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BSE Test

Murgen

Well-known member
being cattle, sheep, deer, elk, 2nd passage medical/dental/surgical, etc. WAKEUP TIMMYBOY! do you actually produce a commodity for the consumer?

Main words in this statement are 2nd Passage. Would it not be possible that BSE and CJD are caused by the same circumstances?

Think cause people, not transmission!

So blaming one on the other is futile, until the original cause is determined.
 

Mike

Well-known member
TimH: According to some, less than 1 gram of "infective" beef can trigger vCJD in humans.

I've not seen the research to support this statement.

In fact, just the opposite is true. From what I've read, the "Cross Species" (human) barrier makes it approximately 100 to 1000 times less likely that a human will be "infected" from eating the same amount of infectivity that cattle do.

The big worry is that cosmetics, the blood supply, and surgical procedures (among others) will transmit PrPsc.

Also, some scientists believe some people might/do have certain genetic immunities to PrPsc that were derived from eating meat back in the Cro-Magnon and Neanderthal days.

It's not the number of actual cases that worry me as much as the "Media Induced Hysteria" that goes along with it.

Just wait until the first case of "Home Grown" nCJD hits North America!

There will be second guessing all to hell and back if the proper precautions are not taken beforehand.
 

TimH

Well-known member
bse-tester said:
part of a study found.

Pathophysiology of peripheral PrPSc deposition in muscles.
In experiments with transgenic mice exhibiting a four- to eightfold overexpression of the cellular isoform of the prion protein (PrPC) in myofibers under the control of myocyte-specific promoters, Bosque et al. (11) showed intrinsic replication of infectivity in muscles injected with scrapie agent. Here, with the more natural route of peroral infection, we have provided direct experimental evidence for the deposition of PrPSc in myofibers, particularly subsarcolemmally, and found that the deposition of this protein in muscles is also endoneurally associated with nerve fibers. Furthermore, the distribution patterns of PrPSc observed in muscles conspicuously resembled those known for the innervation of motor units (31, 32). Taken together, these findings are highly indicative of spread of infection from motor neurons in the spinal cord via axonal projections to neuromuscular junctions and on from there postsynaptically into muscle fibers. The results from the Western blot studies clearly show that PrPSc can be detected in muscles only shortly before the onset of clinical symptoms, that is, at a time point when the CNS is already heavily loaded with PrPSc and infectivity. This, again, would most plausibly be explained by projections of the peripheral nervous system mediating centrifugal infection of muscles from the spinal cord. Further time-course studies will be necessary, however, to validate the proposed neural spreading pathway and to elucidate, or rule out, the relevance of alternative mediators for muscle invasion such as lymph and blood or neural pathways linked with the centripetal ascension of TSE agent to the CNS.

Risks for public health and protection of consumers and patients.
What conclusions regarding the risks for public health emanating from "Prions in skeletal muscle" can be drawn from the findings described in this report?

For the reasons outlined above, the experimental paradigm used in our study is likely to provide a relevant model on the issue of muscle contamination. It probably reflects a worst-case rather than a best-case scenario, however, because the levels of infectivity and PrPSc produced in the CNS and other neural tissues of 263K scrapie-infected hamsters generally tend to be higher than in nonexperimental TSEs. Indeed, other than for caprine scrapie (7) or sporadic CJD (14), no infectivity or PrPSc has been detected so far in skeletal muscles of cattle with BSE (10).

If the findings in the hamster model were confirmed, to a varying degree, in ovine scrapie, BSE, or CWD, significant deposition of PrPSc in muscles of affected sheep, cattle, and elk or deer would be expected to occur only at relatively late stages of preclinical infection, with the bulk of muscle-associated PrPSc accumulating during clinical disease. At those stages of incubation, however, the TSE routine tests currently in use should allow both an identification of affected animals (34) with high reliability and their safe disposal. Thus, our findings strongly support the schemes for active TSE testing of animals that have already been implemented in several countries as an efficient precautionary measure to prevent muscle tissue potentially contaminated with infectious TSE agent from entering the food chain.

In addition to recent findings in patients with sporadic CJD (14), our observations in the hamster model corroborate concerns that muscles from inconspicuous patients preclinically incubating CJD or vCJD may represent a potential risk tissue for the nosocomial transmission of human TSE agents. Epidemiological evidence substantiating this hypothetical hazard has not been reported, however, and in any case the risk can be considered as effectively addressed by comprehensive precautionary measures already implemented for infection control in nosocomial settings. Particularly, recommended procedures for the routine maintenance (cleaning, disinfection, and sterilization) of surgical instruments (35, 36) aimed at the reliable removal and inactivation of unrecognized contaminations with infectious agents are expected to substantially reduce iatrogenic risks possibly originating from TSE agents in muscles — if they are firmly observed.

Since 2002, a considerable body of new data on PrPSc and TSE infectivity in muscles of experimentally infected rodents and patients with sporadic CJD has been compiled (11-14). It remains to be examined in future studies whether, and to what extent, these findings can be transferred to nonexperimental scrapie, BSE, and CWD, or to vCJD in humans. Special attention should be paid to food products containing ruminant or cervid tongue, given the observations reported here and elsewhere (13). In any case, the data available so far leave no doubt that, 40 years after Pattison and Millson (7) reported finding the scrapie agent in muscle tissue of an experimentally infected goat, a topical field of TSE research has been (re)opened.

More to follow.

Did you miss this part Ron?

no infectivity or PrPSc has been detected so far in skeletal muscles of cattle with BSE
 

bse-tester

Well-known member
TimH wrote:

Did you miss this part Ron?

no infectivity or PrPSc has been detected so far in skeletal muscles of cattle with BSE

The results from the Western blot studies clearly show that PrPSc can be detected in muscles only shortly before the onset of clinical symptoms, that is, at a time point when the CNS is already heavily loaded with PrPSc and infectivity.

Perhaps their test was not sensitive enough to detect PrPsc in muscle tissue? I doubt that to be the case though as they identified PrPsc in nervous fibrils that were in the muscle tissue itself. So where do we determine that PrPsc is not present within muscle when it is obviously within the nervous fibrils that are within the muscle. The evidence is clear TimH, but you chose not to see it. Having said that, I shall endeavor to find the studies for you. Ron.
 

flounder

Well-known member
TimH said:
flounder wrote
tim, I don't believe i ever stated that BSE HAD been documented yet in the muscle of the bovine, and if so, maybe old tim can produce this.

How about this........
BMR wrote
So where is this proof that Tim is looking for. I would like to see it as
> well.

flounder responded

it's there, and it's been posted on this board multiple times.

That was easy. It was in this very thread.

Your turn.



nice try timmy, but i was speaking of muscle tissue in infectivity in the species i have already documented. you cannot snip things out of context, but nice try. you'll do anything when you know your wrong i suppose. even lie and cheat. but i never said that about bse, or maybe you could show us the post where i said bse infectivity in muscle tissue has been _documented_. ...


TSS
 

flounder

Well-known member
Mike said:
TimH: According to some, less than 1 gram of "infective" beef can trigger vCJD in humans.

I've not seen the research to support this statement.

In fact, just the opposite is true. From what I've read, the "Cross Species" (human) barrier makes it approximately 100 to 1000 times less likely that a human will be "infected" from eating the same amount of infectivity that cattle do.

The big worry is that cosmetics, the blood supply, and surgical procedures (among others) will transmit PrPsc.

Also, some scientists believe some people might/do have certain genetic immunities to PrPsc that were derived from eating meat back in the Cro-Magnon and Neanderthal days.

It's not the number of actual cases that worry me as much as the "Media Induced Hysteria" that goes along with it.

Just wait until the first case of "Home Grown" nCJD hits North America!

There will be second guessing all to hell and back if the proper precautions are not taken beforehand.




Second, a single unit of vCJD-prion-infected

blood is sufficient to cause transmission of the

disease. This fact is particularly unsettling, as it

can only be taken to signify that the concentration

of ID50 units in blood is relatively high.

One ID50 unit is defined as the infectious

dose sufficient to establish infection in 50% of

recipients; animal experiments indicate that the

amount of prion infectivity needed to reach

one ID50 unit is much higher when prions are

administered intravenously than when they

are inoculated intracerebrally. ...


snip... full text ;


JUNE 2006 VOL 2 NO 6 AGUZZI AND GLATZEL NATURE CLINICAL PRACTICE NEUROLOGY 329

www.nature.com/clinicalpractice/neuro


TSS
 

TimH

Well-known member
bse-tester said:
TimH wrote:

Did you miss this part Ron?

no infectivity or PrPSc has been detected so far in skeletal muscles of cattle with BSE

The results from the Western blot studies clearly show that PrPSc can be detected in muscles only shortly before the onset of clinical symptoms, that is, at a time point when the CNS is already heavily loaded with PrPSc and infectivity.

