RESEARCH ARTICLE bpa_526 265..279
Use of Murine Bioassay to Resolve Ovine Transmissible Spongiform Encephalopathy Cases Showing a Bovine Spongiform Encephalopathy Molecular Profile
Spongiform Encephalopathy Molecular Profile Katy E. Beck1; Rosemary E. Sallis1; Richard Lockey1; Christopher M. Vickery1; Vincent Béringue2; Hubert Laude2; Thomas M. Holder1; Leigh Thorne1; Linda A. Terry1; Anna C. Tout1; Dhanushka Jayasena1; Peter C. Griffiths1; Saira Cawthraw1; Richard Ellis1; Anne Balkema-Buschmann3; Martin H. Groschup3; Marion M. Simmons1; John Spiropoulos1
1 Animal Health and Veterinary Laboratories Agency, Addlestone, Surrey, UK.
2 Virologie Immunologie Moléculaires, U892, Institut National de la Recherche Agronomique, Jouy-en-Josas, France. 3 Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Greifswald-Insel Riems, Germany.
Two cases of unusual transmissible spongiform encephalopathy (TSE) were diagnosed on the same farm in ARQ/ARQ PrP sheep showing attributes of both bovine spongiform encephalopathy (BSE) and scrapie. These cases, UK-1 and UK-2, were investigated further by transmissions to wild-type and ovine transgenic mice. Lesion profiles (LP) on primary isolation and subpassage, incubation period (IP) of disease, PrPSc immunohistochemical (IHC) deposition pattern and Western blot profiles were used to characterize the prions causing disease in these sheep. Results showed that both cases were compatible with scrapie. The presence of BSE was contraindicated by the following: LP on primary isolation in RIII and/or MR (modified RIII) mice; IP and LP after serial passage in wild-type mice; PrPSc deposition pattern in wild-type mice; and IP andWestern blot data in transgenic mice. Furthermore, immunohistochemistry (IHC) revealed that each case generated two distinct PrPSc deposition patterns in both wild-type and transgenic mice, suggesting that two scrapie strains coexisted in the ovine hosts. Critically, these data confirmed the original differential IHC categorization that these UK-1 and UK-2 cases were not compatible with BSE.
In conclusion, results from the mouse bioassay confirm that UK-1 and UK-2 are consistent with classical scrapie. Since bioassay data for ovine passaged atypical (H- and L- type) BSE are not available at this time, conclusions cannot be made regarding these BSE variants. While BSE has not been identified in sheep to date, further investigation of ambiguous cases remains vital as experimental transmission of BSE to sheep is possible (5, 22). Utilizing a combined approach and analyzing biological and biochemical aspects of transmission, we have not only concluded that UK-1 and UK-2 are scrapie, but we are able to hypothesize that each isolate comprised two strains that most likely coexisted in the natural host based on the wild-type and particularly the transgenic mouse data. In our view, the use of transgenic mouse lines is preferable for such investigations. This is because there is no transmission barrier if appropriate lines have been selected, giving a higher attack rate and a greater assurance that what is isolated in the mouse better reflects the agent in the sheep. While different isolates will undoubtedly present different challenges, further expanding our current knowledge regarding the discrimination of TSE agents in wild-type and transgenic mouse models will permit the continued, effective discrimination of TSE strains via the bioassay, using the most suitable mouse lines to reach an unequivocal diagnosis.
How did top scientists mix up brains from cattle and sheep?
By Steve Connor Science Editor Saturday 20 October 2001
The mystery of how a world-class team of scientists could have spent nearly five years and £217,000 mistakenly testing cow brains instead of sheep brains for signs of BSE deepened yesterday.
Labour 'buried report of BSE inquiry'
The mystery of how a world-class team of scientists could have spent nearly five years and £217,000 mistakenly testing cow brains instead of sheep brains for signs of BSE deepened yesterday.
The study was meant to assess whether bovine spongiform encephalopathy had infected sheep at the end of the 1980s when they were fed the same contaminated feed as cattle, but the results have now been declared uninterpretable because of the mix-up.
