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From: TSS ()
Subject: sporadic CJD TSE USA and the UK BSE nvCJD only theory
Date: November 24, 2005 at 8:13 am PST

Diagnosis and Reporting of Creutzfeldt-Jakob Disease

Singeltary, Sr et al. JAMA.2001; 285: 733-734.

http://jama.ama-assn.org/cgi/content/full/285/6/733



Diagnosis and Reporting of Creutzfeldt-Jakob Disease
Singeltary, Sr et al. JAMA.2001; 285: 733-734.

Full Text

Tue, 13 Feb 2001 JAMA Vol. 285 No. 6, February 14, 2001 Letters

Diagnosis and Reporting of Creutzfeldt-Jakob Disease

To the Editor:

In their Research Letter in JAMA. 2000;284:2322-2323, Dr Gibbons and colleagues1 reported that the annual US death rate due to Creutzfeldt-Jakob disease (CJD) has been stable since 1985. These estimates, however, are based only on reported cases, and do not include misdiagnosed or preclinical cases. It seems to me that misdiagnosis alone would drastically change these figures. An unknown number of persons with a diagnosis of Alzheimer disease in fact may have CJD, although only a small number of these patients receive the postmortem examination necessary to make this diagnosis. Furthermore, only a few states have made CJD reportable. Human and animal transmissible spongiform encephalopathies should be reportable nationwide and internationally.

Terry S. Singeltary, Sr Bacliff, Tex

To the Editor:

At the time of my mother's death, various diagnoses were advanced such as "rapid progressive Alzheimer disease," psychosis, and dementia. Had I not persisted and personally sought and arranged a brain autopsy, her death certificate would have read cardiac failure and not CJD.

Through CJD Voice1 I have corresponded with hundreds of grief-stricken families who are so devastated by this horrific disease that brain autopsy is the furthest thing from their minds. In my experience, very few physicians suggest it to the family. After the death and when families reflect that they never were sure what killed their loved one it is too late to find the true cause of death. In the years since my mother died I think that the increasing awareness of the nature of CJD has only resulted in fewer pathologists being willing to perform an autopsy in a suspected case of CJD.

People with CJD may die with incorrect diagnoses of dementia, psychosis, Alzheimer disease, and myriad other neurological diseases. The true cause of death will only be known if brain autopsies are suggested to the families. Too often the physician's comment is, "Well, it could be CJD but that is so rare it isn't likely."

Until CJD is required to be reported to state health departments, as other diseases are, there will be no accurate count of CJD deaths in the United States and thus no way to know if the number of deaths is decreasing, stable, or increasing as it has recently in the United Kingdom.

Dorothy E. Kraemer Stillwater, Okla

In Reply:

Mr Singeltary and Ms Kraemer express an underlying concern that our recently reported mortality surveillance estimate of about 1 CJD case per million population per year in the United States since 1985 may greatly underestimate the true incidence of this disease. Based on evidence from epidemiologic investigations both within and outside the United States, we believe that these national estimates are reasonably accurate.

Even during the 1990s in the United Kingdom, where much attention and public health resources have been devoted to prion disease surveillance, the reported incidence of classic CJD is similar to that reported in the United States.

In addition, in 1996, active US surveillance for CJD and new variant (nv) CJD in 5 sites detected no evidence of the occurrence of nvCJD and showed that 86% of the CJD cases in these sites were identifiable through routinely collected mortality data.

Our report provides additional evidence against the occurrence of nvCJD in the United States based on national mortality data analyses and enhanced surveillance. It specifically mentions a new center for improved pathology surveillance. We hope that the described enhancements along with the observations of Singeltary and Kraemer will encourage medical care providers to suggest brain autopsies for more suspected CJD cases to facilitate the identification of potentially misdiagnosed CJD cases and to help monitor the possible occurrence of nvCJD.

Creutzfeldt-Jakob disease is not on the list of nationally notifiable diseases. In those states where surveillance personnel indicate that making this disease officially notifiable would meaningfully facilitate collection of data that are needed to monitor the incidence of CJD and nvCJD, including the obtaining of brain autopsy results, we encourage such a change. However, adding CJD to the notifiable diseases surveillance system may lead to potentially wasteful, duplicative reporting because the vast majority of the diagnosed cases would also be reported through the mortality surveillance system.

Furthermore, making CJD a notifiable disease may not necessarily help identify undiagnosed CJD cases. The unique characteristics of CJD make mortality data a useful surrogate for ongoing surveillance. Unlike many other neurologic diseases, CJD is invariably fatal and in most cases rapidly progressive and distinguishable clinically from other neurologic diseases.

Because CJD is least accurately diagnosed early in the course of the illness, notifiable disease surveillance of CJD could be less accurate than mortality surveillance of CJD. In addition, because death as a condition is more completely and consistently reported, mortality surveillance has the advantage of being ongoing and readily available.

The absence of CJD and nvCJD from the list of nationally notifiable diseases should not be interpreted to mean that they are not important to public health; this list does not include all such diseases. We encourage medical caregivers to report to or consult with appropriate public health authorities about any diagnosed case of a transmissible disease for which a special public health response may be needed, including nvCJD, and any patient in whom iatrogenic transmission of CJD may be suspected.

Robert V. Gibbons, MD, MPH Robert C. Holman, MS Ermias D. Belay, MD Lawrence B. Schonberger, MD, MPH Division of Viral and Rickettsial Diseases National Center for Infectious Diseases Centers for Disease Control and Prevention Atlanta, Ga

http://jama.amaassn.org/cgi/content/full/285/6/733maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&fulltext=dignosing+and+reporting+creutzfeldt+jakob+disease&searchid=1048865596978_1528&stored_search=&FIRSTINDEX=0&journalcode=jama

Singeltary, Sr et al. JAMA

Neurology 2003;60:176-181
© 2003 American Academy of Neurology


--------------------------------------------------------------------------------


VIEWS & REVIEWS:
Ermias D. Belay, Ryan A. Maddox, Pierluigi Gambetti, and Lawrence B. Schonberger

Monitoring the occurrence of emerging forms of Creutzfeldt-Jakob disease in the United States
Neurology 2003; 60: 176-181 [Abstract] [Full text] [PDF]


Correspondence published:

Reply to Singletary
Ryan A. Maddox, MPH, Ermias D. Belay, MD, Lawrence B. Schonberger, MD (26 March 2003)

RE-Monitoring the occurrence of emerging forms of Creutzfeldt-Jakob disease in the United States
Terry S. Singeltary (26 March 2003)

--------------------------------------------------------------------------------

Send Post-Publication Peer Review to journal:


Re: RE-Monitoring the occurrence of emerging forms of Creutzfeldt-Jakob

disease in the United States


Email Terry S. Singeltary:


flounder@wt.net

I lost my mother to hvCJD (Heidenhain Variant CJD). I would like to

comment on the CDC's attempts to monitor the occurrence of emerging

forms of CJD. Asante, Collinge et al [1] have reported 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. However, CJD and all human TSEs are not reportable

nationally. CJD and all human TSEs must be made reportable in every

state and internationally. I hope that the CDC does not continue to

expect us to still believe that the 85%+ of all CJD cases which are

sporadic are all spontaneous, without route/source. We have many TSEs in

the USA in both animal and man. CWD in deer/elk is spreading rapidly and

CWD does transmit to mink, ferret, cattle, and squirrel monkey by

intracerebral inoculation. With the known incubation periods in other

TSEs, oral transmission studies of CWD may take much longer. Every

victim/family of CJD/TSEs should be asked about route and source of this

agent. To prolong this will only spread the agent and needlessly expose

others. In light of the findings of Asante and Collinge et al, there

should be drastic measures to safeguard the medical and surgical arena

from sporadic CJDs and all human TSEs. I only ponder how many sporadic

CJDs in the USA are type 2 PrPSc?


http://www.neurology.org/cgi/eletters/60/2/176#535

http://www.neurology.org/cgi/eletters/60/2/176#535


LANCET INFECTIOUS DISEASE JOURNAL


Volume 3, Number 8 01 August 2003


Newsdesk


Tracking spongiform encephalopathies in North America


Xavier Bosch

My name is Terry S Singeltary Sr, and I live in Bacliff, Texas. I lost

my mom to hvCJD (Heidenhain variant CJD) and have been searching for

answers ever since. What I have found is that we have not been told the

truth. CWD in deer and elk is a small portion of a much bigger problem.


49-year-old Singeltary is one of a number of people who have remained

largely unsatisfied after being told that a close relative died from a

rapidly progressive dementia compatible with spontaneous

Creutzfeldt-Jakob disease (CJD). So he decided to gather hundreds of

documents on transmissible spongiform encephalopathies (TSE) and

realised that if Britons could get variant CJD from bovine spongiform

encephalopathy (BSE), Americans might get a similar disorder from

chronic wasting disease (CWD) the relative of mad cow disease seen among

deer and elk in the USA. Although his feverish search did not lead him

to the smoking gun linking CWD to a similar disease in North American

people, it did uncover a largely disappointing situation.


Singeltary was greatly demoralised at the few attempts to monitor the

occurrence of CJD and CWD in the USA. Only a few states have made CJD

reportable. Human and animal TSEs should be reportable nationwide and

internationally, he complained in a letter to the Journal of the

American Medical Association (JAMA 2003; 285: 733). I hope that the CDC

does not continue to expect us to still believe that the 85% plus of all

CJD cases which are sporadic are all spontaneous, without route or source.


Until recently, CWD was thought to be confined to the wild in a small

region in Colorado. But since early 2002, it has been reported in other

areas, including Wisconsin, South Dakota, and the Canadian province of

Saskatchewan. Indeed, the occurrence of CWD in states that were not

endemic previously increased concern about a widespread outbreak and

possible transmission to people and cattle.


To date, experimental studies have proven that the CWD agent can be

transmitted to cattle by intracerebral inoculation and that it can cross

the mucous membranes of the digestive tract to initiate infection in

lymphoid tissue before invasion of the central nervous system. Yet the

plausibility of CWD spreading to people has remained elusive.


Part of the problem seems to stem from the US surveillance system. CJD

is only reported in those areas known to be endemic foci of CWD.

Moreover, US authorities have been criticised for not having performed

enough prionic tests in farm deer and elk.


Although in November last year the US Food and Drug Administration

issued a directive to state public-health and agriculture officials

prohibiting material from CWD-positive animals from being used as an

ingredient in feed for any animal species, epidemiological control and

research in the USA has been quite different from the situation in the

UK and Europe regarding BSE.