Perhaps their test was not sensitive enough to detect PrPsc in muscle tissue? I doubt that to be the case though as they identified PrPsc in nervous fibrils that were in the muscle tissue itself. So where do we determine that PrPsc is not present within muscle when it is obviously within the nervous fibrils that are within the muscle. The evidence is clear TimH, but you chose not to see it. Having said that, I shall endeavor to find the studies for you. Ron.

Ron, I can't believe you are still flogging this dead horse even after your own post clearly stated,quote- "no infectivity or PrPSc has been detected so far in skeletal muscles of cattle with BSE".

Here is the rest of the study that you left out of your previous post. I have bolded the pertinent parts. Read it several times if you need to , Ron.


Pathophysiology of peripheral PrPSc deposition in muscles.
In experiments with transgenic mice exhibiting a four- to eightfold overexpression of the cellular isoform of the prion protein (PrPC) in myofibers under the control of myocyte-specific promoters, Bosque et al. (11) showed intrinsic replication of infectivity in muscles injected with scrapie agent. Here, with the more natural route of peroral infection, we have provided direct experimental evidence for the deposition of PrPSc in myofibers, particularly subsarcolemmally, and found that the deposition of this protein in muscles is also endoneurally associated with nerve fibers. Furthermore, the distribution patterns of PrPSc observed in muscles conspicuously resembled those known for the innervation of motor units (31, 32). Taken together, these findings are highly indicative of spread of infection from motor neurons in the spinal cord via axonal projections to neuromuscular junctions and on from there postsynaptically into muscle fibers. The results from the Western blot studies clearly show that PrPSc can be detected in muscles only shortly before the onset of clinical symptoms, that is, at a time point when the CNS is already heavily loaded with PrPSc and infectivity

Gosh-darn it Ron, they inject the scrapie agent into the muscles(of mice), and then they find PrPsc......in the muscles. No $hit Sherlock.

Here it is again Ron, from your own post, I might add.....

".....no infectivity or PrPSc has been detected so far in skeletal muscles of cattle with BSE".

What part of this is it that you don't get??

I'm almost starting to feel sorry for you.......almost. :roll:
 

TimH

Well-known member
flounder said:
TimH said:
flounder wrote
tim, I don't believe i ever stated that BSE HAD been documented yet in the muscle of the bovine, and if so, maybe old tim can produce this.

How about this........
BMR wrote
So where is this proof that Tim is looking for. I would like to see it as
> well.

flounder responded

it's there, and it's been posted on this board multiple times.

That was easy. It was in this very thread.

Your turn.



nice try timmy, but i was speaking of muscle tissue in infectivity in the species i have already documented. you cannot snip things out of context, but nice try. you'll do anything when you know your wrong i suppose. even lie and cheat. but i never said that about bse, or maybe you could show us the post where i said bse infectivity in muscle tissue has been _documented_. ...


TSS

Ho-hum, BMR clearly asked to see the evidence that I was looking for, which was CLEARLY PROOF THAT PrPsc HAD BEEN FOUND IN THE MUSCLE TISSUE OF CATTLE. To which you replied, "It's there...."
Anyone reading this thread can clearly see that. Pretty plain to see who is lying and cheating.
Sucks to be you ,huh Teary??
 

flounder

Well-known member
TimH said:
flounder said:
TimH said:
flounder wrote

How about this........
BMR wrote
> well.

flounder responded



That was easy. It was in this very thread.

Your turn.



nice try timmy, but i was speaking of muscle tissue in infectivity in the species i have already documented. you cannot snip things out of context, but nice try. you'll do anything when you know your wrong i suppose. even lie and cheat. but i never said that about bse, or maybe you could show us the post where i said bse infectivity in muscle tissue has been _documented_. ...


TSS

Ho-hum, BMR clearly asked to see the evidence that I was looking for, which was CLEARLY PROOF THAT PrPsc HAD BEEN FOUND IN THE MUSCLE TISSUE OF CATTLE. To which you replied, "It's there...."
Anyone reading this thread can clearly see that. Pretty plain to see who is lying and cheating.
Sucks to be you ,huh Teary??



not at all, just sucks when you are wrong timmyboy, you still cannot produce where i ever stated that. however i still do firmly believe that they will document BSE infectivity in the muscle tissue of cattle once they start validating these more sensitive test. you don't look, you dont find, you dont test with proper testing protocol, you dont find either. your going to have to do better than this timmyboy. your all talk, no wang cowboy :cboy:


IN FACT, lets look at the games people play.

WHAT exactly did timmyboy say, and who really cares ???



TimH
Rancher



Joined: 10 Feb 2005
Posts: 1035
Location: Southwest Manitoba


snip...

Posted: Mon Aug 14, 2006 9:16 pm Post subject: Re: TimH




I'm pretty sure that flounder, who is as well read on BSE as anyone, just admitted that PrPsc has NEVER been found in muscle tissues of cattle.
You can read, can't you Ron? Wishing don't make it so.
It's pretty simple, Slick. Find another way to create demand for your te$t. ...END

http://ranchers.net/forum/viewtopic.php?t=11693&postdays=0&postorder=asc&start=120



THERE you have it timmyboy, your own words. ..............TSS
 

flounder

Well-known member
Mike said:
TimH: According to some, less than 1 gram of "infective" beef can trigger vCJD in humans.

I've not seen the research to support this statement.

In fact, just the opposite is true. From what I've read, the "Cross Species" (human) barrier makes it approximately 100 to 1000 times less likely that a human will be "infected" from eating the same amount of infectivity that cattle do.

The big worry is that cosmetics, the blood supply, and surgical procedures (among others) will transmit PrPsc.

Also, some scientists believe some people might/do have certain genetic immunities to PrPsc that were derived from eating meat back in the Cro-Magnon and Neanderthal days.

It's not the number of actual cases that worry me as much as the "Media Induced Hysteria" that goes along with it.

Just wait until the first case of "Home Grown" nCJD hits North America!

There will be second guessing all to hell and back if the proper precautions are not taken beforehand.



NO studies on humans have every been done to document load infectivity of BSE or any TSE to humans. ITS against the law to do those studies i suppose. i think instead of primates a good study would be with human volunteers on death row. boy that's another can of worms, but until then, we must look at these studies with great concern.

IT seems to take very little of BSE to infect primates in the only few studies they have every done. my opinion, inoculation of the TSE agent may be a more efficient route though. what the threshold from sub-clinical to clinical disease, well, that's still another million dollar question that is unanswered for humans. ...tss







Research Letters

Up to 400 000 cows with undiagnosed bovine spongiform

encephalopathy (BSE) infection are estimated to

have been slaughtered for food before brain and spinal

cord were banned from human consumption in 1989.

More restricted exposure to BSE could have continued

through 1995 from consumption of processed meat

products containing mechanically recovered meat

contaminated with central nervous system (CNS) tissue

and spinal ganglia.1 The discovery of BSE in Canada and

the USA, where consumption of brain and other viscera

was allowed until 2003, and of secondary cases of variant

Creutzfeldt-Jakob disease (vCJD) in the UK, possibly

attributable to contaminated blood donated by people

with pre-clinical primary infection, reinforces the need

for an experimental assessment of the risk of oral

exposure to BSE. We therefore investigated oral

transmission of BSE to non-human primates.

We chose cynomolgus macaques for the study because

these old-world monkeys have a digestive physiology

similar to that of human beings, are methionine

homozygous at codon 129 of the PRNP gene, and have a

BSE neuropathology similar to that of vCJD.2,3 We gave

two 4-year-old adult macaques a 5 g oral dose of brain

homogenate from a BSE-affected cow. We tested for

proteinase-resistant prion protein (PrPres) in this

homogenate with a commercial BSE-testing ELISA kit

(Bio-Rad, Marnes-la-Coquette, France). A sample of the

100% homogenate brain paste inoculum that was fed to

the primates was rehomogenised at 20% weight-pervolume

in the kit buffer. Serial dilutions were made with

a pool of 20% weight-per-volume BSE-negative brain

homogenate in the same buffer. Testing was done

according to the manufacturer’s instructions and results

were confirmed by a western blot test (Bio-Rad) with a

similar process of PrPres dilution. With both methods,

dilutions of up to 1 in 300 provided a positive signal

(figure A).

One macaque developed neurological disease

60 months after exposure and was killed at 63 months

because of recumbency. Histopathological examination

of the brain of this animal showed the typical pathology

of vCJD (figure B) and an accumulation of PrPres

associated with the follicular dendritic cells in tonsils

(figure C), spleen, and intestine. A western blot showed

similar patterns of PrPres in a brain sample from the

macaque and the BSE-infected bovine inoculum

(figure D). The other macaque remained free of clinical

signs 76 months after exposure, and a tonsil biopsy done

at 72 months was negative (figure E).