On a scale of laboratory blunders the error ranks about as high as they come, given that the fate of Britain's 40 million sheep might have rested on its outcome. Finding BSE in sheep could have led to the culling of the entire national flock.
How scientists could have confused brain material from cattle and sheep is now the subject of two inquiries, one by the Institute for Animal Health, the government-funded laboratory where the research was done, and another by the Department for the Environment, Food and Rural Affairs (Defra), which funded the study.
Both inquiries will attempt to explain how two tests early in the experiment appeared to confirm that the material was the mashed-up brains of sheep while a third, more definitive test, which was made public on Wednesday night, found only bovine brain and no evidence of sheep brain at all.
The roots of the story lie in an experiment conceived and run at the height of the BSE epidemic in the late 1980s and early 1990s. At that time, scientists wanted to know what effect different meat-rendering practices would have on the infectious agents behind both BSE and scrapie, a similar brain disease of sheep.
During 1990, scientists from the Government's Central Veterinary Laboratory at Weybridge, Surrey, collected the diseased brains of more than 800 cows with BSE to "pool" the material and test how it would survive different rendering practices. A second phase of the study took place over the next two years when 2,860 brains of sheep affected by scrapie were collected by 18 veterinary laboratories around the country and pooled for a similar rendering experiment.
Material left over from both these experiments was frozen and stored. Not until 1996, when the link was established between BSE and a variant of human Creutzfeldt-Jakob disease (vCJD), did scientists think the stored sheep material might be useful in answering another question: could BSE have got into sheep?
There has always been a theoretical possibility that BSE infected sheep. Laboratory experiments show the agent can be transmitted through feeding and some sheep were known to have eaten the same BSE-contaminated feed as cattle.
The big question is whether infection had actually happened in the late 1980s, before measures were fully enforced to ban the feeding of ruminant- derived feed to ruminants such as sheep and cattle. Realising that they had a store of sheep brains from the early 1990s, scientists suggested "testing" this material for BSE.
However, a possible complication was that the pooled sheep brains may have been cross-contaminated with infected cattle brains. Both sets of brains were removed by veterinary scientists in the same centres using the same instruments and surgical slabs.
With cross-contamination a possibility, the Institute for Animal Health and the Central Veterinary Laboratory did two sets of tests to see whether the material – which looks like porridge – was fundamentally ovine rather than bovine in origin.
Professor Chris Bostock, the institute's director, said one test involved looking for the presence of the amino acid arginine at "position 171" on the molecules of the prion protein present in the sample. The "arginine 171" signature is unique to sheep and the test proved positive, he said. The Central Veterinary Laboratory was also unable to detect bovine material. "This was psychologically good news as far as contamination was concerned," Professor Bostock said.
Convinced that the pooled material was largely if not entirely sheep brains, the complicated experiment took place within the institute's neuropathogenesis unit in Edinburgh, the research centre that did the work proving the link between BSE and vCJD. The research involved injecting the pooled brain material into different strains of laboratory mice, which incubate the disease in a precise pattern depending on whether it is scrapie or BSE. The object was to see if the sheep that had died with "scrapie" were in fact suffering from BSE.
Preliminary results of the experiment were sent to the Food Standards Agency, which issued a statement two months ago to coincide with a separate initiative by the sheep industry to boost the consumption of lamb. The agency warned there was still a "theoretical risk" of BSE infecting sheep and said early results from the experiment involving the pooled brains from the early 1990s "could be compatible with BSE having been in sheep at that time".
Professor Bostock, who was on a holiday when the agency made its statement, was not happy. He said at the time: "I personally think that it is completely unhelpful to start discussing results until they are complete and in the public domain in a way that everyone can see what we are talking about."
The issue of possible cross-contamination had still not been resolved so Defra organised independent DNA tests of the brain material, which began in September. The results were unequivocal: the material was entirely bovine with no trace of sheep tissue. "Extraordinary," declared Professor Peter Smith of the Government's spongiform encephalopathy advisory committee. Professor Bostock said he was "flabbergasted".