Getting data on TSEs in the USA from the government is like pulling

teeth, Singeltary argues. You get it when they want you to have it,

and only what they want you to have.


Norman Foster, director of the Cognitive Disorders Clinic at the

University of Michigan (Ann Arbor, MI, USA), says that current

surveillance of prion disease in people in the USA is inadequate to

detect whether CWD is occurring in human beings; adding that, the

cases that we know about are reassuring, because they do not suggest the

appearance of a new variant of CJD in the USA or atypical features in

patients that might be exposed to CWD. However, until we establish a

system that identifies and analyses a high proportion of suspected prion

disease cases we will not know for sure. The USA should develop a

system modelled on that established in the UK, he points out.

Ali Samii, a neurologist at Seattle VA Medical Center who recently

reported the cases of three hunters two of whom were friends who died

from pathologically confirmed CJD, says that at present there are

insufficient data to claim transmission of CWD into humans; adding that

[only] by asking [the questions of venison consumption and deer/elk

hunting] in every case can we collect suspect cases and look into the

plausibility of transmission further. Samii argues that by making both

doctors and hunters more aware of the possibility of prions spreading

through eating venison, doctors treating hunters with dementia can

consider a possible prion disease, and doctors treating CJD patients

will know to ask whether they ate venison.


CDC spokesman Ermias Belay says that the CDC will not be investigating

the [Samii] cases because there is no evidence that the men ate

CWD-infected meat. He notes that although the likelihood of CWD

jumping the species barrier to infect humans cannot be ruled out 100%

and that [we] cannot be 100% sure that CWD does not exist in humans&

the data seeking evidence of CWD transmission to humans have been very

limited.


http://infection.thelancet.com/journal/journal.isa

BRITISH MEDICAL JOURNAL

SOMETHING TO CHEW ON

BMJ


Terry S Singeltary,
NA
medically retired
Send response to journal:
Re: Re: vCJD in the USA * BSE in U.S.


In reading the recent article in the BMJ about the potential BSE tests being developed in the U.S. and Bart Van Everbroeck reply. It does not surprize me, that the U.S. has been concealing vCJD. There have been people dying from CJD, with all the symptoms and pathological findings that resemble U.K. vCJD for some time. It just seems that when there is one found, they seem to change the clarical classification of the disease, to fit their agenda. I have several autopsies, stating kuru type amyloid plaques, one of the victims was 41 years of age. Also, my Mom died a most hideous death, Heidenhain Variant Creutzfeldt Jakob disease. Her symptoms resemble that of all the U.K. vCJD victims. She would jerk so bad at times, it would take 3 of us to hold her down, while she screamed "God, what's wrong with me, why can't I stop this." 1st of symptoms to death, 10 weeks, she went blind in the first few weeks. But, then they told me that this was just another strain of sporadic CJD. They can call it what ever they want, but I know what I saw, and what she went through. Sporadic, simply means, they do not know. My neighbors Mom also died from CJD. She had been taking a nutritional supplement which contained the following; vacuum dried bovine BRAIN, bone meal, bovine EYE, veal bone, bovine liver powder, bovine adrenal, vacuum dried bovine kidney, and vacuum dried porcine stomach. As I said, this woman taking these nutritional supplements, died from CJD. The particular batch of pills that was located, in which she was taking, was tested. From what I have heard, they came up negative, for the prion protein. But, in the same breath, they said their testing, may not have been strong enough to pick up the infectivity. Plus, she had been taking these type pills for years, so, could it have come from another batch?

CWD is just a small piece of a very big puzzle. I have seen while deer hunting, deer, squirrels and birds, eating from cattle feed troughs where they feed cattle, the high protein cattle by products, at least up until Aug. 4, 1997. So why would it be so hard to believe that this is how they might become infected with a TSE. Or, even by potentially infected land. It's been well documented that it could be possible, from scrapie. Cats becoming infected with a TSE. Have you ever read the ingredients on the labels of cat and dog food? But, they do not put these tissues from these animals in pharmaceuticals, cosmetics, nutritional supplements, hGH, hPG, blood products, heart valves, and the many more products that come from bovine, ovine, or porcine tissues and organs. So, as I said, this CWD would be a small piece of a very big puzzle. But, it is here, and it most likely has killed. You see, greed is what caused this catastrophe, rendering and feeding practices. But, once Pandora's box was opened, the potential routes of infection became endless.

No BSE in the U.S.A.? I would not be so sure of that considering that since 1990;

Since 1990 the U.S. has raised 1,250,880,700 cattle;

Since 1990 the U.S. has ONLY checked 8,881 cattle brains for BSE, as of Oct. 4, 1999;

There are apprx. 100,000 DOWNER cattle annually in the U.S., that up until Aug. 4, 1997 went to the renders for feed;

Scrapie running rampant for years in the U.S., 950 infected FLOCKS, as of Aug. 1999;

Our feeding and rendering practices have mirrored that of the U.K. for years, some say it was worse. Everything from the downer cattle, to those scrapie infected sheep, to any roadkill, including the city police horse and the circus elephant went to the renders for feed and other products for consumption. Then they only implemented a partial feed ban on Aug. 4, 1997, but pigs, chickens, dogs, and cats, and humans were exempt from that ban. So they can still feed pigs and chickens those potentially TSE tainted by-products, and then they can still feed those by-products back to the cows. I believe it was Dr. Joe Gibbs, that said, the prion protein, can survive the digestinal track. So you have stopped nothing. It was proven in Oprah Winfrey's trial, that Cactus Cattle feeders, sent neurologically ill cattle, some with encephalopathy stamped on the dead slips, were picked up and sent to the renders, along with sheep carcasses. Speaking of autopsies, I have a stack of them, from CJD victims. You would be surprised of the number of them, who ate cow brains, elk brains, deer brains, or hog brains.

I believe all these TSE's are going to be related, and originally caused by the same greedy Industries, and they will be many. Not just the Renders, but you now see, that they are re-using medical devices that were meant for disposal. Some medical institutions do not follow proper auto- claving procedures (even Olympus has put out a medical warning on their endescopes about CJD, and the fact you cannot properly clean these instruments from TSE's), and this is just one product. Another route of infection.

Regardless what the Federal Government in the U.S. says. It's here, I have seen it, and the longer they keep sweeping it under the rug and denying the fact that we have a serious problem, one that could surpass aids (not now, but in the years to come, due to the incubation period), they will be responsible for the continued spreading of this deadly disease.

It's their move, it's CHECK, but once CHECKMATE has been called, how many thousands or millions, will be at risk or infected or even dead. You can't play around with these TSE's. I cannot stress that enough. They are only looking at body bags, and the fact the count is so low. But, then you have to look at the fact it is not a reportable disease in most states, mis-diagnosis, no autopsies performed. The fact that their one-in-a- million theory is a crude survey done about 5 years ago, that's a joke, under the above circumstances. A bad joke indeed........

The truth will come, but how many more have to die such a hideous death. It's the Government's call, and they need to make a serious move, soon. This problem, potential epidemic, is not going away, by itself.

Terry S. Singeltary Sr.
P.O. Box 42, Bacliff, Texas 77518 USA
flounder@wt.net


http://www.bmj.com/cgi/eletters/319/7220/1312/b#EL2

BMJ



2 January 2000

Terry S Singeltary
retired
Send response to journal:
Re: U.S. Scientist should be concerned with a CJD epidemic in the U.S., as well...

In reading your short article about 'Scientist warn of CJD epidemic' news in brief Jan. 1, 2000. I find the findings in the PNAS old news, made famous again. Why is the U.S. still sitting on their butts, ignoring the facts? We have the beginning of a CJD epidemic in the U.S., and the U.S. Gov. is doing everything in it's power to conceal it.

The exact same recipe for B.S.E. existed in the U.S. for years and years. In reading over the Qualitative Analysis of BSE Risk Factors-1, this is a 25 page report by the USDA:APHIS:VS. It could have been done in one page. The first page, fourth paragraph says it all;

"Similarities exist in the two countries usage of continuous rendering technology and the lack of usage of solvents, however, large differences still remain with other risk factors which greatly reduce the potential risk at the national level."

Then, the next 24 pages tries to down-play the high risks of B.S.E. in the U.S., with nothing more than the cattle to sheep ratio count, and the geographical locations of herds and flocks. That's all the evidence they can come up with, in the next 24 pages.

Something else I find odd, page 16;

"In the United Kingdom there is much concern for a specific continuous rendering technology which uses lower temperatures and accounts for 25 percent of total output. This technology was _originally_ designed and imported from the United States. However, the specific application in the production process is _believed_ to be different in the two countries."

A few more factors to consider, page 15;

"Figure 26 compares animal protein production for the two countries. The calculations are based on slaughter numbers, fallen stock estimates, and product yield coefficients. This approach is used due to variation of up to 80 percent from different reported sources. At 3.6 million tons, the United States produces 8 times more animal rendered product than the United Kingdom."

"The risk of introducing the BSE agent through sheep meat and bone meal is more acute in both relative and absolute terms in the United Kingdom (Figures 27 and 28). Note that sheep meat and bone meal accounts for 14 percent, or 61 thousand tons, in the United Kingdom versus 0.6 percent or 22 thousand tons in the United States. For sheep greater than 1 year, this is less than one-tenth of one percent of the United States supply."

"The potential risk of amplification of the BSE agent through cattle meat and bone meal is much greater in the United States where it accounts for 59 percent of total product or almost 5 times more than the total amount of rendered product in the United Kingdom."

Considering, it would only take _one_ scrapie infected sheep to contaminate the feed. Considering Scrapie has run rampant in the U.S. for years, as of Aug. 1999, 950 scrapie infected flocks. Also, Considering only one quarter spoonful of scrapie infected material is lethal to a cow. Considering all this, the sheep to cow ration is meaningless. As I said, it's 24 pages of B.S.e.

To be continued...

Terry S. Singeltary Sr. P.O. Box 42 Bacliff, Texas USA


--------------------------------------------------------------------------------

http://www.bmj.com/cgi/eletters/320/7226/8/b#EL1

HUMAN TSE USA 2005

Animal Prion Diseases Relevant to Humans (unknown types?)
Thu Oct 27, 2005 12:05
71.248.128.109

About Human Prion Diseases /
Animal Prion Diseases Relevant to Humans

Bovine Spongiform Encephalopathy (BSE) is a prion disease of cattle. Since 1986, when BSE was recognized, over 180,000 cattle in the UK have developed the disease, and approximately one to three million are likely to have been infected with the BSE agent, most of which were slaughtered for human consumption before developing signs of the disease. The origin of the first case of BSE is unknown, but the epidemic was caused by the recycling of processed waste parts of cattle, some of which were infected with the BSE agent and given to other cattle in feed. Control measures have resulted in the consistent decline of the epidemic in the UK since 1992. Infected cattle and feed exported from the UK have resulted in smaller epidemics in other European countries, where control measures were applied later.