In a previous study, two macaques orally dosed with

5 g of brain from a macaque with terminal clinical BSE

became ill after 44 and 47 months.4 The results of the

present study suggest that the incubation period for

interspecies transmission of BSE can be considerably

Published online

January 27, 2005

http://image.thelancet.com/

extras/05let1056web.pdf

Commissariat à l’Energie

Atomique/Direction des

Sciences du Vivant/Départment

de Recherche Médicale,

18 Route du Panorama, 92265

Fontenay-aux-Roses, France

(C I Lasmézas DrMedVet,

E Comoy DrMedVet,

C Herzog DipBiol,

F Mouthon DipBiol, F Auvré,

E Correia,

N Lescoutra-Etchegaray DipBiol,

Prof N Salès PhD, J-P Deslys MD);

Veterinary Laboratories

Agency, New Haw, Addlestone,

UK (S Hawkins MIBiol,

T Konold DrMedVet,

G Wells BVetMed); and 7815

Exeter Road, Bethesda, MD

20814, USA (P Brown PhD)

Correspondence to:

Dr Jean-Philippe Deslys

e-mail: [email protected]

www.thelancet.com Published online January 27, 2005 http://image.thelancet.com/extras/05let1056web.pdf 1

Risk of oral infection with bovine spongiform

encephalopathy agent in primates

Corinne Ida Lasmézas, Emmanuel Comoy, Stephen Hawkins, Christian Herzog, Franck Mouthon, Timm Konold, Frédéric Auvré, Evelyne Correia,

Nathalie Lescoutra-Etchegaray, Nicole Salès, Gerald Wells, Paul Brown, Jean-Philippe Deslys

The uncertain extent of human exposure to bovine spongiform encephalopathy (BSE)—which can lead to variant

Creutzfeldt-Jakob disease (vCJD)—is compounded by incomplete knowledge about the efficiency of oral infection

and the magnitude of any bovine-to-human biological barrier to transmission. We therefore investigated oral

transmission of BSE to non-human primates. We gave two macaques a 5 g oral dose of brain homogenate from a

BSE-infected cow. One macaque developed vCJD-like neurological disease 60 months after exposure, whereas the

other remained free of disease at 76 months. On the basis of these findings and data from other studies, we made a

preliminary estimate of the food exposure risk for man, which provides additional assurance that existing public

health measures can prevent transmission of BSE to man.

B

C

E

A

Dilution

D

3·215

1·989

0·984

0·302

0·131

0·065

0·052

1/10

1/30

1/100

1/300

1/1000

1/3000

Neg

36 kDa

36 kDa

22 kDa

22 kDa

16 kDa

1 2 3 4

ELISA detection of PrPres (absorbance units)

Figure: PrPres content of brain homogenate and histopathological assessment of macaque tissues

(A) Results of in-vitro testing for PrPres in BSE-infected inoculum by ELISA and western blot. Neg=normal bovine

brain material. (B) Typical florid plaque in the occipital cortex of the macaque that developed disease.

PrPres detected by proteinase K treatment with SAF32 anti PrP monoclonal antibody (kindly provided by Jacques

Grassi, CEA Saclay). The dense core of PrPres is surrounded by several vacuoles in a fibrillar proteinaceous corona;

bar=10 m. (C) Positive PrPres staining in tonsil (80% of follicules stained positive) of the macaque that developed

disease; bar=50 m. (E) Negative PrPres staining in tonsil of the macaque that did not develop disease; bar=50 m.

(D) Western blot showing similar PrPres patterns in samples from a patient with vCJD (lane 1), the macaque that

developed disease (lane 3), and the bovine BSE inoculum (lane 4). By contrast, a macaque inoculated intracerebrally

with material from a patient with sporadic CJD showed a different PrPres pattern (lane 2).

For personal use. Only reproduce with permission from Elsevier Ltd

Research Letters

longer than that of intraspecies transmission (60 months

vs 44 and 47 months, representing 36% and 28%

increases, respectively). The interval between the period of

peak exposure to infectious BSE tissue and the hitherto

peak incidence of vCJD is about 10–15 years, but

incubation periods of up to 40 years have followed oral

infection with kuru between human beings.5 Therefore,

maximum incubation periods might exceed 50 years in

cases of oral transmission of BSE from cattle to man.

The present data do not provide a definitive minimum

infective dose for transmission of cattle BSE to primates,

but they do give enough information for a preliminary

assessment of the adequacy of existing measures to

protect the human food chain. Results of ongoing

experiments provide a rough estimation of the intraspecies

transmission rates in cattle. The BSE brain

inoculum to which the cattle were exposed had an

infectivity titre of 103·5 mouse infectious (intracerebral

and intraperitoneal) units ID50 per g (ID50 is the dose at

which 50% of animals become infected). Interim results

at 6 years after exposure suggest that the oral ID50 in

cattle may be between 100 mg and 1 g (table 1; S A C

Hawkins, T Konold, G A H Wells, unpublished data).

Since the brain of a cow weighs 500 g and a spinal cord

200 g, CNS tissues from a cow with clinical signs of BSE

could contain enough infective agent to transmit disease

orally to 490–1400 cows (70% of 700 g if 1g is needed, or

20% of 700 g if 100 mg is sufficient), or to 70 primates

(50% of 700 g if 5 g represents the oral ID50).

The accuracy of estimates of the oral ID50 for man will

not be improved until completion, several years from

now, of a large dose-response European study (QLK1-

2002-01096) in macaques, in which the minimum dose

is 50 mg. However, because similar inocula were used in

both the cattle and macaque studies,6 a tentative comparison

can be made between the efficiency of oral infection

in cattle and that in primates. On this basis, a factor of

7–20 could be considered as the range of magnitude of a

bovine-to-primate species barrier for oral BSE infection

(70 primates infected compared with 490 or 1400 cows,

with a similar dose).

Elimination from the human food chain of CNS

tissues from cows with clinical BSE is estimated to have

reduced the risk of human exposure to the disease by

about 90%.7 Risk was further reduced in continental

Europe by systematic screening for the diagnostic

presence of PrPres in the brainstem of all cattle older than

30 months, and in the UK by the total interdiction of

cows older than 30 months. In an oral exposure study to

assess the pathogenesis of BSE in cattle, in which the

same European Union-evaluated test as we used in the

present study was applied to CNS tissues, some

preclinical cases of the disease were diagnosed.8

Using the same test, pooled brainstem from cows with

clinical BSE has yielded a endpoint titre of PrPres

corresponding to a 1-in-300 to 1-in-1000 dilution of

positive brainstem.6,9 If people were to eat CNS tissues

from a cow with preclinical BSE with a concentration of

PrPres just below the test detection limit of 1 in 300, they

would need to ingest at least 1·5 kg to reach the degree

of exposure equivalent to that in the 5 g of brain used for

oral transmission to the macaque in the present study. If

the oral ID50 for man was one log below this dose (ie,

similar to that in cattle, and not accounting for any

species barrier between cattle and man; see table), 150 g

of CNS tissue that tested falsely negative could represent

an infective dose. Because use of cattle brain and spinal

cord for human consumption is prohibited, and in view

of the existing mechanically recovered meat regulations,

a person would be very unlikely to ingest this amount of

cattle CNS tissue.

The minimum sensitivity of screening tests to detect

100% of BSE-infected animals has yet to be ascertained.

However, our results provide reassurance that BSE

screening procedures combined with CNS removal are

effective measures to protect the human food chain.

Contributors

J-P Deslys, C Lasmézas, and E Comoy were responsible for design and

management of this study. G Wells, S Hawkins, and T Konold were

responsible for the pathogenesis study in ruminants. C Lasmézas,

C Herzog, and N Lescoutra-Etchegaray were in charge of the primate

experiments. F Auvré undertook the biochemical analyses. N Salès was

responsible for the immunohistochemical analyses, which were done

by E Correia. C Lasmézas, E Comoy, F Mouthon, G Wells, P Brown, and

J-P Deslys drafted the manuscript.

Conflict of interest statement

Commissariat à l’Energie Atomique owns a patent covering the BSE

diagnostic test commercialised by Bio-Rad. All authors had full access to

all data and had responsibility to submit for publication. The funding

sources had no role in the collection, analysis, and interpretation of

data, writing of the report, or decision to submit the paper for

publication.

