Distinct Molecular Signature of Bovine Spongiform Encephalopathy Prion in Pigs
To the Editor: In a recent article in Emerging Infectious Diseases, Espinosa et al. (1) investigated the porcine transmission barrier to infection with bovine and ovine transmissible spongiform encephalopathies (TSEs) in transgenic mice expressing the porcine prion protein. Bovine spongiform encepatholopathy of the classical type (BSE) derived from cattle and sheep, as well as atypical scrapie, transmitted to these mice, although with different effi - ciencies. Whereas sheep BSE showed a 100% attack rate, cattle BSE and atypical scrapie showed a higher transmission barrier in the fi rst passage. Unexpectedly, the electrophoretic profi le of the proteinase K–resistant prion protein (PrPres) in Western immunoblot (WB) analysis of all 3 TSEs shifted toward a common signature upon transmission. This was a 3-band pattern with a predominant monoglycosylated PrPres moiety and, therefore, clearly differed from those of the BSE and atypical scrapie inocula. The authors speculated that the porcine cellular prion protein (PrPc) might allow only for few options as it changes its conformation to the disease-associated prion protein. However, whether this effect is attributable to the porcine PrPc transgene or to the genetic background of the mouse model remains unknown.
To our knowledge, BSE has been successfully transmitted to pigs in 1 study, but WB data were not reported (2). We had access to central nervous system tissues of 1 of these animals (kindly provided by the Veterinary Laboratories Agency TSE Archive, Weybridge, UK) and aimed at assessing whether a similar effect occurs when cattle BSE affects pigs. Our results show a PrPres signature in BSEinfected pigs similar to that described for the porcine PrPc transgenic mice and clearly different from that in cattle (Figure). These fi ndings support the fi nding by Espinosa et al. that the molecular shift most likely was due to intrinsic properties of the porcine PrPc. Therefore, in this respect the mouse model appears to refl ect the situation in the pig.
BSE prions are considered to transmit to other species, such as exotic ruminants, cats, macaques, humans, sheep, and goats, without any obvious alterations of the molecular phenotype (3,4). Our study provides evidence that the molecular phenotype of classical BSE also may shift upon genuine interspecies transmission. Attempts to discriminate BSE from other prion diseases in humans and animals often rely at fi rst on the analysis of the PrPres signature in WB. Consequently, the situation described in our study complicates the interpretation of such disease surveillance data to assess public health risks for animal TSEs. Whether this applies to other TSEs and species remains to be addressed.
Figure. Molecular signature of bovine spongiform encephalopathy (BSE) in pigs. A) Comparative Western immunoblot analysis of the proteinase K–resistant core fragment (PrPres) of the pathologic prion protein in BSE in cattle and in an experimentally BSE-infected pig using the monoclonal antibody 6H4 (Prionics, Schlieren, Switzerland). B) Average relative intensities of the diglycosylated (black bars), monoglycosylated (gray bars), and unglycosylated (white bars) PrPres moieties as determined by the Quantity One software package (Bio-Rad, Rheinach, Switzerland). Data are based on 4 independent runs, and error bars indicate SD. Note the different extent of PrPres glycosylation in bovine and porcine BSE. By contrast, the molecular masses of the unglycosylated PrPres were similar and scored 18.89 kDa (SD ± 0.28 kDa) and 18.90 kDa (SD ± 0.42 kDa) in bovine and porcine BSE, respectively. Molecular masses of the standards are indicated on the left in panel A.
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 16, No. 1, January 2010
http://www.cdc.gov/eid/content/16/1/pdfs/164.pdf
Torsten Seuberlich and Andreas Zurbriggen Author affi liation: University of Berne, Berne, Switzerland DOI: 10.3201/eid1601.091104
References
1. Espinosa JC, Herva ME, Andreoletti O, Padilla D, Lacroux C, Cassard H, et al. Transgenic mice expressing porcine prion protein resistant to classical scrapie but susceptible to sheep bovine spongiform encephalopathy and atypical scrapie. Emerg Infect Dis. 2009;15:1214–21. DOI: 10.3201/eid1508.081218
2. Wells GA, Hawkins SA, Austin AR, Ryder SJ, Done SH, Green RB, et al. Studies of the transmissibility of the agent of bovine spongiform encephalopathy to pigs. J Gen Virol. 2003;84:1021–31.
3. Collinge J, Sidle KC, Meads J, Ironside J, Hill AF. Molecular analysis of prion strain variation and the aetiology of ‘new variant’ CJD. Nature. 1996;383:685–90.
4. Hill AF, Desbruslais M, Joiner S, Sidle KC, Gowland I, Collinge J, et al. The same prion strain causes vCJD and BSE. Nature. 1997;389:448–50.
Address for correspondence: Torsten Seuberlich, NeuroCentre, Reference Laboratory for TSE in Animals, University of Berne, Bremgartenstrasse 109a, CH-3001 Berne, Switzerland; email: torsten.seuberlich@itn.unibe.ch
http://www.cdc.gov/eid/content/16/1/pdfs/164.pdf
Thursday, October 15, 2009 Transmissibility studies of vacuolar changes in the rostral colliculus of pigs Research article Open Access T
Transmissibility studies of vacuolar changes in the rostral colliculus of pigs
Timm Konold*1,2, John Spiropoulos1, Melanie J Chaplin3, Leigh Thorne3, Yvonne I Spencer1, Gerald AH Wells1 and Steve AC Hawkins1 Address: 1Department of Pathology, Veterinary Laboratories Agency Weybridge, Woodham Lane, Addlestone, UK, 2Royal Veterinary College, Infection and Immunity Research Group, North Mymms, Hatfield, UK and 3Department of Molecular Pathogenesis and Genetics, Veterinary Laboratories Agency Weybridge, Woodham Lane, Addlestone, UK Email: Timm Konold* - t.konold@vla.defra.gsi.gov.uk; John Spiropoulos - j.spiropoulos@vla.defra.gsi.gov.uk; Melanie J Chaplin - m.j.chaplin@vla.defra.gsi.gov.uk; Leigh Thorne - l.thorne@vla.defra.gsi.gov.uk; Yvonne I Spencer - y.i.spencer@vla.defra.gsi.gov.uk; Gerald AH Wells - g.a.h.wells@vla.defra.gsi.gov.uk; Steve AC Hawkins - s.a.c.hawkins@vla.defra.gsi.gov.uk * Corresponding author
Abstract Background:
Histopathological examinations of brains from healthy pigs have revealed localised vacuolar changes, predominantly in the rostral colliculus, that are similar to the neuropil vacuolation featured in the transmissible spongiform encephalopathies and have been described in pigs challenged parenterally with the agent causing bovine spongiform encephalopathy (BSE). Feedstuff containing BSE-contaminated meat and bone meal (MBM) may have been fed to pigs prior to the ban of mammalian MBM in feed of farmed livestock in the United Kingdom in 1996, but there is no evidence of the natural occurrence of a transmissible spongiform encephalopathy (TSE) in the domestic pig. Furthermore, experimental transmission of BSE to pigs by the oral route has been unsuccessful. A study was conducted to investigate whether the localised vacuolar changes in the porcine brain were associated with a transmissible aetiology and therefore biologically significant. Two groups of ten pigs were inoculated parenterally with vacuolated rostral colliculus from healthy pigs either born before 1996 or born after 1996. Controls included ten pigs similarly inoculated with rostral colliculus from New Zealand-derived pigs and nine pigs inoculated with a bovine BSE brain homogenate. Results: None of the pigs inoculated with rostral colliculus developed a TSE-like neurological disease up to five years post inoculation when the study was terminated, and disease-associated prion protein, PrPd, was not detected in the brains of these pigs. By contrast, eight of nine BSE-inoculated pigs developed neurological signs, two of which had detectable PrPd by postmortem tests. No significant histopathological changes were detected to account for the clinical signs in the PrPd-negative, BSE-inoculated pigs. Conclusion: The findings in this study suggest that vacuolation in the porcine rostral colliculus is not caused by a transmissible agent and is probably a clinically insignificant change. The presence of neurological signs in pigs inoculated with BSE without detectable PrPd raises the possibility that the BSE agent may produce a prion disease in pigs that remains undetected by the current postmortem tests.
SNIP...SEE FULL TEXT ;
http://madporcinedisease.blogspot.com/2009/10/transmissibility-studies-of-vacuolar.html
SNIP...SEE FULL TEXT ;
7 OF 10 LITTLE PIGGIES WENT ON TO DEVELOP BSE;
1: J Comp Pathol. 2000 Feb-Apr; 122(2-3): 131-43. Related Articles,
Links
Click here to read
The neuropathology of experimental bovine spongiform encephalopathy in the pig.
Ryder SJ, Hawkins SA, Dawson M, Wells GA.
Veterinary Laboratories Agency Weybridge, Woodham Lane, New Haw, Addlestone, Surrey, KT15 3NB, UK.
In an experimental study of the transmissibility of BSE to the pig, seven of 10 pigs, infected at 1-2 weeks of age by multiple-route parenteral inoculation with a homogenate of bovine brain from natural BSE cases developed lesions typical of spongiform encephalopathy. The lesions consisted principally of severe neuropil vacuolation affecting most areas of the brain, but mainly the forebrain. In addition, some vacuolar change was identified in the rostral colliculi and hypothalamic areas of normal control pigs. PrP accumulations were detected immunocytochemically in the brains of BSE-infected animals. PrP accumulation was sparse in many areas and its density was not obviously related to the degree of vacuolation. The patterns of PrP immunolabelling in control pigs differed strikingly from those in the infected animals.
