Friday, February 15, 2008

SCRAPIE and TSE to human UPDATE 2008 (ambiguous terms of transition and reality set in)

SCRAPIE and TSE to human UPDATE 2008 (ambiguous terms of transition and reality set in)

The EFSA Journal (2008) 626, 1-11

© European Food Safety Authority, 2008

Scientific and technical clarification in the interpretation and consideration of some facets of the conclusions of its Opinion of 8 March 2007 on certain aspects related to the risk of Transmissible Spongiform Encephalopathies (TSEs) in ovine and caprine animals1

Scientific Report of the Scientific Panel on Biological Hazards

January 2008 - 37th Plenary meeting

Question EFSA-Q-2007-193


Olivier Andreoletti, Herbert Budka, Sava Buncic, Pierre Colin, John D Collins, Aline De Koeijer, John Griffin, Arie Havelaar, James Hope, Günter Klein, Hilde Kruse, Simone Magnino, Antonio Martínez López, James McLauchlin, Christophe Nguyen-The, Karsten Noeckler, Birgit Noerrung, Miguel Prieto Maradona, Terence Roberts, Ivar Vågsholm, Emmanuel Vanopdenbosch.


The European Food Safety Authority wishes to thank the members of the Working Group for their contribution to this Scientific and Technical clarification: Olivier Andreoletti, Herbert Budka, Martin Groschup, James Hope, Danny Matthews, Marion Simmons, Emmanuel Vanopdenbosch (Chairman) and Kathy Webster.

1 For citation purposes: Scientific Report of the Panel on Biological Hazards on a request from the European Commission on “Scientific and technical clarification in the interpretation and consideration of some facets of the conclusions of its Opinion of 8 March 2007 on certain aspects related to the risk of Transmissible Spongiform Encephalopathies (TSEs) in ovine and caprine animals”. The EFSA Journal (2008), 626, 1-11. The EFSA Journal (2008) 626, 2-11


Scientific uncertainty results usually from five characteristics of the scientific method (EC, 2000): the variable chosen, the measurements made, the samples drawn, the models used and the causal relationship employed. This may also arise from a controversy on existing data or lack of some relevant data. Following appropriate scientific risk assessment, the expression of such uncertainty in complex settings, by simple and concise wording without sacrificing scientific exactness, is one of the major challenges of risk communication.

The following report is strictly limited to replying to the terms of reference as received from the European Commission by re-addressing the uncertainties that were reflected in the conclusions of the concerned EFSA 2007 opinion.


1. In relation to conclusion 2 in page 7 the Authority is invited to clarify and substantiate if there is at this stage any scientific evidence about the presence of TSEs agents other than BSE, Classical and Atypical Scrapie agents in ovine or caprine animals.

Conclusion 2 in page 7 of the EFSA 2007 opinion states that: “The BSE agent is the only TSE agent identified as zoonotic. However, in view of their diversity it is currently not possible to exclude transmissibility to humans of other animal TSE agents.”

In the preamble of the EFSA 2007 opinion it is stated that:

• “Any positive case of TSE in small ruminants is termed as “TSE in small ruminants” which encompasses Classical Scrapie, Atypical Scrapie including Nor98 in sheep and goats as well as BSE in these species if found.”

It should be noted that, under 3.2.1 of the EFSA 2007 opinion it is stated that:

• “The introduction of active surveillance programmes in the European Union has led to the recognition of isolates that do not conform to previous phenotypes of BSE in cattle and Classical Scrapie in sheep. These are currently termed for operational reasons as “Atypical” BSE or Scrapie, but probably reflect part of a wider spectrum of isolates not previously recognised, and in the case of Atypical Scrapie affecting genotypes highly resistant to clinical Scrapie (Biacabe et al., 2004; Casalone et al., 2004; Benestad et al., 2003; Gavier- Widen et al., 2004; Buschmann et al., 2004).” Scrapie is a disease of ovine and caprine animals caused by a variety of TSE agents harbouring different biological properties that are still incompletely characterised, rather than by one specific transmissible entity. ‘Classical Scrapie’ and ‘Atypical Scrapie’ are operational rather than purely biological terms. (EFSA, 2005; Saegerman et al., 2007;

Benestad et al., 2008). The EFSA Journal (2008) 626, 3-11

Further on the EFSA 2007 opinion, under 3.2.3 the Panel acknowledges that there is a:

• “[…] lack of understanding of the true biodiversity of TSEs in small ruminants in terms of both Classical and Atypical agents […]”

This is in accordance to the approach taken by the Panel in the EFSA 2005 opinion when classifying the possible TSE cases in small ruminants. In fact, the EFSA 2007 opinion stresses that:

• “Practical definitions of TSE in small ruminants are summarized in the EFSA opinion on the classification of Atypical and Classical Scrapie and BSE in small ruminants (EFSA, 2005) in the Table “Criteria for the categorisation of TSEs in small ruminants” (in annex 1 of the said EFSA opinion).”

In Annex 1 of the EFSA 2005 opinion, a table for the categorisation of TSEs in small ruminants can be found. This opinion concludes that the definition of Atypical Scrapie:

• “[…] is provided in juxtaposition with similar definitions for Scrapie and BSE in small ruminants. Sub-categorisation of Scrapie and Atypical Scrapie is premature although this may become possible when more data are available.” Recent studies have identified TSE agents in cattle that differ from the original TSE agent type, which causes the disease commonly known as BSE that was detected since 1986 in the United Kingdom and is referred to as ‘Classical BSE’ in this document.

