your wasting your time there bse-tester with some here that cannot see the forest due to the trees i.e. $$$ you see, the srm regulations will have to change when they document BSE in muscle tissue, so why document it.
AS with blood, where they once thought it too be free of TSE infection, but was wrong there as well, give em time, and more sensitive testing and they will find BSE in muscle tissue of cattle, as they have found with other species once thought to be free of the TSE agent in muscle i.e. humans, sheep, deer, and now in cattle, they have already found it in the peripheral nerves (sciatic nerve, tibial nerve, vagus nerve), this too was once thought to be not possible, as testing comes forward that is much more sensitive, the tide will turn again. and once again these na-sayers will be proven wrong again. it's happened time and time again with human and animal TSEs. ...TSS
Alice Trinkl, News Director
Source: Jennifer O'Brien
[email protected]
415-476-2557
14 March 2002
UCSF prion finding in mice leads team to urge similar study in cattle
University of California, San Francisco researchers have made a finding regarding prions in mice that they say warrants similar study in cattle.
In their investigation, published in the March 19 issue of Proceedings of the National Academy of Sciences, the researchers report that mice exposed to the lethal prion (PREE-on) pathogen develop high levels of the infectious agent in some skeletal muscle. Until now, scientists have thought skeletal muscle was neither susceptible to high levels of infection, nor infectious. The researchers have not investigated whether high levels of prions amass in the skeletal muscle of cattle, sheep, deer or elk infected with prions. Substantial evidence has suggested that prions only accumulate at high levels in brain, spinal cord and lymphatic tissue. As a result, Great Britain – where the epidemic of the prion disease known as bovine spongiform encephalopathy (BSE)1. developed in cattle in the mid 1980s – and countries of the European Union have banned brain, spinal cord and lymphatic tissues from the human food supply.2. This measure is the most extreme step these countries have taken in light of the considerable evidence that consumption of BSE-contaminated beef products causes a human form of the neurodegenerative disease, known as new variant Creutzfeldt-Jakob disease (nvCJD).
However, researchers have not reported whether the skeletal muscle of BSE-infected cattle transmits to mammals. And the UCSF researchers say that in light of their finding in mice this investigation should be carried out in both livestock and game.
“Whether prions accumulate in the skeletal muscle of other animals remains to be established. But our findings indicate the need to carry out a comprehensive and systematic investigation of the distribution of prions in the skeletal muscle of animals who develop prion diseases,” says the senior author of the study, Stanley B. Prusiner,3. MD, UCSF professor of neurology and biochemistry and director of the UCSF Institute for Neurodegenerative Diseases.
Notably, no cases of BSE have been reported in the United States,4. which, since 1989, has banned the importation of cattle, cattle products and sheep from Great Britain and all other European countries with cases of BSE, and which maintains strict surveillance of cattle within its borders. And in Great Britain strong measures to curtail the incidence of BSE have significantly decreased the incidence of BSE. (In Great Britain, cases of BSE have dropped from 36,680 in 1992 to fewer than 1,500 cattle in 2000 and are much lower in all European countries except Portugal and Ireland.) Moreover, the rate of transmission of BSE to humans appears to be low, presumably due to a species barrier. (As of Feb. 4, 2002, 106 had died of nvCJD and eight were still living with the disease in Great Britain,5. and three people had died in France. One person died of CJD in Ireland but as he lived in England for a time, the source of his disease is unclear.) In the UCSF study, the level of prions varied significantly among the different skeletal muscles of the mice, reaching high levels only in the hind limb. The researchers do not know what factors cause the variation. Nor do they know if the distribution would be expected to be the same within the skeletal muscles of other species or among different strains of prions.
“It’s possible that if prions do develop at high levels in the skeletal muscle of other animals that the distribution within the muscles varies even within breeds, varieties and lines of a species, as well as with the strain of prions,” says the lead author of the study, Patrick J. Bosque , MD, assistant professor of neurology at the University of Colorado Health Sciences Center. Bosque conducted the work while he was a postdoctoral fellow in the Prusiner laboratory.
While there is no clear evidence that deer and elk with chronic wasting disease or sheep with different strains of scrapie have ever transmitted these prion diseases to humans, the researchers say the investigation should extend to these animals, as well.
In their study, the researchers note that several variables related to the nature of prions could affect whether the findings in mice would be seen in livestock or humans. In the mouse study, particular strains of mouse prions were studied, and the animals were infected through inoculation.
However, the efficiency with which various prion strains accumulate in muscle may vary. Moreover, the efficiency with which various strains cross the “species barrier,” a barrier that precludes the transmission of most diseases between species, also varies.