PMID: 10684682 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=10684682&dopt=Abstract
IN CONFIENCE
EXPERIMENTAL PORCINE SPONGIFORM ENCEPHALOPATHY
1. CMO should be aware that a pig inoculated experimentally (ic, iv, and ip) with BSE brain suspension has after 15 months developed an illness, now confirmed as a spongiform encephalopathy. This is the first ever description of such a disease in a pig, although it seems there ar no previous attempts at experimental inoculation with animal material. The Southwood group had thought igs would not be susceptible. Most pigs are slaughtered when a few weeks old but there have been no reports of relevant neurological illness in breeding sows or other elderly pigs. ...see full text ;
http://web.archive.org/web/20040302031004/www.bseinquiry.gov.uk/files/yb/1990/08/23001001.pdf
IN CONFIDENCE
So it is plausible pigs could be preclinically affected with BSE but since so few are allowed to reach adulthood this has not been recognised through clinical disease. ...
http://web.archive.org/web/20040904150118/www.bseinquiry.gov.uk/files/yb/1990/08/23002001.pdf
CONFIDENTIAL
EXPERIMENTAL PORCINE SPONGIFORM ENCEPHALOPATHY
While this clearly is a cause for concern we should not jump to the conclusion that this means that pigs will necessarily be infected by bone and meat meal fed by the oral route as is the case with cattle. ...
http://web.archive.org/web/20031026000118/www.bseinquiry.gov.uk/files/yb/1990/08/23004001.pdf
we cannot rule out the possibility that unrecognised subclinical spongiform encephalopathy could be present in British pigs though there is no evidence for this: only with parenteral/implantable pharmaceuticals/devices is the theoretical risk to humans of sufficient concern to consider any action.
http://web.archive.org/web/20030822031154/www.bseinquiry.gov.uk/files/yb/1990/09/10007001.pdf
May I, at the outset, reiterate that we should avoid dissemination of papers relating to this experimental finding to prevent premature release of the information. ...
http://web.archive.org/web/20030822052332/www.bseinquiry.gov.uk/files/yb/1990/09/11005001.pdf
3. It is particularly important that this information is not passed outside the Department, until Ministers have decided how they wish it to be handled. ...
http://web.archive.org/web/20030822052438/www.bseinquiry.gov.uk/files/yb/1990/09/12002001.pdf
But it would be easier for us if pharmaceuticals/devices are not directly mentioned at all. ...
http://web.archive.org/web/20030518170213/www.bseinquiry.gov.uk/files/yb/1990/09/13004001.pdf
Our records show that while some use is made of porcine materials in medicinal products, the only products which would appear to be in a hypothetically ''higher risk'' area are the adrenocorticotrophic hormone for which the source material comes from outside the United Kingdom, namely America China Sweden France and Germany. The products are manufactured by Ferring and Armour. A further product, ''Zenoderm Corium implant'' manufactured by Ethicon, makes use of porcine skin - which is not considered to be a ''high risk'' tissue, but one of its uses is described in the data sheet as ''in dural replacement''. This product is sourced from the United Kingdom.....
http://web.archive.org/web/20030822054419/www.bseinquiry.gov.uk/files/yb/1990/09/21009001.pdf
BSE TO PIGS NEWS RELEASE
http://web.archive.org/web/20030822162313/www.bseinquiry.gov.uk/files/yb/1990/09/24001001.pdf
CONFIDENTIAL
BSE: PRESS PRESENTATION
http://web.archive.org/web/20030822160958/www.bseinquiry.gov.uk/files/yb/1990/09/20003001.pdf
http://web.archive.org/web/20040623191707/www.bseinquiry.gov.uk/files/yb/1990/09/24013001.pdf
http://web.archive.org/web/20030820195733/http://www.bseinquiry.gov.uk/files/yb/1990/09/20010001.pdf
http://web.archive.org/web/20030820195733/http://www.bseinquiry.gov.uk/files/yb/1990/09/25013001.pdf
http://web.archive.org/web/20030820195733/http://www.bseinquiry.gov.uk/files/yb/1990/09/25015001.pdf
INDUSTRY RESPONSE TYPICAL
http://web.archive.org/web/20030822055917/www.bseinquiry.gov.uk/files/yb/1990/09/25007001.pdf
DEFENSIVE BRIEFING
http://web.archive.org/web/20030820195733/http://www.bseinquiry.gov.uk/files/yb/1990/09/25016001.pdf
CONFIDENTIAL
pigs & pharmaceuticals
http://web.archive.org/web/20010305223234/www.bseinquiry.gov.uk/files/yb/1990/09/10007001.pdf
http://web.archive.org/web/20010305223234/www.bseinquiry.gov.uk/files/yb/1990/08/23002001.pdf
http://web.archive.org/web/20010305223234/www.bseinquiry.gov.uk/files/yb/1990/08/23004001.pdf
http://web.archive.org/web/20010305223234/www.bseinquiry.gov.uk/files/yb/1990/08/29003001.pdf
COMMERCIAL IN CONFIDENCE COMMITTEE ON SAFETY OF MEDICINE NOT FOR PUBLICATION BOVINE SPONGIFORM ENCEPHALOPATHY WORKING GROUP
There are only two products using porcine brain and these use corticotrophin BP, made from porcine pituitary, source from outside the UK.............
http://web.archive.org/web/20040622220349/www.bseinquiry.gov.uk/files/yb/1990/10/31003001.pdf
SEE FULL TEXT ;
http://madporcinedisease.blogspot.com/
TSS
Monday, December 21, 2009
Thursday, October 15, 2009
Transmissibility studies of vacuolar changes in the rostral colliculus of pigs
Research article Open Access T
Transmissibility studies of vacuolar changes in the rostral colliculus of pigs
Timm Konold*1,2, John Spiropoulos1, Melanie J Chaplin3, Leigh Thorne3, Yvonne I Spencer1, Gerald AH Wells1 and Steve AC Hawkins1 Address: 1Department of Pathology, Veterinary Laboratories Agency Weybridge, Woodham Lane, Addlestone, UK, 2Royal Veterinary College, Infection and Immunity Research Group, North Mymms, Hatfield, UK and 3Department of Molecular Pathogenesis and Genetics, Veterinary Laboratories Agency Weybridge, Woodham Lane, Addlestone, UK Email: Timm Konold* - t.konold@vla.defra.gsi.gov.uk; John Spiropoulos - j.spiropoulos@vla.defra.gsi.gov.uk; Melanie J Chaplin - m.j.chaplin@vla.defra.gsi.gov.uk; Leigh Thorne - l.thorne@vla.defra.gsi.gov.uk; Yvonne I Spencer - y.i.spencer@vla.defra.gsi.gov.uk; Gerald AH Wells - g.a.h.wells@vla.defra.gsi.gov.uk; Steve AC Hawkins - s.a.c.hawkins@vla.defra.gsi.gov.uk * Corresponding author
Abstract Background:
Histopathological examinations of brains from healthy pigs have revealed localised vacuolar changes, predominantly in the rostral colliculus, that are similar to the neuropil vacuolation featured in the transmissible spongiform encephalopathies and have been described in pigs challenged parenterally with the agent causing bovine spongiform encephalopathy (BSE). Feedstuff containing BSE-contaminated meat and bone meal (MBM) may have been fed to pigs prior to the ban of mammalian MBM in feed of farmed livestock in the United Kingdom in 1996, but there is no evidence of the natural occurrence of a transmissible spongiform encephalopathy (TSE) in the domestic pig. Furthermore, experimental transmission of BSE to pigs by the oral route has been unsuccessful. A study was conducted to investigate whether the localised vacuolar changes in the porcine brain were associated with a transmissible aetiology and therefore biologically significant. Two groups of ten pigs were inoculated parenterally with vacuolated rostral colliculus from healthy pigs either born before 1996 or born after 1996. Controls included ten pigs similarly inoculated with rostral colliculus from New Zealand-derived pigs and nine pigs inoculated with a bovine BSE brain homogenate. Results: None of the pigs inoculated with rostral colliculus developed a TSE-like neurological disease up to five years post inoculation when the study was terminated, and disease-associated prion protein, PrPd, was not detected in the brains of these pigs. By contrast, eight of nine BSE-inoculated pigs developed neurological signs, two of which had detectable PrPd by postmortem tests. No significant histopathological changes were detected to account for the clinical signs in the PrPd-negative, BSE-inoculated pigs. Conclusion: The findings in this study suggest that vacuolation in the porcine rostral colliculus is not caused by a transmissible agent and is probably a clinically insignificant change. The presence of neurological signs in pigs inoculated with BSE without detectable PrPd raises the possibility that the BSE agent may produce a prion disease in pigs that remains undetected by the current postmortem tests.
SNIP...