Based on their diagnostic phenotypes these more recently identified TSE agents in cattle are called L- and H-type BSE agents (Biacabe et al., 2004; Casalone et al., 2004; Baron et al., 2007). In conclusion, the reply to the ToR number 1 is:

• In ovine animals, no TSE agents other than those causing Classical Scrapie and Atypical Scrapie have been identified.

• In caprine animals, no TSE agents other than those causing BSE, Classical Scrapie and Atypical Scrapie have been identified.

• The operational term ‘BSE’ covers a TSE of bovine animals that could be caused by at least three distinct TSE agents with heterogeneous biological properties.

• The operational term ‘Classical Scrapie’ covers a TSE of ovine and caprine animals caused by several TSE agents with heterogeneous biological properties.

• The operational term ‘Atypical Scrapie’ covers a TSE of ovine and caprine animals that differs from Classical Scrapie. Currently, it is a subject for debate whether it is caused by one or more TSE agents. The EFSA Journal (2008) 626, 4-11

2. In relation to conclusion 2 in page 7 the Authority is invited to specify the scientific evidences which do not allow to exclude transmissibility to humans of “other TSE agents” other than BSE. Conclusion 2 in page 7 of the EFSA 2007 opinion states that: “The BSE agent is the only TSE agent identified as zoonotic. However, in view of their diversity it is currently not possible to exclude transmissibility to humans of other animal TSE agents.”

In the EFSA 2007 opinion under 3.2.3. the Panel states that:

• “There are significant uncertainties associated with the question whether TSE agents in their whole spectrum may cross the human transmission barrier under natural conditions”.

This statement is supported both by scientific evidence and considerations, referenced in the EFSA 2007 opinion:

• Scientific evidence from transmission studies to primates: - Transmission of Classical Scrapie from a TSE agent adapted in hamster was demonstrated by oral challenge in squirrel monkey (Saimiri sciureus) (Gibbs et al., 1980);

- Transmission of Classical Scrapie from two distinct sheep sources by intracerebral challenge in cynomologus monkey (Macaca fascicularis) and marmoset monkey (Callithrix jacchus) (Gibbs and Gajdusek, 1972; Baker et al., 1988).

• Scientific considerations on TSE epidemiology: - “The assumed lack of association between TSEs in humans and those in small ruminants […] may be biased by a number of factors: (i) The lack of a data on the historical real prevalence and distribution of small ruminant TSEs, at a time where only passive surveillance was performed; (ii) the lack of understanding of the true biodiversity of TSEs in small ruminants in terms of both Classical and Atypical agents; (iii) the lack of understanding of the diversity of TSEs in humans due to the limited molecular and bioassay characterisation of human TSEs also in relation to the number and spectrum of neurodegenerative diseases of humans; (iv) the predicted phenotype of disease that might arise should an animal derived TSE transmit to humans.” The EFSA Journal (2008) 626, 5-11

Further evidence is provided by:

• In vitro conversion assays: Raymond et al. (1997) studied whether there is a correlation between in vitro conversion efficiencies and known transmissibility of BSE, sheep Scrapie and CJD, and found limited conversion of human PrP-sen to PrP-res driven by PrP-res associated with both Scrapie (PrPSc) and BSE (PrPBSE). They concluded that “the inherent ability of these infectious agents of BSE and Scrapie to affect humans following equivalent exposure may be finite but similarly low”. Nevertheless, uncertainty arises from the fact that this is a simple in vitro model of a complex in vivo situation.

• Laboratory transmission studies with animal models: Since the publication of the Opinion new data have become available with regards to L type of BSE, which has now been identified in various EU members states (Biacabe et al., 2004; Casalone et al., 2004; Baron et al., 2007). This TSE agent, differing from that causing Classical BSE by its biochemical signature and transmission features in mouse models, has been transmitted to a Tg mouse model expressing Human M129 PRP gene (Beringue et al. 2007)2. Here again, uncertainty arises from the limitations of these animal models for the estimation of the human species barrier. These ‘proof of principle’ experiments provide data supporting the ability of TSE agents other than those causing Classical BSE to cross the human species barrier.

Even so, it is important to remember that as mentioned in the EFSA 2007 opinion, transmission to primates:

• “… does not allow to take into account the human gene PRNP polymorphisms (in particular the M/V 129), that have been identified to play a major role on relative susceptibility towards prion disease. In addition, genes other than the PrP gene may also be influential in determining overall susceptibility to TSEs.” Despite the interests in the area, studies of the transmissibility of currently known TSE agents using animal models will remain incomplete for several years.

In conclusion, the reply to the ToR number 2 is:

• Experimental transmissions to primate and to transgenic (Tg) mouse models expressing the human PrP gene, are currently used as to evaluate the potential capacity of a TSE agent to cross the human species barrier.

• TSE agents other than the Classical BSE agent from three field TSE cases (two Classical Scrapie cases and one L type BSE case) have been demonstrated to cross the modelled human species barrier.