Compelling evidence indicates that BSE can be transmitted to humans through the consumption of prion contaminated meat. BSE-infected individuals eventually develop vCJD with an incubation time believed to be on average 10 years. As of November 2004, three cases of BSE have been reported in North America. One had been imported to Canada from the UK, one was grown in Canada, and one discovered in the USA but of Canadian origin. There has been only one case of vCJD reported in the USA, but the patient most likely acquired the disease in the United Kingdom. If current control measures intended to protect public and animal health are well enforced, the cattle epidemic should be largely under control and any remaining risk to humans through beef consumption should be very small. (For more details see Smith et al. British Medical Bulletin, 66: 185. 2003.)

Chronic Wasting Disease (CWD) is a prion disease of elk and deer, both free range and in captivity. CWD is endemic in areas of Colorado, Wyoming, and Nebraska, but new foci of this disease have been detected in Nebraska, South Dakota, New Mexico, Wisconsin, Mississippi Kansas, Oklahoma, Minnesota, Montana, and Canada. Since there are an estimated 22 million elk and deer in the USA and a large number of hunters who consume elk and deer meat, there is the possibility that CWD can be transmitted from elk and deer to humans. As of November 2004, the NPDPSC has examined 26 hunters with a suspected prion disease. However, all of them appeared to have either typical sporadic or familial forms of the disease. The NPDPSC coordinates with the Centers for Disease Control and state health departments to monitor cases from CWD-endemic areas. Furthermore, it is doing experimental research on CWD transmissibility using animal models. (For details see Sigurdson et al. British Medical Bulletin. 66: 199. 2003 and Belay et al. Emerging Infectious Diseases. 10(6): 977. 2004.)

http://www.cjdsurveillance.com/abouthpd-animal.html

SEE STEADY INCREASE IN SPORADIC CJD IN THE USA FROM 1997 TO 2004. SPORADIC CJD CASES TRIPLED, and that is with a human TSE surveillance system that is terrible flawed. in 1997 cases of the _reported_ cases of cjd were at 54, to 163 _reported_ cases in 2004. see stats here;

p.s. please note the 47 PENDING CASES to Sept. 2005

p.s. please note the 2005 Prion D. total 120(8) 8=includes 51 type pending, 1 TYPE UNKNOWN ???

p.s. please note sporadic CJD 2002(1) 1=3 TYPE UNKNOWN???

p.s. please note 2004 prion disease (6) 6=7 TYPE UNKNOWN???

http://www.cjdsurveillance.com/resources-casereport.html

CWD TO HUMANS = sCJD ???

AS implied in the Inset 25 we must not _ASSUME_ that
transmission of BSE to other species will invariably
present pathology typical of a scrapie-like disease.

snip...

http://www.bseinquiry.gov.uk/files/yb/1991/01/04004001.pdf

ATYPICAL TSEs in USA CATTLE AND SHEEP ?

http://www.bseinquiry.gov.uk/files/sc/seac17/tab03.pdf

Infected and Source Flocks

As of August 31, 2005, there were 115 scrapie infected and source flocks (figure 3). There were 3 new infected and source flocks reported in August (Figure 4) with a total of 148 flocks reported for FY 2005 (Figure 5). The total infected and source flocks that have been released in FY 2005 are 102 (Figure 6), with 5 flocks released in August. The ratio of infected and source flocks released to newly infected and source flocks for FY 2005 = 0.69 :
1. In addition, as of August 31, 2005, 574 scrapie cases have been confirmed and reported by the National Veterinary Services Laboratories (NVSL), of which 122 were RSSS cases (Figure 7). This includes 55 newly confirmed cases in August 2005 (Figure 8). Fifteen cases of scrapie in goats have been reported since 1990 (Figure 9). The last goat case was reported in May 2005.

snip...

full text ;

http://www.aphis.usda.gov/vs/nahps/scrapie/monthly_report/monthly-report.html


SCRAPIE USA JULY 2005 UPDATE

AS of July 31, 2005, there were 120 scrapie infected soure flocks (figure 3). There were 16 new infected and source flocks reorted in July (Figure 4) with a total of 143 flocks reported for FY 2005 (Figure 5). The total infected and source flocks that have been released in FY 2005 are 89 (Figure 6), with 8 flocks released in July. The ratio of infected and source flocks released to newly infected and source flocks for FY = 0.62 : 1. IN addition, as of July 31, 2005, 524 scrapie cases have been confirmed and reported by the National Veterinary Services Laboratories (NVSL), of which 116 were RSSS cases (Figure 7). This includes 76 newly confirmed cases in July 2005 (Figure 8). Fifteen cases of scrapie in goats have been reported since 1990 (Figure 9). The last goat case was reported in May 2005. ...........

snip...

http://www.aphis.usda.gov/vs/nahps/scrapie/monthly_report/monthly-report.html


SCRAPIE USA JUNE 2005 UPDATE


AS of June 30, 2005, there were 114 scrapie infected and source flocks
(Figure 3). There were 14 new infected and source flocks reported in June
(Figure 4) with a total of 123 flocks reported for FY 2005 (Figure 5).


snip...


In addition, as of June 30, 2005, 448 scrapie cases have been confirmed and
reported by the National Veterinary Services Laboratories (NVSL), of which
106 were RSSS cases (Figure 7). This includes 81 newly confirmed cases in
June 2005 (Figure 8). Fifteen cases of scrapie in goats have been reported
since 1990 (Figure 9). The last goat case was reported in May 2005.


snip...end


http://www.aphis.usda.gov/vs/nahps/scrapie/monthly_report/monthly-report.html


From: TSS ()
Subject: SCRAPIE USA UPDATE MARCH - JUNE 2005
Date: August 24, 2005 at 7:03 pm PST

SCRAPIE USA MONTHLY REPORT 2005

AS of March 31, 2005, there were 70 scrapie infected source flocks (Figure
3). There were 11 new infected and source flocks reported in March (Figure
4) with a total of 51 flocks reported for FY 2005 (Figure 5). The total
infected and source flocks that have been released in FY 2005 are 39 (Figure
6), with 1 flock released in March. The ratio of infected and source flocks
released to newly infected and source flocks for FY 2005 = 0.76 : 1. IN
addition, as of March 31, 2005, 225 scrapie cases have been confirmed and
reported by the National Veterinary Services Laboratories (NVSL), of which
53 were RSSS cases (Figure 7). This includes 57 newly confirmed cases in
March 2005 (Figure 8). Fourteen cases of scrapie in goats have been reported
since 1990 (Figure 9). The last goat cases was reported in January 2005. New
infected flocks, source flocks, and flocks released or put on clean-up plans
for FY 2005 are depicted in Figure 10. ...

FULL TEXT ;

http://www.aphis.usda.gov/vs/nahps/scrapie/monthly_report/monthly-report.html


Published online before print October 20, 2005

Proc. Natl. Acad. Sci. USA, 10.1073/pnas.0502296102
Medical Sciences

A newly identified type of scrapie agent can naturally infect sheep with resistant PrP genotypes

( sheep prion | transgenic mice )

Annick Le Dur *, Vincent Béringue *, Olivier Andréoletti , Fabienne Reine *, Thanh Lan Laď *, Thierry Baron , Bjřrn Bratberg ¶, Jean-Luc Vilotte ||, Pierre Sarradin **, Sylvie L. Benestad ¶, and Hubert Laude *
*Virologie Immunologie Moléculaires and ||Génétique Biochimique et Cytogénétique, Institut National de la Recherche Agronomique, 78350 Jouy-en-Josas, France; Unité Mixte de Recherche, Institut National de la Recherche Agronomique-Ecole Nationale Vétérinaire de Toulouse, Interactions Hôte Agent Pathogčne, 31066 Toulouse, France; Agence Française de Sécurité Sanitaire des Aliments, Unité Agents Transmissibles Non Conventionnels, 69364 Lyon, France; **Pathologie Infectieuse et Immunologie, Institut National de la Recherche Agronomique, 37380 Nouzilly, France; and ¶Department of Pathology, National Veterinary Institute, 0033 Oslo, Norway


Edited by Stanley B. Prusiner, University of California, San Francisco, CA, and approved September 12, 2005 (received for review March 21, 2005)

Scrapie in small ruminants belongs to transmissible spongiform encephalopathies (TSEs), or prion diseases, a family of fatal neurodegenerative disorders that affect humans and animals and can transmit within and between species by ingestion or inoculation. Conversion of the host-encoded prion protein (PrP), normal cellular PrP (PrPc), into a misfolded form, abnormal PrP (PrPSc), plays a key role in TSE transmission and pathogenesis. The intensified surveillance of scrapie in the European Union, together with the improvement of PrPSc detection techniques, has led to the discovery of a growing number of so-called atypical scrapie cases. These include clinical Nor98 cases first identified in Norwegian sheep on the basis of unusual pathological and PrPSc molecular features and "cases" that produced discordant responses in the rapid tests currently applied to the large-scale random screening of slaughtered or fallen animals. Worryingly, a substantial proportion of such cases involved sheep with PrP genotypes known until now to confer natural resistance to conventional scrapie. Here we report that both Nor98 and discordant cases, including three sheep homozygous for the resistant PrPARR allele (A136R154R171), efficiently transmitted the disease to transgenic mice expressing ovine PrP, and that they shared unique biological and biochemical features upon propagation in mice. These observations support the view that a truly infectious TSE agent, unrecognized until recently, infects sheep and goat flocks and may have important implications in terms of scrapie control and public health.

--------------------------------------------------------------------------------

Author contributions: H.L. designed research; A.L.D., V.B., O.A., F.R., T.L.L., J.-L.V., and H.L. performed research; T.B., B.B., P.S., and S.L.B. contributed new reagents/analytic tools; V.B., O.A., and H.L. analyzed data; and H.L. wrote the paper.

A.L.D. and V.B. contributed equally to this work.

To whom correspondence should be addressed.