Conclusion The findings suggest that vacuolation in the rostral colliculus is a common feature of porcine brains without causing evident clinical signs and does not represent a localised form of a transmissible spongiform encephalopathy. The presence of neurological signs in pigs challenged with BSE in the absence of detectable disease-associated prion protein or other visible pathological changes raises the possibility that the BSE agent may cause a chronic disease that remains undetected by current prion disease phenotypic definitions and postmortem tests.
http://www.biomedcentral.com/content/pdf/1746-6148-5-35.pdf
CONFIDENTIAL TRANSMISSION (Day 4)
6.1 BSE to pigs
SE1802/SE1816 - Transmissibility of BSE to pigs by injection with brain homogenate;
SE1803/SE1817 - Transmissibility of BSE to pigs by oral exposure with brain homogenate.
6.1.3 The possibility that pigs could be recycling the BSE agent through
rendered material, which was then passed back to cattle via the
spreading of pig manure, was discussed. However, it was agreed that no
action was required (see para. 2.1.3).
6.2 BSE to chickens
SE1805/SE1806 - Transmissibility of BSE to domestic fowl
6.2.1 Material from a number of tissues including spleen in the
experimental chicken should be sub-passaged through further birds and
mice: by the intracerebral routes in birds and the intracerebral route
in mice. CSG would ask CVL to prepare a proposal.
6.3 Scrapie to pigs SE1813/SE1822 - Transmissibility of scrapie to pigs by oral exposure to brain homogenate.
6.3.1 SE1822 will replace SE1813. No amendment of this project was required.
6.4 BSE to sheep
SE1402/SE1418 - Transmission of BSE and natural scrapie in sheep and goats by intracerebral and oral routes
6.5 BSE to cattle via exposure to placenta
95/5.11/1.19
http://www.mad-cow.org/00/aug00_late_news.html#ggg
7 OF 10 LITTLE PIGGIES WENT ON TO DEVELOP BSE;
1: J Comp Pathol. 2000 Feb-Apr; 122(2-3): 131-43. Related Articles,
Links
Click here to read
The neuropathology of experimental bovine spongiform encephalopathy in the pig.
Ryder SJ, Hawkins SA, Dawson M, Wells GA.
Veterinary Laboratories Agency Weybridge, Woodham Lane, New Haw, Addlestone, Surrey, KT15 3NB, UK.
In an experimental study of the transmissibility of BSE to the pig, seven of 10 pigs, infected at 1-2 weeks of age by multiple-route parenteral inoculation with a homogenate of bovine brain from natural BSE cases developed lesions typical of spongiform encephalopathy. The lesions consisted principally of severe neuropil vacuolation affecting most areas of the brain, but mainly the forebrain. In addition, some vacuolar change was identified in the rostral colliculi and hypothalamic areas of normal control pigs. PrP accumulations were detected immunocytochemically in the brains of BSE-infected animals. PrP accumulation was sparse in many areas and its density was not obviously related to the degree of vacuolation. The patterns of PrP immunolabelling in control pigs differed strikingly from those in the infected animals.
PMID: 10684682 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=10684682&dopt=Abstract
confindential
pigs & pharmaceuticals
http://web.archive.org/web/20010305223234/www.bseinquiry.gov.uk/files/yb/1990/09/10007001.pdf
http://web.archive.org/web/20010305223234/www.bseinquiry.gov.uk/files/yb/1990/08/23002001.pdf
http://web.archive.org/web/20010305223234/www.bseinquiry.gov.uk/files/yb/1990/08/23004001.pdf
http://web.archive.org/web/20010305223234/www.bseinquiry.gov.uk/files/yb/1990/08/29003001.pdf
SEE FULL TEXT ;
http://chronic-wasting-disease.blogspot.com/2009/03/chronic-wasting-disease-prions-in-elk.html
The case for mad pigs in the US
From the Consumer Policy Institute and Consumers Union: March 24, 1997
Stephen F. Sundlof, D.V.M., Ph.D
Center for Veterinary Medicine
Food and Drug Administration
7500 Standish Place, Room 482, HFV 1
RockvLIle, MD 20855
Dear Dr. Sundlof:
We are writing to you to submit information that has recently come to our attention which suggests that a TSE like disease (transmissible spongiform encephalopathy) might exist in pigs in the U.S. We believe this new informantion calls for intensive research and makes it urgent to ban the use of all mammalian proteins, including swine, in the feed of all food animals, until better answers are found.
The evidence for the potential PSE (porcine spongiform encephalopathy ) is as follows. In 1979, an FSQS veternarian, Dr. Masuo Doi, noticed some unusual central nervous system (CNS) symptoms in young (about 6 months old) hogs coming into a slaughter plant In Albany, New York. Since the plant received hogs from a wide variety of sources (New York, Canada, Indiana, Illinois, Ohio, and other Midwestern states) and was not a plant used to dealing with diseased animals, Dr. Doi thought that the problem might be affecting hogs slaughtered nationwide. So, he decided to conduct a detailed study on central nervous system (CNS) symptoms/disease in young hogs coming into that slaughter plant. The study ran for 15 months (January, 1979 to March, 1980) and consisted of extended observations of the behavior of animals with suspected CNS symptoms at the plant, followed by pathological, histopatholpgical, and microbiological work on tissues from various organs of particular animals after slaughter.
For his behavioral observational work, Dr. Doi extended the usual two day observation period to three to four days, during which he took careful notes on the animals' behavior and other vital signs. During the 15 month period of the study, some 106 animals exhibiting CNS symptoms were retained during antemortem inspection.
A 1980 paper that summarized Dr. Doi's findings on the clinical symptoms and incidence of the 'disease," contained descriptions of these symptoms that sound remarkably similar to the symptoms noted for bovine spongiform encephalopathy (BSE):
"Excitable or nervous temperament to external stimuli such as touch to the skin, handling and menacing approach to the animals is a common characteristic sign among swine affected with the disease.... In the advanced stage of the disease, manifestation of neurological signs are evidenced in the form of general ataxia . . . Many animals have been found to be "downers' at first observation; if the hindquarters of these downers are raised they may be able to walk one or two steps and then fall to the ground" (Doi et al., 1980: 2, 4).
Indeed, a table of symptoms includes, for the early stage: "excitability and nervousness (squealing, smacking of lips, grinding of teath, chewing, gnawing ant foaming at mouth); stiffness of limbs . . . 'tic'; weakness of hindquarters; focal tremors of skeletal muscles"; and for the advanced stage: depression; ataxia; crossing over of limbs . . . kneeling posture . . . crawling". In addition to his clinical observations, Dr. Doi also made an 8 mm film of thirteen of the affected animals; film of two of the pigs was shown at the MPI National Pathology Meeting in Seattle, Washington on flay 20, 1979.
Dr. Doi sent tissue samples from suspect cases to the USDA's Eastern Laboratory in Athens, GA for pathological, histopathogical and microbiological work. Known infectious diseases were ruled out. As Dr. Doi points out, "Histopathological studies of tissue collected from the brain and spinal cord of these animals in the early stage of the disease show congestion, hemorrhage and neuronal degeneration. All animals in the advanced stage of the disease have been confined to have Encephalitis or Meningitis by MPI laboratory" (Doi et al., 1980: 5). Eventually some 60 animals were confirmed by the MPI Laboratory to have encephalitis or meningitis, with no ldentifiable cause. As pointed out in a paper presented at the 1979 MPI National Pathology Meetings,
"Since January, a number of hogs in this establishment have been found, in antemortem, to show what appears to be CNS. Sets of tissue samples were sent to the laboratory for examination, various tests were done which include histological study (E H stain), fluorescence antibody technique, virus neutralization and viral and bacteriological isolation. Differential diagnosis was also done to exclude vitamin B deficiency, post vaccination reaction, chlorinated hydrocarbon, arthritis, and transport stress" (Doi et al., 1979).
The brains of the 60 animals were examined. The brain of one of these pigs, on histopathological analysis, exhibited signs reminiscent of a TSE. This histopathological work was performed by Dr. Karl Langheinrich, Pathologist-In-Charge at USDA's Eastern Laboratory in Athens, Georgia. According to the USDA FSQS laboratory report, dated early November, 1979, Dr. Langheinrich noted:
"Microscopic examination of the barrow tissues revealed a encephalopathy and diffuse gliosis characterized by vacuolated neurons, loss of neurons and gliosis in a confined region (nucleus) of the brain stem (anterior ventral midbrain). Only an empty sometimes divided vacuole was present instead of the normal morphology of a nerve cell. Occasionally a shriveled neuron was seen. According to . . . Pathology of Domestic Animals, . . . 'The degeneration of neurons, the reactivity of the glia .... are the classical hallmarks of viral infection of the central nervous system' .... Scrapie of sheep, and encephalopathy of mink, according to the literature, all produce focal vacuolation of the neurons similar to the kind as described for this pig. I was unable to locate any lead as to the cause of this interesting phenomenon in other species including swine'' (Langheinrich, 1979).
Indeed, Dr. Langheinrich's main diagnosis was, " Encephalopathy and diffuse gliosis of undetermined etiology." Portions of the brain were sent for microbiological testing to a neurologist at the University of Georgia, where they came up negative for pseudo-rabies. The brain was unique enough that USDA scientists, such as Dr. Langheinrich and Or. Dot, mentioned it to student and scientific colleagues over the years.
