Wednesday, January 18, 2017

EU-approved rapid tests might underestimate bovine spongiform encephalopathy infection in goats

EU-approved rapid tests might underestimate bovine spongiform encephalopathy infection in goats

Journal of Veterinary Diagnostic Investigation 1–5 © 2017 The Author(s) Reprints and permissions: sagepub.com/journalsPermissions.nav DOI: 10.1177/1040638716688045 journals.sagepub.com/home/jvdi

Daniela Meloni,1 Elena Bozzetta, Jan P. M. Langeveld, Martin H. Groschup, Wilfred Goldmann, Olivier Andrèoletti, Isabelle Lantier, Lucien Van Keulen, Alex Bossers, Danilo Pitardi, Romolo Nonno, Theodoros Sklaviadis, Francesco Ingravalle, Simone Peletto, Silvia Colussi, Pier Luigi Acutis 

Abstract. 

We report the diagnostic sensitivity of 3 EU-approved rapid tests (ELISAs; 1 from IDEXX and 2 from BioRad) for the detection of transmissible spongiform encephalopathy diseases in goats. Ninety-eight goat brainstem samples were tested. All the rapid tests had 100% specificity and ≥80% sensitivity, with the IDEXX test significantly more sensitive than the 2 Bio-Rad tests. All tests detected 100% of samples from goats with clinical scrapie, but missed 8% (IDEXX) to 33% (Bio-Rad SG) of samples from preclinical goats. Importantly, only IDEXX picked up all samples from clinical bovine spongiform encephalopathy (BSE)-infected goats, whereas the other 2 rapid tests missed 15% (Bio-Rad SG) to 25% (Bio-Rad SAP). These results show that a fraction of preclinical scrapie infections are likely missed by EU surveillance, with sensitivity of detection strongly dependent on the choice of the rapid test. Moreover, a significant proportion of clinical BSE infections are underestimated by using either Bio-Rad test. Assuming that the same sensitivity on preclinical goats would also occur in BSE-infected goats, our data suggest that IDEXX is likely the most sensitive test for detecting preclinical field cases of BSE infection in goats, although with an 8% failure rate. These results raise some concerns about the reliability of current EU surveillance figures on BSE infection in goats. 

Key words: Bovine spongiform encephalopathy; diagnosis; EU; goats; rapid tests; scrapie; sensitivity; surveillance.

snip...

Finally, we compared the sensitivity of rapid tests in recognizing goats with scrapie in the preclinical or clinical phase of disease. Although all rapid tests were systematically able to pick up both natural and experimental scrapie samples from symptomatic goats (Table 3), the IDEXX test missed 2 of 24 samples with “natural” scrapie in the preclinical phase of disease, the Bio-Rad SAP test missed 4 samples, and the Bio-Rad SG test missed 7 (Table 3). ROC curve analysis showed that IDEXX and Bio-Rad SAP tests were significantly more sensitive than the Bio-Rad SG test (Table 4) in detecting positive samples from preclinical animals. 

Several important features of our study should be considered for the surveillance of TSE diseases in goats. All tests detected 100% of samples from goats with clinical scrapie, regardless of whether they were experimentally or naturally infected. In contrast, sensitivity was lower in goats with preclinical scrapie, and rapid tests missed 8% (IDEXX) to 33% (Bio-Rad SG) of these samples. A second important consideration is that only IDEXX detected all samples from clinical BSE-infected goats, whereas the other 2 rapid tests missed 15% (Bio-Rad SG) to 25% (Bio-Rad SAP) of samples. These results suggest that a consistent fraction of preclinical scrapie infection is likely missed by EU surveillance, mostly in areas where Bio-Rad tests are in use, and that BSE infection in goats may also be underreported in areas using the BioRad rapid tests (Tables 2, 4). Assuming that the same sensitivity in preclinical goats would also occur in BSE-infected goats, our data show that the IDEXX test may detect eventual preclinical field cases of BSE infection in goats, although with a disappointing 8% failure rate. Although the analytical sensitivity of some TSE rapid tests might be reduced by the method used to prepare our samples,1,21 the results raise some concerns in relation to the current figures on BSE infections in goats deriving from EU surveillance. 

In goats, the difference in performance of rapid tests between scrapie and BSE infection might depend on the use of PK digestion, the choice of the primary anti-PrP antibodies, or both. PK digestion is used by both Bio-Rad tests but not by the IDEXX test, and it is likely that antibodies used in each kit recognize different PrP epitopes. This last hypothesis, however, is purely speculative because the details on anti-PrP antibodies are covered by patents and are therefore not publicly available. 

The other interesting result, although based solely on 2 samples, is that only the IDEXX and Bio-Rad SAP tests were able to fully recognize samples from goats with the atypical Nor98 scrapie infection, suggesting that surveillance in countries using the Bio-Rad SG test would miss a proportion of atypical scrapie infections in the goat population. The small number of samples, however, is too low to allow a firm conclusion. 

All rapid tests in our study failed to recognize the same 2 samples of “natural” preclinical scrapie. This finding is of concern because it might indicate that there is a small subpopulation of “naturally” scrapie-infected goats (e.g., early preclinical animals) that would be missed by all available rapid tests, and thus by the surveillance system. PRNP polymorphisms might reduce the sensitivity of the assays in goats carrying specific genotypes by reducing antibodybinding epitopes.18,20 In our samples, however, statistical analysis did not show any association between failure of each test and goat genotypes (data not shown). The reason for this finding remains therefore unknown and might simply depend on low levels of PrPSc. Ultimately, none of the 3 rapid tests produced any false-positive results, showing a reassuring 100% specificity.



Bovine spongiform encephalopathy in a goat in France

The occurrence of the first case of bovine spongiform encephalopathy (BSE) in a goat in France was confirmed on 28 January 2005 by a panel of scientific experts from specialised laboratories in the European Union including the OIE Reference Laboratory for BSE, the Veterinary Laboratories Agency at Weybridge in the United Kingdom.

The French Veterinary Authorities had informed the OIE in November 2004 that the animal, which was two and a half years old at the time of its slaughter in 2002, formed part of a flock of 600 goats including 300 adults. The entire herd had been culled and all the adult goats were tested for scrapie at that time, with negative results. All the carcasses, including that of the affected goat, were destroyed. The scrapie isolate from the infected goat was subjected to further diagnostic testing by inoculation in transgenic mice which is the reference test to distinguish BSE from scrapie. The necessity to wait for several months to obtain such diagnosis explains the long delay in confirming the case.

Through its network of Reference Laboratories and experts, the OIE maintains a close watch on all events related to BSE and is well aware of the importance of reaching a full understanding of this case.

It has therefore called a meeting of internationally renowned BSE experts at the OIE Headquarters, Paris, France on 17-18 March 2005. During this meeting, the experts will discuss the possible existence of BSE phenotypes, including the BSE situation in goats. The potential impact of BSE in goats on public health will also be discussed, although it is noted that any possible risk to consumers is currently to be considered as negligible in view of the extremely low prevalence of transmissible spongiform encephalopathies in goats, and the public health measures already in place in the EU. The experts will also discuss whether the OIE needs to modify its international standards on BSE and scrapie to address this new situation. The outcomes of this meeting will be discussed by all OIE Member Countries at the annual OIE General Session in May 2005.

The OIE works in close collaboration with the European Union (EU) regarding BSE. The surveillance standards on BSE proposed by the OIE to its 167 Member Countries take on board the expertise provided by the EU.


Isolation of Prion with BSE Properties from Farmed Goat 

John Spiropoulos, Richard Lockey, Rosemary E. Sallis, Linda A. Terry, Leigh Thorne, Thomas M. Holder, Katy E. Beck, and Marion M. Simmons

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases that include variant Creutzfeldt-Jakob disease in humans, scrapie in small ruminants, and bovine spongiform encephalopathy (BSE) in cattle. Scrapie is not considered a public health risk, but BSE has been linked to variant Creutzfeldt-Jakob disease. Small ruminants are susceptible to BSE, and in 2005 BSE was identified in a farmed goat in France. We confirm another BSE case in a goat in which scrapie was originally diagnosed and retrospectively identified as suspected BSE. The prion strain in this case was further characterized by mouse bioassay after extraction from formaldehyde-fixed brain tissue embedded in paraffin blocks. Our data show that BSE can infect small ruminants under natural conditions and could be misdiagnosed as scrapie. Surveillance should continue so that another outbreak of this zoonotic transmissible spongiform encephalopathy can be prevented and public health safeguarded.

snip...

Discussion We confirmed that the agent responsible for TSE in a UK goat, which was initially reported as scrapie in 1990 and subsequently as suspected BSE in 2006 (16), was a BSE agent. This conclusion was based on bioassay of nervous tissue in mice demonstrating similarities of histopathologic lesions, PrPSc mapping in the brain, and WB of PrPSc with those of mice inoculated with BSE from various ovine, caprine, and bovine sources.

snip...

The BSE case we have confirmed was 1 of 26 historic goat samples examined in the United Kingdom collected during 1984–2002 (16,17). Since 1993, scrapie in goats has been a notifiable disease in the United Kingdom, and since 2005, samples from all suspected cases of TSE in small ruminants are required to be tested for BSE-like features by using WB (19). No BSE cases have been identifi ed, although an intermediate case in a goat was reported and is under investigation by bioassay for fi nal resolution (35,36). This screening of brain samples from all small ruminant cases offers reassurance that BSE is not present in the contemporary small ruminant population. However, application of WB to sheep experimentally co-infected with BSE and scrapie detected only the scrapie agent (37). Also, in contrast to cattle, where infectivity is mainly confi ned to the nervous system, in small ruminants the BSE agent is widely distributed in peripheral tissues and can be transmitted horizontally (11,38). Therefore, feed ban measures alone would be inadequate to control a BSE outbreak in small ruminants. Also, it would be impossible to prevent BSE from entering the human food chain through consumption of food products derived from small ruminants. Because TSEs in goats are still a problem, particularly in Mediterranean countries, our data suggest that extensive surveillance and breeding schemes must remain in place to prevent a BSE outbreak in small ruminants and to safeguard public health. This report also highlights several issues regarding the use of mouse bioassay to identify TSE strains. As governing bodies seek confirmation of equivocal cases that are identified worldwide, they must be aware of the limitations, cost, and timescale demands of confirming such cases.

see full text ;


I have been concerned with TSE Prion testing in the USA for years and years, and have proven that the TSE Prion testing used at times to test for TSE Prion in the USA has been indeed terribly flawed and corrupt i.e. the infamous mad sheep of mad river valley and those follies there from. So, i was curious as to the testing regime used by the USA to detect TSE Prion in Goats, and would the USDA/APHIS/FSIS/FDA et al would be able to detect any typical or atypical BSE in Goats in the USA. i reached out to someone that would know, and here was their reply;

-----Original Message-----

From: Terry Singeltary <flounder9@verizon.net>

To: 

Sent: Tue, Jan 17, 2017 2:10 pm

Subject: tse prion diagnostic test for goat ?

Greetings Dr. 

Happy New Year for 2017!

i was curious as to the types of tse prion testing used on goats here in the US?

i know there is a link, i have changed computers, and have apparently lost those links. 

any help will be appreciated.

many thanks, kindest regards, terry

===========================

With the exception of third eyelid biopsy which are not done on goats we use the same tissues and tests for goats as we do for sheep primarily immunohistochemistry on obex, lymph nodes and tonsil in dead animals and rectal biopsy in live animals. A few western blots are done on poor quality tissue from suspect animals and to further characterizing positive tissues.  ELISA may be used on rare occasions usually to get quicker results on exposed animals that have been depopulated.

=========================

are you concerned with this? i am. should i be?


kind regards, terry

============================

While a sensitivity of less than 100% is not ideal for establishing the disease status of an individual animal, on a population basis the tests should be adequate for determining if BSE is spreading within a goat population by testing rapid test positive animals with discriminatory tests to differentiate BSE from scrapie. Based on the testing that has been reported to date there is no evidence that BSE is spreading in goats.  No test is 100% sensitive on preclinical animals.

END...TSS

=========================

Thursday, December 08, 2016

USDA APHIS National Scrapie Eradication Program October 2016 Monthly Report Fiscal Year 2017 atypical NOR-98 Scrapie


Tuesday, June 07, 2016 

Comparison of two US sheep scrapie isolates supports identification as separate strains Research Project: TRANSMISSION, DIFFERENTIATION, AND PATHOBIOLOGY OF TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES 


Evidence That Transmissible Mink Encephalopathy Results from Feeding Infected Cattle

Over the next 8-10 weeks, approximately 40% of all the adult mink on the farm died from TME.

snip...