Finally, oral transmission of prions – the mode by which transmission would naturally occur – is inefficient compared to transmission by inoculation.
To determine whether cattle develop prions in their skeletal muscle, says Bosque, scientists need to investigate the various skeletal muscle groups of slaughtered animals suspected of having BSE, using highly sensitive and quantitative techniques.
Obtaining consistent results from some 50 BSE-infected cattle representing the various breeds would probably be sufficient to draw a conclusion, he says.
Possible new diagnostic tool Bosque says that if high levels of prions propagate and accumulate early in muscle, conducting muscle biopsies on live animals could prove an effective diagnostic tool in asymptomatic animals. Muscle biopsy might even be used to diagnose humans suspected of having any of the various forms of prion diseases, he says. As brain biopsies are a high-risk procedure and are not 100 percent accurate, most patients are diagnosed definitively only on autopsy. The discovery that prions propagate in the skeletal muscle of mice could put researchers a step closer to understanding how the inscrutable prion carries out its scourge. The only known component of the prion (PrPSc)6. is an aberrant form of a normal protein, known as the cellular prion protein (PrPC), which exists in a healthy state in humans and many animals. The protein becomes lethal when some of the spirals that make up a portion of the protein molecule lose their normal conformation and flatten into so-called beta sheets. (Prions can arise spontaneously, be inherited through a genetic mutation or develop through infectious transmission.) Once the conversion occurs, the prion moves on to normal prion proteins, pinning and flattening their spirals, as well. The accumulation and aggregation of the flattened beta sheets within a cell leads to structural damage that causes cell degeneration. The destruction spreads quickly through the brain, generally causing death in less than a year. One explanation for prions propagating at high levels only in the brain and lymphatic tissue has been that there simply might be more normal prion protein in these regions, and therefore more protein that would be susceptible to conversion. Another possibility has been that some essential, as-yet-unidentified, molecules other than the prion might be involved in the process of converting normal prion proteins, and that these other factors are found only in sufficient quantity in brain, spinal cord and lymphatic tissues. The current study sheds light on these possibilities. In their study, the researchers inoculated the brains of normal mice with either of two strains of mouse prions, known as the Me7 and the Rocky Mountain Laboratory strains. They discovered that the animals did, in fact, develop high levels of prions in some skeletal muscle, specifically in muscles from the hind limb. Then, to determine whether prions were actually produced in the muscle or merely migrated and accumulated there, the team developed mice genetically engineered to express either mouse or Syrian hamster PrPC (normal cellular prion protein) exclusively in hind limb muscle. When they inoculated the muscles with prions, the mice developed high levels of new prions in that muscle. In contrast, when prions were injected into mice genetically engineered to express PrPC exclusively in liver cells, the liver cells did not develop high levels of new prions. This finding demonstrates, the researchers say, that factors other than the amount of normal prion protein expressed in a given tissue contribute to prion propagation. And this conclusion supports the possibility that only certain tissues contain these elusive factors. Prusiner has long proposed that the prion requires the help of an as-yet-unidentified “protein X” to latch on to the normal, corkscrew-shaped tendrils of the prion protein and pin them flat. “One possibility,” says Prusiner, “is that protein X is not expressed, or is only expressed at very low levels, in certain muscle tissues. And that could be why we are seeing this wide disparity from brain to muscle to liver, as well as between one muscle group and another.” Moreover, the finding could explain why previous studies have not detected high levels of prions in skeletal muscle, the researchers say. “Previous studies might not have been done using sensitive enough assays. But in other studies scientists perhaps looked at the wrong skeletal muscle,” says Bosque.
Other co-authors of the UCSF study were Chongusuk Ryou, PhD, postdoctoral scholar in the Prusiner lab; Glenn Telling, PhD, formerly a postdoctoral scholar in the Prusiner lab and currently associate professor of microbiology, immunology and molecular genetics, and neurology, and a member of the Sanders Brown Center on Aging, University of Kentucky; David Peretz, PhD, a postdoctoral fellow in the Prusiner lab and a member of the UCSF Institute of Neurodegenerative Diseases, and Giuseppe Legname, PhD, UCSF adjunct assistant professor of neurology and a member of the UCSF Institute of Neurodegenerative Diseases.
The study was funded by grants from the National Institutes of Health and by a gift from the G. Harold and Leila Y. Mathers Foundation.
###
Link to normal prion protein image and photograph of Stanley B. Prusiner, MD: http://pub.ucsf.edu/imagedb/imsearch.php?keywords=prusiner
1. The outbreak of BSE in Great Britain is believed to have occurred as a result of cattle being fed ground sheep offal that was infected with scrapie, a prion disease.