Hubert Laude, E-mail: laude@jouy.inra.fr

www.pnas.org/cgi/doi/10.1073/pnas.0502296102


http://www.pnas.org/cgi/content/abstract/0502296102v1

From: TSS ()
Subject: Interspecies Transmission of Chronic Wasting Disease Prions to Squirrel Monkeys (Saimiri sciureus)
Date: October 19, 2005 at 8:33 am PST

0022-538X/05/$08.00+0 doi:10.1128/JVI.79.21.13794-13796.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Interspecies Transmission of Chronic Wasting Disease Prions to Squirrel Monkeys (Saimiri sciureus)
Richard F. Marsh,1, Anthony E. Kincaid,2 Richard A. Bessen,3 and Jason C. Bartz4*
Department of Animal Health and Biomedical Sciences, University of Wisconsin, Madison 53706,1 Department of Physical Therapy,2 Department of Medical Microbiology and Immunology, Creighton University, Omaha, Nebraska 68178,4 Department of Veterinary Molecular Biology, Montana State University, Bozeman, Montana 597183

Received 3 May 2005/ Accepted 10 August 2005

Chronic wasting disease (CWD) is an emerging prion disease of deer and elk. The risk of CWD transmission to humans following exposure to CWD-infected tissues is unknown. To assess the susceptibility of nonhuman primates to CWD, two squirrel monkeys were inoculated with brain tissue from a CWD-infected mule deer. The CWD-inoculated squirrel monkeys developed a progressive neurodegenerative disease and were euthanized at 31 and 34 months postinfection. Brain tissue from the CWD-infected squirrel monkeys contained the abnormal isoform of the prion protein, PrP-res, and displayed spongiform degeneration. This is the first reported transmission of CWD to primates.


--------------------------------------------------------------------------------

* Corresponding author. Mailing address: Department of Medical Microbiology and Immunology, Creighton University, 2500 California Plaza, Omaha, NE 68178. Phone: (402) 280-1811. Fax: (402) 280-1875. E-mail: jbartz@creighton.edu .

Deceased.


--------------------------------------------------------------------------------

Journal of Virology, November 2005, p. 13794-13796, Vol. 79, No. 21
0022-538X/05/$08.00+0 doi:10.1128/JVI.79.21.13794-13796.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.


http://jvi.asm.org/cgi/content/abstract/79/21/13794?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&fulltext=cwd&searchid=1129736446553_4280&stored_search=&FIRSTINDEX=0&volume=79&issue=21&journalcode=jvi


Research Project: Transmission, Differentiation, and Pathobiology of Transmissible Spongiform Encephalopathies
Location: Virus and Prion Diseases of Livestock

Title: Experimental Second Passage of Chronic Wasting Disease (Cwd-Mule Deer) Agent to Cattle


Authors

Hamir, Amirali
Kunkle, Robert - bob
Miller, Janice - ARS RETIRED
Greenlee, Justin
Richt, Juergen


Submitted to: Journal Of Comparative Pathology
Publication Acceptance Date: July 25, 2005
Publication Date: N/A


Interpretive Summary: To compare the findings of experimental first and second passage of chronic wasting disease (CWD) in cattle, 6 calves were inoculated into the brain with CWD-mule deer agent previously (first) passaged in cattle. Two other uninoculated calves served as controls. Beginning 10-12 months post inoculation (PI), all inoculates lost appetite and weight. Five animals subsequently developed clinical signs of central nervous system (CNS) abnormality. By 16.5 months PI, all cattle had been euthanized because of poor prognosis. None of the animals showed microscopic lesions of spongiform encephalopathy (SE) but the CWD agent was detected in their CNS tissues by 2 laboratory techniques (IHC and WB). These findings demonstrate that inoculated cattle amplify CWD agent but also develop clinical CNS signs without manifestation of microscopic lesions of SE. This situation has also been shown to occur following inoculation of cattle with another TSE agent, namely, sheep scrapie. The current study confirms previous work that indicates that the diagnostic tests currently used for confirmation of bovine spongiform encephalopathy (BSE) in the U.S. would detect CWD in cattle, should it occur naturally. Furthermore, it raises the possibility of distinguishing CWD from BSE in cattle due to the absence of microscopic lesions and a unique multifocal distribution of PrPres, as demonstrated by IHC, which in this study, appears to be more sensitive than the WB.
Technical Abstract: To compare clinicopathological findings of first and second passage of chronic wasting disease (CWD) in cattle, a group of calves (n=6) were intracerebrally inoculated with CWD-mule deer agent previously (first) passaged in cattle. Two other uninoculated calves served as controls. Beginning 10-12 months post inoculation (PI), all inoculates lost appetite and lost weight. Five animals subsequently developed clinical signs of central nervous system (CNS) abnormality. By 16.5 months PI, all cattle had been euthanized because of poor prognosis. None of the animals showed microscopic lesions of spongiform encephalopathy (SE) but PrPres was detected in their CNS tissues by immunohistochemistry (IHC) and Western blot (WB) techniques. These findings demonstrate that intracerebrally inoculated cattle not only amplify CWD PrPres but also develop clinical CNS signs without manifestation of morphologic lesions of SE. This situation has also been shown to occur following inoculation of cattle with another TSE agent, scrapie. The current study confirms previous work that indicates the diagnostic techniques currently used for confirmation of bovine spongiform encephalopathy (BSE) in the U.S. would detect CWD in cattle, should it occur naturally. Furthermore, it raises the possibility of distinguishing CWD from BSE in cattle due to the absence of neuropathologic lesions and a unique multifocal distribution of PrPres, as demonstrated by IHC, which in this study, appears to be more sensitive than the WB.

http://www.ars.usda.gov/research/publications/publications.htm?seq_no_115=178318


TSS
Coexistence of multiple PrPSc types in individuals with CJD
Fri Nov 18, 2005 10:05
70.110.89.13

Lancet Neurology 2005; 4:805-814

DOI:10.1016/S1474-4422(05)70225-8

Coexistence of multiple PrPSc types in individuals with Creutzfeldt-Jakob disease

Magdalini Polymenidou a, Katharina Stoeck a, Markus Glatzel a b, Martin Vey c, Anne Bellon c and Adriano Aguzzi a

Summary
Background
The molecular typing of sporadic Creutzfeldt-Jakob disease (CJD) is based on the size and glycoform ratio of protease-resistant prion protein (PrPSc), and on PRNP haplotype. On digestion with proteinase K, type 1 and type 2 PrPSc display unglycosylated core fragments of 21 kDa and 19 kDa, resulting from cleavage around amino acids 82 and 97, respectively.

Methods
We generated anti-PrP monoclonal antibodies to epitopes immediately preceding the differential proteinase K cleavage sites. These antibodies, which were designated POM2 and POM12, recognise type 1, but not type 2, PrPSc.

Findings
We studied 114 brain samples from 70 patients with sporadic CJD and three patients with variant CJD. Every patient classified as CJD type 2, and all variant CJD patients, showed POM2/POM12 reactivity in the cerebellum and other PrPSc-rich brain areas, with a typical PrPSc type 1 migration pattern.

Interpretation
The regular coexistence of multiple PrPSc types in patients with CJD casts doubts on the validity of electrophoretic PrPSc mobilities as surrogates for prion strains, and questions the rational basis of current CJD classifications.

Affiliations

a Institute of Neuropathology, University Hospital Zurich, Switzerland
b Present address: Institute of Neuropathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
c ZLB Behring, Marburg, Germany

Correspondence to: Dr Adriano Aguzzi, Institute of Neuropathology, University Hospital of Zürich, Schmelzbergstrasse 12, CH-8091 Zürich, Switzerland

http://www.thelancet.com/journals/laneur/article/PIIS1474442205702258/abstract


Lancet Neurology 2005; 4:805-814

DOI:10.1016/S1474-4422(05)70225-8

Coexistence of multiple PrPSc types in individuals with Creutzfeldt-Jakob disease

Magdalini Polymenidou a, Katharina Stoeck a, Markus Glatzel a b, Martin Vey c, Anne Bellon c and Adriano Aguzzi a


see full text here ;


Articles

Introduction

Prion diseases are invariably fatal neurodegenerative

disorders of infectious, sporadic, or genetic origin, that

affect human beings and many species of animals.

Biochemically, these diseases are characterised by the

accumulation of a pathological protein, called the scrapie

prion protein (PrPSc). This protein is a conformational

isoform of a cellular protein, PrPC, and is thought to be

the infectious agent. Conversion of PrPC into its

pathological isoform involves a structural modification

that results in an increase in -sheet content,1–3

aggregation,4,5 and partial resistance to proteolytic

cleavage.6 Incubation of a prion-infected brain

homogenate with proteinase K (PK) under defined

conditions results in complete degradation of PrPC, but

not PrPSc. PrPSc loses approximately 65 N-terminal amino

acids, but maintains its resistant core, often referred to

as PrP27–30 which indicates the altered molecular weight

range of the partially digested molecule.6,7 This truncated

molecule of PrPSc serves as a well-established marker of

prion infection.

Prion strains are defined as infectious prion isolates

that show distinct disease phenotypes, such as

incubation times and histopathological lesion profiles,

which persist on serial transmission.8–10 The existence of

prion strains was originally taken as evidence against the

protein-only hypothesis, since it was difficult to imagine

how the distinct information of each strain could be

captured in a protein-only infectious agent, without any

contribution by nucleic acids. Experiments with two

strains of transmissible mink encephalopathy suggested

that the prion diversity could indeed be conferred by a

single protein, and that the information could be

enciphered within distinct protein structures.11,12

Although distinct prion strains can only be identified

by bioassays with confirmatory transmission of the

strain characteristics to new hosts, variations in the

banding pattern of PK-digested PrPSc can serve as a

biochemical prion strain indicator.13 Differences in the

level of glycosylation, as well as in the size of

the PK-digested PrPSc, are widely used as surrogates of

prion strain typing (eg, to distinguish sporadic from

variant Creutzfeldt-Jakob disease [CJD]).14 However, the

molecular basis for these differences and their relation

to disease characteristics remain unknown. The

differences in the size of PK-digested PrPSc molecules

are thought to result from strain-specific conformational

states that, in turn, lead to exposure of distinct cleavage

sites for the enzyme.11

The latter phenomenon is thought to produce the

observed variation in PrPSc from CJD patients. These

biochemically distinguishable types of sporadic CJD

(sCJD) are believed to represent distinct human prion

strains, although they formally do not qualify as such,

unless serial passage in experimental animals is done

and persistence of strain characteristics in new hosts is

shown. Since transmission is not practical for each case,

the biochemical appearance of PrPSc serves as a

surrogate strain-typing marker for CJD, which allows

molecular classification of CJD types. In addition, the

host genotype at the polymorphic codon 129 (which can

be either methionine [M] or valine [V]) of the prion

protein gene PRNP influences the course of disease and

the CJD type.15–18 However, investigators deviate in their

nomenclature, and to date at least two different CJD

classifications have been proposed19,20 (figure 1). In the

classification proposed by Gambetti and colleagues,20

two distinct PrPSc were identified after PK digestion: one

Published online

October 31, 2005

DOI:10.1016/S1474-4422(05)