The rancher was a ''dead stock'' feeder using mostly (>95%) downer or dead dairy cattle...




In Confidence - Perceptions of unconventional slow virus diseases of animals in the USA - APRIL-MAY 1989 - G A H Wells

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 fanatical incident to be avoided in the US at all costs. ...


The occurrence of CWD must be viewed against the contest of the locations in which it occurred. It was an incidental and unwelcome complication of the respective wildlife research programmes. Despite it’s subsequent recognition as a new disease of cervids, therefore justifying direct investigation, no specific research funding was forthcoming. The USDA veiwed it as a wildlife problem and consequently not their province! ...page 26.


*** Spraker suggested an interesting explanation for the occurrence of CWD. The deer pens at the Foot Hills Campus were built some 30-40 years ago by a Dr. Bob Davis. At or abut that time, allegedly, some scrapie work was conducted at this site. When deer were introduced to the pens they occupied ground that had previously been occupied by sheep.



SPONTANEOUS ATYPICAL BOVINE SPONGIFORM ENCEPHALOPATHY

***Moreover, sporadic disease has never been observed in breeding colonies or primate research laboratories, most notably among hundreds of animals over several decades of study at the National Institutes of Health25, and in nearly twenty older animals continuously housed in our own facility.***


Primate Biol., 3, 47–50, 2016 www.primate-biol.net/3/47/2016/ doi:10.5194/pb-3-47-2016 © Author(s) 2016. CC

Attribution 3.0 License.

Prions

Walter Bodemer German Primate Center, Kellnerweg 4, 37077 Göttingen, Germany Correspondence to: Walter Bodemer (wbodemer@dpz.eu)

Received: 15 June 2016 – Revised: 24 August 2016 – Accepted: 30 August 2016 – Published: 7 September 2016

SNIP...


3 Conclusion

Most importantly, early signs of an altered circadian rhythm, sleep–wake cycle, and activity and body temperature were recorded in prion-infected animals. This experimental approach would have never been feasible in studies with human CJD cases. After 4–6 years animals developed clinical symptoms highly similar to those typical for CJD. Clinicians confirmed how close the animal model and the human disease matched. Non-neuronal tissue like cardiac muscle and peripheral blood with abnormal, disease-related prion protein were detected in rhesus monkey tissues. 

Molecular changes in RNA from repetitive Alu and BC200 DNA elements were identified and found to be targets of epigenetic editing mechanisms active in prion disease. To conclude, our results with the rhesus monkey model for prion disease proved to be a valid model and increased our knowledge of pathogenic processes that are distinctive to prion disease.


SEE FULL TEXT ;



O.05: Transmission of prions to primates after extended silent incubation periods: Implications for BSE and scrapie risk assessment in human populations

 Emmanuel Comoy, Jacqueline Mikol, Valerie Durand, Sophie Luccantoni, Evelyne Correia, Nathalie Lescoutra, Capucine Dehen, and Jean-Philippe Deslys Atomic Energy Commission; Fontenay-aux-Roses, France

 Prion diseases (PD) are the unique neurodegenerative proteinopathies reputed to be transmissible under field conditions since decades. The transmission of Bovine Spongiform Encephalopathy (BSE) to humans evidenced that an animal PD might be zoonotic under appropriate conditions. Contrarily, in the absence of obvious (epidemiological or experimental) elements supporting a transmission or genetic predispositions, PD, like the other proteinopathies, are reputed to occur spontaneously (atpical animal prion strains, sporadic CJD summing 80% of human prion cases). Non-human primate models provided the first evidences supporting the transmissibiity of human prion strains and the zoonotic potential of BSE. Among them, cynomolgus macaques brought major information for BSE risk assessment for human health (Chen, 2014), according to their phylogenetic proximity to humans and extended lifetime. We used this model to assess the zoonotic potential of other animal PD from bovine, ovine and cervid origins even after very long silent incubation periods.

 *** We recently observed the direct transmission of a natural classical scrapie isolate to macaque after a 10-year silent incubation period,

 ***with features similar to some reported for human cases of sporadic CJD, albeit requiring fourfold long incubation than BSE. Scrapie, as recently evoked in humanized mice (Cassard, 2014),

 ***is the third potentially zoonotic PD (with BSE and L-type BSE),

 ***thus questioning the origin of human sporadic cases. We will present an updated panorama of our different transmission studies and discuss the implications of such extended incubation periods on risk assessment of animal PD for human health.

 ===============

***thus questioning the origin of human sporadic cases***

 ***our findings suggest that possible transmission risk of H-type BSE to sheep and human. Bioassay will be required to determine whether the PMCA products are infectious to these animals. 
  

Saturday, April 23, 2016

PRION 2016 TOKYO

Saturday, April 23, 2016

 SCRAPIE WS-01: Prion diseases in animals and zoonotic potential 2016

 Prion. 10:S15-S21. 2016 ISSN: 1933-6896 printl 1933-690X online

Taylor & Francis

Prion 2016 Animal Prion Disease Workshop Abstracts

WS-01: Prion diseases in animals and zoonotic potential

Juan Maria Torres a, Olivier Andreoletti b, J uan-Carlos Espinosa a. Vincent Beringue c. Patricia Aguilar a,

Natalia Fernandez-Borges a. and Alba Marin-Moreno a

"Centro de Investigacion en Sanidad Animal ( CISA-INIA ). Valdeolmos, Madrid. Spain; b UMR INRA -ENVT 1225 Interactions Holes Agents Pathogenes. ENVT. Toulouse. France: "UR892. Virologie lmmunologie MolécuIaires, Jouy-en-Josas. France

 Dietary exposure to bovine spongiform encephalopathy (BSE) contaminated bovine tissues is considered as the origin of variant Creutzfeldt Jakob (vCJD) disease in human. To date, BSE agent is the only recognized zoonotic prion. Despite the variety of Transmissible Spongiform Encephalopathy (TSE) agents that have been circulating for centuries in farmed ruminants there is no apparent epidemiological link between exposure to ruminant products and the occurrence of other form of TSE in human like sporadic Creutzfeldt Jakob Disease (sCJD). However, the zoonotic potential of the diversity of circulating TSE agents has never been systematically assessed. The major issue in experimental assessment of TSEs zoonotic potential lies in the modeling of the ‘species barrier‘, the biological phenomenon that limits TSE agents’ propagation from a species to another. In the last decade, mice genetically engineered to express normal forms of the human prion protein has proved essential in studying human prions pathogenesis and modeling the capacity of TSEs to cross the human species barrier.

 To assess the zoonotic potential of prions circulating in farmed ruminants, we study their transmission ability in transgenic mice expressing human PrPC (HuPrP-Tg). Two lines of mice expressing different forms of the human PrPC (129Met or 129Val) are used to determine the role of the Met129Val dimorphism in susceptibility/resistance to the different agents.

These transmission experiments confirm the ability of BSE prions to propagate in 129M- HuPrP-Tg mice and demonstrate that Met129 homozygotes may be susceptible to BSE in sheep or goat to a greater degree than the BSE agent in cattle and that these agents can convey molecular properties and neuropathological indistinguishable from vCJD. However homozygous 129V mice are resistant to all tested BSE derived prions independently of the originating species suggesting a higher transmission barrier for 129V-PrP variant.

 Transmission data also revealed that several scrapie prions propagate in HuPrP-Tg mice with efficiency comparable to that of cattle BSE. While the efficiency of transmission at primary passage was low, subsequent passages resulted in a highly virulent prion disease in both Met129 and Val129 mice. Transmission of the different scrapie isolates in these mice leads to the emergence of prion strain phenotypes that showed similar characteristics to those displayed by MM1 or VV2 sCJD prion. These results demonstrate that scrapie prions have a zoonotic potential and raise new questions about the possible link between animal and human prions. 


why do we not want to do TSE transmission studies on chimpanzees $

5. A positive result from a chimpanzee challenged severely would likely create alarm in some circles even if the result could not be interpreted for man. I have a view that all these agents could be transmitted provided a large enough dose by appropriate routes was given and the animals kept long enough. Until the mechanisms of the species barrier are more clearly understood it might be best to retain that hypothesis.

 snip...

 R. BRADLEY


Title: Transmission of scrapie prions to primate after an extended silent incubation period          

*** In complement to the recent demonstration that humanized mice are susceptible to scrapie, we report here the first observation of direct transmission of a natural classical scrapie isolate to a macaque after a 10-year incubation period. Neuropathologic examination revealed all of the features of a prion disease: spongiform change, neuronal loss, and accumulation of PrPres throughout the CNS.

 *** This observation strengthens the questioning of the harmlessness of scrapie to humans, at a time when protective measures for human and animal health are being dismantled and reduced as c-BSE is considered controlled and being eradicated.

 *** Our results underscore the importance of precautionary and protective measures and the necessity for long-term experimental transmission studies to assess the zoonotic potential of other animal prion strains.


SCRAPIE WS-01: Prion diseases in animals and zoonotic potential 2016

Prion. 10:S15-S21. 2016 ISSN: 1933-6896 printl 1933-690X online



Tuesday, July 21, 2009

Transmissible mink encephalopathy - review of the etiology


Saturday, December 01, 2007

Phenotypic Similarity of Transmissible Mink Encephalopathy in Cattle and L-type Bovine Spongiform Encephalopathy in a Mouse Model


Sunday, December 10, 2006

Transmissible Mink Encephalopathy TME



Saturday, June 25, 2011

Transmissibility of BSE-L and Cattle-Adapted TME Prion Strain to Cynomolgus Macaque

"BSE-L in North America may have existed for decades"


Wednesday, April 25, 2012

4th MAD COW DISEASE U.S.A. CALIFORNIA ATYPICAL L-TYPE BSE 2012


2017

TUESDAY, JANUARY 17, 2017 

FDA PART 589 -- SUBSTANCES PROHIBITED FROM USE IN ANIMAL FOOD OR FEEDVIOLATIONS OFFICIAL ACTION INDICATED OAI UPDATE 2016 to 2017 BSE TSE PRION


MONDAY, JANUARY 16, 2017 

APHIS Bovine Spongiform Encephalopathy (BSE): Ongoing Surveillance Program Last Modified: Jan 5, 2017


SUNDAY, JANUARY 15, 2017 

US lifts French beef MAD COW BSE import embargo, France says… LOL!


WEDNESDAY, JANUARY 20, 2016

Exportation of Live Animals, Hatching Eggs, and Animal Germplasm From the United States [Docket No. APHIS-2012-0049] RIN 0579-AE00 2016-00962


MONDAY, MARCH 21, 2011

Sheep and Goat BSE Propagate More Efficiently than Cattle BSE in Human PrP Transgenic Mice



Thursday, October 22, 2015

Former Ag Secretary Ann Veneman talks women in agriculture and we talk mad cow disease USDA and what really happened


***see history of BSE in sheep, goat, and pigs...tss

-------- Original Message -------- 

Subject: DEFRA INVESTIGATES AN UNUSUAL SCRAPIE CASE (similar to exp. BSE in sheep) 

Date: Wed, 7 Apr 2004 08:56:36 -0500 

From: "Terry S. Singeltary Sr." <flounder@wt.net

Reply-To: Bovine Spongiform Encephalopathy  

To: BSE-L@uni-karlsruhe.de

######## Bovine Spongiform Encephalopathy #########

Date: April 07, 2004 Time: 13:45

DEFRA INVESTIGATES AN UNUSUAL SCRAPIE CASE

The Veterinary Laboratories Agency (VLA) have informed Defra, the Devolved Administrations and the Food Standards Agency of a type of scrapie not previously seen in the UK.

The VLA and other European laboratories with expertise in scrapie-like diseases have now applied several rapid diagnostic methods to tissue samples from a sheep with suspected scrapie. Some of the methods have indicated that the case does not appear to resemble previously recognised cases of scrapie and, although there were differences, it had some characteristics similar to experimental BSE in sheep and also to an experimental strain of sheep scrapie. More importantly, though, microscopic analysis of brain material showed that the case neither resembled previously recognised types of scrapie or experimental BSE in sheep.

A meeting of the scientific experts who performed these analyses, held on the 30th March, concluded that this case could not be considered to be BSE in sheep, although it does not behave like known types of scrapie either. Further investigation will be needed before more can be said about how this unusual result should be described.

Defra's Chief Scientific Adviser, Professor Howard Dalton, said "The UK, and especially the VLA, have played an important part in improving the diagnostic methods available for identifying TSEs in sheep. As we continue to assess more samples with these improved methods it is likely that we will continue to find samples, such as this, which fall outside our current knowledge of the disease. Defra, as it does with all research, will continue to consult scientific experts to ensure that we are investigating these cases using the best available techniques and methods."