• Some limitations to these models have to be considered, which include:

(i) The uncertainty of how well they represent the human species barrier. (ii) The uncertainty of how well the experimental inoculation route employed represents exposure under natural conditions. 2 Transmission of this TSE agent by intracerebral challenge to primates (Macaques) has been reported by Comoy et al. at the 2006 Prion Congress held in Torino. A scientific paper reporting this finding has been submitted for peer review publication. The EFSA Journal (2008) 626, 6-11

3. In relation to conclusion 3 in page 7, the Authority is invited:

a. To clarify if, although the diagnostic sensitivity and specificity of the discriminatory tests can not be assumed to be perfect, they are at this moment the diagnostic tool fit for the differentiation of the different TSE strains which can be present in ovine and caprine animals if they are performed according to the procedure described in point 3.2 (c) of Chapter C of Annex X to Regulation (EC) 999/2001.

Clarification of the ToR 3.a.: During the discussion of the WG it was clarified with the Commission services that when referring to differentiation of different TSE strains it was meant “identification of the TSE agent that causes Classical BSE versus any other TSE agent that can be present in ovine or caprine animals”.

Conclusion 3 in page 7 of the EFSA 2007 opinion states that:

• “Current discriminatory tests as described in the EC legislation to be used for discrimination between Scrapie and BSE appear, up to now, to be reliable for the differentiation of BSE from Classical and Atypical Scrapie. However, at the current stage of scientific knowledge, neither their diagnostic sensitivity nor their specificity can be assumed to be perfect.” Characterisation of TSE agents is based on biological and/or molecular methods. Molecular methods are based on the properties of the disease-specific proteaseresistant fragments of PrPSc or physico-chemical behaviour (e.g. proteinase K cleavage site, PrPSc glycosylation, relative proteinase K resistance, molecular conformation, etc). Biological methods identify the reproducibility and stability of the disease phenotype, including lesion profile and incubation period, on serial transmission in a specified mouse line, but also consider the phenotype of the disease in the host species. Discriminatory biochemical tests, as referred here, only represent a group of standardised methods (reagent protocols) allowing reproducible identification of certain abnormal PrP biochemical features associated with the TSE agent causing Classical BSE experimentally propagated in sheep. These biochemical methods cannot be considered to be appropriate tools for the differentiation of the different TSE agents (excluding the agent that causes Classical BSE), which can be present in ovine and caprine animals in the wider sense, as they have not been designed for that purpose.

In page 7 of the EFSA 2007 opinion, it is stated that based on the EFSA Opinion of 25 January 2007 on Quantitative Risk Assessment on the residual BSE risk in sheep meat and meat products (EFSA-Q-2005-235),

• “The Scrapie/BSE discriminatory tests are robust judging by their performance against a small number of samples in a blinded ring trial organised by the EU TSE Community Reference Laboratory (Stack et al., in preparation) and their application as part of small ruminant surveillance was continuing to improve the accuracy of these prevalence estimates. However, balanced against this optimistic scenario, the BIOHAZ Panel accepted that the sensitivity and specificity of the discriminatory tests had, for logistical reasons, not been experimentally evaluated and potential confounding factors, such as

The EFSA Journal (2008) 626, 7-11

concomitant infection of the same animal with Scrapie and BSE, remained to be investigated.” The result of that ring trial employing a limited number of different categories of samples3shows that all tests were able to discriminate between field cases of Classical Scrapie in sheep and the types of BSE in sheep available from experimental animals (Stack et al., 2008).

Moreover, it should be noted that as previously reported under 3.2.3 of the 2007 EFSA opinion, the Panel acknowledged that there is a:

• “[…]lack of understanding of the true biodiversity of TSEs in small ruminants in terms of both Classical and Atypical agents […]”

In field cases of Classical Scrapie, more than one TSE agent can be isolated from a single animal (Pattison et al., 1961; Bruce, et al., 2002). The presence of one TSE agent can mask the presence of another as well as its manifestation with disease, when co-infecting the same individual. This phenomenon of “interference” has been studied in experimental models using different TSE agents (Manuelidis and Lu, 2003; Nishida et al., 2005; Bartz et al., 2007). Data reported by Baron and Biacabe (2001) indicated that intracerebral inoculation of C57Bl6 mice with a mixture of Scrapie and BSE agents, which were both already adapted to this mouse strain (i.e. species barrier effects had been ablated), resulted in the development of a TSE which presented with a Scrapie Western blot profile. Despite the fact that direct extrapolation of these observations to small ruminants is not possible, these results raise the possibility that the presence of BSE in sheep, if it occurred as a co-infecting TSE agent following established Scrapie, may remain undetected. As the likelihood of such a situation is uncertain at this moment, experiments designed to answer specifically to this question are currently ongoing in an EU funded program (QLK – CT –2001- 01309 BSE in sheep). The Panel recognises the uncertainty of performance of the discriminatory tests applied to small ruminants but also that it is pivotal to have some estimate of the likelihood of co-infection of BSE and Scrapie in the same animal. This would aid the understanding of the practical impact of this uncertainty.

In conclusion, the reply to the ToR number 3.a. is:

• Based on the limited data available, the discriminatory tests as implemented at EU level are practicable tools for screening of field TSE cases (as mentioned in Regulation 999/2001, Annex X, Chapter C, point 3.2. (c)), fulfilling the objective of rapid and reproducible identification of TSE cases that have a signature compatible with Classical BSE agent.