In 1979-1980, BSE was completely unknown. However, both the behavior of the pigs, as well as the histopathology on at least one pig, both showed sign consistent with a porcine TSE. This raises particular concern became the affected animal was only 6 months old; in an animal this young, one would rust expect to see any physical signs of TSE in the brain. Histopathology of TSEs can be very variable, so that spongiform appearance (i.e. vacuolated neurons) are not always present. Behavioral changes can be seen in TSE-infected animals before any changes in brain morphology are visible. Dr. Clarence Gibbs, in testimony before a Congressional hearing on the TSE issue on January 29, 1997 made just this point:
''In the mid-1960s, we demonstrated with our French and English collaborators that during the early incubation of the TSEs, when the virus titer in the brain was very low, there were already marked functional changes, even though no pathology was yet detectable, even ultrastructurally. A month or hero later, polynucleation of neurons appeared in spider monkeys, incubating kuru, and somewhat later, microvacuolation and membrane changes visible only by electron microscopy. This preceded the pest appearance of astrogliosis and spongiform change. It was only much later that the classical scrapie TSE pathology appeared with virus titers in brain of 10 -5 or higher" (Gibbs, 1997; pg. 4).
Given that TSEs can cause behavioral changes in infected animals before any physical changes in the brain can be seen, that the manifestation of TSE in the brain can be quite variable, and that changes in brain morphology are not usually seen in 6 month old animals, we are concerned that the brain of one pig actually showed physical evidence consistent with a TSE.
Following the announcement In March, 1996 of ten cases of new variant CJD (Creutzfeldt-Jakob Disease) in the United Kingdom and their possible connection to BSE, Drs. Doi, Langheinrich and others urged reinvestigation of this case.
In August, 1996, the USDA sent five slides, one of which was a histopathology slide, to Dr. Janice Miller of USDA's Agricultural Research Servicer . Dr. Miller stained four of the slides for prion protein (she didn't stain the H&E slide). Dr. Miller told Consumers Union that Dr. Patrick McCaskey, USDA/FSIS, in charge of the Research Center at Athens, GA, called her, told her that he had five slides that all showed "problems" and asked her to stain four of them. The H&E slide, which clearly show vacuoles in the neurons (one sign of TSE), wasn't stained because to stain for PrP entails removing the slide cover, baking the slide to destain it and then restaining it for PrP; they didn't want to risk destroying the H&E slide.
Dr. Doi had kept frozen samples of the brain and spinal chord of the suspect PSE pig in case the Eastern lab wanted more material for analysis. Unfortunately, these samples were discarded when the packing plant in Albany, NY closed in 1991. It appears that the brain material sent to the Univcrsity of Georgia may have been discarded. [pers com.. Dr. Doi 3/13/97]
Dr. Miller found that the PrP stained in the four pig slides was found only on the inside of neurons, while a positive control slide from a scrapie sheep showed massive amounts of extraneuronal staining. In a letter summarizing her results (copy attached), she concludes that the PrP stained in this pig was normal: "In the pig sections you will see a small particulate type of staining that is confined to neurons and as I indicated on the phone, I would interpret as normal PrP. It is in marked contrast to the massive amount of extraneuronal staining seen in the scrapie section" (Miller, 1996).
Unfortunately, Dr. Miller's finding toes not conclusively rule out a TSE. We are concerned that while British BSE and serapie create a massive amount of extraneuronal staining, there are TSEs where this isn't the case. Three experiments were done in He U.S. -- in Mission, TX (APHIS work), Pullman, Washington (ARS work), and Ames, Iowa (ARS work) -- to see whether sheep scrapie can possibly infect cows. In all the experiments, cattle were inoculated with tissue from scrapie -infected sheep primarily by intra-cranial injection, but in the case of the Texas and Iowa studies also by oral feeding -- to see if cattle were susceptible to scrapie at all. In all three experiments, the majority of cows injected in the brain with scrapie-infected sheep material (usually brains) also developed a fatal spongiform encephalopathy.
However, in all three examples, the symptoms of the spongifonn encephalopathy differed from "mad cow" disease ~ England, as did the appearances of slides from their brains. The brain lesions seen in all these animals were more variable than those seen in England. When Dr. Miller did similar staining for PrP from these brains (what she called "bovine scrapie") she only found PrP stains on the inside of the neurons, not the massive extraneuronal staining seen in BSE (Miller, pers. comm., March 7, 1997). Thus, Dr. Miller's finding of PrP stains only inside the neurons in the suspect pigs is not particularly reassuring.
In November 1996, USDA sent the single histopathology slide to Dr. William Hadlow, one of the foremost spongiform encephalopathy pathologists in the world. (For unknown reasons, Dr. Hadlow was only sent the one slide; he was not told of the existence of the other slides, nor of Dr. Miller's findings, nor was he told or given the behavioral report from Dr. Doi or the morphology work by Dr. Langheinrich, or shown film of the affected pigs [Dr. Hadlow, pers. com., 3/13/97] From this single slide, Dr. Hadlow found some evidence consistent with TSEs but not enough for a conclusive diagnosis. He noted that the slide contained vacuoles inside neurons, one of the signs of a TSE (Dr. Langheinrich had noted this as well).
However, since such vacuoles occasionally occur normally in pigs, he thought that was not something special: "About twelve (12) neurons in the parasympathetic nucleus have unilocular optically empty vacuoles in the perikaryon. This is the site where such vacuolated neurons have been seen in the swine (as well as in cats and sheep) as an incidental finding. So I do not think such cells have any significance in this pig" (Hadlow, 1996). However, he did see evidence, Including changes in astrocytes, that suggested a TSE, but without examining other parts of the brain to look for other evidence of TSE, he couldn't be sure:
"I am impressed, though, with what seems to be an increase in the number of astrocytes in the section. Some astrocytes are in clusters, some are enlarged and vesicular. Where they are most numerous, a few rod cells (activated microglia) are seen. These findings suggest some perturbation of the nervous tissue. Although such a global response occurs in the transmissible spongifonn encephalopathies, I do no! know its significance in this case without examining other parts of the brain for changes characteristic of these diseases. Thus, from looking; at this one (1) section of brain, I cannot conclude that the pig was affected with a scrapie-like spongiform encephalopathy" (Hadlow, 1996).
In sum, Dr. Hadlow~s letter does not rule out the possibility of a TSE. He says that there is suggestive evidence, but that he would need to look at other slides/sections of the brain, to make a conclusive diagnosis.
In our view, the implications of this data are extremely serious. Experiments in the United Kingdom have shown that pigs are susceptible to BSE. Pigs inoculated with BSE develop a TSE (Dawson et al., 1990). Feeding experiments are underway in the UK to see if BSE can be orally transmitted to pigs; as of March, 1997, some 6 years after the start of the experiment, none of the pigs fed BSE brain have come down with a TSE. Unfortunately the design of this experiment severely limits what we will learn from it, and will most likely not tell us conclusively if pigs can get BSE from feed. It turns out that the pigs were not fed BSE brain continuously. Rather, the pigs were only fed BSE brain material on three days, over a three week period (i.e.. one day each week). Following these three doses, the pigs were never fed contaminated material again. The total amount of infective material given to the pigs was therefore quite small. Thus, a negative finding would be hard to interpret and would not mean that BSE is not orally active in pigs.
We believe that as a top priority USDA should conduct follow-up studies to look for potential CNS/PSE cases in pigs (we plan to communicate about this to USDA separately). In brief, we feel that the following kinds of studies need to be done:
i) TSE pathology experts should examine all the slides from the suspect pig (2709). To our knowledge, at least 12 separate slides exist.
ii) Determine if any brain material from the suspect pig (2709) still exists at the Unlverslty of Georgia. If so, this material should be retrieved and used for transmission studies. In particular, suckling pigs should be inoculated with the material and then permitted to live unto they die of a disease or old age, at which point their brains should be examined for physical signs of a TSE as well as for immunchistochemical evidence (i.e. staining looking for the abnormal PrP).
iii) Increase antemortem inspection for CNS symptoms at hog facilities. Inspectors should be trained to detect the subtle CNS symptoms seen in the Doi et al. study. At a select number of slaughter facilities, animals exhibiting CNS symptoms should be removed and held for observation until they die, at which time their brains should be examined for evidence of a TSE.
iv) Research on CNS symptoms among Me 6,000 or so breeding sows which are permitted to live for 3+ years. Sows exhibiting CNS symptoms should be removed and held for observation until they die, at which time then brains should be exernined for evidence of a TSE.
While such work is underway, given the above inforrnabon, we believe that as a precutionary measure the FDA must expand the proposed ruminant plus mink-to-ruminnant feed ban to prevent protein from any material, including hogs, being fed to any food animal.
Sincerely,
Michael Hansen, Ph.D Research Associate
Jean Halloran Director
References
Dawson, M., Wells, G.A.H., Parker, B.N;J. and A.C Scott. 1990. Primary parental transmission of bovine spongiform encephalopathy to the pig. Veternary Record, pg. 338.
Doi, M., Matzner, N.D. and C. Rothaug. 1979. Observation of CNS disease in market hogs at Est. 893 Tobin Packing Co., Inc. Albany, New York. United States Department of Agriculture, Food Safety and Quality.Service, Meat and Poultry Inspection Service. 7pp.
Doi, M, Langheinrich, K. and F. Rellosa. 1980. Observations of CNS signs in hogs at Est. 893 Tobin Packing C:o., Inc. Presented by Dr. Lngheinrich at the MPI National Pathology Meeting in Seattle, Washington on July 20, 1979.