The National Scrapie Plan remains unaffected by this new result and SEAC will be consulted in the near future.

Notes to editors

1. Scrapie is a fatal neurological sheep disease belonging to a group of diseases called transmissible spongiform encephalopathies (TSEs), including BSE in cattle and CJD in humans. It has been present in the national flock for over 250 years. It is not considered to be transmissible to humans.

2. There is a theoretical risk that BSE could be present in sheep, masked by scrapie, but it has not been found naturally occurring in sheep.

3. There is as yet no definitive diagnostic method that can rapidly distinguish between different TSEs for example scrapie from BSE. Consequently, from time to time the scrapie surveillance programmes in EU member states throw up unusual results that merit further investigations (Defra press release 371/03 refers 


4. The VLA have applied several different methods to the sample to compare it to a wide range of previously detected scrapie cases, experimental BSE in sheep and an experimental strain of scrapie, termed CH1461. Two main methods have been used in this analysis:-

a. Western blot (WB) This involves taking a sample of the brain and treating it with an enzyme proteinase k to destroy the normal prion protein (PrPC). The diseased form of the protein (PrPSc) is able to withstand this treatment and is then separated from other cellular material on a gel. A blot is taken of the gel and the PrPSc is visualised using specific antibodies.

b. Immunohistochemistry (IHC) This involves taking thin slices of the brain, and by using special (antibody) markers to detect the PrPSc it is possible to see disease specific patterns of PrPSc distribution in the brain under a microscope. The Western blot method found that the sample did not appear to resemble previously recognised cases of scrapie and, although there were some differences, some characteristics were similar to experimental BSE in sheep and also the experimental strain of sheep scrapie, CH1461. IHC found that it neither resembled previously recognised types of scrapie or experimental BSE in sheep

5. The tissue sample has now been analysed using a total of 5 different diagnostic methods claiming to be able to differentiate between scrapie and experimental BSE in sheep. Two were performed at the VLA and three were performed in other European laboratories.

6. The VLA is the European Reference Laboratory for TSEs and is responsible for co-ordinating such investigations into unusual cases. Their findings will be considered by the European Food Safety Authority's committee of TSE experts and in the UK by the Spongiform Encephalopathy Advisory Committee (SEAC).

7. The genotype of the suspect sheep was ARQ/ARQ which is known to be susceptible to some strains of scrapie and, in experiments, to BSE. Background information on scrapie, scrapie genotyping, and the National Scrapie Plan is published on the Defra internet at www.defra.gov.uk/nsp.

8. For information and advice on BSE in sheep from the FSA please consult their web site at www.foodstandards.gov.uk

Public enquiries 08459 335577; Press notices are available on our website www.defra.gov.uk Defra's aim is sustainable development

End

Nobel House 17 Smith Square London SW1P 3JR Website www.defra.gov.uk ;


TSS


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; 

HOUND STUDY 

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) 


*** DEFRA TO SINGELTARY ON HOUND STUDY AND BSE 2001 ***

DEFRA Department for Environment, Food & Rural Affairs

Area 307, London, SW1P 4PQ Telephone: 0207 904 6000 Direct line: 0207 904 6287 E-mail: h.mcdonagh.defra.gsi.gov.uk

GTN: FAX:

Mr T S Singeltary P.O. Box 42 Bacliff Texas USA 77518

21 November 2001

Dear Mr Singeltary

TSE IN HOUNDS

Thank you for e-mail regarding the hounds survey. I am sorry for the long delay in responding.
As you note, the hound survey remains unpublished. However the Spongiform Encephalopathy Advisory Committee (SEAC), the UK Government's independent Advisory Committee on all aspects related to BSE-like disease, gave the hound study detailed consideration at their meeting in January 1994. As a summary of this meeting published in the BSE inquiry noted, the Committee were clearly concerned about the work that had been carried out, concluding that there had clearly been problems with it, particularly the control on the histology, and that it was more or less inconclusive. However was agreed that there should be a re-evaluation of the pathological material in the study.
Later, at their meeting in June 95, The Committee re-evaluated the hound study to see if any useful results could be gained from it. The Chairman concluded that there were varying opinions within the Committee on further work. It did not suggest any further transmission studies and thought that the lack of clinical data was a major weakness.

Overall, it is clear that SEAC had major concerns about the survey as conducted. As a result it is likely that the authors felt that it would not stand up to peer review and hence it was never published. As noted above, and in the detailed minutes of the SEAC meeting in June 95, SEAC considered whether additional work should be performed to examine dogs for evidence of TSE infection. Although the Committee had mixed views about the merits of conducting further work, the Chairman noted that when the Southwood Committee made their recommendation to complete an assessment of possible spongiform disease in dogs, no TSEs had been identified in other species and hence dogs were perceived as a high risk population and worthy of study. However subsequent to the original recommendation, made in 1990, a number of other species had been identified with TSE ( e.g. cats) so a study in hounds was less critical. For more details see- 
As this study remains unpublished, my understanding is that the ownership of the data essentially remains with the original researchers. Thus unfortunately, I am unable to help with your request to supply information on the hound survey directly. My only suggestion is that you contact one of the researchers originally involved in the project, such as Gerald Wells. He can be contacted at the following address.

Dr Gerald Wells, Veterinary Laboratories Agency, New Haw, Addlestone, Surrey, KT 15 3NB, UK
You may also wish to be aware that since November 1994 all suspected cases of spongiform encephalopathy in animals and poultry were made notifiable. Hence since that date there has been a requirement for vets to report any suspect SE in dogs for further investigation. To date there has never been positive identification of a TSE in a dog.

I hope this is helpful
Yours sincerely 4
HUGH MCDONAGH BSE CORRESPONDENCE SECTION

======================================

HOUND SURVEY

I am sorry, but I really could have been a co-signatory of Gerald's minute.
I do NOT think that we can justify devoting any resources to this study, especially as larger and more important projects such as the pathogenesis study will be quite demanding.

If there is a POLITICAL need to continue with the examination of hound brains then it should be passed entirely to the VI Service.

J W WILESMITH Epidemiology Unit 18 October 1991

Mr. R Bradley

cc: Mr. G A H Wells

3.3. Mr R J Higgins in conjunction with Mr G A Wells and Mr A C Scott would by the end of the year, indentify the three brains that were from the ''POSITIVE'' end of the lesion spectrum.

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

TSE ; HOUNDS

GAH WELLS (very important statement here...TSS)

HOUND STUDY

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

snip...

76 pages on hound study;

snip...

The spongiform changes were not pathognomonic (ie. conclusive proof) for prion disease, as they were atypical, being largely present in white matter rather than grey matter in the brain and spinal cord. However, Tony Scott, then head of electron microscopy work on TSEs, had no doubt that these SAFs were genuine and that these hounds therefore must have had a scrapie-like disease. I reviewed all the sections myself (original notes appended) and although the pathology was not typical, I could not exclude the possibility that this was a scrapie-like disorder, as white matter vacuolation is seen in TSEs and Wallerian degeneration was also present in the white matter of the hounds, another feature of scrapie.

38.I reviewed the literature on hound neuropathology, and discovered that micrographs and descriptive neuropathology from papers on 'hound ataxia' mirrored those in material from Robert Higgins' hound survey. Dr Tony Palmer (Cambridge) had done much of this work, and I obtained original sections from hound ataxia cases from him. This enabled me provisionally to conclude that Robert Higgins had in all probability detected hound ataxia, but also that hound ataxia itself was possibly a TSE. Gerald Wells confirmed in 'blind' examination of single restricted microscopic fields that there was no distinction between the white matter vacuolation present in BSE and scrapie cases, and that occurring in hound ataxia and the hound survey cases.

39.Hound ataxia had reportedly been occurring since the 1930's, and a known risk factor for its development was the feeding to hounds of downer cows, and particularly bovine offal. Circumstantial evidence suggests that bovine offal may also be causal in FSE, and TME in mink. Despite the inconclusive nature of the neuropathology, it was clearly evident that this putative canine spongiform encephalopathy merited further investigation.

40.The inconclusive results in hounds were never confirmed, nor was the link with hound ataxia pursued. I telephoned Robert Higgins six years after he first sent the slides to CVL. I was informed that despite his submitting a yearly report to the CVO including the suggestion that the hound work be continued, no further work had been done since 1991. This was surprising, to say the very least.
41.The hound work could have provided valuable evidence that a scrapie-like agent may have been present in cattle offal long before the BSE epidemic was recognised. The MAFF hound survey remains unpublished.

Histopathological support to various other published MAFF experiments
42.These included neuropathological examination of material from experiments studying the attempted transmission of BSE to chickens and pigs (CVL 1991) and to mice (RVC 1994).

It was thought likely that at least some, and probably all, of the cases in zoo animals were caused by the BSE agent. Strong support for this hypothesis came from the findings of Bruce and others (1994) ( Bruce, M.E., Chree, A., McConnell, I., Foster, J., Pearson, G. & Fraser, H. (1994) Transmission of bovine spongiform encephalopathy and scrapie to mice: strain variation and species barrier. Philosophical Transactions of the Royal Society B 343, 405-411: J/PTRSL/343/405 ), who demonstrated that the pattern of variation in incubation period and lesion profile in six strains of mice inoculated with brain homogenates from an affected kudu and the nyala, was similar to that seen when this panel of mouse strains was inoculated with brain from cattle with BSE. The affected zoo bovids were all from herds that were exposed to feeds that were likely to have contained contaminated ruminant-derived protein and the zoo felids had been exposed, if only occasionally in some cases, to tissues from cattle unfit for human consumption.

snip...

NEW URL ;

TSEs TRANSMISSION STUDIES 

what a coincedence , CONVENIENTLY, MORE FLUBBED UP BRAINS; 

HOUND STUDY 

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) 


EP-021 Canine Prions: A New Form of Prion Disease

Mourad Tayebi1, Monique A David2, Brian Summers3

1 University of Melbourne, Veterinary Sciences, Australia; 2Ausbiologics, Sydney, Australia; 3Royal Veterinary College, London, UK

The origin of bovine spongiform encephalopathy (BSE), which rapidly evolved into a major epidemic remains unresolved and was initially widely attributed to transmission of sheep scrapie to cattle with contaminated feed prepared from rendered sheep carcasses. Alternative transmission hypotheses also include feed contaminated with unrecognized subclinical case(s) of bovine prion disease or with prion-infected human remains. However, following the demonstration of a BSE case exhibiting the novel mutation E211 K, similar to the E200K mutation associated with most genetic CJD in humans, support for a genetic origin of prion disease in cattle is gaining momentum. In contrast to other animal species such as feline, the canine species seems to be resistant to prion disease as no canine prion cases were previously reported.

We describe here three cases of Rottweiler puppy (called RWD cases) with neurological deficits and spongiform change. We used animal bioassays and in vitro studies to show efficient interspecies transmission of this novel canidae prion isolate to other species.

Biochemical studies revealed the presence of partially proteinase K (PK)-resistant fragment and immunohistochemistry displayed staining for PrPSc in the cerebral cortex. Importantly, interspecies transmission of canine PrPSc derived from RWD3 brain homogenates following inoculation of hamsters led to signs of prion disease and replication of PrPSc in brains, spinal cords and spleens of these animals.

These findings if confirmed by further cases of prion disease in canidae and regardless of the origin of the disease would have a major impact on animal and public health.

PRION 2016 TOKYO

OR-09: Canine spongiform encephalopathy—A new form of animal prion disease

Monique David, Mourad Tayebi UT Health; Houston, TX USA

It was also hypothesized that BSE might have originated from an unrecognized sporadic or genetic case of bovine prion disease incorporated into cattle feed or even cattle feed contaminated with prion-infected human remains.1 However, strong support for a genetic origin of BSE has recently been demonstrated in an H-type BSE case exhibiting the novel mutation E211K.2 Furthermore, a specific prion protein strain causing BSE in cattle is believed to be the etiological agent responsible for the novel human prion disease, variant Creutzfeldt-Jakob disease (vCJD).3 Cases of vCJD have been identified in a number countries, including France, Italy, Ireland, the Netherlands, Canada, Japan, US and the UK with the largest number of cases. Naturally occurring feline spongiform encephalopathy of domestic cats4 and spongiform encephalopathies of a number of zoo animals so-called exotic ungulate encephalopathies5,6 are also recognized as animal prion diseases, and are thought to have resulted from the same BSE-contaminated food given to cattle and humans, although and at least in some of these cases, a sporadic and/or genetic etiology cannot be ruled out. The canine species seems to display resistance to prion disease and no single case has so far been reported.7,8 Here, we describe a case of a 9 week old male Rottweiler puppy presenting neurological deficits; and histological examination revealed spongiform vacuolation characteristic of those associated with prion diseases.9 Initial biochemical studies using anti-PrP antibodies revealed the presence of partially proteinase K-resistant fragment by western blotting. Furthermore, immunohistochemistry revealed spongiform degeneration consistent with those found in prion disease and displayed staining for PrPSc in the cortex.