• These discriminatory tests cannot be considered to be perfect because of the current lack of understanding of both the true biodiversity of TSE agents in ovine and caprine animals and how the agents interact in case of co-infection. 3 These were: 4 Ovine BSE samples from ovine oral primary challenge with bovine BSE brain; 10 Ovine BSE samples, from intracerebral challenge with Cheviot AHQ/AHQ ovine BSE (1st sheep to sheep challenge); 12 Sheep Scrapie, positive by statutory tests; 9 Scrapie clinical suspects, negative by statutory tests; 2 Control sheep, negative samples; 2 confirmed bovine BSE positives; 2 Bovine negative controls.

The EFSA Journal (2008) 626, 8-11

b. To clarify if, although the diagnostic sensitivity and specificity of the discriminatory tests can not be assumed to be perfect, the absence of statistically sufficient data on the performance of the tests is not compensated by the procedure in place including a ring trial with additional molecular testing methods in different laboratories and an evaluation by an expert panel chaired by the Community Reference Laboratory for TSEs and taken into account the conclusion of the BIOHAZ Panel in its opinion dated 13 July 2006 that the proportion of sheep being infected by BSE is likely to be extremely small. In page 7 of the Opinion, it is stated that based in the EFSA Opinion of 25 January 2007 on Quantitative Risk Assessment on the residual BSE risk in sheep meat and meat products (EFSA-Q-2005-235, The EFSA Journal, 442, 1-44),

• “[…] the BIOHAZ Panel accepted that the sensitivity and specificity of the discriminatory tests had, for logistical reasons, not been experimentally evaluated and potential confounding factors, such as concomitant infection of the same animal with Scrapie and BSE, remained to be investigated.”

By definition the term diagnostic specificity means the number of correctly identified negative samples divided by the sum of the correctly identified negative and of the false positive samples. If it is uncertain to some degree whether the samples which are negative (this is non-BSE) in the discriminatory tests are correctly identified, an enlargement of the sample size does not add to the already existing data on their real diagnostic specificity.

By definition the term diagnostic sensitivity means the number of correctly identified positive samples divided by the sum of the correctly identified positive and of the false negative samples. Therefore, it is clear that in this case any increase of the number of negative samples is completely irrelevant for the determination of the diagnostic sensitivity of an assay.

To date, the discriminatory tests as described in Regulation (EC) 999/2001, Annex X, Chapter C, point 3.2 (c) have been only validated in the framework of a ring trial employing TSE agents isolated from sheep experimentally infected with Classical BSE agent. No confirmed ovine BSE field case has been detected through this process. Moreover, the tests have not been validated against a possible Classical BSE/other TSE agent co-infection.

In conclusion and also considering the content of the answer to ToR 3.a, the answer to ToR 3.b is:

• Despite consistent performance in ring trials employing samples from experimental ovine BSE cases, there is uncertainty on their performance in the field (because of the lack of detection of natural BSE in ovine or caprine animal).

• TSE positive cases go through the full discriminatory process (including bioassay) only when biochemical discriminatory testing is compatible with BSE signature. Therefore, data obtained through this process can not be used for the evaluation of the sensitivity or the specificity of the discriminatory tests.

The EFSA Journal (2008) 626, 9-11

• Increasing the number of negative results during the TSE discriminatory testing of ovine or caprine animals can not compensate for the absence of statistically sufficient data on the performance of the tests.

The EFSA Journal (2008) 626, 10-11


Baker, H. F., Ridley, R. M., Wells, G. A. and Ironside, J. W. 1988. Prion protein immunohistochemical staining in the brains of monkeys with transmissible spongiform encephalopathy. Neuropathol. Appl. Neurobiol. 24 (6): 476-86.

Baron, T. G. M. and Biacabe, A. G. 2001. Molecular analysis of the abnormal prion protein during coinfection of mice by bovine spongiform encephalopathy and a Scrapie agent. Journal Of Virology 75 (1): 107-114.

Baron, T., Biacabe, A. G., Arsac, J. N., Benestad, S. and Groschup, M. H. 2007. Atypical transmissible spongiform encephalopathies (TSEs) in ruminants. Vaccine 25 (30): 5625-5630.

Bartz, J. C., Kramer, M. L., Sheehan, M. H., Hutter, J. A. L., Ayers, J. I., Bessen, R. A. and Kincaid, A. E. 2007. Prion interference is due to a reduction in strain-specific PrPSc levels. Journal Of Virology 81 (2): 689-697.

Benestad, S. L., Sarradin, P., Thu, B., Schonheit, J., Tranulis, M. A. and Bratberg, B. 2003. Cases of Scrapie with unusual features in Norway and designation of a new type, Nor98. Veterinary Record 153 (7): 202-+.

Benestad, S. L., Arsac, J. N., Goldmann, W. and Nöremark, M. 2008.Atypical-Nor98 Scrapie: properties of the agent, genetics, and epidemiology. Veterinary Research, 39:19. DOI: 10.1051- vetres:2007056.

Beringue, V., Andreoletti, O., Le Dur, A., Essalmani, R., Vilotte, J. L., Lacroux, C., Reine, F., Herzog, L., Biacabe, A. G., Baron, T., Caramelli, M., Casalone, C. and Laude, H. 2007. A bovine prion acquires an epidemic bovine spongiform encephalopathy strain-like phenotype on interspecies transmission. Journal Of Neuroscience 27: 6965-6971.

Biacabe, A. G., Laplanche, J. L., Ryder, S. and Baron, T. 2004. Distinct molecular phenotypes in bovine prion diseases. Embo Reports 5 (1): 110-114.