2 www.thelancet.com Published online January 27, 2005 http://image.thelancet.com/extras/05let1056web.pdf

BSE bovine brain inoculum

100 g 10 g 5 g 1 g 100 mg 10 mg 1 mg 0·1 mg 0·01 mg

Primate (oral route)* 1/2 (50%)

Cattle (oral route)* 10/10 (100%) 7/9 (78%) 7/10 (70%) 3/15 (20%) 1/15 (7%) 1/15 (7%)

RIII mice (icip route)* 17/18 (94%) 15/17 (88%) 1/14 (7%)

PrPres biochemical detection

The comparison is made on the basis of calibration of the bovine inoculum used in our study with primates against a bovine brain inoculum with a similar PrPres concentration that was

inoculated into mice and cattle.8 *Data are number of animals positive/number of animals surviving at the time of clinical onset of disease in the first positive animal (%). The accuracy of

bioassays is generally judged to be about plus or minus 1 log. icip=intracerebral and intraperitoneal.

Table 1: Comparison of transmission rates in primates and cattle infected orally with similar BSE brain inocula



Research Letters

Acknowledgments

We gratefully acknowledge the expert care of the primate animals

provided by René Rioux, Sébastien Jacquin, and Anthony Fort, and the

technical expertise of Dominique Marcé, Capucine Dehen,

Sophie Freire, and Aurore Jolit Charbonnier. This work has received

financial support from the French Ministry of Research (GIS Prion). It is

now continued within the framework of the EU consortium QLK1-2002-

01096 and the European network of Excellence NeuroPrion. Ongoing

studies by the Veterinary Laboratories Agency in cattle are funded by the

UK Department for Environment, Food, and Rural Affairs.

References

1Anderson RM, Donnelly CA, Ferguson NM, et al. Transmission

dynamics and epidemiology of BSE in British cattle. Nature 1996;

382: 779–88.

2 Lasmézas CI, Deslys JP, Demaimay R, et al. BSE transmission to

macaques. Nature 1996; 381: 743–44.

3 Lasmézas CI, Fournier JG, Nouvel V, et al. Adaptation of the bovine

spongiform encephalopathy agent to primates and comparison with

Creutzfeldt-Jakob disease: implications for human health. Proc Natl

Acad Sci USA 2001; 98: 4142–47.

4 Herzog C, Salès N, Etchegaray N, et al. Tissue distribution of bovine

spongiform encephalopathy agent in primates after intravenous or

oral infection. Lancet 2004; 363: 422–28.

5 Klitzman RL, Alpers MP, Gajdusek DC. The natural incubation

period of kuru and the episodes of transmission in three clusters of

patients. Neuroepidemiol 1984; 3: 3–20.

6 Deslys JP, Comoy E, Hawkins S, et al. Screening slaughtered cattle

for BSE. Nature 2001; 409: 476–78.

7 European Commission. Opinion of the Scientific Steering

Committee on the Human Exposure Risk via food with respect to

BSE. Adopted on 10 December 1999. http://europa.eu.int./comm/

food/fs/sc/ssc/out67_en.pdf (accessed Jan 17, 2004).

8 Grassi J, Comoy E, Simon S, et al. Rapid test for the preclinical

postmortem diagnosis of BSE in central nervous system tissue.

Vet Rec 2001; 149: 577–82.

9 Moynagh J, Schimmel H. Tests for BSE evaluated. Bovine

spongiform encephalopathy. Nature 1999; 400: 105.

www.thelancet.com Published online January 27, 2005 http://image.thelancet.com/extras/05let1056web.pdf 3





Vol. 96, Issue 7, 4046-4051, March 30, 1999


Neurobiology
Natural and experimental oral infection of nonhuman primates by bovine spongiform encephalopathy agents
Nöelle Bons*,, Nadine Mestre-Frances*, Patrick Belli, Françoise Cathala§, D. Carleton Gajdusek¶, and Paul Brown
* Ecole Pratique des Hautes Etudes, Laboratoire de Neuromorphologie Fonctionnelle, Université Montpellier II, 34095-Montpellier cedex 5, France; Centre National d'Etudes Veterinaires et Alimentaires, Pathologie Bovine, 31 Av. Tony Garnier, 69342-Lyon cedex 07, France; § 68 Bd Saint-Michel, 75006-Paris, France; ¶ Institut Alfred Fessard, Centre National de la Recherche Scientifique, 91198-Gif-sur-Yvette, France; and Laboratory of Central Nervous System Studies, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892

Contributed by D. Carleton Gajdusek, December 21, 1998



ABSTRACT



Experimental lemurs either were infected orally with the agent of bovine spongiform encephalopathy (BSE) or were maintained as uninfected control animals. Immunohistochemical examination for proteinase-resistant protein (prion protein or PrP) was performed on tissues from two infected but still asymptomatic lemurs, killed 5 months after infection, and from three uninfected control lemurs. Control tissues showed no staining, whereas PrP was detected in the infected animals in tonsil, gastrointestinal tract and associated lymphatic tissues, and spleen. In addition, PrP was detected in ventral and dorsal roots of the cervical spinal cord, and within the spinal cord PrP could be traced in nerve tracts as far as the cerebral cortex. Similar patterns of PrP immunoreactivity were seen in two symptomatic and 18 apparently healthy lemurs in three different French primate centers, all of which had been fed diets supplemented with a beef protein product manufactured by a British company that has since ceased to include beef in its veterinary nutritional products. This study of BSE-infected lemurs early in their incubation period extends previous pathogenesis studies of the distribution of infectivity and PrP in natural and experimental scrapie. The similarity of neuropathology and PrP immunostaining patterns in experimentally infected animals to those observed in both symptomatic and asymptomatic animals in primate centers suggests that BSE contamination of zoo animals may have been more widespread than is generally appreciated.



MATERIALS AND METHODS



Epidemiological Study. A detailed study was undertaken of 61 primates belonging to 11 species housed in the Montpellier Zoological Park to evaluate the possible role of diet on the longevity of the animals. The animals live in very large cages spread out in a natural garrigue (Mediterranean forest). Depending on animal size, no more than three simians or five lemurians live in any one cage. A questionnaire also was mailed to other zoos and primate breeding facilities in France, asking for information about neurological or unexplained primate deaths and dietary practices. In the course of this inquiry, we were informed that a number of apparently healthy lemurs in the Besançon zoo and the Strasbourg breeding facility were going to be euthanized because of a new French regulation concerning hybrid primates, and so we obtained an additional group of 18 animals (six from Besançon and 12 from Strasbourg).

These 79 animals were all large-sized, long-lived monkeys and lemurs (over 1,000 g in body weight and more than 20 years longevity), who were fed a daily diet of vegetables and fruits supplemented by 20-40 g/kg of commercial food products containing animal-derived proteins (Singe 107, MP, or Marex). According to the manufacturers, this food contained various items, including gross protein (19.2-25.4%), fats (5.7-7.5%), corn, soya, carob bean, alfalfa, mineral, yeasts, vitamins A, C, D3, and E, and cracklings (the so-called "fifth quarter of beef" suitable for human consumption).


Experimental Study. This study involved a group of five lemurs belonging to the small-sized and short-lived species Microcebus murinus (around 100 g in body weight, 8-10 years longevity). These animals, from a colony housed at the Center for Laboratory Animals of the Montpellier University of Science, were 1-year-old adults and had never been fed commercial food containing meat. Three lemurs (control animals nos. 538, 593, and 655) were allowed to remain in the colony. Two lemurs (nos. 654 and 656) were reared in a locale protected under French law, one animal (no. 654) having been fed a single 0.5-g dose of a BSE-infected cattle brain (obtained from Centre National d'Etudes Veterinaires et Alimentaires, Lyon, France), and the other (no. 656) having been fed two 0.5-g doses, spaced 2 months apart, of the same cattle brain. The brain fragments were mixed with apple compote and given to the animals before their customary daily diet.

Immunohistology. Animals were anaesthetized by an i.p. injection of pentobarbital (0.5 ml/kg). The various organs were dissected, and samples were fixed by immersion in paraformaldehyde (4% in 0.1 M phosphate buffer, pH 7.4) and Carnoy's liquid. After routine histological protocols, 6-µm microscopic sections of different parts of the gastrointestinal tract, spleen, tonsil, thymus, spinal cord, and brain were prepared for PrP immunohistological study as follows: sections were immersed in 85% formic acid for 45 min, washed in distilled water, immersed in 5% hydrogen peroxide for 10 min, immersed in distilled water, and autoclaved for 10 min at 121°C.

The sections then were rinsed in Tris-buffered saline (TBS) before overnight incubation at 4°C with either of two mouse monoclonal primary antibodies: anti-PrP106-126 (dilution 1:2) or anti-PrP 3F4 (dilutions 1:200, 1:500, or 1:1,000). Sections then were incubated for 1 h with a secondary anti-mouse IgG antibody coupled to peroxidase (Boehringer Mannheim). Color was developed with 0.2% diaminobenzidine (Sigma) in TBS containing 0.02% hydrogen peroxide and counterstained with Harris' hematoxylin. Histological sections of brain, spleen, and gastrointestinal tract from several different Eulemur spp. were independently studied in the laboratory of P. Belli, using the laboratory's own rabbit polyclonal antibody RS1 and revealed by the kit Duet (Dako) according to the protocol of Tagliavini et al. (3).