Gibbs, C. 1997. Statement to the Committee on Governnent Reform and Oversight, Subcommittee on Human Resources and Intergovernmental Relations, U.S. House of Representatives. January 29,1997.
Hadlow, WJ. 1996. Letter to Patrick McCaskey, USDA/FSIS/Eastem Lab, dated November 13, 1996.
Langheinrich, KA. 1979. USDA/FSQS Laboratory report on specimen 2709. Dated November 8, 1979
Miller, J. 1996. Letter to Patrick McCaskey, USDA/ESIS/Eastern Lab, dated September 6, 1996.
Dr. Janice Miller, ARS
http://www.mad-cow.org/~tom/mad_pigs.html
Subject: EXPERIMENTAL INTRACEREBRAL AND ORAL INOCULATION OF SCRAPIE TO SWINE: PRELIMINARY REPORT
Date: February 6, 2006 at 12:33 pm PST
Title: EXPERIMENTAL INTRACEREBRAL AND ORAL INOCULATION OF SCRAPIE TO SWINE: PRELIMINARY REPORT
Authors
Greenlee, Justin
Kunkle, Robert - bob
Hamir, Amirali
Submitted to: American Association Of Veterinary Laboratory Diagnosticians
Publication Acceptance Date: November 5, 2005
Publication Date: November 5, 2005
Citation: Greenlee, J.J., Kunkle, R.A., Hamir, A.N. 2005. Experimental Intracerebral And Oral Inoculation Of Scrapie To Swine: Preliminary Report [abstract]. Proceedings Of The American Association Of Veterinary Laboratory Diagnosticians 48th Annual Conference. P. 38.
Technical Abstract: Transmissible spongiform encephalopathies (TSEs, prion diseases) are chronic neurodegenerative diseases that occur in humans, cattle, sheep, goats, cervids, and a number of laboratory animal models. In a laboratory setting, the host range of a given TSE can be tested by inoculating animals with brain tissue from affected animals through various routes including oral and intracranial. There is no evidence of the natural occurrence of any form of TSE in the pig, but pigs have been shown to be susceptible to bovine spongiform encephalopathy (BSE) infection by multiple-route parenteral challenge. However, pigs orally exposed at eight weeks of age to large amounts of brain from cattle clinically affected with BSE did not support infection after seven years of observation. In the United States, feeding of ruminant by-products to ruminants is prohibited, but feeding of ruminant materials to swine and poultry still occurs. The potential for swine to have access to scrapie-contaminated feedstuffs exists, but the potential for swine to serve as a host for replication/accumulation of the agent of scrapie is unknown. The purpose of this study was to perform oral and intracerebral inoculation of the U.S. scrapie agent to determine the potential of swine as a host for the scrapie agent and their clinical susceptibility. This study utilized 26 swine randomly divided into three groups: controls (n=6), oral inoculates (n=8), and intracranial inoculates (n=12). Brain homogenate (10%) derived from scrapie-affected sheep was given by a single intracranial injection of 0.75 ml or by oral inoculation of 15 ml on four consecutive days. Scrapie inoculum was derived from clinically ill sheep inoculated with material derived from 13 sheep in seven source flocks. A sample of this material was also inoculated back into sheep to assure infectivity. Necropsies were planned for six months post inoculation, at approximately the time the pigs were expected to reach market weight. Samples collected were examined microscopically after routine staining (hematoxylin and eosin) and staining by standard immunohistochemical methods for prion protein (PrP**Sc). After approximately six months incubation time, no histologic lesions
suggestive of spongiform encephalopathy or immunohistochemical evidence of prion infection were obtained. No evidence of scrapie infection was demonstrated in this short-term study, but positive results after an incubation period of only six months would be uncharacteristic. The only TSE with an incubation of six months or less known at this time is transmissible mink encephalopathy in mink, skunk, or raccoon hosts. However, scrapie in the raccoon model has a two-year incubation period. A replicate of littermate pigs has been inoculated and will be studied after long-term (3-7 years) incubation, and a similar study is underway with pigs inoculated with material derived from elk, mule deer, and whitetail deer affected by chronic wasting disease (CWD).
Project Team
Kehrli, Marcus
Nicholson, Eric
Greenlee, Justin
Hamir, Amirali
Richt, Juergen
Kunkle, Robert - Bob
http://www.ars.usda.gov/research/publications/publications.htm?SEQ_NO_115=191555
DOI: 10.3201/eid1508.081218
Suggested citation for this article: Espinosa J-C, Herva M-E, Andréoletti O, Padilla D, Lacroux C, Cassard H, et al. Transgenic mice expressing porcine prion protein resistant to classical scrapie but susceptible to sheep bovine spongiform encephalopathy and atypical scrapie. Emerg Infect Dis. 2009 Aug; [Epub ahead of print]
Transgenic Mice Expressing Porcine Prion Protein Resistant to Classical Scrapie but Susceptible to Sheep Bovine Spongiform Encephalopathy and Atypical Scrapie
Juan-Carlos Espinosa,1 María-Eugenia Herva,1 Olivier Andréoletti, Danielle Padilla, Caroline Lacroux, Hervé Cassard, Isabelle Lantier, Joaquin Castilla, and Juan-María Torres
Author affiliations: Centro de Investigación en Sanidad Animal, Madrid, Spain (J.-C. Espinosa, M.-E. Herva, D. Padilla, J. Castilla, J.-M. Torres); École Nationale Vétérinaire de Toulouse, Toulouse, France (O. Andréoletti, C. Lacroux, H. Cassard); and Centre Institut National de la Recherche Agronomique de Tours, Nouzilly, France (I. Lantier)
1These authors contributed equally to this article.
How susceptible pigs are to infection with sheep prions is unknown. We show, through transmission experiments in transgenic mice expressing porcine prion protein (PrP), that the susceptibility of this mouse model to bovine spongiform encephalopathy (BSE) can be enhanced after its passage in ARQ sheep, indicating that the pathogenicity of the BSE agent is modified after passage in sheep. Transgenic mice expressing porcine PrP were, nevertheless, completely resistant to infection with a broad panel of classical scrapie isolates from different sheep PrP genotypes and with different biochemical characteristics. The atypical (Nor98 like) isolate (SC-PS152) was the only scrapie isolate capable of transmission in these mice, although with a marked transmission barrier. Unexpectedly, the atypical scrapie agent appeared to undergo a strain phenotype shift upon transmission to porcine-PrP transgenic mice and acquired new strain properties, suggesting that atypical scrapie agent may exhibit different phenotypes depending on the host cellular PrP or other genetic factors.
SNIP...
The atypical isolate SC-PS152 was the only scrapie isolate able to infect the Po-PrP mouse model after intracerebral inoculation (Table), albeit with a low efficiency of infection in the first passage (attack rate 16%). These results suggest the potential ability of atypical scrapie prions to infect pigs, although with a strong transmission barrier. Given the increasing number of atypical scrapie cases found in Europe and in North America, the potential ability of atypical scrapie to adapt to the pig becoming more easily transmitted could raise concerns about the potential danger of feeding ruminant meat and bone meal to swine.
http://www.cdc.gov/eid/content/15/8/pdfs/08-1218.pdf
http://nor-98.blogspot.com/2009/07/transgenic-mice-expressing-porcine.html
Wednesday, July 1, 2009
Nor98 scrapie identified in the United States J Vet Diagn Invest 21:454-463 (2009)
http://nor-98.blogspot.com/2009/07/nor98-scrapie-identified-in-united.html
TSS
Transmissibility studies of vacuolar changes in the rostral colliculus of pigs
Timm Konold*1,2, John Spiropoulos1, Melanie J Chaplin3, Leigh Thorne3, Yvonne I Spencer1, Gerald AH Wells1 and Steve AC Hawkins1 Address: 1Department of Pathology, Veterinary Laboratories Agency Weybridge, Woodham Lane, Addlestone, UK, 2Royal Veterinary College, Infection and Immunity Research Group, North Mymms, Hatfield, UK and 3Department of Molecular Pathogenesis and Genetics, Veterinary Laboratories Agency Weybridge, Woodham Lane, Addlestone, UK Email: Timm Konold* - t.konold@vla.defra.gsi.gov.uk; John Spiropoulos - j.spiropoulos@vla.defra.gsi.gov.uk; Melanie J Chaplin - m.j.chaplin@vla.defra.gsi.gov.uk; Leigh Thorne - l.thorne@vla.defra.gsi.gov.uk; Yvonne I Spencer - y.i.spencer@vla.defra.gsi.gov.uk; Gerald AH Wells - g.a.h.wells@vla.defra.gsi.gov.uk; Steve AC Hawkins - s.a.c.hawkins@vla.defra.gsi.gov.uk * Corresponding author
Abstract Background:
Histopathological examinations of brains from healthy pigs have revealed localised vacuolar changes, predominantly in the rostral colliculus, that are similar to the neuropil vacuolation featured in the transmissible spongiform encephalopathies and have been described in pigs challenged parenterally with the agent causing bovine spongiform encephalopathy (BSE). Feedstuff containing BSE-contaminated meat and bone meal (MBM) may have been fed to pigs prior to the ban of mammalian MBM in feed of farmed livestock in the United Kingdom in 1996, but there is no evidence of the natural occurrence of a transmissible spongiform encephalopathy (TSE) in the domestic pig. Furthermore, experimental transmission of BSE to pigs by the oral route has been unsuccessful. A study was conducted to investigate whether the localised vacuolar changes in the porcine brain were associated with a transmissible aetiology and therefore biologically significant. Two groups of ten pigs were inoculated parenterally with vacuolated rostral colliculus from healthy pigs either born before 1996 or born after 1996. Controls included ten pigs similarly inoculated with rostral colliculus from New Zealand-derived pigs and nine pigs inoculated with a bovine BSE brain homogenate. Results: None of the pigs inoculated with rostral colliculus developed a TSE-like neurological disease up to five years post inoculation when the study was terminated, and disease-associated prion protein, PrPd, was not detected in the brains of these pigs. By contrast, eight of nine BSE-inoculated pigs developed neurological signs, two of which had detectable PrPd by postmortem tests. No significant histopathological changes were detected to account for the clinical signs in the PrPd-negative, BSE-inoculated pigs. Conclusion: The findings in this study suggest that vacuolation in the porcine rostral colliculus is not caused by a transmissible agent and is probably a clinically insignificant change. The presence of neurological signs in pigs inoculated with BSE without detectable PrPd raises the possibility that the BSE agent may produce a prion disease in pigs that remains undetected by the current postmortem tests.