Bruce, ME, Boyle, A, Cousens, S, McConnell, I, Foster, J, Goldmann, W and Fraser, H, 2002. Strain characterization of natural sheep Scrapie and comparison with BSE. Journal of General Virology, 83 (3): 695-704

Buschmann, A., Biacabe, A. G., Ziegler, U., Bencsik, A., Madec, J. Y., Erhardt, G., Luhken, G., Baron, T. and Groschup, M. H. 2004. Atypical Scrapie cases in Germany and France are identified by discrepant reaction patterns in BSE rapid tests. Journal Of Virological Methods 117 (1): 27-36.

Casalone, C., Zanusso, G., Acutis, P., Ferrari, S., Capucci, L., Tagliavini, F., Monaco, S. and Caramelli, M. 2004. Identification of a second bovine amyloidotic spongiform encephalopathy: Molecular similarities with sporadic Creutzfeldt-Jakob disease. Proceedings Of The National Academy Of Sciences Of The United States Of America 101 (9): 3065-3070. EC 2000. Communication from the Commission on the precautionary principle. COMM (2000)


EFSA 2005. Opinion of the Scientific Panel on Biological Hazards on the request from the European Commission on classification of Atypical Transmissible Spongiform Encephalopathy (TSE) cases in small ruminants. The EFSA Journal 276: 1-30.

The EFSA Journal (2008) 626, 11-11

EFSA 2007. Opinion of the Scientific Panel on Biological Hazards on a request from the European Commission on certain aspects related to the risk of Transmissible Spongiform Encephalopathies (TSEs) in ovine and caprine animals. The EFSA Journal 466: 1-10.

Gavier-Widen, D., Noremark, M., Benestad, S., Simmons, M., Renstrom, L., Bratberg, B., Elvander, M. and af Segerstad, C. H. 2004. Recognition of the Nor98 variant of Scrapie in the Swedish sheep population. Journal Of Veterinary Diagnostic Investigation 16 (6): 562-567.

Gibbs, C. J., Amyx, H. L., Bacote, A., Masters, C. L. and Gajdusek, D. C. 1980. Oral- Transmission Of Kuru, Creutzfeldt-Jakob Disease, And Scrapie To Nonhuman-Primates. Journal Of Infectious Diseases 142 (2): 205-208.

Gibbs, C. J. J. and Gajdusek, D. C. 1972. Transmission of Scrapie to the cynomolgus monkey (Macaca fascicularis). Nature 236 (5341): 73-4.

Manuelidis, L. and Lu, Z. Y. 2003. Virus-like interference in the latency and prevention of Creutzfeldt-Jakob disease. Proceedings Of The National Academy Of Sciences Of The United States Of America 100 (9): 5360-5365.

Nishida, N., Katamine, S. and Manuelidis, L. 2005. Reciprocal interference between specific CJD and Scrapie agents in neural cell cultures. Science 310 (5747): 493-496.

Pattison, I.H. and Millson, G.C., 1961.Further experimental observations on Scrapie. Journal of Comparative Pathology. 71:350-9.

Raymond, G. J., Hope, J., Kocisko, D. A., Priola, S. A., Raymond, L. D., Bossers, A., Ironside, J., Will, R. G., Chen, S. G., Petersen, R. B., Gambetti, P., Rubenstein, R., Smits, M. A., Lansbury, P. T. and Caughey, B. 1997. Molecular assessment of the potential transmissibilities of BSE and Scrapie to humans. Nature 388 (6639): 285-288.

Saegerman, C., Vanopdenbosch, E. and Berkvens, D. 2007. Current status of Scrapie. CAB Reviews: Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources 2 (027): 20 pp.

Stack, M., Jeffrey, M., Deslys, J.P., Grassi, J., Baron, T., Safar, J., Groschup, M., Agrimi, U., Langeveld, J., Matthews, D., Hope, J. and Bostock, C., 2008. An evaluation of techniques for the discrimination of natural scrapie and experimental bovine spongiform encephalopathy in sheep. Document available at the Community Reference Laboratory for Transmissible Spongiform Encephalopathies.


7 February 2008 - A Statement on the potential human health risk from changes to classical scrapie controls(41 KB) has been published.

16. There is no firm evidence for a link between human TSEs and classical scrapie. Although a link cannot be ruled out, even if there is a link, the human health risk from classical scrapie must be very low and result in very few human TSE cases per annum. This is because the incidence of human TSEs is very low and relatively constant world-wide (around one case per million people per year) showing that there must be at least a substantial, if not complete, barrier to transmission of classical scrapie to humans. Although it is not possible to quantify any increase in risk that would arise from the changes to classical scrapie controls, the increased risk is

6 © SEAC 2008

highly unlikely to be greater than the risk before classical scrapie controls were introduced. Indeed, given the apparent effect of the National Scrapie Plan (NSP) on reducing the incidence of classical scrapie in the UK, the risk, if it exists, is likely to be appreciably lower than prior to the implementation of the NSP controls. 17. It is possible that the changes to the EU controls may increase the level of atypical scrapie entering the food chain from classical scrapie affected flocks with concurrent atypical scrapie infections. However, given the low occurrence of atypical scrapie within flocks, any increase in the potential human health risk from atypical scrapie is likely to be very low.