Selected brain and spinal cord sections also were treated with the polyclonal antibody 961S28T (4) (1:200 dilution for 5 days), which stains abnormal neuronal Tau proteins, and the polyclonal glial fibrillary acidic protein antibody (GFAP) (Dako, 1:100 dilution overnight), which stains reactive astrocytes. The protocol was identical to that used for anti-PrP antibodies, except for the omission of formic acid and autoclaving pretreatment. Quantitative studies were performed on brain sections chosen with reference to the microcebe brain atlas (5); the distribution of cortical neurons containing abnormal aggregated Tau proteins was mapped with an image analysis computer (Biocom Histo 200, Paris).


Because no anti-PrP antibody is capable of distinguishing between the normal and pathological isoforms of PrP in fixed tissue, and because discrimination by proteinase K partial digestion also is rendered ineffective by fixation, it is essential that a number of methodological criteria be met for a proper interpretation of immunostaining results. These criteria include: unequivocal staining having a characteristic morphological appearance, with little or no background noise; and the absence of such staining in parallel sections treated with (i) preimmune serum from the animal in which the primary antibody was raised, (ii) immune serum preabsorbed with its corresponding PrP antigen, (iii) secondary antibody without previous incubation with the anti-PrP antibody, and (iv) at least one other antibody unrelated to PrP. In addition, staining must not occur in identically prepared sections from tissues of healthy control animals, and the results should be duplicated by an independent laboratory using the same or different immunohistochemical techniques and antibodies. Our study meets all of these criteria, and we therefore have accepted positive staining results as representing the presence of the pathological isoform of PrP.



snip...



Epidemiological Study. Among the primates in the Montpellier zoo, 26 deaths were recorded between 1989-1998, of which 23 occurred between 1989 and 1993 (Table 1). The date of arrival of each primate at the zoo was always known, but the date and the locality of its birth were often unknown (many animals came from other zoological parks). Although detailed clinical information rarely was recorded in the zoo registers, clinical signs were observed before death in 14 animals, of which 12 were characterized as having had serious neurological abnormalities.





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Table 1. Epidemiological summary of primates housed in the Montpellier Zoological Park during the period 1989-1998




In view of the multiple geographic origins of the animals dying at the Montpellier zoo, it is not possible to state that infection in all animals occurred in this locale. However, three animals dying from spongiform encephalopathy must certainly have been infected in Montpellier: two lemurs (nos. 481 and 586) came directly from Madagascar to Montpellier in 1974 and 1979, well before the era of BSE, and one animal (no. 474) was born and raised in the Montpellier zoo.


We received nine responses (representing only about a 10% response rate) from our mailed questionnaire to other primate holding facilities: one respondent zoo had no primates, and of the eight respondent zoos with primates, seven denied any suspicious or neurological deaths, and one (Lille) noted three deaths in January 1996 in primates after neurological illnesses similar to those seen in the Montpellier primates.


All of the primates in Lille, Strasbourg, Besançon, and Montpellier, as well as animals in the seven zoos that reported no neurological deaths, had diets that included nutritional supplements containing meat meal, sold under the names Singe 107, MP, or Marex. The supplements are produced by two different companies (one of which is based in the United Kingdom), which distribute them through a French company to zoos and animal breeding facilities. It is highly likely that British beef was included in the source of meat powder, especially as the British manufacturer announced that as of June 1996 it ceased to use beef meal in its nutritional supplements.


Immunohistological Studies. We studied two lemurs (microcebes) that were experimentally fed with BSE-infected brain tissue and three unexposed control lemurs. After the killing of one of the BSE-fed lemurs (no. 654) by its cage mates, we sacrificed one of the two remaining BSE-fed animals (no. 656) to have optimally preserved tissues for examination from at least one animal during the incubation phase of disease (5 months postinfection). Other animals are being held under observation until such time as they may show signs of neurological disease.

We also studied two additional symptomatic lemurs in the Montpellier zoo (nos. 456 and 586), and 18 asymptomatic lemurs (nos. 700-717) in captivity in either Besançon or Strasbourg. All of these animals were 6-16 years of age (except for two animals 25 years of age), with body weights of 1,500-1,800 g. The presence and distribution of PrP immunoreactivity described in the following paragraphs was similar in the captive lemurs and in the two microcebes that had been experimentally infected with BSE (Tables 2 and 3). Uninfected control animals showed no PrP immunoreactivity.





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Table 2. PrP immunostaining in non-nervous system tissues of spontaneous cases of spongiform encephalopathy in eulemurs and in microcebes fed with BSE-infected brain tissue (nos. 654 and 656)







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Table 3. PrP, Tau, and GFAP immunopositivity, and micro-vacuolation in nervous system tissues of spontaneous cases of spongiform encephalopathy in eulemurs, and in microcebes fed with BSE-infected brain tissue (nos. 654 and 656)




In the tonsils, PrP was seen in the peripheral epithelium, lymphoid nodules, and in scattered cells inside the glands. In the esophagus, PrP was present in the stratified epithelial cells, but not in the mucigen-secreting esophageal glands. Immunoreactive lymphocytes were scattered throughout the connective tissue of the lamina propria and infiltrating the muscularis mucosae and the submucosa. An abrupt transition between the esophagus and the stomach was conspicuous by a different PrP distribution starting at the cardia: the gastric columnar epithelium bordering the lumen and the gastric pits were PrP-negative but the gastric glands were positive. The underlying lymphoreticular tissue in the lamina propria also was labeled (Fig. 1 E and F).





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Fig. 1. (A) Zoo lemur no. 703. PrP deposits in large vacuolated fibers of the ventral funiculus of the cervical spinal cord. Arrows point to fiber membranes. Anti-PrP 3F4, 1:200. (B) Zoo lemur no. 712. Nerve fibers showing PrP immunoreactivity (brown) in layer IV of the cerebral cortex. Anti-PrP 3F4, 1:200. (C) Experimental BSE-infected microcebe no. 656. Microvacuolation in the neuropil of the parietal cortex (layer V). Hematoxylin and eosin. (D) Experimental BSE-infected microcebe no. 656. Abnormal Tau proteins inside pyramidal neurons of the parietal cortex layer III. Anti-tau 961S28T, 1:200. (E) Experimental control microcebe no. 593. High magnification of the stomach wall: no PrP immunoreactivity is detected in the epithelium, secretory glands, or various lymphoreticular tissue elements (arrows). Star indicates luminal surface. Anti-PrP 3F4, 1:200. (F) Experimental BSE-infected microcebe no. 656. PrP distribution in the stomach wall. Arrows point to reticulolymphatic elements; star indicates luminal surface. Anti-PrP 3F4, 1:500. (G) Experimental BSE-infected microcebe no. 656. PrP localization in an intestinal villus. Note the interrupted epithelium at the level of M cells containing a lymphocyte, and the immunoreactivity of lymphoid reticular structures. Stars indicate luminal surfaces. Anti-PrP106-126, 1:2. (H) Experimental BSE-infected microcebe no. 656. Peyer's patch with PrP immunoreactive lymphoid structures. Anti-PrP106-126, 1:2. (I) Experimental BSE-infected microcebe no. 656. PrP labeling in splenic red pulp. Anti-PrP 3F4, 1:500. (J) Experimental BSE-infected microcebe no. 656. Small intestine. Anti-PrP 3F4, 1:200, pre-adsorbed with PrP antigen.




In the small intestine, including the duodenum, finely particulate PrP was spread throughout the cytoplasm of the epithelial cells (except in goblet cells), located close to the lumen as well in the villi. The PrP was located within the striated border cells, the glandular cells located at the base of the villi, and the specialized M cells associated with lymphocytes infiltrating the epithelium (Fig. 1 G and J). The lamina propria and the submucosa contained labeled lymphocytes as did the wall of the lymph and blood vessels. In these areas, PrP-labeled cellular elements also were observed at the periphery of both lymphoid structures associated with the intestine: the elongated Peyer's patches (Fig. 1H) and the spherical lymph nodes. In the colon, PrP immunoreactivity was noted in the columnar epithelial cells near the lumen but not in the crypts. The tunica muscularis of the different regions of the gastrointestinal tract never exhibited immunoreactivity. The spleen showed an obvious staining of numerous cells located in the red pulp (Fig. 1I) and, in lower number, at the periphery of the white pulp.