One possibility being investigated is that the pool of cattle brains collected in 1990 was confused with the pool of sheep brains collected two years later.
Professor Bostock said: "There has to be an explanation for this discrepancy. We'll have to wait to see what [it] is."
Meanwhile, we are still no nearer to knowing whether BSE has infected sheep.
TSE in Sheep Contingency Planning Assessment of Risk due to BSE Infectivity from Disposal of Sheep A report for DEFRA November 2001
Management Summary It has been recognised for a considerable time that sheep in the United Kingdom may have been infected with BSE. To date no evidence has been found to demonstrate that the national flock is actually infected with the disease. DEFRA have prepared a draft contingency plan in the event that BSE were to be identified in UK sheep. The worst case scenario under this plan is the disposal of the entire UK flock, some 40 million sheep and lambs. This study has estimated the potential exposure of the UK population to BSE infectivity present in sheep in the event that this plan had to be put into effect.
but who would have guessed that such an important experiment/study would have gotton so screwed up, by not being able to tell a sheep brain from a cow brain;
© DEFRA 2002 Item 3- Scrapie Brain pool experiments- Update on current position and audits of samples 3.1 Members were updated on experiments conducted at the Institute of Animal Health (IAH) to examine a pool of scrapie brains collected in the early 1990 s for evidence of BSE. SEAC had previously recommended that the material should be examined by DNA analysis to assess whether the pooled brain material may have been contaminated with bovine tissue. The Laboratory of the Government Chemist (LGC) had been asked to perform the work. Their results were completely unexpected as the analysis detected only bovine material in the sample. SEAC had intended to meet on the 19 October to Agreed version consider the experiment in detail. However, in view of the result, the meeting was cancelled.
Executive Summary An audit of the sample handling procedures at IAH-E was carried out on 24 October 2001 at the request of the Department of the Environment, Food and Rural Affairs (DEFRA), by a team of two UKAS auditors. The scope of the audit was limited to the traceability of cow and sheep brain samples used in several experiments relating to transmissible spongiform encephalopathy (TSE) agents. In particular, the team focused on the audit trail of samples that had been sent to LGC, Teddington, the audit trail of brains collected in 1990/92 by Veterinary Investigation Centres and the audit trail for archived material held by IAH-E. In addition the audit team evaluated the IAH-E procedures against the specific requirements for sampling handling of international standard, ISO 17025 and identified opportunities for improvement. The audit established that there was no formal documented quality system covering this work at IAH-E and that record keeping was inadequate to give confidence in the chain of custody of samples used in the various rendering, genotyping and strain typing experiments audited. It was not possible to establish clear traceability between the samples that had been used in the individual experiments carried out by IAH-E or IAH-C with those analysed at LGC or with those that had been collected in 1990/92. The sample handling procedures covered by this audit at IAH-E did not meet the requirements of ISO 17025.