18. Although the changes to the classical scrapie controls may increase the potential risks to human health from small ruminant TSEs, any risk that is present is currently very low or negligible and any increased risk likely to be very low or negligible. For classical scrapie, any increase in potential risk will be less than the longterm historic risk prior to the introduction of small ruminant TSE controls. A risk-benefit analysis is required to determine the proportionality of the changes in controls with respect to the increased potential risks. Such an analysis is not within the remit of SEAC.

SEAC February 2008

Thursday, January 31, 2008

SPONGIFORM ENCEPHALOPATHY ADVISORY COMMITTEE Draft minutes of the 99th meeting held on 14th December 2007



40. The Chair explained that the purpose of the question and answer session was to give members of the public an opportunity to ask questions related to the work of SEAC. Mr Terry Singeltary (Texas, USA) had submitted a question prior to the meeting, asking: “With the Nor-98 now documented in five different states so far in the USA in 2007, and with the two atypical BSE H-base

13 © SEAC 2007

cases in Texas and Alabama, with both scrapie and chronic wasting disease (CWD) running rampant in the USA, is there any concern from SEAC with the rise of sporadic CJD in the USA from ''unknown phenotype'', and what concerns if any, in relations to blood donations, surgery, optical, and dental treatment, do you have with these unknown atypical phenotypes in both humans and animals in the USA? Does it concern SEAC, or is it of no concern to SEAC? Should it concern USA animal and human health officials?”

41. A member considered that this question ............

snip... please see full text, sources, and comments here ;



USA BASE CASE, (ATYPICAL BSE), AND OR TSE (whatever they are calling it today), please note that both the ALABAMA COW, AND THE TEXAS COW, both were ''H-TYPE'', personal communication Detwiler et al Wednesday, August 22, 2007 11:52 PM. ...TSS

P02.35 Molecular Features of the Protease-resistant Prion Protein (PrPres) in H- type BSE

Biacabe, A-G1; Jacobs, JG2; Gavier-Widén, D3; Vulin, J1; Langeveld, JPM2; Baron, TGM1 1AFSSA, France; 2CIDC-Lelystad, Netherlands; 3SVA, Sweden

Western blot analyses of PrPres accumulating in the brain of BSE- infected cattle have demonstrated 3 different molecular phenotypes regarding to the apparent molecular masses and glycoform ratios of PrPres bands. We initially described isolates (H-type BSE) essentially characterized by higher PrPres molecular mass and decreased levels of the diglycosylated PrPres band, in contrast to the classical type of BSE. This type is also distinct from another BSE phenotype named L-type BSE, or also BASE (for Bovine Amyloid Spongiform Encephalopathy), mainly characterized by a low representation of the diglycosylated PrPres band as well as a lower PrPres molecular mass. Retrospective molecular studies in France of all available BSE cases older than 8 years old and of part of the other cases identified since the beginning of the exhaustive surveillance of the disease in 20001 allowed to identify 7 H- type BSE cases, among 594 BSE cases that could be classified as classical, L- or H-type BSE. By Western blot analysis of H-type PrPres, we described a remarkable specific feature with antibodies raised against the C-terminal region of PrP that demonstrated the existence of a more C-terminal cleaved form of PrPres (named PrPres#2 ), in addition to the usual PrPres form (PrPres #1). In the unglycosylated form, PrPres #2 migrates at about 14 kDa, compared to 20 kDa for PrPres #1. The proportion of the PrPres#2 in cattle seems to by higher compared to the PrPres#1. Furthermore another PK–resistant fragment at about 7 kDa was detected by some more N-terminal antibodies and presumed to be the result of cleavages of both N- and C- terminal parts of PrP. These singular features were maintained after transmission of the disease to C57Bl/6 mice. The identification of these two additional PrPres fragments (PrPres #2 and 7kDa band) *** reminds features reported respectively in sporadic Creutzfeldt-Jakob disease and in Gerstmann-Sträussler-Scheinker (GSS) syndrome in humans.

FC5.5.1 BASE Transmitted to Primates and MV2 sCJD Subtype Share PrP27-30 and PrPSc C-terminal Truncated Fragments

Zanusso, G1; Commoy, E2; Fasoli, E3; Fiorini, M3; Lescoutra, N4; Ruchoux, MM4; Casalone, C5; Caramelli, M5; Ferrari, S3; Lasmezas, C6; Deslys, J-P4; Monaco, S3 1University of Verona, of Neurological and Visual Sciences, Italy; 2CEA, IMETI/SEPIA, France; 3University of Verona, Neurological and Visual Sciences, Italy; 4IMETI/SEPIA, France; 5IZSPLVA, Italy; 6The Scripps Research Insitute, USA

The etiology of sporadic Creutzfeldt-Jakob disease (sCJD), the most frequent human prion disease, remains still unknown. The marked disease phenotype heterogeneity observed in sCJD is thought to be influenced by the type of proteinase K- resistant prion protein, or PrPSc (type 1 or type 2 according to the electrophoretic mobility of the unglycosylated backbone), and by the host polymorphic Methionine/Valine (M/V) codon 129 of the PRNP. By using a two-dimensional gel electrophoresis (2D-PAGE) and imunoblotting we previously showed that in sCJD, in addition to the PrPSc type, distinct PrPSc C-terminal truncated fragments (CTFs) correlated with different sCJD subtypes. Based on the combination of CTFs and PrPSc type, we distinguished three PrPSc patterns: (i) the first was observed in sCJD with PrPSc type 1 of all genotypes,;