Of major importance, PrPSc isolated from the Rottweiler was able to cross the species barrier transmitted to hamster in vitro with PMCA and in vivo (one hamster out of 5). Futhermore, second in vivo passage to hamsters, led to 100% attack rate (n = 4) and animals displayed untypical lesional profile and shorter incubation period.

In this study, we show that the canine species might be sensitive to prion disease and that PrPSc isolated from a dog can be transmitted to dogs and hamsters in vitro using PMCA and in vivo to hamsters.

If our preliminary results are confirmed, the proposal will have a major impact on animal and public health and would certainly lead to implementing new control measures for ‘canine spongiform encephalopathy’ (CSE).

References

1. Colchester AC, Colchester NT. The origin of bovine spongiform encephalopathy: the human prion disease hypothesis. Lancet 2005; 366:856-61; PMID:16139661; http:// dx.doi.org/10.1016/S0140-6736(05)67218-2.

2. Richt JA, Hall SM. BSE case associated with prion protein gene mutation. PLoS Pathog 2008; 4:e1000156; PMID:18787697; http://dx.doi.org/10.1371/journal. ppat.1000156.

3. Collinge J. Human prion diseases and bovine spongiform encephalopathy (BSE). Hum Mol Genet 1997; 6:1699-705; PMID:9300662; http://dx.doi.org/10.1093/ hmg/6.10.1699.

4. Wyatt JM, Pearson GR, Smerdon TN, Gruffydd-Jones TJ, Wells GA, Wilesmith JW. Naturally occurring scrapie-like spongiform encephalopathy in five domestic cats. Vet Rec 1991; 129:233-6; PMID:1957458; http://dx.doi.org/10.1136/vr.129.11.233.

5. Jeffrey M, Wells GA. Spongiform encephalopathy in a nyala (Tragelaphus angasi). Vet Pathol 1988; 25:398-9; PMID:3232315; http://dx.doi.org/10.1177/030098588802500514.

6. Kirkwood JK, Wells GA, Wilesmith JW, Cunningham AA, Jackson SI. Spongiform encephalopathy in an arabian oryx (Oryx leucoryx) and a greater kudu (Tragelaphus strepsiceros). Vet Rec 1990; 127:418-20; PMID:2264242.

7. Bartz JC, McKenzie DI, Bessen RA, Marsh RF, Aiken JM. Transmissible mink encephalopathy species barrier effect between ferret and mink: PrP gene and protein analysis. J Gen Virol 1994; 75:2947-53; PMID:7964604; http://dx.doi.org/10.1099/0022-1317- 75-11-2947.

8. Lysek DA, Schorn C, Nivon LG, Esteve-Moya V, Christen B, Calzolai L, et al. Prion protein NMR structures of cats, dogs, pigs, and sheep. Proc Natl Acad Sci U S A 2005; 102:640-5; PMID:15647367; http://dx.doi.org/10.1073/pnas.0408937102.

9. Budka H. Neuropathology of prion diseases. Br Med Bull 2003; 66:121-30; PMID:14522854; http://dx.doi.org/10.1093/bmb/66.1.121.

=======================================

DOGS AND RABBITS AND BSE

2013

Strain characteristics of the in vitro-adapted rabbit and dog BSE agent remained invariable with respect to the original cattle BSE prion, suggesting that the naturally low susceptibility of rabbits and dogs to prion infections should not alter their zoonotic potential if these animals became infected with BSE.

=======================================

Bovine Spongiform Encephalopathy Induces Misfolding of Alleged Prion-Resistant Species Cellular Prion Protein without Altering Its Pathobiological Features

Enric Vidal3, Natalia Fernández-Borges1, Belén Pintado4, Montserrat Ordóñez3, Mercedes Márquez6, Dolors Fondevila5,6, Juan María Torres7, Martí Pumarola5,6, and Joaquín Castilla1,2 + Author Affiliations

1CIC bioGUNE, 48160 Derio, Bizkaia, Spain,

2IKERBASQUE, Basque Foundation for Science, 48011 Bilbao, Bizkaia, Spain,

3Centre de Recerca en Sanitat Animal, Campus de la Universitat Autònoma de Barcelona (UAB)-IRTA, 08193 Bellaterra, Barcelona, Spain,
4Centro Nacional de Biotecnología, Campus de Cantoblanco, 28049 Cantoblanco, Madrid, Spain,
5Department of Animal Medicine and Surgery, Veterinary Faculty, UAB, 08193 Bellaterra (Cerdanyola del Vallès), Barcelona, Spain,
6Murine Pathology Unit, Centre de Biotecnologia Animal i Teràpia Gènica, UAB, 08193 Bellaterra (Cerdanyola del Vallès), Barcelona, Spain, and
7Centro de Investigación en Sanidad Animal-Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, 28130 Valdeolmos, Madrid, Spain
Author contributions: E.V., N.F.-B., and J.C. designed research; E.V., N.F.-B., B.P., M.O., M.M., D.F., and J.C. performed research; E.V., N.F.-B., B.P., and J.C. contributed unpublished reagents/analytic tools; E.V., N.F.-B., B.P., M.O., M.M., D.F., J.M.T., M.P., and J.C. analyzed data; E.V. and J.C. wrote the paper.

Abstract

Bovine spongiform encephalopathy (BSE) prions were responsible for an unforeseen epizootic in cattle which had a vast social, economic, and public health impact. This was primarily because BSE prions were found to be transmissible to humans. Other species were also susceptible to BSE either by natural infection (e.g., felids, caprids) or in experimental settings (e.g., sheep, mice). However, certain species closely related to humans, such as canids and leporids, were apparently resistant to BSE. In vitro prion amplification techniques (saPMCA) were used to successfully misfold the cellular prion protein (PrPc) of these allegedly resistant species into a BSE-type prion protein. The biochemical and biological properties of the new prions generated in vitro after seeding rabbit and dog brain homogenates with classical BSE were studied. Pathobiological features of the resultant prion strains were determined after their inoculation into transgenic mice expressing bovine and human PrPC. Strain characteristics of the in vitro-adapted rabbit and dog BSE agent remained invariable with respect to the original cattle BSE prion, suggesting that the naturally low susceptibility of rabbits and dogs to prion infections should not alter their zoonotic potential if these animals became infected with BSE. This study provides a sound basis for risk assessment regarding prion diseases in purportedly resistant species.

Received January 18, 2013. Revision received March 7, 2013. Accepted March 23, 2013. Copyright © 2013 the authors 0270-6474/13/337778-09$15.00/0

Friday, March 8, 2013

Dogs may have been used to make Petfood and animal feed

Monday, March 26, 2012

CANINE SPONGIFORM ENCEPHALOPATHY: A NEW FORM OF ANIMAL PRION DISEASE



THE RISK TO HUMANS FROM SHEEP

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. 

snip... 

IN CONFIDENCE 

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." 

'NOT'...tss 


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 BSE 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. 

snip... 

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. 

snip... 

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... 


*** In the US, scrapie is reported primarily in sheep homozygous for 136A/171Q (AAQQ) and the disease phenotype is similar to that seen with experimental strain CH1641.


snip...see ;


SHEEP AND BSE

PERSONAL AND CONFIDENTIAL

SHEEP AND BSE

A. The experimental transmission of BSE to sheep.

Studies have shown that the ''negative'' line NPU flock of Cheviots can be experimentally infected with BSE by intracerebral (ic) or oral challenge (the latter being equivalent to 0.5 gram of a pool of four cow brains from animals confirmed to have BSE).


RB264

BSE - TRANSMISSION STUDIES


 *** CH1641 SEE ;


IN CONFIDENCE 

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 

IN CONFIDENCE 

PERCEPTIONS OF UNCONVENTIONAL SLOW VIRUS DISEASES OF ANIMALS IN THE UNITED USA

G A H WELLS

REPORT OF A VISIT TO THE USA APRIL - MAY 1989


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. 

snip... 


STATEMENT OF DR HELEN GRANT MD FRCP ISSUED 13/05/1999 

BSE INQUIRY 



-------- Original Message -------- 

Subject: DEFRA INVESTIGATES AN UNUSUAL SCRAPIE CASE (similar to exp. BSE in sheep) 

Date: Wed, 7 Apr 2004 08:56:36 -0500 

From: "Terry S. Singeltary Sr." <flounder@wt.net

Reply-To: Bovine Spongiform Encephalopathy  

To: BSE-L@uni-karlsruhe.de

######## Bovine Spongiform Encephalopathy #########

Date: April 07, 2004 Time: 13:45

DEFRA INVESTIGATES AN UNUSUAL SCRAPIE CASE

The Veterinary Laboratories Agency (VLA) have informed Defra, the Devolved Administrations and the Food Standards Agency of a type of scrapie not previously seen in the UK.

The VLA and other European laboratories with expertise in scrapie-like diseases have now applied several rapid diagnostic methods to tissue samples from a sheep with suspected scrapie. Some of the methods have indicated that the case does not appear to resemble previously recognised cases of scrapie and, although there were differences, it had some characteristics similar to experimental BSE in sheep and also to an experimental strain of sheep scrapie. More importantly, though, microscopic analysis of brain material showed that the case neither resembled previously recognised types of scrapie or experimental BSE in sheep.

A meeting of the scientific experts who performed these analyses, held on the 30th March, concluded that this case could not be considered to be BSE in sheep, although it does not behave like known types of scrapie either. Further investigation will be needed before more can be said about how this unusual result should be described.

Defra's Chief Scientific Adviser, Professor Howard Dalton, said "The UK, and especially the VLA, have played an important part in improving the diagnostic methods available for identifying TSEs in sheep. As we continue to assess more samples with these improved methods it is likely that we will continue to find samples, such as this, which fall outside our current knowledge of the disease. Defra, as it does with all research, will continue to consult scientific experts to ensure that we are investigating these cases using the best available techniques and methods."

The National Scrapie Plan remains unaffected by this new result and SEAC will be consulted in the near future.

Notes to editors

1. Scrapie is a fatal neurological sheep disease belonging to a group of diseases called transmissible spongiform encephalopathies (TSEs), including BSE in cattle and CJD in humans. It has been present in the national flock for over 250 years. It is not considered to be transmissible to humans.

2. There is a theoretical risk that BSE could be present in sheep, masked by scrapie, but it has not been found naturally occurring in sheep.

3. There is as yet no definitive diagnostic method that can rapidly distinguish between different TSEs for example scrapie from BSE. Consequently, from time to time the scrapie surveillance programmes in EU member states throw up unusual results that merit further investigations (Defra press release 371/03 refers 


4. The VLA have applied several different methods to the sample to compare it to a wide range of previously detected scrapie cases, experimental BSE in sheep and an experimental strain of scrapie, termed CH1461. Two main methods have been used in this analysis:-

a. Western blot (WB) This involves taking a sample of the brain and treating it with an enzyme proteinase k to destroy the normal prion protein (PrPC). The diseased form of the protein (PrPSc) is able to withstand this treatment and is then separated from other cellular material on a gel. A blot is taken of the gel and the PrPSc is visualised using specific antibodies.

b. Immunohistochemistry (IHC) This involves taking thin slices of the brain, and by using special (antibody) markers to detect the PrPSc it is possible to see disease specific patterns of PrPSc distribution in the brain under a microscope. The Western blot method found that the sample did not appear to resemble previously recognised cases of scrapie and, although there were some differences, some characteristics were similar to experimental BSE in sheep and also the experimental strain of sheep scrapie, CH1461. IHC found that it neither resembled previously recognised types of scrapie or experimental BSE in sheep

5. The tissue sample has now been analysed using a total of 5 different diagnostic methods claiming to be able to differentiate between scrapie and experimental BSE in sheep. Two were performed at the VLA and three were performed in other European laboratories.

6. The VLA is the European Reference Laboratory for TSEs and is responsible for co-ordinating such investigations into unusual cases. Their findings will be considered by the European Food Safety Authority's committee of TSE experts and in the UK by the Spongiform Encephalopathy Advisory Committee (SEAC).