explaining the brain mixup blunder;
An Investigation of the Substitution of Scrapie Brain Pool Samples A report for DEFRA November 2001
Risk Solutions Page 19 Why did the experimenters not notice that they were working with cow brains not sheep brains? The simple answer is because for the most part they were working with brain pool macerate (minced brain material) not brains. It is not credible that staff collecting brains at VICs would have uniformly supplied cow brains or cow brain parts in mistake for sheep. We have interviewed staff at VICs and we understand from the VLA that records do not support the possibility that significant numbers of cow brains were sent to PDM in place of sheep brains. It is also very unlikely that the people preparing the scrapie brain pool would not have noticed if they were for the most part handling cow brains or cow brain parts in place of sheep brains. We cannot rule out the possibility that some cow brain material entered the brain pool at this stage but it is not feasible that the majority of the material was bovine. The substitution, if substitution occurred, must have involved brain pool macerate or rendered products. Why can t the results of the experiments tell us what material was used? The experiments had a number of features that make the results of the mouse bioassay difficult to interpret unambiguously and lead to the possibility that substitution of the samples would be difficult to detect by examining the results of the experiments: 1. The original experiments were not designed to determine whether BSE was present in sheep. Reasonable efforts were taken to ensure that the brain pool remained free from D5055 02 Issue 1 Risk Solutions Page 20 contamination during preparation but the level of control applied during the earlier experiments (272R and 372R) was not to the standard applied later. 2. Mouse bioassay as a method of diagnosing TSEs is not based on a full understanding of biochemical and physical processes. It is an empirical technique that has been widely applied, for example to show v-CJD is similar to BSE and different from scrapie. It is a complex process and the results need to be interpreted by experts. It can take several years to generate a firm result. The principal data collected in the experiments are lesion profiles (patterns of lesions in the mice brains) and incubation period (time from injection of mice to onset of clinical symptoms. The type of TSE is identified by comparing the results with those of known provenance. There is no good agreed test of sameness of lesion profile , so in marginal cases we are reduced to using subjective observations of the form somewhat similar and interpretation is difficult. The incubation times in principle give a more objective signal, but the effect of concentration has to be controlled. The mouse bioassay data that we understand has been collected and analysed at each stage of the experiments is summarised in Table 4.1. Several features of these experiments are not commonly encountered in mouse bioassay of TSEs and this makes determining the origin of the original material from the experimental results extremely difficult. They include: a. Mouse bioassay is generally carried out on individual brains; experience of working with brain pools is very limited. b. The BBP exhibited a low titre of infectivity, which can confound interpretation of results. c. The BBP comprised bovine brains with the hindbrains removed. By contrast most of the BSE strain typing has been carried out on the hindbrains, which may give a different pattern of results. d. The 272R titrations used a different strain of mice than the 372R titrations, so direct comparison of the resulting lesion profiles cannot be made. e. The 246 experiments used brain pool which was in an unsatisfactorily autolysed state. f. The strain typing data collected (incubation time and lesion profiles) are very sparse. Judging the sameness or difference of samples is a less challenging task for strain typing than identifying a strain and it may be possible to compare data from the 246 experiments with both the 272R and 372R experiments to determine whether the samples are similar or clearly different. However, the data are sparse and the result is unlikely to be clear cut. Much of this work is currently unpublished.
RESPONSE TO THE UKAS REPORT FROM THE INSTITUTE FOR ANIMAL HEALTH
The Institute is concerned, therefore, that the authors of this UKAS report may have based their findings on an unrepresentative and limited examination of procedures in place at IAH-E.
Transmission of prion diseases by blood transfusion
Nora Hunter,1 James Foster,1 Angela Chong,1 Sandra McCutcheon,2 David
Parnham,1 Samantha Eaton,1 Calum MacKenzie1 and Fiona Houston2
TSEs TRANSMISSION STUDIES
what a coincedence , CONVENIENTLY, MORE FLUBBED UP BRAINS;
b) Fibrillar material closely similar to SAF, found in BSE/Scrapie, was observed in 19 (4.3%) cases, all of which were hounds > 7 years of age. 14/19 of these suspected SAF results correlated with cases in the unresolveable histopathological catergory...
HOUND SURVEY (about 72 pages)
Also, at paragraph 17, it is noted that BSE had transmitted to the NPU negative line sheep (please not that as at January 1996, only one of six challenged sheep was clinically affected after oral challenge, four others have since died, and one remains alive. Following intracerebral challenge, three out of six were clinically affected, two confirmed only on pathology, while one was negative.)
4. Meeting 16, on 26/1/94 - the update on research (16/5) confirmed that BSE had been transmitted to sheep, and that there was clinical evidence of transmission to mice from the spleen of the affected sheep.
A STUDY AIMED AT DETERMINING WHETHER OR NOT THERE HAVE BEEN SIGNIFICANT CHANGES IN THE NEUROPATHOLOGY OF SCRAPIE IN SHEEP AND GOATS DURING THE LAST TWO DECADES IN MATERIAL SUBMITTED TO CVL PATHOLOGY DEPARTMENT
EXPERIMENTAL TRANSMISSION OF BSE TO SHEEP
THE RISK OF TRANSMISSION OF BSE TO SHEEP VIA FEED
hell, they knew they were screwing up the sheep brains with cow brains in 1992;
"The sensitivity of the project may be partially compromised by pooling of brains, but it is considered that the success of transmission to mice with BSE will prove advantageous."