SNIP...
Conclusion The findings suggest that vacuolation in the rostral colliculus is a common feature of porcine brains without causing evident clinical signs and does not represent a localised form of a transmissible spongiform encephalopathy. The presence of neurological signs in pigs challenged with BSE in the absence of detectable disease-associated prion protein or other visible pathological changes raises the possibility that the BSE agent may cause a chronic disease that remains undetected by current prion disease phenotypic definitions and postmortem tests.
http://www.biomedcentral.com/content/pdf/1746-6148-5-35.pdf
CONFIDENTIAL TRANSMISSION (Day 4)
6.1 BSE to pigs
SE1802/SE1816 - Transmissibility of BSE to pigs by injection with brain homogenate;
SE1803/SE1817 - Transmissibility of BSE to pigs by oral exposure with brain homogenate.
6.1.3 The possibility that pigs could be recycling the BSE agent through
rendered material, which was then passed back to cattle via the
spreading of pig manure, was discussed. However, it was agreed that no
action was required (see para. 2.1.3).
6.2 BSE to chickens
SE1805/SE1806 - Transmissibility of BSE to domestic fowl
6.2.1 Material from a number of tissues including spleen in the
experimental chicken should be sub-passaged through further birds and
mice: by the intracerebral routes in birds and the intracerebral route
in mice. CSG would ask CVL to prepare a proposal.
6.3 Scrapie to pigs SE1813/SE1822 - Transmissibility of scrapie to pigs by oral exposure to brain homogenate.
6.3.1 SE1822 will replace SE1813. No amendment of this project was required.
6.4 BSE to sheep
SE1402/SE1418 - Transmission of BSE and natural scrapie in sheep and goats by intracerebral and oral routes
6.5 BSE to cattle via exposure to placenta
95/5.11/1.19
http://www.mad-cow.org/00/aug00_late_news.html#ggg
7 OF 10 LITTLE PIGGIES WENT ON TO DEVELOP BSE;
1: J Comp Pathol. 2000 Feb-Apr; 122(2-3): 131-43. Related Articles,
Links
Click here to read
The neuropathology of experimental bovine spongiform encephalopathy in the pig.
Ryder SJ, Hawkins SA, Dawson M, Wells GA.
Veterinary Laboratories Agency Weybridge, Woodham Lane, New Haw, Addlestone, Surrey, KT15 3NB, UK.
In an experimental study of the transmissibility of BSE to the pig, seven of 10 pigs, infected at 1-2 weeks of age by multiple-route parenteral inoculation with a homogenate of bovine brain from natural BSE cases developed lesions typical of spongiform encephalopathy. The lesions consisted principally of severe neuropil vacuolation affecting most areas of the brain, but mainly the forebrain. In addition, some vacuolar change was identified in the rostral colliculi and hypothalamic areas of normal control pigs. PrP accumulations were detected immunocytochemically in the brains of BSE-infected animals. PrP accumulation was sparse in many areas and its density was not obviously related to the degree of vacuolation. The patterns of PrP immunolabelling in control pigs differed strikingly from those in the infected animals.
PMID: 10684682 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=10684682&dopt=Abstract
confindential
pigs & pharmaceuticals
http://web.archive.org/web/20010305223234/www.bseinquiry.gov.uk/files/yb/1990/09/10007001.pdf
http://web.archive.org/web/20010305223234/www.bseinquiry.gov.uk/files/yb/1990/08/23002001.pdf
http://web.archive.org/web/20010305223234/www.bseinquiry.gov.uk/files/yb/1990/08/23004001.pdf
http://web.archive.org/web/20010305223234/www.bseinquiry.gov.uk/files/yb/1990/08/29003001.pdf
SEE FULL TEXT ;
http://chronic-wasting-disease.blogspot.com/2009/03/chronic-wasting-disease-prions-in-elk.html
The case for mad pigs in the US
From the Consumer Policy Institute and Consumers Union: March 24, 1997
Stephen F. Sundlof, D.V.M., Ph.D
Center for Veterinary Medicine
Food and Drug Administration
7500 Standish Place, Room 482, HFV 1
RockvLIle, MD 20855
Dear Dr. Sundlof:
We are writing to you to submit information that has recently come to our attention which suggests that a TSE like disease (transmissible spongiform encephalopathy) might exist in pigs in the U.S. We believe this new informantion calls for intensive research and makes it urgent to ban the use of all mammalian proteins, including swine, in the feed of all food animals, until better answers are found.
The evidence for the potential PSE (porcine spongiform encephalopathy ) is as follows. In 1979, an FSQS veternarian, Dr. Masuo Doi, noticed some unusual central nervous system (CNS) symptoms in young (about 6 months old) hogs coming into a slaughter plant In Albany, New York. Since the plant received hogs from a wide variety of sources (New York, Canada, Indiana, Illinois, Ohio, and other Midwestern states) and was not a plant used to dealing with diseased animals, Dr. Doi thought that the problem might be affecting hogs slaughtered nationwide. So, he decided to conduct a detailed study on central nervous system (CNS) symptoms/disease in young hogs coming into that slaughter plant. The study ran for 15 months (January, 1979 to March, 1980) and consisted of extended observations of the behavior of animals with suspected CNS symptoms at the plant, followed by pathological, histopatholpgical, and microbiological work on tissues from various organs of particular animals after slaughter.
For his behavioral observational work, Dr. Doi extended the usual two day observation period to three to four days, during which he took careful notes on the animals' behavior and other vital signs. During the 15 month period of the study, some 106 animals exhibiting CNS symptoms were retained during antemortem inspection.
A 1980 paper that summarized Dr. Doi's findings on the clinical symptoms and incidence of the 'disease," contained descriptions of these symptoms that sound remarkably similar to the symptoms noted for bovine spongiform encephalopathy (BSE):
"Excitable or nervous temperament to external stimuli such as touch to the skin, handling and menacing approach to the animals is a common characteristic sign among swine affected with the disease.... In the advanced stage of the disease, manifestation of neurological signs are evidenced in the form of general ataxia . . . Many animals have been found to be "downers' at first observation; if the hindquarters of these downers are raised they may be able to walk one or two steps and then fall to the ground" (Doi et al., 1980: 2, 4).
Indeed, a table of symptoms includes, for the early stage: "excitability and nervousness (squealing, smacking of lips, grinding of teath, chewing, gnawing ant foaming at mouth); stiffness of limbs . . . 'tic'; weakness of hindquarters; focal tremors of skeletal muscles"; and for the advanced stage: depression; ataxia; crossing over of limbs . . . kneeling posture . . . crawling". In addition to his clinical observations, Dr. Doi also made an 8 mm film of thirteen of the affected animals; film of two of the pigs was shown at the MPI National Pathology Meeting in Seattle, Washington on flay 20, 1979.
Dr. Doi sent tissue samples from suspect cases to the USDA's Eastern Laboratory in Athens, GA for pathological, histopathogical and microbiological work. Known infectious diseases were ruled out. As Dr. Doi points out, "Histopathological studies of tissue collected from the brain and spinal cord of these animals in the early stage of the disease show congestion, hemorrhage and neuronal degeneration. All animals in the advanced stage of the disease have been confined to have Encephalitis or Meningitis by MPI laboratory" (Doi et al., 1980: 5). Eventually some 60 animals were confirmed by the MPI Laboratory to have encephalitis or meningitis, with no ldentifiable cause. As pointed out in a paper presented at the 1979 MPI National Pathology Meetings,
"Since January, a number of hogs in this establishment have been found, in antemortem, to show what appears to be CNS. Sets of tissue samples were sent to the laboratory for examination, various tests were done which include histological study (E H stain), fluorescence antibody technique, virus neutralization and viral and bacteriological isolation. Differential diagnosis was also done to exclude vitamin B deficiency, post vaccination reaction, chlorinated hydrocarbon, arthritis, and transport stress" (Doi et al., 1979).