7. The genotype of the suspect sheep was ARQ/ARQ which is known to be susceptible to some strains of scrapie and, in experiments, to BSE. Background information on scrapie, scrapie genotyping, and the National Scrapie Plan is published on the Defra internet at www.defra.gov.uk/nsp.

8. For information and advice on BSE in sheep from the FSA please consult their web site at www.foodstandards.gov.uk

Public enquiries 08459 335577; Press notices are available on our website www.defra.gov.uk Defra's aim is sustainable development

End

Nobel House 17 Smith Square London SW1P 3JR Website www.defra.gov.uk ;


TSS


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 


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. 

snip... 

IN CONFIDENCE 

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 


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. 

snip... 



Published Date: 2004-12-11 23:50:00

Subject: PRO/AH> BSE, goats - France 2002 (03): susp 

Archive Number: 20041211.3279

BSE, GOATS - FRANCE 2002 (03): SUSPECTED

****************************************

A ProMED-mail post


ProMED-mail is a program of the International Society for Infectious Diseases


Date: Sat, 11 Dec 2004

From: Terry S Singeltary Sr. <flounder@wt.net>

Source: UK Food Standards Agency - Consultations, 10 Dec 2004 [edited]

Proposed amendments to EC Regulation 999/2001 on transmissible spongiform 
encephalopathies -- goat specified risk material

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

Comments are sought on proposed possible changes to EC Regulation 999/2001 
following an announcement by the European Commission of a possible finding 
of bovine spongiform encephalopathy (BSE) in a French goat. It is proposed 
that these changes will only be implemented if BSE is confirmed in the 
suspect animal.

EC Regulation 999/2001 (as amended) lays down community-wide rules for the 
prevention, control and eradication of certain transmissible spongiform 
encephalopathies (TSEs). These rules are amended as necessary in light of 
new findings and emerging scientific evidence.

On 28 Oct 2004, the European Commission announced a possible finding of BSE 
in a French goat. In accordance with EU procedures, the finding was 
submitted to the Community Reference Laboratory (CRL) in Weybridge for 
evaluation by an expert panel. The Commission issued a subsequent 
announcement on 26 Nov 2004 stating that the expert panel had met and 
concluded that currently available results were incomplete, and a further 2 
months were required before all the results could be properly interpreted. 
The results of the CRL evaluation are therefore expected in the New Year.
Although BSE had never before been found naturally occurring in goats, the 
possibility that it could exist, masked by scrapie, has been acknowledged 
for some time. This is because it is likely that some goats ate the same 
feed that spread BSE in cattle, and laboratory experiments have shown that 
BSE can be transmitted to sheep and goats. As a result, precautionary 
measures, such as SRM controls, have been in place for some time to 
minimise any potential risk to public health.

In its opinion of April 2002, the EC Scientific Steering Committee (SSC) 
recommended additional measures to protect public health in the event that 
BSE should become likely in small ruminants. In view of the possible 
finding of BSE in a French goat, the Commission is proposing that the 
additional measures recommended in the SSC opinion should be implemented, 
if the finding of BSE is confirmed.

The draft EC proposal was discussed at an EU TSE Working Group meeting on 
30 Nov 2004 and will be subject to further discussions at SCoFCAH (Standing 
Committee of Food Chain and Animal Health) in December and January. It is 
unlikely that SCoFCAH will vote on the proposal until the views of the CRL 
expert panel are known. These are expected at the end of January 2005 [full 

One effect of the proposal will be a change to the list of tissues that are 
designated as specified risk material (SRM) in goats. As the proposal 
currently stands, the list of tissues designated SRM in goats will be 
extended to include:

- the whole alimentary canal;

- the organs, including lymph nodes, of the thoracic and abdominal cavities;

- the pre-femoral and pre-scapular lymph nodes;

- the entire head;

- the tonsils.

These tissues would be designated SRM in goats of all ages. Spinal cord 
will remain SRM in goats over 12 months of age. At present, only the spleen 
and ileum are designated SRM in goats of all ages, while the skull 
(including the brain and eyes), tonsils and spinal cord are designated SRM 
in goats aged over 12 months of age.

At this stage, the FSA is inclined to support the extension of the tissues 
to be classified as SRM. The EC proposals would remove most of the tissues 
that are associated with BSE infectivity in an infected animal. Unlike in 
cattle, the lymph nodes in sheep and goats have been shown to carry TSE 
infectivity in infected animals. However, removal of all lymph nodes would 
not be practicable.

We would welcome your views on any benefits or drawbacks likely to result 
from the changes, in particular, on the potential cost of the proposals and 
practical aspects of removing the additional SRM. Your views will help to 
formulate the UK negotiating position on this issue. It will not be 
possible to accurately assess the impact of the measures without this feedback.
If the proposals do become European law then domestic legislation, in this 
case the TSE (Scotland) Regulations 2002 (and equivalent legislation in 
England, Wales, and Northern Ireland), will require amendment to take 
account of the new European Regulation. In addition to informing you of the 
Commission proposals, we are also informing you of a possible new domestic 
requirement, relating to goats, if the Commission proposals receive a 
positive vote. At present, there is no difference between tissues 
designated SRM in goats and tissues designated SRM in sheep. That position 
will change if the proposals relating to goat SRM are accepted. As it would 
be difficult to differentiate between sheep and goats once carcasses have 
been dressed, it would be necessary to introduce an additional measure to 
enable such differentiation for enforcement purposes. We would therefore 
need to consider introducing a "goat stamp" to aid correct identification 
and removal of SRM. This stamp would probably be applied by the Meat 
Hygiene Service. We would welcome your views on this proposal and any 
effects -- positive or negative -- it might be likely to have.

-- 
ProMED-mail

[The rapid response of the EU to the published results of the French 
research on suspected BSE infection in goats reflects the significant 
change which international and national animal- and public-health agencies 
have undergone in view of the sad BSE history, especially since 1996. Such 
a response was almost unimaginable beforehand.

The total number of adult goats in EU's 25 member countries is about 9.5 
million, compared with 66 million adult sheep. The impact of the amendment 
in the UK will be rather limited. Being one of the 2 leading sheep breeding 
EU countries (the other one is Spain), UK's adult goat population is 
reportedly only 49 000. The leading goat breeding countries are (millions 
of adult animals): Greece (3.9), Spain (2.33), France (1.03), Italy (0.82), 
Portugal (0.39), Cyprus (0.3) and Netherlands (0.2).

Responses to the above consultation are required by 6 Mar 2005. Since a 
meeting of SCoFCAH is likely to be held in mid-January 2005, however, 
preliminary views have been requested, if possible, by 7 Jan 2005. Further 
details, including address for responses, conditions for their publication, 
etc, are to be seen at the above URL. - Mod.AS]

See Also

Scrapie, atypical, sheep - UK and Ireland 20041210.3274
BSE, goats - France 2002 (02): susp 20041119.3097
BSE, goats - France 2002: susp. 20041030.2929
Scrapie, sheep, presence in muscle tissue 20040525.1398
BSE - France: distinct molecular phenotypes 20040107.0076
2002
---
TSE, sheep - Belgium 20021028.5662
BSE, potential for emergence in sheep - France 20020314.3742
1999
---
TSE, goats & sheep - Italy 19990227.0278
.................arn/pg/sh

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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

We confirmed that the agent responsible for TSE in a UK goat, which was initially reported as scrapie in 1990 and subsequently as suspected BSE in 2006 (16), was a BSE agent. This conclusion was based on bioassay of nervous tissue in mice demonstrating similarities of histopathologic lesions, PrPSc mapping in the brain, and WB of PrPSc with those of mice inoculated with BSE from various ovine, caprine, and bovine sources.

snip...

ecause TSEs in goats are still a problem, particularly in Mediterranean countries, our data suggest that extensive surveillance and breeding schemes must remain in place to prevent a BSE outbreak in small ruminants and to safeguard public health. This report also highlights several issues regarding the use of mouse bioassay to identify TSE strains. As governing bodies seek confirmation of equivocal cases that are identified worldwide, they must be aware of the limitations, cost, and timescale demands of confirming such cases.

Dr Spiropoulos is a veterinary researcher at Veterinary Laboratories Agency with a particular interest in animal pathology. He is the head of the Mouse Bioassay Team that specializes in pathology of experimental animals. His research interests include neurodegenerative disorders and animal diseases of policy relevance, particularly zoonoses.

Acknowledgments

We thank John Sheehan for tissue retrieval from wax-impregnated tissue blocks; Angel Ortiz-Pelaez for epidemiologic assistance; histopathology employees at Veterinary Laboratories Agency for expert technical support in histopathology and immunohistochemistry; and Animal Services Unit employees at Veterinary Laboratories Agency for expert support with animal procedures and care.

This work was supported by a Department of Environment, Food and Rural Affairs grant (project SE1849).



Sunday, October 3, 2010

Scrapie, Nor-98 atypical Scrapie, and BSE in sheep and goats North America, who's looking ?



Thursday, November 18, 2010

Increased susceptibility of human-PrP transgenic mice to bovine spongiform encephalopathy following passage in sheep


WEDNESDAY, JANUARY 18, 2012

BSE IN GOATS CAN BE MISTAKEN FOR SCRAPIE

February 1, 2012


-------- Original Message --------
Subject: USDA advised against mad cow test in 2002
Date: Tue, 13 Jul 2004 08:07:52 -0500
From: "Terry S. Singeltary Sr." 
Reply-To: Bovine Spongiform Encephalopathy 
To: BSE-L@uni-karlsruhe.de

######## Bovine Spongiform Encephalopathy #########

USDA advised against mad cow test in 2002

By Steve Mitchell

United Press International

Published 7/13/2004 7:57 AM

WASHINGTON, July 13 (UPI) -- The U.S. Department of Agriculture in late
2002 warned against using the same mad cow disease test the agency now
is using in its expanded surveillance program for the deadly disorder,
United Press International has learned.

The USDA said governments should not authorize the test, which is
manufactured by Bio-Rad Laboratories in Hercules, Calif., because it can
give false positives -- results that are ruled negative on follow-up
testing -- and "will cause loss of consumer confidence in beef and beef
products," the agency wrote in a letter to the World Organization for
Animal Health in Paris.

The OIE, as it is known by its French initials, establishes
international standards for animal disease issues.

The USDA recommended countries employ a different test manufactured by
the Swiss firm Prionics -- a test the agency has licensed but has not
yet put into use. The USDA's reason for the delay is the Prionics test,
which has not yielded a false positive in more than 20 million tests in
Europe, still must pass through the agency's validation procedures.
Concerns about false positives with the Bio-Rad test became a reality
recently during the first month of the USDA's expanded surveillance
plan, launched June 1 in response to the only confirmed U.S. case of mad
cow last December. The agency reported two preliminary positive results,
which caused concern among the public and havoc in the cattle futures
markets until both were ruled negative on follow-up testing several days
later.

So far, seven of the 12 state laboratories participating in the USDA's
mad cow surveillance plan are using the Bio-Rad test and the remaining
five are expected to opt for the test when they begin testing operations.
Experts on testing and mad cow disease have suggested one reason the
USDA might have opted for Bio-Rad is the same reason it advised against
it in 2002: its potential to yield false positives.

By releasing preliminary positives -- or inconclusives, as the USDA has
deemed them -- that are later ruled negative, the agency could
desensitize markets, consumers and foreign trading partners to real
positive cases when and if they occur, the sources said.

"Bio-Rad was approved as a way of getting people used to a possible case
if there ever was one," a veterinarian with expertise in mad cow disease
told UPI.

"They (USDA officials) know it has a high false positive rate ... The
more inconclusives they have, the easier it is to 'mix something up' and
have all negative tests," said the veterinarian, who requested anonymity.
The veterinarian's comments were echoed by other experts in this field,
who also declined to be named.

USDA spokeswoman Julie Quick did not respond to UPI's question of
whether this was the agency's intended strategy. However, John Clifford,
USDA's chief veterinary officer, acknowledged at a recent news
conference that release of the inconclusive results could have that affect.
"We want to minimize the impacts upon the markets," Clifford said. "We
feel like that after we get this information out there a couple of times
that hopefully it will continue to minimize that impact."

To date, more than 15,000 cows have been tested under the surveillance
plan and USDA officials have said they expect many more false positives
as the agency seeks to conduct thousands of mad cow tests per week over
the next 12 to 18 months.