2.http://www.fda.gov/oc/opacom/hottopics/bse.html
3. Prusiner won the Nobel Prize in Physiology or Medicine in 1997 for discovering that a class of neurodegenerative diseases known as spongiform encephalopathies was caused by prions.
4.http://www.cdc.gov/travel/madcow.htm
5. http://www.cjd.ed.ac.uk/index.htm
6. The prion, for proteinacious infectious particle, is unlike any other disease –causing agent. All other pathogens – bacteria, viruses, protozoans or fungi – contain nucleic acid that allows them to transmit their pathogenic code.
The prion, in contrast, is devoid of nucleic acid, and instead in composed of amino acids. Prion diseases are rare in humans. One in a million people each year develop a sporadic form of the condition, known as Creutzfeldt-Jakob disease (CJD), for which there is no known cause.
Approximately 5 to 15 percent of all cases are inherited. The disease can also occur in humans through infectious transmission, from ingestion of prion-contaminated meats and contamination through biological and pharmaceutical products, as seen with the development of new variant CJD in 1990. Other infectious prion diseases include kuru, which arose among New Guinea natives engaged in ritualistic cannibalism, and iatrogenic CJD, caused by prion-contaminated cadaveric growth hormone and dura mater grafts. Prion diseases also develop in sheep, deer, elk and mink.
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Link to normal prion protein image and photograph of Stanley B. Prusiner, MD
==================
Journal of Virology, November 2001, p. 10106-10112, Vol. 75, No. 21 0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.21.10106-10112.2001 Long-Term Subclinical Carrier State Precedes Scrapie Replication and Adaptation in a Resistant Species: Analogies to Bovine Spongiform Encephalopathy and Variant Creutzfeldt-Jakob Disease in Humans Richard Race, Anne Raines, Gregory J. Raymond, Byron Caughey, and Bruce Chesebro* Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, Hamilton, Montana 59840 Received 24 May 2001/Accepted 31 July 2001 9/13/2005
Page 9 of 17 Cattle infected with bovine spongiform encephalopathy (BSE) appear to be a reservoir for transmission of variant Creutzfeldt-Jakob disease (vCJD) to humans. Although just over 100 people have developed clinical vCJD, millions have probably been exposed to the infectivity by consumption of BSE-infected beef. It is currently not known whether some of these individuals will develop disease themselves or act as asymptomatic carriers of infectivity which might infect others in the future. We have studied agent persistence and adaptation after cross-species infection using a model of mice inoculated with hamster scrapie strain 263K. Although mice inoculated with hamster scrapie do not develop clinical disease after inoculation with 10 million hamster infectious doses, hamster scrapie infectivity persists in brain and spleen for the life span of the mice. In the present study, we were surprised to find a 1-year period postinfection with hamster scrapie where there was no evidence for replication of infectivity in mouse brain. In contrast, this period of inactive persistence was followed by a period of active replication of infectivity as well as adaptation of new strains of agent capable of causing disease in mice. In most mice, neither the early persistent phase nor the later replicative phase could be detected by immunoblot assay for protease-resistant prion protein (PrP). If similar asymptomatic carriers of infection arise after exposure of humans or animals to BSE, this could markedly increase the danger of additional spread of BSE or vCJD infection by contaminated blood, surgical instruments, or meat. If such subclinical carriers were negative for protease-resistant PrP, similar to our mice, then the recently proposed screening of brain, tonsils, or other tissues of animals and humans by present methods such as immunoblotting or immunohistochemistry might be too insensitive to identify these individuals. * Corresponding author. Mailing address: Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, 903 South Fourth St., Hamilton, MT 59840-2999. Phone: (406) 363-9354. Fax: (406) 363-9286. E-mail:
[email protected] . Journal of Virology, November 2001, p. 10106-10112, Vol. 75, No. 21 0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.21.10106-10112.2001 http://jvi.asm.org/cgi/content/abstract/75/21/10106 From: TSS () Subject: PrPSc distribution of a natural case of bovine spongiform encephalopathy Date: August 8, 2005 at 12:28 pm PST PrPSc distribution of a natural case of bovine spongiform encephalopathy Yoshifumi Iwamaru, Yuka Okubo, Tamako Ikeda, Hiroko Hayashi, Mori- kazu Imamura, Takashi Yokoyama and Morikazu Shinagawa Priori Disease Research Center, National Institute of Animal Health, 3-1-5 Kannondai, Tsukuba 305-0856 Japan