The brains of the 60 animals were examined. The brain of one of these pigs, on histopathological analysis, exhibited signs reminiscent of a TSE. This histopathological work was performed by Dr. Karl Langheinrich, Pathologist-In-Charge at USDA's Eastern Laboratory in Athens, Georgia. According to the USDA FSQS laboratory report, dated early November, 1979, Dr. Langheinrich noted:
"Microscopic examination of the barrow tissues revealed a encephalopathy and diffuse gliosis characterized by vacuolated neurons, loss of neurons and gliosis in a confined region (nucleus) of the brain stem (anterior ventral midbrain). Only an empty sometimes divided vacuole was present instead of the normal morphology of a nerve cell. Occasionally a shriveled neuron was seen. According to . . . Pathology of Domestic Animals, . . . 'The degeneration of neurons, the reactivity of the glia .... are the classical hallmarks of viral infection of the central nervous system' .... Scrapie of sheep, and encephalopathy of mink, according to the literature, all produce focal vacuolation of the neurons similar to the kind as described for this pig. I was unable to locate any lead as to the cause of this interesting phenomenon in other species including swine'' (Langheinrich, 1979).
Indeed, Dr. Langheinrich's main diagnosis was, " Encephalopathy and diffuse gliosis of undetermined etiology." Portions of the brain were sent for microbiological testing to a neurologist at the University of Georgia, where they came up negative for pseudo-rabies. The brain was unique enough that USDA scientists, such as Dr. Langheinrich and Or. Dot, mentioned it to student and scientific colleagues over the years.
In 1979-1980, BSE was completely unknown. However, both the behavior of the pigs, as well as the histopathology on at least one pig, both showed sign consistent with a porcine TSE. This raises particular concern became the affected animal was only 6 months old; in an animal this young, one would rust expect to see any physical signs of TSE in the brain. Histopathology of TSEs can be very variable, so that spongiform appearance (i.e. vacuolated neurons) are not always present. Behavioral changes can be seen in TSE-infected animals before any changes in brain morphology are visible. Dr. Clarence Gibbs, in testimony before a Congressional hearing on the TSE issue on January 29, 1997 made just this point:
''In the mid-1960s, we demonstrated with our French and English collaborators that during the early incubation of the TSEs, when the virus titer in the brain was very low, there were already marked functional changes, even though no pathology was yet detectable, even ultrastructurally. A month or hero later, polynucleation of neurons appeared in spider monkeys, incubating kuru, and somewhat later, microvacuolation and membrane changes visible only by electron microscopy. This preceded the pest appearance of astrogliosis and spongiform change. It was only much later that the classical scrapie TSE pathology appeared with virus titers in brain of 10 -5 or higher" (Gibbs, 1997; pg. 4).
Given that TSEs can cause behavioral changes in infected animals before any physical changes in the brain can be seen, that the manifestation of TSE in the brain can be quite variable, and that changes in brain morphology are not usually seen in 6 month old animals, we are concerned that the brain of one pig actually showed physical evidence consistent with a TSE.
Following the announcement In March, 1996 of ten cases of new variant CJD (Creutzfeldt-Jakob Disease) in the United Kingdom and their possible connection to BSE, Drs. Doi, Langheinrich and others urged reinvestigation of this case.
In August, 1996, the USDA sent five slides, one of which was a histopathology slide, to Dr. Janice Miller of USDA's Agricultural Research Servicer . Dr. Miller stained four of the slides for prion protein (she didn't stain the H&E slide). Dr. Miller told Consumers Union that Dr. Patrick McCaskey, USDA/FSIS, in charge of the Research Center at Athens, GA, called her, told her that he had five slides that all showed "problems" and asked her to stain four of them. The H&E slide, which clearly show vacuoles in the neurons (one sign of TSE), wasn't stained because to stain for PrP entails removing the slide cover, baking the slide to destain it and then restaining it for PrP; they didn't want to risk destroying the H&E slide.
Dr. Doi had kept frozen samples of the brain and spinal chord of the suspect PSE pig in case the Eastern lab wanted more material for analysis. Unfortunately, these samples were discarded when the packing plant in Albany, NY closed in 1991. It appears that the brain material sent to the Univcrsity of Georgia may have been discarded. [pers com.. Dr. Doi 3/13/97]
Dr. Miller found that the PrP stained in the four pig slides was found only on the inside of neurons, while a positive control slide from a scrapie sheep showed massive amounts of extraneuronal staining. In a letter summarizing her results (copy attached), she concludes that the PrP stained in this pig was normal: "In the pig sections you will see a small particulate type of staining that is confined to neurons and as I indicated on the phone, I would interpret as normal PrP. It is in marked contrast to the massive amount of extraneuronal staining seen in the scrapie section" (Miller, 1996).
Unfortunately, Dr. Miller's finding toes not conclusively rule out a TSE. We are concerned that while British BSE and serapie create a massive amount of extraneuronal staining, there are TSEs where this isn't the case. Three experiments were done in He U.S. -- in Mission, TX (APHIS work), Pullman, Washington (ARS work), and Ames, Iowa (ARS work) -- to see whether sheep scrapie can possibly infect cows. In all the experiments, cattle were inoculated with tissue from scrapie -infected sheep primarily by intra-cranial injection, but in the case of the Texas and Iowa studies also by oral feeding -- to see if cattle were susceptible to scrapie at all. In all three experiments, the majority of cows injected in the brain with scrapie-infected sheep material (usually brains) also developed a fatal spongiform encephalopathy.
However, in all three examples, the symptoms of the spongifonn encephalopathy differed from "mad cow" disease ~ England, as did the appearances of slides from their brains. The brain lesions seen in all these animals were more variable than those seen in England. When Dr. Miller did similar staining for PrP from these brains (what she called "bovine scrapie") she only found PrP stains on the inside of the neurons, not the massive extraneuronal staining seen in BSE (Miller, pers. comm., March 7, 1997). Thus, Dr. Miller's finding of PrP stains only inside the neurons in the suspect pigs is not particularly reassuring.
In November 1996, USDA sent the single histopathology slide to Dr. William Hadlow, one of the foremost spongiform encephalopathy pathologists in the world. (For unknown reasons, Dr. Hadlow was only sent the one slide; he was not told of the existence of the other slides, nor of Dr. Miller's findings, nor was he told or given the behavioral report from Dr. Doi or the morphology work by Dr. Langheinrich, or shown film of the affected pigs [Dr. Hadlow, pers. com., 3/13/97] From this single slide, Dr. Hadlow found some evidence consistent with TSEs but not enough for a conclusive diagnosis. He noted that the slide contained vacuoles inside neurons, one of the signs of a TSE (Dr. Langheinrich had noted this as well).
However, since such vacuoles occasionally occur normally in pigs, he thought that was not something special: "About twelve (12) neurons in the parasympathetic nucleus have unilocular optically empty vacuoles in the perikaryon. This is the site where such vacuolated neurons have been seen in the swine (as well as in cats and sheep) as an incidental finding. So I do not think such cells have any significance in this pig" (Hadlow, 1996). However, he did see evidence, Including changes in astrocytes, that suggested a TSE, but without examining other parts of the brain to look for other evidence of TSE, he couldn't be sure:
"I am impressed, though, with what seems to be an increase in the number of astrocytes in the section. Some astrocytes are in clusters, some are enlarged and vesicular. Where they are most numerous, a few rod cells (activated microglia) are seen. These findings suggest some perturbation of the nervous tissue. Although such a global response occurs in the transmissible spongifonn encephalopathies, I do no! know its significance in this case without examining other parts of the brain for changes characteristic of these diseases. Thus, from looking; at this one (1) section of brain, I cannot conclude that the pig was affected with a scrapie-like spongiform encephalopathy" (Hadlow, 1996).
In sum, Dr. Hadlow~s letter does not rule out the possibility of a TSE. He says that there is suggestive evidence, but that he would need to look at other slides/sections of the brain, to make a conclusive diagnosis.
In our view, the implications of this data are extremely serious. Experiments in the United Kingdom have shown that pigs are susceptible to BSE. Pigs inoculated with BSE develop a TSE (Dawson et al., 1990). Feeding experiments are underway in the UK to see if BSE can be orally transmitted to pigs; as of March, 1997, some 6 years after the start of the experiment, none of the pigs fed BSE brain have come down with a TSE. Unfortunately the design of this experiment severely limits what we will learn from it, and will most likely not tell us conclusively if pigs can get BSE from feed. It turns out that the pigs were not fed BSE brain continuously. Rather, the pigs were only fed BSE brain material on three days, over a three week period (i.e.. one day each week). Following these three doses, the pigs were never fed contaminated material again. The total amount of infective material given to the pigs was therefore quite small. Thus, a negative finding would be hard to interpret and would not mean that BSE is not orally active in pigs.