Mad cow disease is otherwise known as bovine spongiform encephalopathy
or BSE. Officials have said they also expect to find additional BSE cases.
Consumer groups and some members of Congress expressed concern about the
USDA's decision to use the Bio-Rad test after the two false positives.
Sen. Conrad Burns, R-Mont., recently sent a letter to Agriculture
Secretary Ann Veneman urging her "to seriously consider the reliability
of your tests and to rigorously evaluate BSE screening tests used
internationally that may offer more accurate results."

The concern to consumers is people can contract an incurable brain
disorder called variant Creutzfeldt Jakob disease from eating meat
infected with the mad cow pathogen. More than 150 people worldwide have
become infected, but none of the cases has been linked to U.S. beef.
"Why are we using Bio-Rad instead of Prionics if they are as bad as the
(USDA) would have us believe with all these 'inconclusives?'" asked
Terry Singletary, coordinator of CJD Watch, an advocacy group for
patients and family members. His mother died of a rare form of CJD
called Heidenhain Variant, which has not been linked with mad cow disease.
"Do they really want to find all the cases, or are we just playing a
(public relations) game?" asked Singletary, who has been sharply
critical of the USDA's approach to mad cow. "How many more are we going
to expose to this deadly pathogen?"

Brad Crutchfield, Bio-Rad's vice president, said the science and
experience with his company's test in Europe establishes its soundness
and reliability. Bio-Rad has yielded false positives only once every
300,000 tests, he said, adding that the false positives will decrease as
the U.S. labs acquire more experience.

"The issue has nothing to do with the test, it has to do with the
notification process," Crutchfield said, referring to the USDA's
decision to notify the public of inconclusive results before they were
confirmed or ruled out with confirmatory tests.

Heads of several state labs contacted by UPI said the agency allowed
them to choose among five licensed rapid tests, including Bio-Rad's.
However, outside testing experts said it was curious that all the labs
chose the same test.

"It is slightly peculiar that it went all to one supplier for no other
reason than you would want different tests because they give different
results," said Roger Rosedale, chairman of Microsens Biotechnologies, a
company in London that manufactures technology used in tests for
detecting mad cow disease and similar disorders. One of Microsens'
clients is Idexx Laboratories, which makes a competing mad cow test to
Bio-Rad and Prionics.

At the time the labs were making their choices, the USDA said Bio-Rad
was the only test the agency had "field tested," which apparently is a
prerequisite for putting the tests into use.

The Bio-Rad test "was the only one that had been field tested, so that's
limiting," said USDA's Quick. So although the USDA had licensed the
other rapid tests, they may not have been available for use even if the
laboratories had selected them.

The department also purchased the equipment needed to run the Bio-Rad
test for the labs, some of which otherwise would not have been able to
afford the machinery due to budget constraints.

A source with an American company that manufactures tests for detecting
animal disease said it was unusual for the USDA to approve a test and
then require field testing. Field trials are usually done before
approval, the source said.

"With other tests, if you get USDA approval, you're all set," the source
said. "This is something new."

Asked why the USDA was not using the Prionics Check test, as it had
recommended in the 2002 OIE letter, Quick said it is still being "field
tested." In the letter, however, the agency did not cite the need for a
field test or validation procedure.

"Certain tests, such as the rapid tests, may not give an accurate
picture of the BSE situation in a country or zone," the USDA wrote. "It
is well known that certain rapid tests such as the Enfer and Bio-Rad
tests have recorded false positive BSE results. For BSE-free countries
or zones, the use of rapid BSE tests that give false positive results
will cause loss of consumer confidence in beef and beef products."
A better approach would be to use the Prionics Check test, the agency
wrote.

"For BSE free countries or zones, the use of histopathology,
immunohistochemistry and the Prionics 'check' immunoblot test would
provide a definitive diagnosis of a BSE suspect case," the letter stated.
Quick insisted the agency's statement was not intended to recommend
against the Bio-Rad test. Instead, she said, it was meant to recommend
that countries not simply rely on rapid screening tests as a way to
confirm a case of mad cow disease.

No other testing experts UPI contacted interpreted the statement that
way and Quick, who acknowledged she was not familiar with the technical
details of the tests, declined to make Clifford or other USDA officials
available to discuss the issue or offer clarification.

The USDA's decision not to release the samples from the two
inconclusives for verification by outside labs has also come under
question. The agency used a test called immunohistochemistry, or IHC, to
determine the animals were not infected with mad cow, but experts said
this is not always a foolproof test and it can miss cases.

Markus Moser, Prionics' chief executive officer and a molecular
biologist, noted that Germany was considered BSE-free when using the IHC
test. When officials there began using the Prionics rapid test in 2000,
he said, they found several cases and so far have detected more than 300
infected animals.

Stuart Wilson, Microsens' scientific director and a molecular
pathologist, noted in a document he recently prepared on false positives
that there have been instances when Bio-Rad was used more than a few
months before the animal developed symptoms and they were found
correctly to be positive, but IHC incorrectly ruled them negative.
"In a cow that you don't know is infected or not, it can always appear
IHC positive (or negative) simply by changing the IHC timing," Wilson
wrote in the document.

"This was one of the biggest problems with investigating tonsils in
asymptomatic humans in the United Kingdom," the document said, referring
to a recent study that indicated as many as 3,800 people in England may
be unwittingly incubating vCJD.

In the December mad cow case, the USDA had the results confirmed by the
Weybridge laboratory in the United Kingdom, which is one of the three
mad cow disease testing reference laboratories recognized by the OIE.
The other two are in Switzerland and Japan.

"It must by definition create doubt if they're not allowing any other
... OIE reference laboratory have access to them," Rosedale said. "It's
not to suggest (the USDA) guys are not competent, but why would they not
release it?"

--
Steve Mitchell is UPI's Medical Correspondent. E-mail sciencemail@upi.com
Copyright © 2001-2004 United Press International


TSS



*** MORE CONCERN HERE, PIGS AND MAD COW DISEASE ;

Scrapie in Swine: a Diagnostic Challenge 

Justin J. Greenlee1, Robert A. Kunkle1, Jodi D. Smith1, and M. Heather West Greenlee2 1Virus and Prion Research Unit, National Animal Disease Center, ARS, USDA, Ames, Iowa, USA 2Iowa State University, Ames, Iowa, USA A naturally occurring prion disease has not been recognized in swine, but the agent of bovine spongiform encephalopathy does transmit to swine by experimental routes. Swine are thought to have a robust species barrier when exposed to the naturally occurring prion diseases of other species, but the susceptibility of swine to the agent of sheep scrapie has not been thoroughly tested. We conducted this experiment to test the susceptibility of swine to U.S. scrapie isolates by intracranial and oral inoculation. Scrapie inoculum was a pooled 10% (w/v) homogenate derived from the brains of clinically ill sheep from the 4th passage of a serial passage study of the U.S scrapie agent (No. 13–7) through susceptible sheep (homozygous ARQ at prion protein residues 136, 154, and 171, respectively). Pigs were inoculated intracranially (n=19) with a single 0.75 mL dose or orally (n=24) with 15 mL repeated on 4 consecutive days. Necropsies were done on a subset of animals at approximately six months post inoculation (PI): the time the pigs were expected to reach market weight. Remaining pigs were maintained and monitored for clinical signs of transmissible spongiform encephalopathies (TSE) until study termination at 80 months PI or when removed due to intercurrent disease (primarily lameness). Brain samples were examined by immunohistochemistry (IHC), western blot (WB), enzyme immunoassay (EIA), and for a subset of pigs in each inoculation group, bioassay in mice expressing porcine prion protein. At six-months PI, no evidence of scrapie infection was noted by any diagnostic method. However, at 51 months of incubation or greater, 5 animals were positive by one or more methods: IHC (n=4), WB (n=3), or EIA (n=4). Furthermore, positive bioassay results were obtained from all inoculated groups (oral and intracranial; market weight and end of study) suggesting that swine are potential hosts for the agent of scrapie. 

Key words: prion, scrapie, swine, transmissible spongiform encephalopathy (6)

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Pigs were exposed to the agent of BSE in contaminated meat and bone meal in many European countries, but no naturally occurring cases have been described and surveys of meat and bone meal fed pigs failed to demonstrate any evidence of TSE32). Pigs challenged orally with BSE failed to develop evidence of infection33) despite observation for up to seven years22). These previously published studies along with results of the current study suggest that swine are capable of harboring a prion disease, although epidemiologic evidence is not in support of this occurring under production conditions. 

*** We have demonstrated that swine are susceptible to the agent of sheep scrapie. While no pigs examined at market weight were positive by traditional diagnostic methods, there were 5 aged pigs with positive IHC, WB, and/or ELISA from brain tissue. These pigs did not have definitive clinical illness or spongiform change during the course of this experiment. Positive bioassay results from the brains of market weight pigs suggest that even orally inoculated pigs do harbor a low level of infectivity. If scrapie were to occur in pigs, it would represent a significant diagnostic challenge because of long incubation periods, failure of pigs to develop clinical signs, and the inability of traditional diagnostic methods to detect infected animals early in the course of disease.


*** We have demonstrated that swine are susceptible to the agent of sheep scrapie.

Transmission of sheep-bovine spongiform encephalopathy to pigs 

Carlos Hedman1 , Rosa Bolea1*, Belén Marín1 , Fabien Cobrière4 , Hicham Filali1 , Francisco Vazquez2 , José Luis Pitarch1 , Antonia Vargas1 , Cristina Acín1 , Bernardino Moreno1 , Martí Pumarola3 , Olivier Andreoletti4 and Juan José Badiola1 

Abstract 

Experimental transmission of the bovine spongiform encephalopathy (BSE) agent has been successfully reported in pigs inoculated via three simultaneous distinct routes (intracerebral, intraperitoneal and intravenous). Sheep derived BSE (Sh-BSE) is transmitted more efficiently than the original cattle-BSE isolate in a transgenic mouse model expressing porcine prion protein. However, the neuropathology and distribution of Sh-BSE in pigs as natural hosts, and susceptibility to this agent, is unknown. In the present study, seven pigs were intracerebrally inoculated with Sh-BSE prions. One pig was euthanized for analysis in the preclinical disease stage. The remaining six pigs developed neurological signs and histopathology revealed severe spongiform changes accompanied by astrogliosis and microgliosis throughout the central nervous system. Intracellular and neuropil-associated pathological prion protein (PrPSc) deposition was consistently observed in different brain sections and corroborated by Western blot. PrPSc was detected by immunohistochemistry and enzyme immunoassay in the following tissues in at least one animal: lymphoid tissues, peripheral nerves, gastrointestinal tract, skeletal muscle, adrenal gland and pancreas. PrPSc deposition was revealed by immunohistochemistry alone in the retina, optic nerve and kidney. These results demonstrate the efficient transmission of Sh-BSE in pigs and show for the first time that in this species propagation of bovine PrPSc in a wide range of peripheral tissues is possible. These results provide important insight into the distribution and detection of prions in non-ruminant animals.

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Discussion 

This study was aimed at investigating the susceptibility and neuropathological features of pigs intracerebrally inoculated with the BSE agent after passage in sheep, as well as describing the PrPSc distribution in peripheral tissues in this species. 

In the present study, seven pigs were intracerebrally inoculated with 0.5  mL of 10% Sh-BSE homogenate. Except in one animal (P-7), which was euthanized for preclinical analysis, the transmission rate was 100%, with an incubation period range of 77–109 wpi. Two previous studies in which bovine BSE has been transmitted to pigs, reported 87.5% and 20% of rate attacks, with incubation period ranges of 74–163 and 148–175 wpi, respectively [19, 20]. Sh-BSE infected pigs show slightly shorter incubation periods. However, it is not possible to compare the incubation period of our inoculated pigs with respect to the incubation period found in the studies mentioned above, due to the lack of titration of the original inoculum. Moreover, the incubation period could also be modified in TSE due to the species barrier, which is modulated by specific polymorphisms of the PRNP gene and plays a key role in susceptibility to prion disease in other species such as sheep [35, 36], and goats [37]. Although some studies show that there are no differences in the sequence of the porcine PRNP gene [38–40], the possibility of changes in other regions of the gene or the involvement of other genes in the incubation periods of BSE in pigs should not be excluded. In addition, the restricted number of animals used does not allow comparing difference on rate attacks in previous studies with the present report. However, transmissible studies in porcine PRNP transgenic mice (Tgpo) has demonstrated that the Sh-BSE agent reached rate attack of 100% and lower survival time when compared to the original bovine BSE (19%) and other BSE isolates at first passage [15]. At two subsequent passages, the transmission rate of both ShBSE and bovine BSE was reported to be similar (100%) but always with a lower survival time of the Sh-BSE infected mice [15]. Recent studies have demonstrated an increase in the PrP-converting potency of Sh-BSE caused by decreases in polymorphism barriers [24] and other specific cellular factors [25], allowing Sh-BSE to be transmitted more efficiently than cattle BSE to other species [16, 17] including supposedly less susceptible hosts such as pigs [15]. The current study was in agreement with previous reports [20–22] involving intracerebral inoculation of BSE prions to pigs demonstrating that this species is susceptible to BSE. However, it is still unknown if pigs can succumb to BSE after oral exposure which is the most likely route of inoculation under natural conditions. 