In the central nervous system of large-size lemurs in the preclinical stage of disease, we observed PrP particles in both dorsal and ventral roots of the spinal cord in the cervical region and scattered along vacuolated fibers in the spinal cord (Fig. 1A). PrP was also visible as dust-like particles in layer IV of the cerebral cortex near PrP-labeled fibers originating from the corpus callosum (Fig. 1B). Moreover, clearly degenerative central nervous system processes were seen in both the zoo eulemurs and the experimental microcebes. This degeneration was manifested by three abnormalities, which were never detected in the brains of control animals.


First, numerous aggregated Tau-containing neurons were present throughout the cerebrum, particularly in the cerebral cortex, the brain stem, the superior colliculus, and the thalamus (Fig. 1D). As the evolution of Tau proteins in the cortical pyramidal neurones is well studied in microcebes (6, 7), we were able to compare their number to those in the experimental microcebe with optimally preserved tissue (the condition of the tissue from the lemur killed by his cage mates was not good enough for quantitative study). The BSE-infected lemur had more than 10 times as many degenerating neurones as aged normal lemurs (8-13 years), and nearly 300 times as many as young lemurs of comparable age (1-2 years). In particular, degeneration of the pyramidal cortical neurones in healthy young adult microcebes begins in the occipital cortex, and aggregated Tau-containing neurones are never observed in the parietal and frontal cortices, whereas, on average, 280 and 269 abnormal neurones were found in these areas of the BSE-infected lemur.


Second, innumerable small vacuoles were present in the cortical parenchyma (Fig. 1C), often in close contact with the hyperphosphorylated Tau-containing neurones. In the brains and spinal cords of all animals, a majority of large nerve tract fibres exhibited vacuolation, and in some large bundle tracts, such as the reticular formation and corpus callosum, it was possible to distinguish between discrete vacuolated and nonvacuolated tracts.


Third, astrocytic gliosis was evident in the large increase of reactive astrocytes showing GFAP immunoreactivity, particularly well developed in the white matter of the brain, in layers I, V, and VI of the cortex, and in proximity to blood vessels. Blood vessesls in the pia matter also were surrounded by reactive astrocytes. In the spinal cord, GFAP-labeled astrocytes were very numerous in the white matter but also scattered in the central gray matter. Aggregated Tau proteins were seen in fibers of the spinal cord tracts and in the axoplasm of myelinated fibers in peripheral nerves near the spinal cord.


DISCUSSION


Pathogenesis has been a continuing subject of importance in the study of transmissible spongiform encephalopathies, having been first addressed systematically by Hadlow et al. (8-10) in a landmark set of experiments in which the sequential appearance of infectivity in different organs was determined in both naturally and experimentally acquired disease, continued by Kimberlin and Walker (11, 12) in a series of experiments on orally infected mice, and most recently extended by Beekes et al. (13, 14) to include parallel studies of PrP in tissues after oral infection and by Klein et al. (15) with particular attention to the role of B cells in neuroinvasion. All of these studies were undertaken by using scrapie as the model of infection, but preliminary investigations also have been reported on BSE in naturally and experimentally infected cattle (16).

From the ensemble of these studies it has become clear that, after oral infection, infectivity and pathologic PrP first appear in the digestive tract and its contained or proximate lymphoid tissues (tonsils, lymph nodes, Peyer's patches, and spleen), before moving, presumably through autonomic nervous system fibers, to the spinal cord and up to the brain. Natural and experimental BSE in bovines is notable in the comparatively limited distribution of infectivity outside the central nervous system, having been demonstrated only in the trigeminal and dorsal root ganglia, distal ileum, and (possibly) bone marrow and retina.

The present study, which extends our earlier investigations of two lemurs and one monkey dying with spongiform encephalopathy in the Montpellier zoo (1, 2), contributes two additional pieces of information about oral infection by transmissible spongiform encephalopathy agents. First, the immunohistochemical results of our experimental study of BSE-fed lemurs has precisely defined the distribution and localization of PrP within a variety of tissues early in the incubation period of disease. PrP (and by implication, the infectious agent) evidently is taken up by epithelial cells lining the lumen of the digestive tract (including those of the tonsil), initiating a reaction of the M cells and lymphocytes within the tissues of the digestive tract and in their lymphatic drainage system (including lymph nodes and spleen). Our observations also show that even before PrP can be detected in the central nervous system in the pattern typical of terminal illness, it can be traced along nerve pathways from ventral and dorsal root ganglia through the spinal cord into the brain cortex. These results are consistent with the observed distribution and progression of infectivity and PrP during the evolution of scrapie, as measured by infectivity assays (12) and Western blots of extracted PrP (14).

Second, the similar neuropathology and distribution of PrP in orally infected experimental lemurs and spontaneously affected zoo lemurs, together with the epidemiological observations confirming the occurrence of spongiform encephalopathy in animals fed a diet supplemented with meat protein that until 1996 had very likely included rendered British beef, leave little room for doubt that cases of spongiform encephalopathy in French primates resulted from infection by BSE-contaminated meat, just as in felines and ungulates in zoos elsewhere. Our unexpected finding that the same patterns of PrP distribution and brain degeneration were present in asymptomatic lemurs from two other French primate facilities suggests that BSE-contaminated diets may have been far more widespread than appreciated and mandates continued surveillance of primates in European zoos and breeding facilities.



snip...



full text ;



http://www.pnas.org/cgi/content/full/96/7/4046





J Infect Dis 1980 Aug;142(2):205-8



Oral transmission of kuru, Creutzfeldt-Jakob disease, and scrapie to nonhuman primates.

Gibbs CJ Jr, Amyx HL, Bacote A, Masters CL, Gajdusek DC.

Kuru and Creutzfeldt-Jakob disease of humans and scrapie disease of sheep and goats were transmitted to squirrel monkeys (Saimiri sciureus) that were exposed to the infectious agents only by their nonforced consumption of known infectious tissues. The asymptomatic incubation period in the one monkey exposed to the virus of kuru was 36 months; that in the two monkeys exposed to the virus of Creutzfeldt-Jakob disease was 23 and 27 months, respectively; and that in the two monkeys exposed to the virus of scrapie was 25 and 32 months, respectively. Careful physical examination of the buccal cavities of all of the monkeys failed to reveal signs or oral lesions. One additional monkey similarly exposed to kuru has remained asymptomatic during the 39 months that it has been under observation.

PMID: 6997404
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=6997404&dopt=Abstract



TSS
 

TimH

Well-known member
flounder wrote-

"....your going to have to do better than this timmyboy. your all talk, no wang cowboy..."

:D :D :D :D :D :D :D

What is a "wang" flounder?? I certainly hope,for your sake, that wasn't meant as a personal attack of some sort.

:D :D :D :D :D :D :D :D :D :D :D :D :D :D :D
 

bse-tester

Well-known member
Flounder, be careful, it appears that Timmy-boy is going to hunt you down and spank your butt??? Talk about a veiled threat Tim. Oh my, what have you become??

Surely you don't mean to threaten Flounder with words like "...I certainly hope, for your sake, that wasn't meant as a personal attack of some sort?"

Talk about paranoid!! Tim, have you ever heard of therapy?? It may help you man!

Flounder, calling TimH a wang was incredibly harsh!!! Especially as he has no idea as to what a "Wang" is!!! No wonder he is peed at you!! I would be too. :eek: :eek: :shock: :shock: :shock: :D :D :D
 

TimH

Well-known member
bse-tester said:
Flounder, be careful, it appears that Timmy-boy is going to hunt you down and spank your butt??? Talk about a veiled threat Tim. Oh my, what have you become??

Surely you don't mean to threaten Flounder with words like "...I certainly hope, for your sake, that wasn't meant as a personal attack of some sort?"

Talk about paranoid!! Tim, have you ever heard of therapy?? It may help you man!

Flounder, calling TimH a wang was incredibly harsh!!! Especially as he has no idea as to what a "Wang" is!!! No wonder he is peed at you!! I would be too. :eek: :eek: :shock: :shock: :shock: :D :D :D

Ronnie,Ronnie,Ronnie....... :roll: :roll: :roll:

#1- I don't make threats, veiled or otherwise. If I was "peed" I would have used this- :mad: - and not this- :D . Obvious enough for you, Oh Wise One??

#2- Anytime Flounder, or better yet YOU and Flounder at the same time, would like to get together with me, I would be happy to hear just what a "no wang cowboy is" in person.

#3- Prove yourself WRONG lately, Ron???? :D :D :D :D
 

Mike

Well-known member
Big Muddy rancher said:
So out of 14 pages has anyone shown that anything that has eaten muscle tissue from a BSE infected cow come down with BSE or nvCJD ? :???:

There are all kinds of oral inoculation tests. From calves to monkeys.