(ii) the second was found in M/M-2 (cortical form); (iii) the third in amyloidogenic M/V- 2 and V/V-2 subtypes (Zanusso et al., JBC 2004) . Recently, we showed that sCJD subtype M/V-2 shared molecular and pathological features with an atypical form of BSE, named BASE, thus suggesting a potential link between the two conditions. This connection was further confirmed after 2D-PAGE analysis, which showed an identical PrPSc signature, including the biochemical pattern of CTFs. To pursue this issue, we obtained brain homogenates from Cynomolgus macaques intracerebrally inoculated with brain homogenates from BASE. Samples were separated by using a twodimensional electrophoresis (2D-PAGE) followed by immunoblotting. We here show that the PrPSc pattern obtained in infected primates is identical to BASE and sCJD MV-2 subtype. *** These data strongly support the link, or at least a common ancestry, between a sCJD subtype and BASE. This work was supported by Neuroprion (FOOD-CT-2004-506579)

FC5.5.2 Transmission of Italian BSE and BASE Isolates in Cattle Results into a Typical BSE Phenotype and a Muscle Wasting Disease

Zanusso, G1; Lombardi, G2; Casalone, C3; D’Angelo, A4; Gelmetti, D2; Torcoli, G2; Barbieri, I2; Corona, C3; Fasoli, E1; Farinazzo, A1; Fiorini, M1; Gelati, M1; Iulini, B3; Tagliavini, F5; Ferrari, S1; Monaco, S1; Caramelli, M3; Capucci, L2 1University of Verona, Neurological and Visual Sciences, Italy; 2IZSLER, Italy; 3IZSPLVA, Italy; 4University of Turin, Animal Pathology, Italy; 5Isituto Carlo Besta, Italy

The clinical phenotype of bovine spongiform encephalopathy has been extensively reported in early accounts of the disorder. Following the introduction of statutory active surveillance, almost all BSE cases have been diagnosed on a pathological/molecular basis, in a pre-symptomatic clinical stage. In recent years, the active surveillance system has uncovered atypical BSE cases, which are characterized by distinct conformers of the PrPSc, named high-type (BSE-H) and low-type (BSE-L), whose clinicopathological phenotypes remain unknown. We recently reported two Italian atypical cases with a PrPSc type similar to BSE-L, pathologically characterized by PrP amyloid plaques. Experimental transmission to TgBov mice has recently disclosed that BASE is caused by a distinct prion strain which is extremely virulent. A major limitation of transmission studies to mice is the lack of reliable information on clinical phenotype of BASE in its natural host. In the present study, we experimentally infected Fresian/Holstein and Alpine/Brown cattle with Italian BSE and BASE isolates by i.c. route. BASE infected cattle showed survival times significantly shorter than BSE, a finding more readily evident in Fresian/Holstein, and in keeping with previous observations in TgBov mice. Clinically, BSE-infected cattle developed a disease phenotype highly comparable with that described in field BSE cases and in experimentally challenged cattle. On the contrary, BASE-inoculated cattle developed an amyotrophic disorder accompanied by mental dullness. The molecular and neuropathological profiles, including PrP deposition pattern, closely matched those observed in the original cases. This study further confirms that BASE is caused by a distinct prion isolate and discloses a novel disease phenotype in cattle, closely resembling the phenotype previous reported in scrapie-inoculated cattle *** and in some subtypes of inherited and sporadic Creutzfeldt-Jakob disease. Oral Abstracts 14


Experimental BSE Infection of Non-human Primates: Efficacy of the Oral Route

Holznagel, E1; Yutzy, B1; Deslys, J-P2; Lasmézas, C2; Pocchiari, M3; Ingrosso, L3; Bierke, P4; Schulz-Schaeffer, W5; Motzkus, D6; Hunsmann, G6; Löwer, J1 1Paul-Ehrlich-Institut, Germany; 2Commissariat à l´Energie Atomique, France; 3Instituto Superiore di Sanità, Italy; 4Swedish Institute for Infectious Disease control, Sweden; 5Georg August University, Germany; 6German Primate Center, Germany


In 2001, a study was initiated in primates to assess the risk for humans to contract BSE through contaminated food. For this purpose, BSE brain was titrated in cynomolgus monkeys.


The primary objective is the determination of the minimal infectious dose (MID50) for oral exposure to BSE in a simian model, and, by in doing this, to assess the risk for humans. Secondly, we aimed at examining the course of the disease to identify possible biomarkers.


Groups with six monkeys each were orally dosed with lowering amounts of BSE brain: 16g, 5g, 0.5g, 0.05g, and 0.005g. In a second titration study, animals were intracerebrally (i.c.) dosed (50, 5, 0.5, 0.05, and 0.005 mg).


In an ongoing study, a considerable number of high-dosed macaques already developed simian vCJD upon oral or intracerebral exposure or are at the onset of the clinical phase. However, there are differences in the clinical course between orally and intracerebrally infected animals that may influence the detection of biomarkers.


Simian vCJD can be easily triggered in cynomolgus monkeys on the oral route using less than 5 g BSE brain homogenate. The difference in the incubation period between 5 g oral and 5 mg i.c. is only 1 year (5 years versus 4 years). However, there are rapid progressors among orally dosed monkeys that develop simian v CJD as fast as intracerebrally inoculated animals.

The work referenced was performed in partial fulfilment of the study “BSE in primates“ supported by the EU (QLK1-2002-01096).