70225-8

Institute of Neuropathology,

University Hospital Zurich,

Switzerland

(M Polymenidou PhD,

K Stoeck MD, M Glatzel MD,

A Aguzzi PhD); present address:

Institute of Neuropathology,

University Medical Center

Hamburg-Eppendorf,

Hamburg, Germany

(M Glatzel MD), and ZLB

Behring, Marburg, Germany

(M Vey PhD, A Bellon PhD)

Correspondence to:

Dr Adriano Aguzzi, Institute of

Neuropathology, University

Hospital of Zürich,

Schmelzbergstrasse 12,

CH-8091 Zürich, Switzerland

adriano@pathol.unizh.ch

http://neurology.thelancet.com Published online October 31, 2005 DOI:10.1016/S1474-4422(05)70225-8 1

Coexistence of multiple PrPSc types in individuals with

Creutzfeldt-Jakob disease

Magdalini Polymenidou, Katharina Stoeck, Markus Glatzel, Martin Vey, Anne Bellon, and Adriano Aguzzi

Summary

Background The molecular typing of sporadic Creutzfeldt-Jakob disease (CJD) is based on the size and glycoform

ratio of protease-resistant prion protein (PrPSc), and on PRNP haplotype. On digestion with proteinase K, type 1 and

type 2 PrPSc display unglycosylated core fragments of 21 kDa and 19 kDa, resulting from cleavage around amino

acids 82 and 97, respectively.

Methods We generated anti-PrP monoclonal antibodies to epitopes immediately preceding the differential proteinase

K cleavage sites. These antibodies, which were designated POM2 and POM12, recognise type 1, but not type 2, PrPSc.

Findings We studied 114 brain samples from 70 patients with sporadic CJD and three patients with variant CJD.

Every patient classified as CJD type 2, and all variant CJD patients, showed POM2/POM12 reactivity in the

cerebellum and other PrPSc-rich brain areas, with a typical PrPSc type 1 migration pattern.

Interpretation The regular coexistence of multiple PrPSc types in patients with CJD casts doubts on the validity of

electrophoretic PrPSc mobilities as surrogates for prion strains, and questions the rational basis of current CJD

classifications.

Articles

with an unglycosylated fragment migrating at 21 kDa,

named PrPSc type 1, and the second, with unglycosylated

fragment of 19 kDa, named PrPSc type 2. Protein

sequencing revealed that PrPSc type 1 results from PK

cleavage at position 82, whereas PrPSc type 2 is generated

by cleavage at amino acid 97.7 Hill and colleagues19

subclassified PrPSc type 1 into two subgroups with core

fragment sizes differing by less than 1 kDa. These cases

were classified as CJD type 1 and CJD type 2 (with

slightly lower molecular weight). Moreover, another

type, observed in only one case, was reported, with a

molecular weight slightly higher than type 1 (CJD

type 6).19 PrPSc type 2 in Gambetti’s classification

represents CJD type 3 according to Hill and colleagues,19

who have distinguished two further subtypes within

this group. Both are detectable with unglycosylated

fragments at 19 kDa, which intensity in comparison with

the monoglycosylated band is either equally strong (in

MV patients) or weaker (in subtype VV or MM

individuals). Herein, we will refer to CJD type 1 and 2 as

defined by the Gambetti classification,20 unless indicated

otherwise.

The controversy is possibly due to very small

differences in PK-digested unglycosylated PrPSc

fragments and to deviations of their apparent molecular

weight due to diverse gel and western blotting systems.

In addition to the main PK digestion sites at positions 82

and 97, several others have been identified, possibly

accounting for the disparity in the classification of PrPSc

type 1 cases.7 Dependence of PK digestion site on the

concentration of metal ions,21 or on the pH of brain

homogenates,22,23 may also contribute to the

discrepancies. The recent identification of CJD cases

with apparent co-occurrence of multiple PrPSc types

indicates that CJD types can combine to increase disease

diversity.24–26

Some studies have suggested that monoclonal

antibodies with epitopes directed against the sequence

of PrP that is differentially cleaved by PK in different

strains can be used for strain typing. For example, an

octarepeat-specific antibody was shown to discriminate

sheep scrapie from bovine spongiform encephalopathy

(BSE).27,28 Here, we have produced and characterised by

epitope mapping several anti-PrP monoclonal antibodies

with specificities that span the entire prion protein. Two

of these, named POM2 and POM12, were found to

detect repetitive epitopes located exactly at the boundary

that is differentially cleaved by PK on PrPSc types 1 and 2.

POM2 and POM12 specifically recognise PrPSc species

that are cleaved at any site N-proximal of amino acid 82,

but not after this position. Comparative western blot

analysis of brain samples from 70 patients with sCJD

and three with variant CJD (vCJD) showed that all

patients classified as CJD type 2 or vCJD, on the basis of

conventional strain typing from cortical samples,

showed POM2/POM12 reactivity and PrPSc type 1

migration pattern in cerebellum and other PrPSc-rich

brain areas.

Methods

Generation of monoclonal antibodies

Prnpo/o mice were immunised with recombinant mouse

PrP23–230 (rmPrP23–230), produced and purified as described

previously.29 For the initial injection, 10 µg of protein

were emulsified in complete Freund’s adjuvant.

Boosting injections were done over a period of 65 days

according to the following schedule: subcutaneous

injection of 10 µg of mPrP23–230 plus complete Freund’s

adjuvant on day 1; subcutaneous injection of 10 µg of

mPrP23–230 on day 22, and of 20 µg of mPrP23–230 on day 43;

intravenous injection of 10 µg of mPrP23–230on day 64, on

day 65, and on day 66. At day 66, the mice were

sacrificed, and splenocytes were fused to the myeloma

cell line Sp2/0-Ag14 with polyethylene glycol by use of a

standard protocol. Fused cells were selected with

hypoxanthine, aminopterin, and thymidine medium.

Clones consisting of small numbers of cells were

transferred into fresh wells and cultured. Based on a first

screen of 55 positive clones, 19 clones were selected for

further characterisation. The clones are hereafter termed

POM1 to POM19.

Antibody purification and labelling

Hybridoma cells were cultured to a log-phase state.

During early scale-up, standard fetal calf serum was

gradually replaced with ultralow-IgG fetal calf serum

while cells were closely monitored for proliferation and

viability. Cells were transferred into production flasks at

high density and kept in static culture. Supernatant was

harvested, centrifuged, and filtered. Antibodies were

purified by affinity chromatography on protein G

columns, eluted with glycine buffer (pH 3·0), and

dialysed against phosphate-buffered saline (PBS;

2 http://neurology.thelancet.com Published online October 31, 2005 DOI:10.1016/S1474-4422(05)70225-8

1 2 3 4 1 2 2b

MM

CJD type

Codon 129 MM MM MV

or

VV

MM,

MV

or

VV

MM,

MV

or

VV

MM,

MV

or

VV

A B

Figure 1: Classifications of Creutzfeldt-Jakob disease (CJD) .

(A) In the classification proposed by Hill and colleagues, 19 three distinct banding

patterns are identified for sporadic CJD, designated CJD types 1 to 3. Type 4

represents variant CJD, with a diglycosylated band. Polymorphisms at codon 129

of PRNP are indicated under each type. (B) According to Gambetti’s

classification, 20 two main PrPSc types are identified: type 1, with an apparent

molecular weight of approximately 21 kDa, and type 2, of approximately

19 kDa. Type 2b is associated to variant CJD . Its electrophoretic migration

pattern is similar to type 2, despite their distinct glycosylation patterns. Grey

boxes represent unglycosylated fragments.

Articles

pH 7·2–7·4). Purity was assessed by sodium dodecyl

sulphate polyacrylamide gel electrophoresis (SDS-PAGE),

and protein concentration was determined by the Lowry

assay. 1 mg of each purified POM1 and POM2 antibodies

was used for labelling with cyanine 2 (Cy2) and cyanine 3

(Cy3), respectively. Conjugation of dyes to antibodies was

done using FluoroLink-Ab labelling kits (Amersham

Biosciences, Little Chalfont, Buckinghamshire, UK),

according to the manufacturer’s instructions.

Patients

All CJD cases included in this study were derived from

an unselected series of patients with clinically,

genetically, and neuropathologically proven sCJD, with

the exception of vCJD samples, which were a kind gift of

Dr J W Ironside (National CJD Surveillance Unit,

University of Edinburgh, Edinburgh, UK). For sCJD,

tissue specimens were retrieved from the tissue bank of

the Swiss National Reference Centre of Prion Diseases

(Zurich, Switzerland). For all tissue specimens, consent

to examine necropsy material had been obtained from

each patient or legal guardian. Clinical data and PRNP

haplotypes were available for all cases. Frozen brain

tissue was stored at –80şC, and samples were taken from

the following brain regions: frontal, parietal, occipital,

and temporal cortices, putamen, thalamus, midbrain,

medulla oblongata, and cerebellum. A neuropathologically

proven sCJD case (codon 129 MM; PrPSc type 2,

thalamus region) was chosen as the positive control, and

a randomly selected necropsied brain from a nondemented

patient (age 79 years, female) and the brain

from a patient with Alzheimer’s disease were used as

negative controls.

Sample preparation and western blotting

Brain homogenates of CJD or control patients were

prepared in PBS, 0·5% (w/v) sodium deoxycholate, and

0·5% (v/v) Nonidet P-40. Total protein concentration

was estimated using a standard colorimetric method

based on bicinchoninic acid (Pierce Biotechnology,

Rockford, IL, USA). 100 µg of protein were incubated

with PK (25 µg/mL, 20 units/mg; Sigma-Aldrich,

St Louis, MO, USA) at 37şC for 30 min. The digestion

was stopped by adding 3 mM phenylmethyl-sulphonyl-

fluoride. For the PrPSc spiking experiment,

homogenates of solely PrPSc type 1 and PrPSc type 2

brain samples were mixed in percentage ratios of 50:50,

60:40, 70:30, 80:20, and 90:10, according to their

protein content, and digested with PK. For

deglycosylation, 50 µg of PK-digested protein was

precipitated with trichloroacetic acid (Sigma-Aldrich),

using a standard protein precipitation protocol.