Personal $ Confidential -- Addressee only TO ALL MEMBERS OF SEAC
THE EXPERIMENTAL TRANSMISSION OF BSE TO SHEEP
a) Summary of transmission studies. b) Update
The only circumstance in which infection with the natural isolate produces an higher incidence of disease compared to BSE, is in intracerebrally (and possibly orally) challenged ''positive'' line sheep. Notwithstanding the possibility of indigenous natural scrapie in some of these sheep, there are still sufficient numbers of transmission cases with PrP genotypes which preclude the natural disease developing i.e. those typed as VA136/RR154/QR171.
As an extension to this study, it has been possible to recover BSE by passage in mice from brain and spleen taken from ''negative'' line sheep infected with BSAE by ic and oral challenge (Foster and others 1996). The close similarity of incubation periods and pathology from the passage of these tissues in mice to those seen in direct BSE transmission studies from cattle to mice suggests that passaging BSE in sheep does not alter its bilogical properties (Bruce and others 1994). IN FACT, because it has been possible to isolate BSE infectivity from ovine spleens, when this proved impossible from the spleens of naturally infected BSE cows (Fraser and Foster 1993), experimentally-induced BSE in sheep appears to behave more like the natural disease of scrapie.Whether this putative similarity to natural scrapie extends to the possibility of maternal transmission of experimentally-induced BSE in sheep, has till to be elucidated...
we have found a link between BSE and CH1641, a C-group of scrapie. Disease susceptibility of sheep to these isolates is associated with different PrP genotypes compared to SSBP/1 scrapie...
Transmission of BSE in sheep, goats and mice.
BSE has been transmitted in two lines of genetically selected sheep (differeing in their susceptibilities to the SSBP/1 source of scrapie), and to goats by intracerebral injection AND BY ORAL DOSING.
Also, intermediate passage of BSE in sheep or goats did not alter these primary transmission properties. Hamsters were susceptible to BSE only after intervening passage through mice...
Perceptions of unconventional slow virus in the USA
3. Prof. A Robertson gave a brief account of BSE. The US approach was to accord it a very low profile indeed. Dr. A Thiermann showed the picture in the ''Independent'' with cattle being incinerated and thought this was a fantical incident to be avoided in the USA AT ALL COSTS. BSE was not reported in the USA...........(some good data on CWD)
> avoided in the USA AT ALL COSTS
and indeed they have and it continues today...TSS
BSE TRANSMISSION STUDIES
Furthermore, we showed that the strain responsible for iCJD is closely related to that of one patient with sCJD, and, more unexpectedly, that these agents were similar to the French scrapie strain studied (but different from the U.S. scrapie strain). This finding requires a cautious interpretation for several reasons, not least because of the inevitably limited number of TSE strains that can be studied by such a cumbersome method as strain typing. Nonetheless, it also prompts reconsideration of the possibility that, in some instances, sheep and human TSEs can share a common origin.
WHAT ABOUT THAT HOUND STUDY AND THOSE BRAINS ???
OR-09 15:10 - 15:25 CANINE SPONGIFORM ENCEPHALOPATHY: A NEW FORM OF ANIMAL PRION DISEASE David
not knew to me. ...TSS
Monday, March 8, 2010
Canine Spongiform Encephalopathy aka MAD DOG DISEASE
TSE in dogs have not been documented simply because OF THE ONLY STUDY, those brain tissue samples were screwed up too. see my investigation of this here, and to follow, later follow up, a letter from defra, AND SEE SUSPICIOUS BRAIN TISSUE SAF's. ...TSS
SEE MORE HERE ;
Monday, March 26, 2012
CANINE SPONGIFORM ENCEPHALOPATHY: A NEW FORM OF ANIMAL PRION DISEASE
Monday, December 1, 2008
When Atypical Scrapie cross species barriers
Andreoletti O., Herva M. H., Cassard H., Espinosa J. C., Lacroux C., Simon S., Padilla D., Benestad S. L., Lantier F., Schelcher F., Grassi J., Torres, J. M., UMR INRA ENVT 1225, Ecole Nationale Veterinaire de Toulouse.France; ICISA-INlA, Madrid, Spain; CEA, IBiTec-5, DSV, CEA/Saclay, Gif sur Yvette cedex, France; National Veterinary Institute, Postboks 750 Sentrum, 0106 Oslo, Norway, INRA IASP, Centre INRA de Tours, 3738O Nouzilly, France.
