPrPSc in white matter (data
not shown). In contrast, the CDI found PrPSc in white matter
in all cases of sCJD and fCJD (Fig. 4 and Tables 1 and 2).

Discussion

The clinical diagnosis of human prion disease is often
difficult until the patient shows profound signs of
neurologic dysfunction. It is widely accepted that the
clinical diagnosis must be provisional until a tissue
diagnosis either confirms or rules out the clinical
assessment. Before the availability of Abs to PrP, a tissue
diagnosis was generally made by histologic evaluation of
neuropil vacuolation. IHC with
anti-glial-fibrillary-acidic-protein Abs in combination with
H&E staining preceded the use of anti-PrP Ab staining.

Recently, the role of IHC in the diagnosis of scrapie in the
brains of eight clinically affected goats inoculated with
the SSBP1 prion isolate has been challenged (14). Thalamic
samples taken from seven of eight goats with scrapie were
positive for PrPSc by Western blotting but negative by IHC.
The eighth goat was negative by Western blotting and IHC.
Consistent with these findings in goats are the data
reported here, in which IHC of formalin-fixed,
paraffin-embedded human brain samples was substantially less
sensitive than the CDI.

The CDI was developed to quantify PrPSc in tissue samples
from mammals producing prions. Concerned that limited PK
digestion was hydrolyzing some or even most of the PrPSc, we
developed a CDI that does not require PK digestion. The CDI
revealed that as much as 90% of PrPSc is sPrPSc; thus, it
was being destroyed during limited proteolytic digestion
used to hydrolyze PrPC. sPrPSc comprises 80% of PrPSc in the
frontal lobe and in the white matter (Fig. 4).

The CDI detected HuPrPSc with a sensitivity comparable to
the bioassay for prion infectivity in Tg(MHu2M) mice (Fig.
1). The high sensitivity achieved by the CDI is due to
several factors (8, 10, 11, 15). First, both sPrPSc and
rPrPSc conformers are specifically precipitated by PTA
(Table 5) (8, 9). PTA has also been used to increase the
sensitivity of Western blots enabling the detection of
rPrPSc in human muscle and other peripheral tissues (16,
17). Second, a sandwich protocol was used with the
high-affinity MAR1 mAb (11) to capture HuPrPSc and
Eu-labeled 3F4 mAb to detect HuPrPSc (12). Third, the CDI
detects PrPSc by Ab-binding to native and denatured forms of
the protein and, therefore, does not depend on proteolytic
degradation of PrPC. We chose not to perform Western blots
on most of the samples used in this study because such
immunoblots require denaturation of the sample, which
eliminates measurement of the native signal corresponding to
PrPC (Table 5). Moreover, a comparison between the CDI and
Western blotting on brain samples from sCJD and variant CJD
patients showed that the CDI was 50- to 100-fold more
sensitive (15). Additionally, Western blots combined with
densitometry are linear over a 10- to 100-fold range of
concentrations, whereas the CDI is linear over a >104-fold
range. The CDI has been automated, which not only improves
accuracy and reproducibility (10) but also allows numerous
samples to be analyzed, as reported here. Western blots are
difficult to automate and are labor intensive.

Our studies show that only the CDI detected PrPSc in all
regions examined in 24 sCJD and 3 fCJD(E200K) brains (Figs.
2 and 6). Comparative analyses demonstrated that the CDI was
vastly superior to histology and IHC. When 18 regions of 8
sCJD and 2 fCJD(E200K) brains were compared, we discovered
that histology and IHC were unreliable diagnostic tools
except for samples from a few brain regions. In contrast,
the CDI was a superb diagnostic procedure because it
detected PrPSc in all 18 regions in 8 of 8 sCJD and 2 of 2
fCJD(E200K) cases (Tables 1 and 2).

Histologic changes in prion disease have been shown to
follow the accumulation of prions as measured by bioassay of
infectivity and by PrPSc accumulation (18–22). Because low
levels of PrPSc are not associated with neuropathologic
changes, some discrepancy between vacuolation and PrPSc was
expected. In contrast to histology, IHC measures PrP
immunostaining after autoclaving tissue sections exposed to
formic acid. Because IHC measures PrP, we expected the
sensitivity of this procedure might be similar to the CDI,
but that proved not to be the case. Whether exposure of
formic acid-treated tissue sections to elevated temperature
destroys not only PrPC but also sPrPSc and only denatures
rPrPSc remains to be determined. Such a scenario could
account for the lower sensitivity of IHC compared with CDI
or bioassay (Tables 1 and 2).

Studies of the white matter in CJD brains were particularly
informative with respect to the sensitivity of the CDI,
where PrPSc levels were low but readily detectable, 10- to
100-fold above the threshold value (Fig. 4). Because animal
studies have shown that PrPSc and infectivity are
transported anterogradely from one brain region to another
along neuroanatomical pathways (23–25), we expected to find
PrPSc in white matter as demonstrated by the CDI but not
IHC. Axonal transport of PrPSc is also suggested by
diffusion-weighted MRI scans of CJD cases, which show
high-intensity signals in analogous neocortical regions of
the right and left cerebral hemispheres (26). This symmetry
of neuroradiological abnormalities is consistent with spread
of PrPSc to the contralateral cortex by means of callosal
commissural pathways.