[email protected] Abstract Bovine spongiform encephalopathy (BSE) is a disease of cattle that causes progressive neurodegeneration of the central nervous system. Infectivity of BSE agent is accompanied with an abnormal isoform of prion protein (PrPSc). The specified risk materials (SRM) are tissues potentially carrying BSE infectivity. The following tissues are designated as SRM in Japan: the skull including the brain and eyes but excluding the glossa and the masse- ter muscle, the vertebral column excluding the vertebrae of the tail, spinal cord, distal illeum. For a risk management step, the use of SRM in both animal feed or human food has been prohibited. However, detailed PrPSc distribution remains obscure in BSE cattle and it has caused controversies
about definitions of SRM. Therefore we have examined PrPSc distribution in a BSE cattle by Western blotting to reassess definitions of SRM. The 11th BSE case in Japan was detected in fallen stock surveillance. The carcass was stocked in the refrigerator. For the detection of PrPSc, 200 mg of tissue samples were homogenized. Following collagenase treatment, samples were digested with proteinase K. After digestion, PrPSc was precipitated by sodium phosphotungstate (PTA). The pellets were subjected to Western blotting using the standard procedure. Anti-prion protein monoclonal antibody (mAb) T2 conjugated horseradish peroxidase was used for the detection of PrPSc. PrPSc was detected in brain, spinal cord, dorsal root ganglia, trigeminal ganglia, sublingual ganglion, retina. In addition, PrPSc was also detected in the peripheral nerves (sciatic nerve, tibial nerve, vagus nerve). Our results suggest that the currently accepted definitions of SRM in 9/13/2005
179
Page 10 of 17 BSE cattle may need to be reexamined. T. Kitamoto (Ed.)
PRIONS
Food and Drug Safety
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ALSO from the International Symposium of Prion Diseases held in Sendai, October 31, to November 2, 2004; Bovine spongiform encephalopathy (BSE) in Japan snip... "Furthermore, current studies into transmission of cases of BSE that are atypical or that develop in young cattle are expected to amplify the BSE prion" NO. Date conf. Farm Birth place and Date Age at diagnosis 8. 2003.10.6. Fukushima Tochigi 2001.10.13. 23 9. 2003.11.4. Hiroshima Hyogo 2002.1.13. 21 Test results # 8b, 9c cows Elisa Positive, WB Positive, IHC negative, histopathology negative b = atypical BSE case c = case of BSE in a young animal b,c, No PrPSc on IHC, and no spongiform change on histology International Symposium of Prion Diseases held in Sendai, October 31, to November 2, 2004. Tetsuyuki Kitamoto Professor and Chairman Department of Prion Research Tohoku University School of Medicine 2-1 SeiryoAoba-ku, Sendai 980-8575, JAPAN TEL +81-22-717-8147 FAX +81-22-717-8148 e-mail;
[email protected] Symposium Secretariat Kyomi Sasaki TEL +81-22-717-8233 FAX +81-22-717-7656 e-mail:
[email protected]ns.tohoku.ac.ip ================================= 9/13/2005
Prions in Skeletal Muscles of Deer with Chronic Wasting Disease
Rachel C. Angers,1* Shawn R. Browning,1*† Tanya S. Seward,2 Christina J.
Sigurdson,4‡ Michael W. Miller,5 Edward A. Hoover,4 Glenn C. Telling1,2,3§
1Department of Microbiology, Immunology and Molecular Genetics, 2Sanders
Brown Center on Aging, 3Department of Neurology, University of Kentucky,
Lexington, KY 40536, USA. 4Department of Microbiology, Immunology and
Pathology, Colorado State University, Fort Collins, CO 80523, USA. 5Colorado
Division of Wildlife, Wildlife Research Center, Fort Collins, CO 80526, USA.
*These authors contributed equally to this work.
†Present address: Department of Infectology, Scripps Research Institute,
5353 Parkside Drive, RF-2, Jupiter, Florida, 33458, USA.
‡Present address: Institute of Neuropathology, University of Zurich,
Schmelzbergstrasse 12, 8091 Zurich, Switzerland.
§To whom correspondence should be addressed: E-mail:
[email protected]
Prions are transmissible proteinaceous agents of mammals that cause fatal
neurodegenerative diseases of the central nervous system (CNS). The presence
of infectivity in skeletal muscle of experimentally infected mice raised the
possibility that dietary exposure to prions might occur through meat
consumption (1). Chronic wasting disease (CWD), an enigmatic and contagious
prion disease of North American cervids, is of particular concern. The
emergence of CWD in an increasingly wide geographic area and the
interspecies transmission of bovine spongiform encephalopathy (BSE) to
humans as variant Creutzfeldt Jakob disease (vCJD) have raised concerns
about zoonotic transmission of CWD.