Know anybody that will volunteer for a human test? :lol:
 

TimH

Well-known member
Mike said:
Big Muddy rancher said:
So out of 14 pages has anyone shown that anything that has eaten muscle tissue from a BSE infected cow come down with BSE or nvCJD ? :???:

There are all kinds of oral inoculation tests. From calves to monkeys.

Know anybody that will volunteer for a human test? :lol:

All kinds?? I only want to see one(1) in which muscle tissue from a BSE infected bovine, has been proven to cause nvCJD or any other TSE for that matter.
Here is another "Hmmmmm "........... if PrPsc is present in blood(and by association, muscle tissue) why in the world would Ronald S. Arnold and BSE Prion Solutions Inc. be farting around trying to test urine(making the animal pee) and trying to figure out how to "sterilize" their urine testing gear. Would it not be easier to just draw some blood??? It is done all the time for other disease tests. Hmmmmmm???? :???: :???:
 

Mike

Well-known member
TimH said:
Mike said:
Big Muddy rancher said:
So out of 14 pages has anyone shown that anything that has eaten muscle tissue from a BSE infected cow come down with BSE or nvCJD ? :???:

There are all kinds of oral inoculation tests. From calves to monkeys.

Know anybody that will volunteer for a human test? :lol:

All kinds?? I only want to see one(1) in which muscle tissue from a BSE infected bovine, has been proven to cause nvCJD or any other TSE for that matter.
Here is another "Hmmmmm "........... if PrPsc is present in blood(and by association, muscle tissue) why in the world would Ronald S. Arnold and BSE Prion Solutions Inc. be farting around trying to test urine(making the animal pee) and trying to figure out how to "sterilize" their urine testing gear. Would it not be easier to just draw some blood??? It is done all the time for other disease tests. Hmmmmmm???? :???: :???:

They are in the UK BSE Inquiry. Look em up yourself. There's plenty of oral inoculation research articles there. You might read the whole thing while you're there and become educated. :lol:


Might be because of the numerous proteins in blood. They do have to separate them for BSE testing, you know.

You know how small a protein is? Clue, think almost "atom" sizes.

All other disease tests aren't dealing with proteins. Usually antibodies of bacteria's and viruses and blood cell abnormalities.
 

Big Muddy rancher

Well-known member
Mike said:
TimH said:
Mike said:
There are all kinds of oral inoculation tests. From calves to monkeys.

Know anybody that will volunteer for a human test? :lol:

All kinds?? I only want to see one(1) in which muscle tissue from a BSE infected bovine, has been proven to cause nvCJD or any other TSE for that matter.
Here is another "Hmmmmm "........... if PrPsc is present in blood(and by association, muscle tissue) why in the world would Ronald S. Arnold and BSE Prion Solutions Inc. be farting around trying to test urine(making the animal pee) and trying to figure out how to "sterilize" their urine testing gear. Would it not be easier to just draw some blood??? It is done all the time for other disease tests. Hmmmmmm???? :???: :???:

They are in the UK BSE Inquiry. Look em up yourself. There's plenty of oral inoculation research articles there. You might read the whole thing while you're there and become educated. :lol:


Might be because of the numerous proteins in blood. They do have to separate them for BSE testing, you know.

You know how small a protein is? Clue, think almost "atom" sizes.

All other disease tests aren't dealing with proteins. Usually antibodies of bacteria's and viruses and blood cell abnormalities.



Gee Tim it's back to look them up yourself. :???: I thought they would be quick to prove us hicks wrong.
 

Mike

Well-known member
OK. I'll fall for it. Here's a smidgen. You bunch of hicks:

Infectious Dose Experiment
This experiment was designed to determine the minimum dose that would cause BSE infection in a cow. It should be noted, however, that in the epidemic itself, cattle did not receive raw brain. Brain would have been heat treated during rendering and subsequently diluted with other feed constituents.

Calves were challenged by mouth with homogenised brain from confirmed cases of BSE. Some received 300g (3 doses of 100g), some 100g, 10g or 1g. They were then left to develop BSE, but were not subjected to the normal stresses that they might have encountered in a dairy herd. Animals in all four groups developed BSE. There has been a considerable spread of incubation period in some of the groups, but it appears as if those in the 1 and 10g challenge groups most closely fit the picture of incubation periods seen in the epidemic. Experiments in progress indicate that oral infection can occur in some animals with doses as low as 0.01g and 0.001g.
 