We believe that as a top priority USDA should conduct follow-up studies to look for potential CNS/PSE cases in pigs (we plan to communicate about this to USDA separately). In brief, we feel that the following kinds of studies need to be done:
i) TSE pathology experts should examine all the slides from the suspect pig (2709). To our knowledge, at least 12 separate slides exist.
ii) Determine if any brain material from the suspect pig (2709) still exists at the Unlverslty of Georgia. If so, this material should be retrieved and used for transmission studies. In particular, suckling pigs should be inoculated with the material and then permitted to live unto they die of a disease or old age, at which point their brains should be examined for physical signs of a TSE as well as for immunchistochemical evidence (i.e. staining looking for the abnormal PrP).
iii) Increase antemortem inspection for CNS symptoms at hog facilities. Inspectors should be trained to detect the subtle CNS symptoms seen in the Doi et al. study. At a select number of slaughter facilities, animals exhibiting CNS symptoms should be removed and held for observation until they die, at which time their brains should be examined for evidence of a TSE.
iv) Research on CNS symptoms among Me 6,000 or so breeding sows which are permitted to live for 3+ years. Sows exhibiting CNS symptoms should be removed and held for observation until they die, at which time then brains should be exernined for evidence of a TSE.
While such work is underway, given the above inforrnabon, we believe that as a precutionary measure the FDA must expand the proposed ruminant plus mink-to-ruminnant feed ban to prevent protein from any material, including hogs, being fed to any food animal.
Sincerely,
Michael Hansen, Ph.D Research Associate
Jean Halloran Director
References
Dawson, M., Wells, G.A.H., Parker, B.N;J. and A.C Scott. 1990. Primary parental transmission of bovine spongiform encephalopathy to the pig. Veternary Record, pg. 338.
Doi, M., Matzner, N.D. and C. Rothaug. 1979. Observation of CNS disease in market hogs at Est. 893 Tobin Packing Co., Inc. Albany, New York. United States Department of Agriculture, Food Safety and Quality.Service, Meat and Poultry Inspection Service. 7pp.
Doi, M, Langheinrich, K. and F. Rellosa. 1980. Observations of CNS signs in hogs at Est. 893 Tobin Packing C:o., Inc. Presented by Dr. Lngheinrich at the MPI National Pathology Meeting in Seattle, Washington on July 20, 1979.
Gibbs, C. 1997. Statement to the Committee on Governnent Reform and Oversight, Subcommittee on Human Resources and Intergovernmental Relations, U.S. House of Representatives. January 29,1997.
Hadlow, WJ. 1996. Letter to Patrick McCaskey, USDA/FSIS/Eastem Lab, dated November 13, 1996.
Langheinrich, KA. 1979. USDA/FSQS Laboratory report on specimen 2709. Dated November 8, 1979
Miller, J. 1996. Letter to Patrick McCaskey, USDA/ESIS/Eastern Lab, dated September 6, 1996.
Dr. Janice Miller, ARS
http://www.mad-cow.org/~tom/mad_pigs.html
Subject: EXPERIMENTAL INTRACEREBRAL AND ORAL INOCULATION OF SCRAPIE TO SWINE: PRELIMINARY REPORT
Date: February 6, 2006 at 12:33 pm PST
Title: EXPERIMENTAL INTRACEREBRAL AND ORAL INOCULATION OF SCRAPIE TO SWINE: PRELIMINARY REPORT
Authors
Greenlee, Justin
Kunkle, Robert - bob
Hamir, Amirali
Submitted to: American Association Of Veterinary Laboratory Diagnosticians
Publication Acceptance Date: November 5, 2005
Publication Date: November 5, 2005
Citation: Greenlee, J.J., Kunkle, R.A., Hamir, A.N. 2005. Experimental Intracerebral And Oral Inoculation Of Scrapie To Swine: Preliminary Report [abstract]. Proceedings Of The American Association Of Veterinary Laboratory Diagnosticians 48th Annual Conference. P. 38.
Technical Abstract: Transmissible spongiform encephalopathies (TSEs, prion diseases) are chronic neurodegenerative diseases that occur in humans, cattle, sheep, goats, cervids, and a number of laboratory animal models. In a laboratory setting, the host range of a given TSE can be tested by inoculating animals with brain tissue from affected animals through various routes including oral and intracranial. There is no evidence of the natural occurrence of any form of TSE in the pig, but pigs have been shown to be susceptible to bovine spongiform encephalopathy (BSE) infection by multiple-route parenteral challenge. However, pigs orally exposed at eight weeks of age to large amounts of brain from cattle clinically affected with BSE did not support infection after seven years of observation. In the United States, feeding of ruminant by-products to ruminants is prohibited, but feeding of ruminant materials to swine and poultry still occurs. The potential for swine to have access to scrapie-contaminated feedstuffs exists, but the potential for swine to serve as a host for replication/accumulation of the agent of scrapie is unknown. The purpose of this study was to perform oral and intracerebral inoculation of the U.S. scrapie agent to determine the potential of swine as a host for the scrapie agent and their clinical susceptibility. This study utilized 26 swine randomly divided into three groups: controls (n=6), oral inoculates (n=8), and intracranial inoculates (n=12). Brain homogenate (10%) derived from scrapie-affected sheep was given by a single intracranial injection of 0.75 ml or by oral inoculation of 15 ml on four consecutive days. Scrapie inoculum was derived from clinically ill sheep inoculated with material derived from 13 sheep in seven source flocks. A sample of this material was also inoculated back into sheep to assure infectivity. Necropsies were planned for six months post inoculation, at approximately the time the pigs were expected to reach market weight. Samples collected were examined microscopically after routine staining (hematoxylin and eosin) and staining by standard immunohistochemical methods for prion protein (PrP**Sc). After approximately six months incubation time, no histologic lesions
suggestive of spongiform encephalopathy or immunohistochemical evidence of prion infection were obtained. No evidence of scrapie infection was demonstrated in this short-term study, but positive results after an incubation period of only six months would be uncharacteristic. The only TSE with an incubation of six months or less known at this time is transmissible mink encephalopathy in mink, skunk, or raccoon hosts. However, scrapie in the raccoon model has a two-year incubation period. A replicate of littermate pigs has been inoculated and will be studied after long-term (3-7 years) incubation, and a similar study is underway with pigs inoculated with material derived from elk, mule deer, and whitetail deer affected by chronic wasting disease (CWD).
Project Team
Kehrli, Marcus
Nicholson, Eric
Greenlee, Justin
Hamir, Amirali
Richt, Juergen
Kunkle, Robert - Bob
http://www.ars.usda.gov/research/publications/publications.htm?SEQ_NO_115=191555
DOI: 10.3201/eid1508.081218
Suggested citation for this article: Espinosa J-C, Herva M-E, Andréoletti O, Padilla D, Lacroux C, Cassard H, et al. Transgenic mice expressing porcine prion protein resistant to classical scrapie but susceptible to sheep bovine spongiform encephalopathy and atypical scrapie. Emerg Infect Dis. 2009 Aug; [Epub ahead of print]
Transgenic Mice Expressing Porcine Prion Protein Resistant to Classical Scrapie but Susceptible to Sheep Bovine Spongiform Encephalopathy and Atypical Scrapie
Juan-Carlos Espinosa,1 María-Eugenia Herva,1 Olivier Andréoletti, Danielle Padilla, Caroline Lacroux, Hervé Cassard, Isabelle Lantier, Joaquin Castilla, and Juan-María Torres
Author affiliations: Centro de Investigación en Sanidad Animal, Madrid, Spain (J.-C. Espinosa, M.-E. Herva, D. Padilla, J. Castilla, J.-M. Torres); École Nationale Vétérinaire de Toulouse, Toulouse, France (O. Andréoletti, C. Lacroux, H. Cassard); and Centre Institut National de la Recherche Agronomique de Tours, Nouzilly, France (I. Lantier)
1These authors contributed equally to this article.
How susceptible pigs are to infection with sheep prions is unknown. We show, through transmission experiments in transgenic mice expressing porcine prion protein (PrP), that the susceptibility of this mouse model to bovine spongiform encephalopathy (BSE) can be enhanced after its passage in ARQ sheep, indicating that the pathogenicity of the BSE agent is modified after passage in sheep. Transgenic mice expressing porcine PrP were, nevertheless, completely resistant to infection with a broad panel of classical scrapie isolates from different sheep PrP genotypes and with different biochemical characteristics. The atypical (Nor98 like) isolate (SC-PS152) was the only scrapie isolate capable of transmission in these mice, although with a marked transmission barrier. Unexpectedly, the atypical scrapie agent appeared to undergo a strain phenotype shift upon transmission to porcine-PrP transgenic mice and acquired new strain properties, suggesting that atypical scrapie agent may exhibit different phenotypes depending on the host cellular PrP or other genetic factors.
SNIP...
The atypical isolate SC-PS152 was the only scrapie isolate able to infect the Po-PrP mouse model after intracerebral inoculation (Table), albeit with a low efficiency of infection in the first passage (attack rate 16%). These results suggest the potential ability of atypical scrapie prions to infect pigs, although with a strong transmission barrier. Given the increasing number of atypical scrapie cases found in Europe and in North America, the potential ability of atypical scrapie to adapt to the pig becoming more easily transmitted could raise concerns about the potential danger of feeding ruminant meat and bone meal to swine.
http://www.cdc.gov/eid/content/15/8/pdfs/08-1218.pdf
http://nor-98.blogspot.com/2009/07/transgenic-mice-expressing-porcine.html
Wednesday, July 1, 2009
Nor98 scrapie identified in the United States J Vet Diagn Invest 21:454-463 (2009)
http://nor-98.blogspot.com/2009/07/nor98-scrapie-identified-in-united.html
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