To test whether skeletal muscle of diseased cervids contained prion
infectivity, Tg(CerPrP)1536 mice (2) expressing cervid prion protein
(CerPrP), were inoculated intracerebrally with extracts prepared from the
semitendinosus/semimembranosus muscle group of CWD-affected mule deer or
from CWD-negative deer. The availability of CNS materials also afforded
direct comparisons of prion infectivity in skeletal muscle and brain. All
skeletal muscle extracts from CWD-affected deer induced progressive
neurological dysfunction in Tg(CerPrP)1536 mice with mean incubation times
ranging between 360 and ~490 d, whereas the incubation times of prions from
the CNS ranged from ~230 to 280 d (Table 1). For each inoculation group, the
diagnosis of prion disease was confirmed by the presence of PrPSc in the
brains of multiple infected Tg(CerPrP)1536 mice (see supporting online
material for examples). In contrast, skeletal muscle and brain material from
CWD-negative deer failed to induce disease in Tg(CerPrP)1536 mice (Table 1)
and PrPSc was not detected in the brains of sacrificed asymptomatic mice as
late as 523 d after inoculation (supporting online material).
Our results show that skeletal muscle as well as CNS tissue of deer with CWD
contains infectious prions. Similar analyses of skeletal muscle BSE-affected
cattle did not reveal high levels of prion infectivity (3). It will be
important to assess the cellular location of PrPSc in muscle. Notably, while
PrPSc has been detected in muscles of scrapie-affected sheep (4), previous
studies failed to detect PrPSc by immunohistochemical analysis of skeletal
muscle from deer with natural or experimental CWD (5, 6). Since the time of
disease onset is inversely proportional to prion dose (7), the longer
incubation times of prions from skeletal muscle extracts compared to matched
brain samples indicated that prion titers were lower in muscle than in CNS
where infectivity titers are known to reach high levels. Although possible
effects of CWD strains or strain mixtures on these incubation times cannot
be excluded, the variable 360 to ~490 d incubation times suggested a range
of prion titers in skeletal muscles of CWD-affected deer. Muscle prion
titers at the high end of the range produced the fastest incubation times
that were ~30% longer than the incubation times of prions from the CNS of
the same animal. Since all mice in each inoculation group developed disease,
prion titers in muscle samples producing the longest incubation times were
higher than the end point of the bioassay, defined as the infectious dose at
which half the inoculated mice develop disease. Studies are in progress to
accurately assess prion titers.
While the risk of exposure to CWD infectivity following consumption of
prions in muscle is mitigated by relatively inefficient prion transmission
via the oral route (8), these
results show that semitendinosus/semimembranosus muscle, which is likely to
be consumed by humans, is a significant source of prion infectivity. Humans
consuming or handling meat from CWD-infected deer are therefore at risk to
prion exposure.
References and Notes
snip...end
=================
(American Journal of Pathology. 2006;168:927-935.)
© 2006 American Society for Investigative Pathology
Detection and Localization of PrPSc in the Skeletal Muscle of Patients with Variant, Iatrogenic, and Sporadic Forms of Creutzfeldt-Jakob Disease
Alexander H. Peden, Diane L. Ritchie, Mark W. Head and James W. Ironside
From the National Creutzfeldt-Jakob Disease Surveillance Unit and Division of Pathology, School of Molecular and Clinical Medicine, University of Edinburgh, Western General Hospital, Edinburgh, United Kingdom
Variant Creutzfeldt-Jakob disease (vCJD) differs from other human prion diseases in that the pathogenic prion protein PrPSc can be detected to a greater extent at extraneuronal sites throughout the body, principally within lymphoid tissues. However, a recent study using a high-sensitivity Western blotting technique revealed low levels of PrPSc in skeletal muscle from a quarter of Swiss patients with sporadic CJD (sCJD). This posed the question of whether PrPSc in muscle could also be detected in vCJD, sCJD, and iatrogenic (iCJD) patients from other populations. Therefore, we have used the same high-sensitivity Western blotting technique, in combination with paraffin-embedded tissue blotting, to screen for PrPSc in muscle tissue specimens taken at autopsy from 49 CJD patients in the United Kingdom. These techniques identified muscle PrPSc in 8 of 17 vCJD, 7 of 26 sCJD, and 2 of 5 iCJD patients. Paraffin-embedded tissue blotting analysis showed PrPSc in skeletal muscle in localized anatomical structures that had the morphological and immunohistochemical characteristics of nerve fibers. The detection of PrPSc in muscle tissue from all forms of CJD indicates the possible presence of infectivity in these tissues, suggesting important implications for assessing the potential risk of iatrogenic spread via contaminated surgical instruments.
TSS