Proteins were then boiled in denaturation buffer,

according to the manufacturer’s protocol (New

England Biolabs, Ipswich, MA, USA), and incubated

with 5000 units of N-Glycosidase F (PNGaseF; New

England Biolabs) for 2 h at 37şC. Bacterially expressed

human prion proteins (a kind gift from Dr K Wuthrich,

Institute for Molecular Biology and Biophysics, Swiss

Federal Institute of Technology, Zurich, Switzerland)

were produced and purified as described previously.29,30

40 µg of total protein (digested or not with PK), or

bacterially expressed human prion protein (100 ng),

were mixed with loading buffer (NuPAGE 4 lithium

dodecyl sulphate sample buffer; Invitrogen, Carlsbad,

CA, USA) and run on 12% NuPAGE gels (MES

running buffer; Invitrogen), transferred onto

nitrocellulose membranes, incubated first with

hybridoma cell supernatants or purified monoclonal

anti-PrP antibodies, and then with horseradish

peroxidase (HRP)-conjugated rabbit anti-mouse IgG

(gamma) antibody (Zymed, Invitrogen). Blots were

developed by using HRP substrate (enhanced

chemiluminescent substrate; Pierce), and signals were

acquired with photosensitive film (Kodak, Rochester,

NT, USA) or with a Versadoc 3000 imaging system

(Bio-Rad, Hercules, CA, USA). Alternatively,

membranes were incubated with fluorescentlyconjugated

antibodies (POM1-Cy2 and POM2-Cy3, as

described above) for 3 h at room temperature, or

overnight at 4şC. In such cases, fluorescence was

scanned with a gel and blot imager (Typhoon 9400;

Amersham Biosciences).

Densitometric and statistical analysis

Densitometric analysis used for the triplot representation

was done with a one-dimensional software

analysis program (Quantity One; Bio-Rad). We used

SPSS version 11 for Macintosh (SPSS Inc, Chicago, IL,

USA) for statistical analysis. To compare type 1 and

type 2 sample groups, we used paired t test for each

glycoform, detected with either non-discriminatory

3F4/POM1 or the type 1-specific POM2/POM12.

Screening and isotyping of clones by ELISA against

recombinant mPrP (rmPrP)

Plates were coated overnight at 4şC with 5 µg/mL of

rmPrP23–230 or rmPrP121-230 in PBS and washed with PBS

containing 0·1% (v/v) Tween-20, blocked with 5%

(w/v) bovine serum albumin (BSA), and incubated for

2 h at room temperature with 30 µL of two-fold serially

diluted hybridoma cell supernatant (1:10 prediluted) in

PBS-Tween containing 1–5% BSA. Plates were

washed with PBS-Tween and probed with a HRPconjugated

anti-mouse antibody (total IgG, or isotypespecific

antibodies IgM, IgG1, IgG2a, IgG2b, and IgG3,

all from Zymed, used at 1:1000 dilution in

PBS-Tween-BSA). Plates were developed with 2,2-

azinodiethyl-benzothiazoline-sulphonate, and optical

density was measured at 405 nm. Titres were defined

as the highest dilution showing an optical density more

than twice the background, which was calculated as the

average density of uncoated wells and wells incubated

without serum.

http://neurology.thelancet.com Published online October 31, 2005 DOI:10.1016/S1474-4422(05)70225-8 3

Articles

Epitope mapping by peptide competition

A PrP peptide library of 100 dodecamers was synthesised

(JPT Peptide Technologies GmbH, Berlin, Germany).

The first peptide of the library represented amino acids

23–34 of the mature mouse prion protein. All other

peptides sequences were shifted by two amino acids of

PrP sequence (eg, 25–36, 27–38, …, 219–230, and

221–232). 384-well plates were coated with 300 ng/mL of

mouse PrP23–230 overnight at 4şC. Plates were washed with

PBS containing 0·1% Tween-20, and blocked with 5%

BSA for 2 h at room temperature. After washing, plates

were incubated with 30 µL of two-fold serially diluted

hybridoma cell supernatant (prediluted 1:1000–1:5000)

in PBS-Tween containing 1% BSA, with or without

peptides in a final concentration of 0·8–8·0 ng/mL. After

2 h at room temperature, plates were washed and probed

with HRP-conjugated rabbit anti-mouse IgG (1:1000

dilution; Zymed) for 1 h at room temperature. The assay

was developed with 2,2-azinodiethyl-benzothiazolinesulphonate.

Optical density was measured at 405 nm.

Surface plasmon resonance measurements

All experiments were performed on a Biacore 3000

(Biacore International AB, Uppsala, Sweden). For pairwise

antibody mapping, monoclonal antibodies (POMs)

or pooled IgG1 (isotype control) were immobilised on

CM-5 chips (Biacore) after activation with N,N-(3-

dimethylaminopropyl)-N’-ethyl-carbodiimide hydrochloride

and N-hydroxysuccinimide at a flow rate of

5 µL/min. A 5 µL aliquot of a 50 µg/mL antibody

solution in acetate buffer (pH 4·5) was used to

immobilise approximately 2500–5000 response units of

covalently bound antibody. After inactivation with

ethanolamine, the surface was washed twice with 20 µL

of 0·5% (w/v) SDS to remove any non-covalently bound

antibody. The system was primed and a new sensogram

was started with HBS-EP (0.01M Hepes pH 7·4, 0·15M

NaCl, 3mM EDTA, 0·005% (v/v) Surfactant P20;

Biacore) as running buffer. 20 µL of HBS-EP were used

at a constant flow rate (5 µL/min) for all injections.

Proteins (BSA or PrP) or antibodies were injected at a

concentration of 50 µg/mL diluted in HBS-EP buffer.

For chip regeneration, several injections of 0·5% SDS

were done until the baseline returned to zero. For

control, injections were done in flow cells 1 and 2, or 3

and 4, for which the first flow cell was coated with pooled

IgG1 (Zymed), and the second with the relevant

antibody.

Analysis of peptide competition for N-terminal POMs

by surface plasmon resonance

The coating and washing of the flow cells with control

IgG or POM antibodies were performed as described

above. A volume of 20 µL was used for all injections at a

constant flow rate of 5 µL/min. rmPrP121–230 (negative

control) and rmPrP23–230 were diluted in HBS-EP buffer at

a concentration of 10 µg/mL. Injection of POM

antibodies (40 µg/mL in HBS-EP) was done with or

without individual peptides (20 µg) from a library of

100 partially overlapping dodecapeptides, each shifted

by two amino acids and covering the entire mature

mouse PrP sequence. For selected epitopes, mutant

undecameric peptides with scanning single amino acid

deletions were used (Jerini AG, Berlin, Germany).

Role of the funding source

The sponsors of this study had no role in study design,

data collection, data analysis, data interpretation, or

writing of the report. The corresponding author had full

access to all the data in the study and had final

responsibility for the decision to submit it for

publication.

Results

By immunising Prnpo/o mice with bacterially produced

rmPrP, we induced potent immune responses towards

various PrP epitopes. By screening with western blot and

ELISA (data not shown), 19 clones were selected for

further characterisation and named POM1 to POM19.

Immunoglobulin-isotype-specific ELISA showed that

most POM hybridomas secreted IgG1 (table 1). In

ELISA assays against immobilised rmPrP23–230 and

rmPrP121–230, POM2, POM3, POM11, POM12, POM14,

and POM18 were found to bind full-length PrP, but not

N-terminally truncated PrP, indicating that their

epitopes were situated N-proximally of position

121 (table 1). By contrast, POM1, POM4–POM10,

POM13, POM15–POM17, and POM19 reacted with both

rmPrP23–230 and rmPrP121–230, indicating that their epitopes

were situated in the globular PrP domain (amino acids

121–230). For the purposes of this study, we focused on

4 http://neurology.thelancet.com Published online October 31, 2005 DOI:10.1016/S1474-4422(05)70225-8

Isotype Amino-acid position on murine PrP

(epitope sequence)

MAbs against unstructured PrP

POM2 IgG1 58–64, 66–72, 74–80, 82–88 (QPXXGG/SW)

POM3 IgG1 95–100 (HNQWNK)

POM11 IgG1 64–72, 72–80 (GQPHGGSW)

POM12 IgG1 58–64, 66–72, 74–80, 82–88 (QPXGGG/SW)

POM14 IgG1 53–64, 61–72, 69–80, 77–88 (GGTWGQPHGGG/SW)

POM18 IgG1 ..

MAbs against the globular PrP domain

POM1 IgG1 121–230

POM4 IgG1 121–134, 218–221

POM5 IgG1 168–174

POM6 IgG2a 121–230

POM7 IgG1/IgG2a 121–230

POM8 IgG1 121–230

POM9 IgG2a 121–230

POM10 IgG1 121–134, 218–221

POM13 IgG1 121–230

POM15 IgG1 121–230

POM16 IgG2b 121–230

POM17 IgG1 121–230

POM19 IgG1 121–134, 218–221

MAbs=monoclonal antibodies; ..=not done.

Table 1: Isotypes and specificity of novel anti-PrP antibodies

Figure 2: Discrimination of PrPSc from different Creutzfeldt-Jakob disease (CJD) types by POM2 and POM12

(A) Representative replica blots containing proteinase-K digested CJD samples, classified as type 1 or 2, probed with five different monoclonal antibodies (as indicated

in the right side of each panel). POM2 and POM12 recognise the samples classified as CJD type 1 only, whereas POM1 and 3F4 detect both. POM7 exclusively detects

the unglycosylated fragments. The red star indicates a type 2 cortical sample with trace amounts of type 1 probed with both POM2 and POM12. (B) Diagram

representing the molecular basis of POM2 and POM12 specificity towards PK-digested PrPSc type 1 CJD. Their epitopes are cleaved by PK on CJD type 2 samples. The

dashed and black boxes represent -helices and -sheets, respectively. The round markers represent the glycan chains. Numbers correspond to the amino acid

sequence of human PrP. (C) Scheme showing the localisation of POM2 and POM12 epitopes with regard to predicted PK cleavage sites on human PrP. Any PrPSc

digested at position 82 or upstream will be detected by POM2 and POM12; digestion at any residue downstream of this position will disrupt the POM2/12 epitope.

The red filled boxes indicate the minimum sequence required for POM2 and POM12 binding on the human PrP sequence. The blue box indicates the 3FA epitope.