Atypical scrapie is a TSE occurring in small ruminants and harbouring peculiar clinical, epidemiological and biochemical properties. Currently this form of disease is identified in a large number of countries. In this study we report the transmission of an atypical scrapie isolate through different species barriers as modeled by transgenic mice (Tg) expressing different species PRP sequence.
The donor isolate was collected in 1995 in a French commercial sheep flock. inoculation into AHQ/AHQ sheep induced a disease which had all neuro-pathological and biochemical characteristics of atypical scrapie. Transmitted into Transgenic mice expressing either ovine or PrPc, the isolate retained all the described characteristics of atypical scrapie.
Surprisingly the TSE agent characteristics were dramatically different v/hen passaged into Tg bovine mice. The recovered TSE agent had biological and biochemical characteristics similar to those of atypical BSE L in the same mouse model. Moreover, whereas no other TSE agent than BSE were shown to transmit into Tg porcine mice, atypical scrapie was able to develop into this model, albeit with low attack rate on first passage.
Furthermore, after adaptation in the porcine mouse model this prion showed similar biological and biochemical characteristics than BSE adapted to this porcine mouse model. Altogether these data indicate.
(i) the unsuspected potential abilities of atypical scrapie to cross species barriers
(ii) the possible capacity of this agent to acquire new characteristics when crossing species barrier
These findings raise some interrogation on the concept of TSE strain and on the origin of the diversity of the TSE agents and could have consequences on field TSE control measures.
Saturday, December 3, 2011
Isolation of Prion with BSE Properties from Farmed Goat
Volume 17, Number 12—December 2011
Wednesday, January 18, 2012
BSE IN GOATS CAN BE MISTAKEN FOR SCRAPIE
February 1, 2012
Wednesday, January 18, 2012
Selection of Distinct Strain Phenotypes in Mice Infected by Ovine Natural Scrapie Isolates Similar to CH1641 Experimental Scrapie
Journal of Neuropathology & Experimental Neurology:
February 2012 - Volume 71 - Issue 2 - p 140–147
Thursday, March 29, 2012
atypical Nor-98 Scrapie has spread from coast to coast in the USA 2012
NIAA Annual Conference April 11-14, 2011San Antonio, Texas
Wednesday, February 16, 2011
SCRAPIE TRANSMISSION TO CHIMPANZEES
Sunday, April 18, 2010
SCRAPIE AND ATYPICAL SCRAPIE TRANSMISSION STUDIES A REVIEW 2010
Monday, April 25, 2011
Experimental Oral Transmission of Atypical Scrapie to Sheep
Volume 17, Number 5-May 2011
Sunday, March 28, 2010
Nor-98 atypical Scrapie, atypical BSE, spontaneous TSE, trade policy, sound science ?
Monday, November 30, 2009
USDA AND OIE COLLABORATE TO EXCLUDE ATYPICAL SCRAPIE NOR-98 ANIMAL HEALTH CODE
I strenuously urge the USDA and the OIE et al to revoke the exemption of the legal global trading of atypical Nor-98 scrapie TSE. ...TSS
Friday, February 11, 2011
Atypical/Nor98 Scrapie Infectivity in Sheep Peripheral Tissues
Sunday, March 11, 2012
APHIS Proposes New Bovine Spongiform Encephalopathy Import Regulations in Line with International Animal Health Standards Proposal Aims to Ensure Health of the U.S. Beef Herd, Assist in Negotiations