The clinical signs observed in the present study were similar to those described in BSE-infected pigs [20]. Animals initially showed progressive confusion, followed by motor deficits [19]. The behavioral and sensory changes were also consistent with those observed in cattle infected naturally [41] and experimentally with BSE [12]. The minimal neuropil vacuoles found in the control pig are in total agreement with previous studies [21] and apparently does not represent a clinical significant change [20]. The main pathological changes observed were neuropil spongiosis, intraneuronal vacuolation and PrPSc deposition, all of which are characteristic of TSE [41]. The lesion distribution pattern resembled that described previously in experimentally BSE- infected pigs [21] and cattle [12]; the thalamus was the most affected area, followed by the cerebellar and cerebral cortices, with the mildest effect observed in the spinal cord. PrPSc deposits were identified in the CNS of all clinically affected pigs. PrPSc deposits were typically associated with lesions in the fourth and fifth layers of the cerebral cortex. Intracellular (ITNR, ITAS and ITMG) and particulate/coalescing type PrPSc deposition were the most commonly observed patterns in the different CNS samples, in line with previous findings in sheep [29, 30] and pigs [21] experimentally infected with BSE. Similarities in the PrPSc deposition types and distribution pattern could be explained by the high stability of the BSE agent reported for different breeds and different genotypes of the prion protein gene (PRNP) in sheep [30]. In addition, the porcine PRNP gene has been described to be very homogenous [38–40]. 

The glial reaction in all affected pigs was characterized by marked astrocytosis and microgliosis. Astrocytosis was diffusely distributed throughout the brain of affected pigs, perhaps caused by the accumulation of PrPSc or by cytokines secreted from astroglial or microglial cells [42]. Microgliosis was present in the deeper layers of the gray matter in the cerebral cortex, which also showed vacuolation and PrPSc deposition, in accordance with previous findings in mice [43]. The most extreme microglial activation was observed in the hippocampus of all affected pigs, as previously described for CJD [44]. Numerous astrocytic processes and reactive microglia have been described in pigs experimentally infected with BSE [45]. Our results suggest that astrogliosis and microgliosis are common neuropathological features of Sh-BSE infection in pigs, as described for TSE in other species [28, 43, 45–47]. 

Histopathological changes indicative of retinal degeneration were observed in all clinically affected pigs. This has not been previously described in pigs experimentally infected with BSE. Neuronal vacuolation in the GCL and disorganization in the plexiform and nuclear layers have been reported in both experimental [48] and natural scrapie infections in sheep [49] and goats [50], chronic wasting disease (CWD) in mule deer [51] and in CJDinfected mice [52]. IHC revealed higher levels of PrPSc in the retina than in the optic nerve, where staining was less intense and more irregularly distributed, as described in both sCJD and nvCJD [53]. 

The presence of PrPSc in the optic nerve and retina is consistent with the centrifugal spread of the agent from the brain, presumably via the optic nerve [52]. This may indicate the existence of other routes of PrPSc migration to the retina (e.g., via the extracellular space [54], the ad-axonal route along the optic nerve, or both [55]). Other authors have suggested that the spread occurs from the subarachnoid space into the perineural space of the optic nerve, and from there to the epichoroidal and episcleral tissues of the eyeball [56]. Alternatively, the increased presence of PrPSc in the retina more than in the optic nerve could be attributed to the higher presence of PrPc in the membranes of retinal neurons. Our detection of PrPSc in different retinal layers is in accordance with previous observations in TSE in mice [52], feline spongiform encephalopathy (FSE) [57], scrapie [49], CWD [51], BSE [56] and in patients with sporadic and nvCJD [53]. 

Western blot revealed a characteristic 3-band pattern that clearly differed from the original inoculum, with a predominant monoclycosylated band. This finding is consistent with previous Western blot findings in BSEinfected pigs [58]. Our results reinforce the hypothesis that this particular signature is associated with the porcine PrPc properties described in Tgpo mice [15]. 

The IDEXX enzyme immunoassay, which is not validated for PrPsc in pigs, detected PrPSc in samples that tested positive in other postmortem assays, but detected no PrPsc in negative control tissues. Analysis of peripheral tissues revealed widespread dissemination of PrPSc in many organs other than the CNS. This finding suggests that unlike in cattle where BSE is confined mainly in the nervous system, in the pig, BSE prions can propagate in peripheral tissues as reported in sheep [59–61]. However, it is not possible to ascertain that the peripheral distribution of the agent is due to centrifugal dissemination from the brain through the nerves as it is also probable that during an ic challenge part of the inoculum enters into the blood circulation and can be disseminated to the periphery where it can propagate in target tissues [62]. 

PrPSc deposition in brachial and sciatic nerves has also been described in cattle experimentally infected with L-type BSE [63] and in BSE-infected sheep [64]. 

Immunohistochemistry demonstrated the presence of PrPSc in the lymphoreticular system of our Sh-BSE infected pigs. The assay revealed sporadic intracytoplasmic accumulation within the tingible body macrophages in some lymph nodes, findings that were subsequently corroborated by IDEXX, in good agreement with previous findings in sheep experimentally infected with BSE [64]. In contrast to our findings, previous studies reported no infectivity of lymphoid tissues in BSEinfected pigs [19]. No PrPSc was detected in the spleen or GALT of our pigs, in line with previous studies of BSEinfected cattle [65] and FSE [57]. 

PrPSc accumulation in the gastrointestinal tract of Sh-BSE infected pigs has not been described in similar experiments using this species. We observed PrPSc deposition in the myenteric plexi without apparent morphological alterations of the enteric neurons, as seen in cattle experimentally infected with BSE [65]. This finding is indicative of a potential centrifugal spread of the Sh-BSE agent from the CNS via the vagus nerve to the peripheral nervous system, and may account for the large deposits of PrPSc observed in the dorsal motor nucleus of the vagus nerve in the medulla oblongata. 

We observed PrPSc deposition in nerve fibers of the oculomotor muscle in two pigs. In cattle naturally infected with BSE [66], PrPSc has been detected in intramuscular nerve fibers and muscle spindles. Although we found no PrPSc in the oculomotor muscle of any other clinically affected pigs, positive labeling was observed in the oculomotor nuclei in the mesencephalon of all clinically affected pigs. 

Pancreatic PrPSc staining was observed in 5 pigs. Analysis of pancreatic nervous tissue has revealed PrPSc deposition in the islets of Langerhans in natural scrapie [34]. In natural BSE [56], PrPSc deposition has been documented in the nerve fibers of the adrenal gland. In agreement with previous findings in natural scrapie [34], one pig showed PrPSc immunolabeling in the medullary region of the adrenal gland, associated with chromaffin cells, which are considered modified sympathetic postganglionic neurons. Similarly, the presence of PrPSc within the epithelial tubular cells of the convoluted tubules and the collecting ducts in the kidney in one pig has been described in FSE [67], suggesting possible prionuria. 

In addition to the large amount of PrPSc observed in the CNS of Sh-BSE-infected pigs, PrPSc was widely distributed in the peripheral tissues, although the extent of this distribution varied between animals. This variation may be related to the distribution of PrPSc within individual organs, the exact anatomical location points at which samples were collected, and the detection limits of the techniques used. More sensitive studies, such as in vitro protein misfolding cyclic amplification (PMCA) and mouse bioassays will be needed to clarify the distribution and infectivity of PrPSc in peripheral tissues of Sh-BSE infected pigs. These assays will most likely indicate a higher number of PrPSc-positive peripheral organs. 

Comparison with previous studies of cattle-BSE in pigs revealed that the incubation period of Sh-BSE in our pigs was generally shorter [20, 21] and that PrPSc was present in more peripheral tissue types [19]. We believe that these differences may be due to a modification in the pathogenicity of the cattle-BSE agent caused by its prior passage in sheep, as previously described in TgPo mice [15]. However, studies of natural routes of transmission (e.g., oral) will be required to determine the real susceptibility of pigs to the ShBSE agent. 

Abbreviations

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SATURDAY, JANUARY 9, 2016

Transmission of sheep-bovine spongiform encephalopathy to pigs

Research article
Transmission of sheep-bovine spongiform encephalopathy to pigs
Carlos Hedman1, Rosa Bolea1*, Belén Marín1, Fabien Cobrière4, Hicham Filali1, Francisco Vazquez2, José Luis Pitarch1, Antonia Vargas1, Cristina Acín1, Bernardino Moreno1, Martí Pumarola3, Olivier Andreoletti4 and Juan José Badiola1
* Corresponding author: Rosa Bolea rbolea@unizar.es
Author Affiliations
1 Veterinary Faculty, Centro de Investigación en Encefalopatías y Enfermedades Transmisibles Emergentes (CIEETE), Universidad de Zaragoza, Zaragoza, 50013, Spain
2 Veterinary Hospital, Universidad de Zaragoza, Zaragoza, 50013, Spain
3 Veterinary Faculty, Department of Animal Medicine and Surgery, Universitat Autònoma de Barcelona, Barcelona, 08193, Spain
4 UMR INRA ENVT 1225, Interactions Hôtes Agents Pathogènes, Ecole Nationale Vétérinaire de Toulouse, Toulouse, 31076, France
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Veterinary Research 2016, 47:14 doi:10.1186/s13567-015-0295-8
The electronic version of this article is the complete one and can be found online at: http://www.veterinaryresearch.org/content/47/1/14
Received: 15 May 2015 Accepted: 21 September 2015 Published: 7 January 2016
© 2016 Hedman et al.
Open Access
This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Abstract
Experimental transmission of the bovine spongiform encephalopathy (BSE) agent has been successfully reported in pigs inoculated via three simultaneous distinct routes (intracerebral, intraperitoneal and intravenous). Sheep derived BSE (Sh-BSE) is transmitted more efficiently than the original cattle-BSE isolate in a transgenic mouse model expressing porcine prion protein. However, the neuropathology and distribution of Sh-BSE in pigs as natural hosts, and susceptibility to this agent, is unknown. In the present study, seven pigs were intracerebrally inoculated with Sh-BSE prions. One pig was euthanized for analysis in the preclinical disease stage. The remaining six pigs developed neurological signs and histopathology revealed severe spongiform changes accompanied by astrogliosis and microgliosis throughout the central nervous system. Intracellular and neuropil-associated pathological prion protein (PrP Sc ) deposition was consistently observed in different brain sections and corroborated by Western blot. PrP Sc was detected by immunohistochemistry and enzyme immunoassay in the following tissues in at least one animal: lymphoid tissues, peripheral nerves, gastrointestinal tract, skeletal muscle, adrenal gland and pancreas. PrP Sc deposition was revealed by immunohistochemistry alone in the retina, optic nerve and kidney. These results demonstrate the efficient transmission of Sh-BSE in pigs and show for the first time that in this species propagation of bovine PrP Sc in a wide range of peripheral tissues is possible. These results provide important insight into the distribution and detection of prions in non-ruminant animals.

SNIP...