(D) Analysis of the antibodies specificity by dual-colour western blot of two different variants of rhPrP. The two arrows indicate PrP degradation products including

POM1 epitope but not POM2 (upper arrow), or POM2 epitope but not POM1 (lower arrow). The low intensity band running approximately at 25 kDa in the hPrP96–230

preparation detected by POM1 only, represents a PrP dimer, which is often detected in non-reducing conditions .

the C-proximal POM1 and POM7 epitopes, and the

N-proximal POM2 and POM12.

The epitopes of POM1 and POM7 were defined by

surface plasmon resonance experiments, which showed

that they both competed for binding with monoclonal

antibody 6H4 (Prionics, Schlieren, Switzerland). This

indicates that their epitopes are located on alpha helix 1

of the structured domain of PrP (data not shown). The

POM7 epitope is sterically hindered by the PrP sugar

chains. Hence POM7 binds preferentially to the

unglycosylated PrP subpopulation. The epitopes

recognised by POM2 and POM12 were characterised

using a library of 100 synthetic dodecapeptides, each

shifted by two amino acids, which spanned the entire

PrP sequence (webappendix, webfigures 1 and 2). Our

analysis shows that POM2 and POM12 recognise similar

but not identical epitopes. The POM12 epitope is

QPXGGGW, whereas the POM2 epitope is QPXXGGW.

By western blotting, we explored the reactivity of POM

hybridomas to human brain samples from patients with

CJD and controls. All patients were classified according

to cortical PrPSc electrophoretic migration using

Articles

monoclonal antibody 3F4 and codon 129 polymorphism.

20 Replica western blots were probed with

five different antibodies: 3F4 or POM1, which bind

indistinguishably to all PrPSc types; POM7, which only

interacts with the unglycosylated PrP band; octarepeatspecific

POM2; and POM12. POM1 and 3F4 showed the

typical migration pattern of CJD type 1 and 2 (Gambetti

classification), or type 1, type 2 (MV2 and VV2), and

type 3 (MM2 and MV2), according to Hill and

colleagues’ classification (figure 2).19 In addition, POM1

recognised a shorter PK-resistant PrPSc C-terminal

fragment, running at about 16–17 kDa, as described

previously for other C-terminal specific antibodies.31

When PK-digested PrPSc from CJD samples were

stained with POM2 and POM12, only PrPSc type 1

samples were detected, whereas replica blots incubated

with 3F4 or POM1 identified all four samples with

typical migration patterns of the respective types

(figure 2). The POM2 and POM12 epitopes are

preserved by PK cleavage at position 82 or upstream,

but destroyed by digestion at any residue downstream

of this position. In the human PrP sequence, the POM2

and POM12 epitopes of CJD type 2 samples were

destroyed by PK (figure 2). Full-length and truncated

recombinant human PrP (rhPrP23–230 and rhPrP96–230)

were co-incubated with Cy2-labelled POM1 and Cy3-

labelled POM2. Two-colour fluorescence detection

confirmed that POM2 detects epitopes upstream of

position 96, whereas POM1 recognizes both PrP

variants (figure 2).

We then screened brain samples from 70 sCJD

patients (41 subtype MM1, six MV1, three MM2,

11 MV2, and nine VV2) and three vCJD patients (all

subtype MM), whose PrPSc had been previously typed by

3F4 western blot at necropsy. As expected, POM2 and

POM12 recognised PK-digested PrPSc from all CJD

type 1 samples. However, in 11 of 23 type 2 cortical

samples, low-intensity POM2 and POM12 reactivity was

observed. The latter signal always showed a typical type 1

migration pattern, indicating that low amounts of PrPSc

type 1 coexist in the cortex of approximately 50% of the

CJD type 2 patients (figure 2, table 2).

Samples from the cerebellum and other PrPSc-rich

areas (as determined by Schoch and colleagues32) were

then probed with POM2/POM12, 3F4, and POM1.

Surprisingly, each of the patients classified as type 2

showed obvious POM2/POM12 reactivity with PrPSc

type 1 electrophoretic migration pattern, either in

cerebellum or in other brain areas (figure 3, table 3).

Although all type 2 cerebellar samples would have been

identified as such based on the 3F4 or POM1 blots,

when probed with POM2 they appeared positive with a

PrPSc type 1 migration pattern. Moreover, brain

samples from frontal cortex of the three vCJD patients

were also found to be positive for POM2 on western

blot with a type 1-like PrPSc electrophoretic migration

(figure 3). Case 59 was atypical, and was diagnosed as

type 2 according to 3F4 western blot analysis of a

cortical sample. However, cerebellar sample analyses

with POM1, 3F4, POM2, and POM12 identified a

typical type 1 pattern without any obvious type 2

fragments.

PrPSc glycoform ratios have been reported to diverge in

various CJD types.19 We therefore analysed PrPSc

glycoform ratios of all cerebellar type 2 samples,

according to 3F4/POM1 western blot and compared

them to replica blots incubated with POM2/POM12.

Moreover, we selected 10 type 1 cortical samples and did

the same type of analysis, using either 3F4/POM1 or

POM2/12 (webfigure 3). Statistical analysis of PrPSc

glycoform ratios detected by 3F4/POM1 and

6 http://neurology.thelancet.com Published online October 31, 2005 DOI:10.1016/S1474-4422(05)70225-8

3F4/POM1 POM2/POM12

(positive/total) (positive/total)

CJD type 1

MM1 41/41 41/41

MV1 6/6 6/6

CJD type 2

MM2 3/3 2/3

MV2 11/11 5/11

VV2 9/9 4/9

Samples typed according to the Gambetti classification.20

Table 2: Summary of cortical samples by Creutzfeldt-Jakob disease

(CJD) subtype screened with POM1/3F4 and POM2/POM12

monoclonal antibodies

MM1 VV2 MM1

 PK

Codon 129

37 kDa–

25 kDa–

20 kDa–

15 kDa–

PK

Codon 129

37 kDa–

25 kDa–

20 kDa–

15 kDa–

3F4 POM1 POM2

POM1 POM2

                   

VV VV MV MV MM1 VV2 MM1 VV VV MV MV MM1 VV2 MM1 VV VV MV MV

Cortex Type 2/cerebellum Cortex Type 2/cerebellum Cortex Type 2/cerebellum



MM MM MM MM MV MV VV VV

      

Cortex

Type 1 Type 2 vCJD

Cortex Cerebellum



MM MM MM MM MV MV VV VV

      

Cortex

Type 1 Type 2 vCJD

Cortex Cerebellum

A

B

Figure 3: Co-existence of Creutzfeldt-Jakob disease (CJD) types

(A) Three replica blots containing conventionally classified CJD type 2 samples (as indicated) blotted with either 3F4

and POM1, which recognise all PrPSc species, or POM2, which only binds to proteinase K (PK)-digested type 1 PrPSc.

POM1 also bound to some C-terminal PrP fragments in the non-PK-digested samples that did not react with either

3F4 or POM2. When probed with POM2, a type 1 PrPSc pattern is detected. (B) The same phenomenon is shown in

cortical samples of three vCJD patients; when probed with POM2, they show type 1 PrPSc migration pattern.

Articles

POM2/POM12 in the same group of samples did not

show any significant variations within the analysed

groups (webtable).

We investigated the concentration of PK to find the

best conditions for our analysis. It has been proposed

that a PK concentration of 100 µg/mL should be used

for the analysis of CJD brain samples.22 However, for

optimum evaluation, the PK concentration for reliable

prion disease diagnosis should be the minimum

concentration that will completely digest PrPC. Any

concentration higher than this will potentially decrease

the sensitivity of the assay and, in the worst-case

scenario, may lead to false negatives due to excessive

digestion of PrPSc. It has been long known than PrPSc is

only partially resistant to PK: it can be degraded by high

PK concentrations or by long incubation times

(figure 4). Any lower concentration is also suboptimum

and may result in false positives due to residual PrPC.

In our study, 25 µg/mL of PK for 30 min at 37şC were

sufficient to degrade PrPC from a healthy control or an

Alzheimer’s disease brain sample (figure 4).

Furthermore, this digestion protocol allows to

positively diagnose CJD from necropsies of patients

with very low PrPSc load. The same patients would have

been falsely diagnosed as negative if a higher PK

concentration (100 µg/mL) had been used. The

differences in signal intensity observed in the blots

were due to the variable PrPSc content in brain samples,

as indicated by probing those samples, before PK

digestion, with control anti-actin antibodies (figure 4).

We also investigated whether differences in pH of the

total homogenate at the time of PK digestion might

interfere with our results, since it has been shown that

pH may affect the size of the PrPSc fragments

generated.22 We found that there was no effect of pH on

the POM2/POM12-positive intrinsic type 1 PrPSc

detected in CJD type 2 brain samples (figure 4).

We then reasoned that analytical limitations intrinsic

to the western blot detection method may have

prevented the detection of intrinsic type 1 PrPSc within

type 2 brain samples in the past. By mixing brain

homogenates that contain solely type 1 or type 2 PrPSc

in various ratios, we determined that western blots

using non-discriminatory antibodies, such as POM1

(or 3F4), consistently fail to detect type 1 PrPSc

whenever it represents less than 40% of total PrPSc

(figure 5). We went on to determine quantitively the

type 1 PrPSc content within a cerebellum sample of a

CJD type 2 patient. To achieve this, we used POM2-

positive PrPSc band-intensity of a bona fide type 1

sample serially diluted within a single type 2 sample in

various ratios to obtain a standard curve of type 1

content (figure 5). We found that PrPSc type 1 in the

CJD type 2 cerebellar sample (VV) was 6·9%, which

was well below detectability of any non-discriminatory

antibody. These results support our notion that,

without a type-specific antibody, the intrinsic type 1

content in every type 2 sample would have been missed

due to low sensitivity.

Deglycosylation allows the detection of different PrPSc

types in the same sample, even with non-discriminatory

antibodies, such as 3F4.24 We investigated whether our

observations using type 1 specific antibodies could have

been discovered by deglycosylation. We found that

despite elimination of alternative glycoforms, POM1

still fails to detect intrinsic type 1 content within the

type 2 sample, whereas it is clearly detected by POM2

(figure 5). Moreover, when the latter blot was reincubated

with biotinylated POM1, the presence of

type 2 PrPSc was revealed. As an alternative to

deglycosylation, we also used POM7 antibody, which is

specific for the unglycosylated PrP band. It also failed to

detect intrinsic type 1 content within the type 2 sample

in these conditions (figure 5).