Most immunoassays that detect HuPrPSc do so only after
subjecting the sample to limited proteolysis to form PrP
27–30, followed by denaturation. Because the CDI measures
the immunoreactivity before and after denaturation to an
epitope that is exposed in native PrPC but buried in PrPSc,
limited proteolysis to eliminate PrPC is unnecessary. Assays
based on limited proteolysis underestimate the level of
PrPSc because they digest sPrPSc, which represents 80–90% of
PrPSc in CJD and scrapie brains (Fig. 4 and Table 5).

Gerstmann–Sträussler–Scheinker, an inherited human prion
disease, is caused by the P102L mutation in the PRNP gene.
In mice expressing the Gerstmann–Sträussler–Scheinker mutant
PrP transgene, the CDI detected high levels of sPrPSc(P101L)
as well as low levels of rPrPSc(P101L) long before
neurodegeneration and clinical symptoms occurred (9).
sPrPSc(P101L) as well as low concentrations of rPrPSc(P101L)
previously escaped detection (27). Whether a similar
situation applies in other genetic forms of prion disease,
sCJD, or variant CJD remains to be determined. Because most
of the PrPSc in the brains of sCJD patients is
protease-sensitive (Fig. 4), it is likely that the lower
sensitivity of IHC is due to its inability to detect sPrPSc.
Presently, we have no information about the kinetics of
either sPrPSc or rPrPSc accumulation in human brain. Limited
information on the kinetics of PrPSc accumulation in
livestock comes from studies of cattle, sheep, and goats
inoculated orally, but most of the bioassays were performed
in non-Tg mice (28–30) in which prion titers were
underestimated by as much as a factor of 104 (10).

The studies reported here are likely to change profoundly
the approach to the diagnosis of prion disease in both
humans and livestock (31–33). The superior performance of
the CDI in diagnosing prion disease compared to routine
neuropathologic examination and IHC demands that the CDI be
used in future diagnostic evaluations of prion disease.
Prion disease can no longer be ruled out by routine
histology or IHC. Moreover, the use of IHC to confirm cases
of bovine spongiform encephalopathy after detection of
bovine PrPSc by the CDI (10) seems an untenable approach in
the future. Clearly, the CDI for HuPrPSc is as sensitive or
more sensitive than bioassays in Tg(MHu2M) mice (Fig. 1).

Our results suggest that using the CDI to test large numbers
of samples for human prions might alter the epidemiology of
prion diseases. At present, there is limited data on the
frequency of subclinical variant CJD infections in the U.K.
population (34). Because appendixes and tonsils were
evaluated only by IHC, many cases might have escaped
detection (Tables 1 and 2). Equally important may be the use
of CDI-like tests to diagnose other neurodegenerative
disorders, such as Alzheimer's disease, Parkinson's disease,
and the frontotemporal dementias. Whether IHC underestimates
the incidence of one or more of these common degenerative
diseases is unknown. Moreover, CDI-like tests may help
determine the frequency with which these disorders and the
prion diseases occurs concomitantly in a single patient (35,
36).

Acknowledgements

We thank the staff of the Hunters Point Animal Facility for
their expert mouse studies and ZLB Behring for making the
MAR1 mAb available. This work was supported by National
Institutes of Health Contract NS02328 and National
Institutes of Health Grants AG02132, AG010770, and AG021601.
M.D.G. is supported by the John Douglas French Foundation
for Alzheimer's research, the McBean Foundation; National
Institute on Aging Grants AG021989, AG019724, and AG023501;
the Alzheimer's Disease Research Center of California; and
National Institutes of Health Grant M01 RR00079 to the
General Clinical Research Center. B.L.M. is supported by
National Institute on Aging Grants AG019724 and AG023501.

Footnotes

Author contributions: J.G.S. and S.B.P. designed research;
M.D.G., C.D., S.D., M.S., H.S., and S.J.D. performed
research; A.S., M.V., and H.B. contributed new
reagents/analytic tools; J.G.S., K.G., B.L.M., S.J.D., and
S.B.P. analyzed data; and J.G.S., S.J.D., and S.B.P. wrote
the paper.

Abbreviations: CDI, conformation-dependent immunoassay; CJD,
Creutzfeldt–Jakob disease; sCJD, sporadic CJD; fCJD,
familial CJD; iCJD, iatrogenic CJD; IHC,
immunohistochemistry; PrP, prion protein; PrPC, normal
cellular PrP; PrPSc, disease-causing PrP; HuPrP, human PrP;
MHu2M, chimeric mouse–human transgene; sPrPSc,
protease-sensitive PrPSc; rPrPSc, protease-resistant PrPSc;
PTA, phosphotungstate; PK, proteinase K; H&E, hematoxylin
and eosin; Tg, transgenic; (D - N), difference in Ab-binding
between native and denatured samples.

J.G.S., S.J.D., A.S., K.G., and S.B.P. have financial
interest in InPro Biotechnology, Inc.

To whom correspondence should be addressed at: Institute for
Neurodegenerative Diseases, University of California, Box
0518, San Francisco, CA 94143-0518. E-mail:
stanley@ind.ucsf.edu.

© 2005 by The National Academy of Sciences of the USA

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