flounder

Well-known member
Short
Communication
Pathological prion protein in muscles of hamsters
and mice infected with rodent-adapted BSE
or vCJD
Achim Thomzig,13 Franco Cardone,23 Dominique Kru¨ ger,1
Maurizio Pocchiari,2 Paul Brown3 and Michael Beekes1
Correspondence
Michael Beekes
[email protected]
1Robert Koch-Institut (P24 – Transmissible Spongiform Encephalopathies), Nordufer 20,
13353 Berlin, Germany
2Department of Cell Biology and Neurosciences, Istituto Superiore di Sanita` , Viale Regina
Elena 299, 00161 Rome, Italy
37815 Exeter Road, Bethesda, MD 20814, USA
Received 22 June 2005
Accepted 12 September 2005
Recently, pathological prion protein (PrPTSE) was detected in muscle from sheep infected with
scrapie, the archetype of transmissible spongiform encephalopathies (TSEs). This finding has
highlighted the question of whether mammalian muscle may potentially also provide a reservoir for
TSE agents related to bovine spongiform encephalopathy (BSE) and variant Creutzfeldt–Jakob
Disease (vCJD). Here, results are reported from studies in hamsters and mice that provide direct
experimental evidence, for the first time, of BSE- and vCJD-associated PrPTSE deposition in
muscles. Our findings emphasize the need for further assessment of possible public-health risks
from TSE involvement of skeletal muscle.
Recently, Andre´oletti et al. (2004) reported the detection of
disease-associated prion protein (hereafter referred to as
PrPTSE; Brown & Cervenakova, 2005), the biochemical
marker for infectious agents causing transmissible spongiform
encephalopathies (TSEs), in muscles from sheep
infected experimentally with scrapie by the intracerebral or
oral route and from sheep with natural scrapie. Although
dietary exposure to ovine meat products contaminated with
the scrapie agent is currently considered non-hazardous to
humans, the report of Andre´oletti et al. (2004) has gained
considerable attention because it calls for a review of whether
muscle of sheep should be considered as a potential source
of infectious material for humans, especially in the case
of spread of bovine spongiform encephalopathy (BSE) to
sheep. Independently of the route of infection, PrPTSE is
also deposited in muscle fibres of hamsters perorally
(Thomzig et al., 2004a), intraperitoneally or intracerebrally
(A. Thomzig, unpublished data) challenged with 263K
scrapie agent, providing further evidence that scrapieinfected
hamsters are relevant model animals mimicking key
features of the spread of infection through the body in ovine
TSEs (McBride et al., 2001; van Keulen et al., 2000, 2002).
Here, we report on PrPTSE deposition in muscle tissue of
hamsters and mice infected experimentally with BSE or
variant Creutzfeldt–Jakob disease (vCJD) agents. We have
examined muscles from terminally ill rodents inoculated
intracerebrally with hamster-adapted BSE (isolate BSE-H;
Thomzig et al., 2004b), mouse-adapted BSE (isolate 6PB1;
Maignien et al., 1999) or mouse-adapted vCJD(Cervenakova
et al., 2003) for the presence of PrPTSE. The recipients
inoculated with these TSE agents [50 or 30 ul 1 % (w/v)
brain homogenate from terminally diseased hamsters or
mice, respectively] showed incubation times of 287±28,
142±9 and 140±11 days until terminal disease (expressed
as means±SD), respectively. Western blot and immunohistochemical
detection of PrPTSE were performed as
described previously (Thomzig et al., 2004a), with the
exception that, in murine samples, we used the monoclonal
anti-PrP antibody ICSM-18 (D-Gen Ltd). The specificity of
this antibody for the detection of PrP has been demonstrated
previously (White et al., 2003). Our study revealed
widespread accumulation of PrPTSE in muscle tissue from
animals in all three experimental models (Table 1).
Staining intensities for PrPTSE in muscle samples from
BSE-infected hamsters corresponding to 20–50 mg tissue
(Fig. 1a) and in muscle specimens from BSE- or vCJDinfected
mice corresponding to 15–40 mg tissue (Fig. 1b)
reached levels similar to or higher than those observed in the
positive-control samples, i.e. in muscle specimens from
uninfected animals that were spiked with 5x10-6 g BSEinfected
hamster brain or 5x10-5 g BSE- (not shown) or
vCJD-infected mouse brain.
The topology of PrPTSE deposition in myocytes of BSEinfected
hamsters was consistent with that described for
scrapie-infected hamsters (Thomzig et al., 2004a) and sheep 3These authors contributed equally to this work.
0008-1277 G 2006 SGM Printed in Great Britain 251
Journal of General Virology (2006), 87, 251–254 DOI 10.1099/vir.0.81277-0
Table 1. Detection of PrPTSE in muscles of rodents infected intracerebrally with BSE or vCJD agents
Data represent the number of animals with PrPTSE in muscles/number of animals examined. Control, specimen from uninfected animals.
ND, Not done.
Muscle sample Hamster-adapted BSE Mouse-adapted BSE Mouse-adapted vCJD
Western blotting Immunohistochemistry Western blotting Western blotting
Control BSE Control BSE Control BSE Control vCJD
M. biceps femoris (hindlimb) 0/3 5/5 ND ND 0/2 1/4 0/2 2/4
M. tibialis cranialis (hindlimb) 0/3 5/5 0/2 2/2 0/2 0/4 0/2 1/4
M. triceps brachii (forelimb) 0/3 5/5 ND ND 0/2 2/4 0/2 2/4
M. extensor carpi radialis (forelimb) 0/3 5/5 ND ND 0/2 0/4 0/2 2/4
M. trapezius (shoulder) 0/3 5/5 ND ND 0/2 1/4 0/2 1/4
M. masseter (head) 0/3 5/5 0/2 2/2 0/2 0/4 0/2 2/4
M. psoas major (back) 0/3 5/5 0/2 2/2 0/2 2/4 0/2 1/4
M. lingualis (tongue) 0/3 5/5 0/2 2/2 0/2 1/4 0/2 0/4
Heart 0/3 5/5* ND ND 0/2 0/4 0/2 0/4
*Only weak signals for PrPTSE.
Fig. 1. (a) Western blot detection of PrP27–30, the protease-resistant core of PrPTSE, extracted from muscles and sciatic
nerve of terminally ill hamsters infected intracerebrally with hamster-passaged BSE agent. Lanes 1 and 8, proteinase
K-digested brain homogenate from BSE-infected hamsters, containing 5x10-7 g brain tissue; lanes 2–7 and 9, skeletal
muscles from hindlimb (2, 3), forelimb (4, 5), shoulder (6), head (7) and back (9); lane 10, tip of tongue; lane 11, heart; lane
12, sciatic nerve; lane 13, skeletal muscles from uninfected control hamster, spiked before extraction with 5x10-6 g brain
homogenate from BSE-infected hamsters; lane 14, skeletal muscle from an uninfected control hamster. All examined hamster
muscle samples corresponded to 20–50 mg tissue. (b) Western blot detection of PrP27–30 extracted from muscles of
terminally ill mice infected intracerebrally with mouse-adapted BSE (lanes 1–3) and vCJD (lanes 4–6) agent. Lane 1, muscle
from hindlimb; lanes 2–3, different muscles from forelimb; lane 4, muscle from hindlimb; lanes 5–6, different muscles from
forelimb; lane 7, skeletal muscle from uninfected control mouse, spiked before extraction with 5x10-5 g brain homogenate
from vCJD-infected mice; lanes 8–9, skeletal muscles from uninfected control mouse. All examined murine muscle samples
corresponded to 15–40 mg tissue. (c, d) Location of PrPTSE in lingual muscle fibres of BSE-infected hamsters visualized by
PrP immunohistochemistry. Brownish granular immunostaining demonstrates PrPTSE deposition predominantly in the region of
the fibre surface (c, arrowheads), but also scattered within myocytes (d, arrows). Insets show higher magnification of muscle
fibres marked by an asterisk. Bars, 20 um.
252 Journal of General Virology 87
A. Thomzig and others
with scrapie (Andre´oletti et al., 2004): in the lingual muscle,
PrPTSE was found in individual muscle fibres predominantly
in the region of the fibre surface (Fig. 1c), but also scattered
within myocytes (Fig. 1d).
Muscle extracts from terminally ill C57Bl mice infected
intracerebrally with the 6PB1 BSE agent showed substantial
PrPTSE deposition in many of the skeletal muscles examined
(Fig. 1b, lanes 1–3). We also observed similar deposits of
PrPTSE in muscles of C57Bl mice inoculated with the mouseadapted
vCJD agent (Fig. 1b, lanes 4–6). Positive muscle
specimens from BSE- or vCJD-infected mice showed an
approximately 500- to 1000-fold lower concentration of
PrPTSE than in the brain, similar to what has been reported
previously for scrapie-infected mice (Bosque et al., 2002).
All of this special interest in muscle involvement derives
from the fact that meat, and particularly beef, is a staple of
the human diet and would thus constitute a public-health
risk if it were found to be infectious in cattle or small
ruminants like sheep and goats (European Food Safety
Authority, 2005) with TSEs. Studies to investigate the
involvement of muscle in the pathogenesis of TSEs date
back to a report on scrapie infectivity in the muscle of an
affected goat in 1962 (Pattison & Millson, 1962) that
remained, for decades, the sole successful transmission
attempt. However, increasingly sensitive methodologies
developed within the past few years have documented the
presence of PrPTSE in rodents infected experimentally with
various strains of TSE and, as noted above, in both natural
and experimental scrapie infections of sheep (Table 2).
Although the detection of PrPTSE cannot be equated directly
to the presence of infectious TSE agent, a consistent association
between PrPTSE and infectivity was found when these
two parameters were assayed in muscles of scrapie-infected
hamsters and mice (Bosque et al., 2002; Thomzig et al.,
2004a).
A large pathogenesis study of BSE-infected cattle in the UK
failed to detect infectivity in muscle tissue at any time during
the course of the disease (European Commission, 2002).
However, at each time point, only approximately 0.5 g
muscle was bioassayed, representing a minute sample of the
>500 kg muscle from the slaughtered cattle, so that any lack
of homogeneity in the distribution of infectivity could easily
have led to a negative result. Moreover, a statistical analysis
of the sampling shows that, even with a homogeneous
distribution, there was only a 50% chance of detecting one
infectious dose. This caveat to pronouncing BSE muscle to
be non-infectious was fully appreciated by the authors of
the study, but may not have been widely understood by its
interpreters.
It must be borne in mind that the data of this study have
been obtained fromintracerebrally infected rodents, whereas
natural transmission of scrapie and BSE probably occurs via
the oral route. However, the relevance of the reported
findings is corroborated by the detection of PrPTSE in
muscles of sheep and hamsters after both oral and intracerebral
infection with scrapie (Andre´oletti et al., 2004;
Table 2. Reports on the detection of TSE infectivity or PrPTSE in muscles
sCJD, Sporadic Creutzfeldt–Jakob disease; ND, not done.
Infected species Infectivity bioassay
(indicator animals)
PrPTSE Reference
Scrapie
Goat + (goat) ND Pattison & Millson (1962)
Mouse + (mouse) + Bosque et al. (2002)
Hamster ND + Bartz et al. (2003)
Hamster ND + Thomzig et al. (2003)
Hamster + (hamster) + Thomzig et al. (2004a)
Sheep ND + Andre´oletti et al. (2004)
Sheep ND + Casalone et al. (2005)
TME
Hamster ND + Bartz et al. (2003)
Hamster ND + Mulcahy et al. (2004)
BSE
Hamster ND + This paper
Mouse ND + This paper
sCJD
Human ND + Glatzel et al. (2003)
Human ND + Kovacs et al. (2004)
vCJD
Mouse ND + This paper
http://vir.sgmjournals.org 253
BSE- and vCJD-associated prion protein in muscles
Thomzig et al., 2004a; A. Thomzig, unpublished data for
intracerebrally infected hamsters). If rodents challenged
intracerebrally with BSE or vCJD agents mirror the pathophysiology
of muscle targeting in ovine BSE and human
vCJD to a similar extent, this would point to muscles as
reservoirs for infectivity in these diseases. Thus, our findings
emphasize the need for further assessment of the risks for
public health that may result from prions in skeletal muscle
(Bosque et al., 2002), either by oral exposure to BSE in ruminant
meat products or by surgical exposure to human vCJD.
This is the first report showing deposition of BSE- and
vCJD-associated PrPTSE in muscle tissue. Kinetic studies in
intracerebrally and perorally challenged animals will be
necessary to further reveal whether BSE- and vCJD-related
agents target muscle tissue prior to the onset of clinical
symptoms after either highly invasive inoculation or alimentary
infection.
Acknowledgements
The authors would like to thank Christine Kratzel and Marion Joncic
for the dissection of muscle samples from hamsters at the Robert Koch-
Institut and Angela Valenzano for collecting muscles from mice at
the Istituto Superiore di Sanita` . The skilful assistance of Maurizio
Bonanno, Nicola Bellizi and Mei Lu in animal husbandry is gratefully
acknowledged. This work was supported in part by grants from the
German Bundesministerium fu¨r Bildung und Forschung.
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