Discussion

The discovery of heritable polymorphic PK cleavage

sites in PrPSc has been used for the molecular

classification of CJD cases.11,13,14,17 In concert with the

codon 129 PRNP haplotype, the different PrPSc types

correlate with distinct disease phenotypes. Most

patients with the MM1 or MV1 subtype present with

so-called classic CJD, and show rapid progressive

dementia, early myoclonus, visual disturbances

including cortical blindness, and a disease duration of

See Lancet Online

for webfigure 3 and webtable

http://neurology.thelancet.com Published online October 31, 2005 DOI:10.1016/S1474-4422(05)70225-8 7TSS

Frontal cortex Cerebellum Other (PrPSc rich)

3F4/POM1 POM2/POM12 3F4/POM1 POM2/POM12 3F4/POM1 POM2/POM12

CJD type MM2

59      

130   .. ..  

221     .. ..

CJD type MV2

98      

145      

154      

155      

166      

172      

214      

228     .. ..

242     .. ..

247      

248     .. ..

CJD type VV2

78      

111     .. ..

116   .. ..  

129      

137      

142     .. ..

161   .. .. .. ..

197      

233      

=weak; =moderate; =high; =no PrP signal; ..=not done.

Table 3: Antibody reactivity of samples from patients classified as type 2 Creutzfeldt-Jakob disease (CJD) .

Figure 4: Effect of proteinase K (PK) concentration and pH on prion diagnosis

(A) Schematic representation of PrPC and PrPSc degradation curves. The optimum concentration for prion diagnosis is the minimum concentration that suffices to digest PrPSc completely. (B) Western

blot showing that 25 g/mL of PK lead to complete digestion of PrPC from a healthy control (indicated by “C”) or an Alzheimer’s disease (AD) brain sample. (C) Western blots showing that 100 g/mL

of PK treatment can lead to false negatives. Upper panel shows CJD diagnosis of cerebellum samples from MM1 and MV1 patients with low PrPSc load when 25 g/mL of PK were used; middle panel

shows apparent PrPSc negativity when 100 g/mL PK were used; lower panel shows the actin control blot of the same samples before PK digestion. (D) Lack of effect of pH on prion diagnosis. Cortical

or cerebellar samples were digested with 25 g/mL of PK in a pH buffer 7 or 8 (as indicated).

approximately 4 months. By contrast, patients with the

MV2 or VV2 subtype show an atypical disease course,

with a longer disease duration (6–18 months), early

ataxia, predominant extrapyramidal symptoms, and

late-onset dementia.18

Because of the limited resolution of conventional

western blot systems, the coexistence of PK-digested

PrPSc bands at 19 kDa and 21 kDa is not recognisable by

conventional antibodies binding to both PrPSc types,

including POM1. By mixing brain homogenates that

contain solely type 1 or type 2 PrPSc, we have found that

3F4-based or POM1-based western blots consistently

fail to detect type 1 PrPSc whenever it represents less

than 30–40% of total PrPSc. We addressed these

problems by developing a set of antibodies binding Nproximal

epitopes of PrP, which recognise PK-digested

type 1 PrPSc (cleaved at amino acid 82 or upstream), but

not type 2 PrPSc (cleaved at amino acids 97 or 86).

8 http://neurology.thelancet.com Published online October 31, 2005 DOI:10.1016/S1474-4422(05)70225-8TSS

On investigation of 70 sporadic CJD cases, we

determined that 50% of the patients formerly classified

as CJD type 2 had low but detectable amounts of PrPSc

type 1 in their cerebral cortex. Investigations of

additional brain areas revealed that significant

amounts of PrPSc type 1 coexist with type 2 in at least

some areas of all patients classified as having type 2

disease. Co-occurrence of CJD types in the same brain

has been previously reported in approximately 30% of

the tested cases.24 Using unambiguous analytical tools,

we have established that type 2 PrPSc does not exist

independently from type 1 PrPSc, at least not in the

Swiss CJD patients included in our study.

Furthermore, using our type 1-specific antibodies, we

found type 1 PrPSc content in three cortical samples of

vCJD patients.

Glycotyping of blots incubated with 3F4/POM1

versus POM2/POM12 showed no significant

differences in glycoform ratios, neither for the same

groups incubated with different antibodies, nor for the

two PrPSc groups, in accordance with previously

published data.33 The homogeneity of glycoforms

suggests that our collective is comparable to those

investigated in other countries, despite the peculiarities

of Swiss CJD epidemiology.34

The results presented here do not question the validity

of the established correlations between PrPSc types and

clinical findings. The coincident presence of PrPSc type 1

may not alter the expected clinical outcome of patients

with predominant PrPSc type 2 deposition. However, the

existence of heritable PrPSc types implies that strainspecific

characteristics of prions are enshrined in the

conformation of PrPSc, and that the different core

fragment sizes of PK-digested PrPSc may be used as

surrogates of such conformational variations. In the

light of the data presented here, the strength of these

arguments becomes somewhat questionable.

Why did the co-existence of type 1 and type 2 PrPSc go

often undetected in the past, despite its ubiquitous

presence? Maybe the 21 kDa band characteristic of

type 1 PrPSc is easily obfuscated by the simultaneous

occurrence of the neighbouring 19 kDa type 2 band and

of the monoglycosylated PrPSc band. We tested this

hypothesis by ascertaining the detectability threshold

of type 1 PrPSc in mixtures of pure type 1 and type 2

samples. Indeed, we found that type 1 PrPSc becomes

undiscernible as soon as 40% or more type 2 PrPSc is

present. This observation provides a plausible

explanation for the failure to appreciate the invariable

coexistence of PrPSc types in previous reports.

To determine whether a similar phenomenon occurs

in patients classified as type 1 would be interesting.

However, this issue is impossible to address at present,

as there is no CJD type-2-specific antibody. The fact

that the type 1 coexistence is also apparent in cortical

samples from CJD patients, indicates that the currently

described phenomenon might be a more general one.

The above results set the existing CJD classifications

into debate and introduce interesting questions about

human CJD types. For example, do human prion types

exist in a dynamic equilibrium in the brains of affected

individuals? Do they coexist in most or even all CJD

cases? Is the biochemically identified PrPSc type simply

the dominant type, and not the only PrPSc species?

http://neurology.thelancet.com Published online October 31, 2005 DOI:10.1016/S1474-4422(05)70225-8 9TSS

% type 1

% type 2

25 kDa–

20 kDa–

15 kDa–

25 kDa–

20 kDa–

15 kDa–

25 kDa–

20 kDa–

15 kDa–

25 kDa–

20 kDa–

15 kDa–

25 kDa–

20 kDa–

15 kDa–

25 kDa–

20 kDa–

15 kDa–

% type 1 100 50 40 30 20

0

PrPSc band-intensity

10 20 30 40 50 60

y=0·5084x

R2=0·783

% of type 1 CJD

30

25

20

15

10

5

0

X 0

100

0

0

100

50

50

60

40

70

30

80

20

90

10

10

100

0

0

100

50

50

60

40

70

30

80

20

90

10

POM1

POM2

POM2



POM1

POM1



POM2

POM1 POM7

POM1 POM2

POM2

Type 1 Type 2

MV VV MM

Type 1 Type 2

MV VV MM

Type 1 Type 2

MV VV VV VV MM

Type 1 Type 2

MV MV MM VV VV

Type 1 Type 2

MV MV MM Codon 129

A

B

C

E F

D

Figure 5: Non-discriminatory antibodies fail to detect coexistance of PrPSc types

Homogenised brain samples containing pure type 1 and type 2 PrPSc were mixed in various ratios (as indicated).

Replica blots with sample mixtures were probed with (A) non-discriminatory antibody POM1, or with (B) type 1-

specific antibody POM2. Type 2 PrPSc is detected when lanes contain 50% or more of the type 2 brain sample, but it

is not detected in lanes with a contribution of 40% or less. In the blots probed with POM2 (type 1 specific

antibody), type 1 PrPSc is detected in all cases , even if it corresponds to 10% of the total sample. (C) Comparison of

mixed type 1 and type 2 samples in various ratios (as indicated) with a cerebellar sample of a Creutzfeldt-Jakob

disease (CJD) type 2 patient (VV, indicated as X) when probed with POM2 (upper panel) and biotinylated POM1

(lower panel). (D) Standard curve of type 1 content , obtained according to the band-intensities of the serially

diluted “bona fide” type 1 in C (upper blot). (E) Replica blots analysing brain homogenates from two type 1

(cortex) and one type 2 (cerebellum) patients treated with PNGase and proteinase K (PK). Despite elimination of

alternative glycoforms, POM1 fails to detect type 1 content within the type 2 sample (left panel), whereas it is

clearly detected by POM2 (middle panel). When the same blot is reincubated with biotinylated POM1, the type 2

PrPSc band is revealed (right panel). (F) Replica blots probed with POM1 and POM7. POM7 specifically detects the

unglycosilated fragment. However, it does not detect type 1 content within type 2 samples. POM1 detection

pattern is shown here for comparison.

Articles

Acknowledgments

We thank all referring physicians, Mike Scott, Ralph Schlapbach, and

the Functional Genomics Center of Zurich for valuable help with the

surface plasmon resonance experiments and development of the

fluorescent blot, and Dieter Zimmerman for genotyping. We thank

Burkhardt Seifert (Department of Biostatistics, University of Zurich) for

advice on the statistical analysis. We also thank Wiegand Lang for

contributing to the production of hybridomas, and Albrecht Gröner for

discussions. MP is supported by a PhD fellowship of the Zentrum für

Neurowissenschaften Zürich, by UBS grant BA29 AKRB-DZZ (675/B),

and by a career award from the Foundation of Research at the Medical

Faculty, University of Zurich. This study was supported by a grant of

the Swiss Federal Office of Health to AA.

Authors’ contributions

MP contributed in the production of the antibodies, performed their

characterisation, contributed in sample analysis, and drafted the

manuscript. KS analysed the CJD samples and contributed in writing

the manuscript. MG obtained the cases and handled samples. MV and

AB contributed in the production of the antibodies. AA supervised this

study and contributed to writing and finalising the manuscript. MP, KS,

MG, and AA contributed in the design of the study. MP and KS

contributed equally to this work.

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10 http://neurology.thelancet.com Published online October 31, 2005 DOI:10.1016/S1474-4422(05)70225-8TSS

what i been saying for years, that the diagnostic criteria differentiating between the nvCJD (i.e. 'the chosen ones') and the sCJD (i.e. 'the forgotten ones') has been terribly flawed from the beginning. ....


Happy Thanksgiving Day.....TSS


Terry S. Singeltary Sr.
P.O. Box 42
Bacliff, Texas USA 77518



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