Discussion This study was aimed at investigating the susceptibility and neuropathological features of pigs intracerebrally inoculated with the BSE agent after passage in sheep, as well as describing the PrP Sc distribution in peripheral tissues in this species.
In the present study, seven pigs were intracerebrally inoculated with 0.5 mL of 10% Sh-BSE homogenate. Except in one animal (P-7), which was euthanized for preclinical analysis, the transmission rate was 100%, with an incubation period range of 77–109 wpi. Two previous studies in which bovine BSE has been transmitted to pigs, reported 87.5% and 20% of rate attacks, with incubation period ranges of 74–163 and 148–175 wpi, respectively [19], [20]. Sh-BSE infected pigs show slightly shorter incubation periods. However, it is not possible to compare the incubation period of our inoculated pigs with respect to the incubation period found in the studies mentioned above, due to the lack of titration of the original inoculum. Moreover, the incubation period could also be modified in TSE due to the species barrier, which is modulated by specific polymorphisms of the PRNP gene and plays a key role in susceptibility to prion disease in other species such as sheep [35], [36], and goats [37]. Although some studies show that there are no differences in the sequence of the porcine PRNP gene [38]–[40], the possibility of changes in other regions of the gene or the involvement of other genes in the incubation periods of BSE in pigs should not be excluded. In addition, the restricted number of animals used does not allow comparing difference on rate attacks in previous studies with the present report. However, transmissible studies in porcine PRNP transgenic mice (Tgpo) has demonstrated that the Sh-BSE agent reached rate attack of 100% and lower survival time when compared to the original bovine BSE (19%) and other BSE isolates at first passage [15]. At two subsequent passages, the transmission rate of both Sh-BSE and bovine BSE was reported to be similar (100%) but always with a lower survival time of the Sh-BSE infected mice [15]. Recent studies have demonstrated an increase in the PrP-converting potency of Sh-BSE caused by decreases in polymorphism barriers [24] and other specific cellular factors [25], allowing Sh-BSE to be transmitted more efficiently than cattle BSE to other species [16], [17] including supposedly less susceptible hosts such as pigs [15]. The current study was in agreement with previous reports [20]–[22] involving intracerebral inoculation of BSE prions to pigs demonstrating that this species is susceptible to BSE. However, it is still unknown if pigs can succumb to BSE after oral exposure which is the most likely route of inoculation under natural conditions.
The clinical signs observed in the present study were similar to those described in BSE-infected pigs [20]. Animals initially showed progressive confusion, followed by motor deficits [19]. The behavioral and sensory changes were also consistent with those observed in cattle infected naturally [41] and experimentally with BSE [12]. The minimal neuropil vacuoles found in the control pig are in total agreement with previous studies [21] and apparently does not represent a clinical significant change [20]. The main pathological changes observed were neuropil spongiosis, intraneuronal vacuolation and PrP Sc deposition, all of which are characteristic of TSE [41]. The lesion distribution pattern resembled that described previously in experimentally BSE- infected pigs [21] and cattle [12]; the thalamus was the most affected area, followed by the cerebellar and cerebral cortices, with the mildest effect observed in the spinal cord. PrP Sc deposits were identified in the CNS of all clinically affected pigs. PrP Sc deposits were typically associated with lesions in the fourth and fifth layers of the cerebral cortex. Intracellular (ITNR, ITAS and ITMG) and particulate/coalescing type PrP Sc deposition were the most commonly observed patterns in the different CNS samples, in line with previous findings in sheep [29], [30] and pigs [21] experimentally infected with BSE. Similarities in the PrP Sc deposition types and distribution pattern could be explained by the high stability of the BSE agent reported for different breeds and different genotypes of the prion protein gene (PRNP) in sheep [30]. In addition, the porcine PRNP gene has been described to be very homogenous [38]–[40].
The glial reaction in all affected pigs was characterized by marked astrocytosis and microgliosis. Astrocytosis was diffusely distributed throughout the brain of affected pigs, perhaps caused by the accumulation of PrP Sc or by cytokines secreted from astroglial or microglial cells [42]. Microgliosis was present in the deeper layers of the gray matter in the cerebral cortex, which also showed vacuolation and PrP Sc deposition, in accordance with previous findings in mice [43]. The most extreme microglial activation was observed in the hippocampus of all affected pigs, as previously described for CJD [44]. Numerous astrocytic processes and reactive microglia have been described in pigs experimentally infected with BSE [45]. Our results suggest that astrogliosis and microgliosis are common neuropathological features of Sh-BSE infection in pigs, as described for TSE in other species [28], [43], [45]–[47].
Histopathological changes indicative of retinal degeneration were observed in all clinically affected pigs. This has not been previously described in pigs experimentally infected with BSE. Neuronal vacuolation in the GCL and disorganization in the plexiform and nuclear layers have been reported in both experimental [48] and natural scrapie infections in sheep [49] and goats [50], chronic wasting disease (CWD) in mule deer [51] and in CJD-infected mice [52]. IHC revealed higher levels of PrP Sc in the retina than in the optic nerve, where staining was less intense and more irregularly distributed, as described in both sCJD and nvCJD [53]. The presence of PrP Sc in the optic nerve and retina is consistent with the centrifugal spread of the agent from the brain, presumably via the optic nerve [52]. This may indicate the existence of other routes of PrP Sc migration to the retina (e.g., via the extracellular space [54], the ad-axonal route along the optic nerve, or both [55]). Other authors have suggested that the spread occurs from the subarachnoid space into the perineural space of the optic nerve, and from there to the epichoroidal and episcleral tissues of the eyeball [56]. Alternatively, the increased presence of PrP Sc in the retina more than in the optic nerve could be attributed to the higher presence of PrP c in the membranes of retinal neurons. Our detection of PrP Sc in different retinal layers is in accordance with previous observations in TSE in mice [52], feline spongiform encephalopathy (FSE) [57], scrapie [49], CWD [51], BSE [56] and in patients with sporadic and nvCJD [53].
Western blot revealed a characteristic 3-band pattern that clearly differed from the original inoculum, with a predominant monoclycosylated band. This finding is consistent with previous Western blot findings in BSE-infected pigs [58]. Our results reinforce the hypothesis that this particular signature is associated with the porcine PrP c properties described in Tgpo mice [15].
The IDEXX enzyme immunoassay, which is not validated for PrP sc in pigs, detected PrP Sc in samples that tested positive in other postmortem assays, but detected no PrP sc in negative control tissues. Analysis of peripheral tissues revealed widespread dissemination of PrP Sc in many organs other than the CNS. This finding suggests that unlike in cattle where BSE is confined mainly in the nervous system, in the pig, BSE prions can propagate in peripheral tissues as reported in sheep [59]–[61]. However, it is not possible to ascertain that the peripheral distribution of the agent is due to centrifugal dissemination from the brain through the nerves as it is also probable that during an ic challenge part of the inoculum enters into the blood circulation and can be disseminated to the periphery where it can propagate in target tissues [62].
PrP Sc deposition in brachial and sciatic nerves has also been described in cattle experimentally infected with L-type BSE [63] and in BSE-infected sheep [64].
Immunohistochemistry demonstrated the presence of PrP Sc in the lymphoreticular system of our Sh-BSE infected pigs. The assay revealed sporadic intracytoplasmic accumulation within the tingible body macrophages in some lymph nodes, findings that were subsequently corroborated by IDEXX, in good agreement with previous findings in sheep experimentally infected with BSE [64]. In contrast to our findings, previous studies reported no infectivity of lymphoid tissues in BSE-infected pigs [19]. No PrP Sc was detected in the spleen or GALT of our pigs, in line with previous studies of BSE-infected cattle [65] and FSE [57].
PrP Sc accumulation in the gastrointestinal tract of Sh-BSE infected pigs has not been described in similar experiments using this species. We observed PrP Sc deposition in the myenteric plexi without apparent morphological alterations of the enteric neurons, as seen in cattle experimentally infected with BSE [65]. This finding is indicative of a potential centrifugal spread of the Sh-BSE agent from the CNS via the vagus nerve to the peripheral nervous system, and may account for the large deposits of PrP Sc observed in the dorsal motor nucleus of the vagus nerve in the medulla oblongata.
We observed PrP Sc deposition in nerve fibers of the oculomotor muscle in two pigs. In cattle naturally infected with BSE [66], PrP Sc has been detected in intramuscular nerve fibers and muscle spindles. Although we found no PrP Sc in the oculomotor muscle of any other clinically affected pigs, positive labeling was observed in the oculomotor nuclei in the mesencephalon of all clinically affected pigs.
Pancreatic PrP Sc staining was observed in 5 pigs. Analysis of pancreatic nervous tissue has revealed PrP Sc deposition in the islets of Langerhans in natural scrapie [34]. In natural BSE [56], PrP Sc deposition has been documented in the nerve fibers of the adrenal gland. In agreement with previous findings in natural scrapie [34], one pig showed PrP Sc immunolabeling in the medullary region of the adrenal gland, associated with chromaffin cells, which are considered modified sympathetic postganglionic neurons. Similarly, the presence of PrP Sc within the epithelial tubular cells of the convoluted tubules and the collecting ducts in the kidney in one pig has been described in FSE [67], suggesting possible prionuria.
In addition to the large amount of PrP Sc observed in the CNS of Sh-BSE-infected pigs, PrP Sc was widely distributed in the peripheral tissues, although the extent of this distribution varied between animals. This variation may be related to the distribution of PrP Sc within individual organs, the exact anatomical location points at which samples were collected, and the detection limits of the techniques used. More sensitive studies, such as in vitro protein misfolding cyclic amplification (PMCA) and mouse bioassays will be needed to clarify the distribution and infectivity of PrP Sc in peripheral tissues of Sh-BSE infected pigs. These assays will most likely indicate a higher number of PrP Sc -positive peripheral organs.
Comparison with previous studies of cattle-BSE in pigs revealed that the incubation period of Sh-BSE in our pigs was generally shorter [20], [21] and that PrP Sc was present in more peripheral tissue types [19]. We believe that these differences may be due to a modification in the pathogenicity of the cattle-BSE agent caused by its prior passage in sheep, as previously described in TgPo mice [15]. However, studies of natural routes of transmission (e.g., oral) will be required to determine the real susceptibility of pigs to the Sh-BSE agent.
*** IN CONFIDENCE ***
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 pigs 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 ;
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. ...
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CONFIDENTIAL EXPERIMENTAL PORCINE SPONGIFORM ENCEPHALOPATHY
PLEASE NOTE, these old BSE Inquiry links take a while to open with the wayback machine, so be patient. ...tss 

Title: Experimental Intracerebral and Oral Inoculation of Scrapie to Swine: Preliminary Report 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. 

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IN CONFIDENCE 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 ;
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.
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. ...
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. ...
But it would be easier for us if pharmaceuticals/devices are not directly mentioned at all. ...
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.....
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It was not until . . . August 1990, that the result from the pig persuaded both SEAC and us to change our view and to take out of pig rations any residual infectivity that might have arisen from the SBOs.
4.303 The minutes of the meeting record that: It was very difficult to draw conclusions from one experimental result for what may happen in the field. However it would be prudent to exclude specified bovine offals from the pig diet. Although any relationship between BSE and the finding of a spongiform encephalopathy in cats had yet to be demonstrated, the fact that this had occurred suggested that a cautious view should be taken of those species which might be susceptible. The 'specified offals' of bovines should therefore be excluded from the feed of all species. 17
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]
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]
Wednesday, July 06, 2011
Swine Are Susceptible to Chronic Wasting Disease by Intracerebral Inoculation snip... In the US, feeding of ruminant by-products to ruminants is prohibited, but feeding of ruminant materials to swine, mink and poultry still occurs. Although unlikely, the potential for swine to have access to TSE-contaminated feedstuffs exists.
snip...
Wednesday, July 06, 2011
Swine Are Susceptible to Chronic Wasting Disease by Intracerebral Inoculation (see tonnage of mad cow feed in commerce USA...tss)

SNIP...SEE MORE HERE ON PORCINE SPONGIFORM ENCEPHALOPATHY TSE PRION DISEASE

SATURDAY, JANUARY 9, 2016

Transmission of sheep-bovine spongiform encephalopathy to pigs

Saturday, January 9, 2016

Transmission of sheep-bovine spongiform encephalopathy to pigs 

Research article 


MONDAY, JANUARY 16, 2017 

*** APHIS Bovine Spongiform Encephalopathy (BSE): Ongoing Surveillance Program Last Modified: Jan 5, 2017 ***


Diagnosis and Reporting of Creutzfeldt-Jakob Disease
Singeltary, Sr et al. JAMA.2001; 285: 733-734. Vol. 285 No. 6, February 14, 2001 JAMA
Diagnosis and Reporting of Creutzfeldt-Jakob Disease
To the Editor: In their Research Letter, Dr Gibbons and colleagues1 reported that the annual US death rate due to Creutzfeldt-Jakob disease (CJD) has been stable since 1985. These estimates, however, are based only on reported cases, and do not include misdiagnosed or preclinical cases. It seems to me that misdiagnosis alone would drastically change these figures. An unknown number of persons with a diagnosis of Alzheimer disease in fact may have CJD, although only a small number of these patients receive the postmortem examination necessary to make this diagnosis. Furthermore, only a few states have made CJD reportable. Human and animal transmissible spongiform encephalopathies should be reportable nationwide and internationally.
Terry S. Singeltary, Sr Bacliff, Tex
1. Gibbons RV, Holman RC, Belay ED, Schonberger LB. Creutzfeldt-Jakob disease in the United States: 1979-1998. JAMA. 2000;284:2322-2323.
Terry S. Singeltary Sr.