Subject: Aspects of the Cerebellar Neuropathology in Nor98

Date: September 26, 2007 at 4:06 pm PST


Aspects of the Cerebellar Neuropathology in Nor98

Gavier-Widén, D1; Benestad, SL2; Ottander, L1; Westergren, E1 1National Veterinary Insitute, Sweden; 2National Veterinary Institute, Norway

Nor98 is a prion disease of old sheep and goats. This atypical form of scrapie was first described in Norway in 1998. Several features of Nor98 were shown to be different from classical scrapie including the distribution of disease associated prion protein (PrPd) accumulation in the brain. The cerebellum is generally the most affected brain area in Nor98. The study here presented aimed at adding information on the neuropathology in the cerebellum of Nor98 naturally affected sheep of various genotypes in Sweden and Norway. A panel of histochemical and immunohistochemical (IHC) stainings such as IHC for PrPd, synaptophysin, glial fibrillary acidic protein, amyloid, and cell markers for phagocytic cells were conducted. The type of histological lesions and tissue reactions were evaluated. The types of PrPd deposition were characterized. The cerebellar cortex was regularly affected, even though there was a variation in the severity of the lesions from case to case. Neuropil vacuolation was more marked in the molecular layer, but affected also the granular cell layer. There was a loss of granule cells. Punctate deposition of PrPd was characteristic. It was morphologically and in distribution identical with that of synaptophysin, suggesting that PrPd accumulates in the synaptic structures. PrPd was also observed in the granule cell layer and in the white matter. *** The pathology features of Nor98 in the cerebellum of the affected sheep showed similarities with those of sporadic Creutzfeldt-Jakob disease in humans.


In April of 1985, a mink rancher in Wisconsin reported a debilitating neurologic disease in his herd which we diagnosed as TME by histopathologic findings confirmed by experimental transmission to mink and squirrel monkeys. The rancher was a ''dead stock'' feeder using mostly (>95%) downer or dead dairy cattle and a few horses. She had never been fed.

We believe that these findings may indicate the presence of a previously unrecognized scrapie-like disease in cattle and wish to alert dairy practitioners to this possibility.



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

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

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

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

PMID: 6997404



A The Present Position with respect to Scrapie A] The Problem

Scrapie is a natural disease of sheep and goats. It is a slow and inexorably progressive degenerative disorder of the nervous system and it ia fatal. It is enzootic in the United Kingdom but not in all countries.

The field problem has been reviewed by a MAFF working group (ARC 35/77). It is difficult to assess the incidence in Britain for a variety of reasons but the disease causes serious financial loss; it is estimated that it cost Swaledale breeders alone $l.7 M during the five years 1971-1975. A further inestimable loss arises from the closure of certain export markets, in particular those of the United States, to British sheep.

It is clear that scrapie in sheep is important commercially and for that reason alone effective measures to control it should be devised as quickly as possible.

Recently the question has again been brought up as to whether scrapie is transmissible to man. This has followed reports that the disease has been transmitted to primates. One particularly lurid speculation (Gajdusek 1977) conjectures that the agents of scrapie, kuru, Creutzfeldt-Jakob disease and transmissible encephalopathy of mink are varieties of a single "virus". The U.S. Department of Agriculture concluded that it could "no longer justify or permit scrapie-blood line and scrapie-exposed sheep and goats to be processed for human or animal food at slaughter or rendering plants" (ARC 84/77)" The problem is emphasised by the finding that some strains of scrapie produce lesions identical to the once which characterise the human dementias"

Whether true or not. the hypothesis that these agents might be transmissible to man raises two considerations. First, the safety of laboratory personnel requires prompt attention. Second, action such as the "scorched meat" policy of USDA makes the solution of the acrapie problem urgent if the sheep industry is not to suffer grievously.



Epidemiology of Scrapie in the United States 1977


Prepared by National Center for Animal Health Programs Ruminant Health Programs Team November 15, 2007


Infected and Source Flocks

During FY 2007, there were a total of 76 new infected or source flocks identified. Of those new flocks identified, 30 were infected flocks and 46 were source flocks (Figure 2). As of September 30, 2007, there were 38 scrapie infected and source flocks with open statuses (Figure 3). ...


In FY 2007, 331 scrapie cases have been confirmed and reported by the National Veterinary Services Laboratories (NVSL), including 59* Regulatory Scrapie Slaughter Surveillance (RSSS) cases (Figure 5 and Slide 16). In FY 2007, two field cases, one validation case, and two RSSS cases were consistent with Nor-98 scrapie. The Nor98-like cases originated from flocks in California, Minnesota, Colorado, Wyoming and Indiana respectively. Nineteen cases of scrapie in goats have been reported since 1990 (Figure 6). The last goat case was reported in September 2007.


see full report here ;

Like lambs to the slaughter

31 March 2001 Debora MacKenzie Magazine issue 2284

FOUR years ago, Terry Singeltary watched his mother die horribly from a degenerative brain disease. Doctors told him it was Alzheimer's, but Singeltary was suspicious. The diagnosis didn't fit her violent symptoms, and he demanded an autopsy. It showed she had died of sporadic Creutzfeldt-Jakob disease.

Most doctors believe that sCJD is caused by a prion protein deforming by chance into a killer. But Singeltary thinks otherwise. He is one of a number of campaigners who say that some sCJD, like the variant CJD related to BSE, is caused by eating meat from infected animals. Their suspicions have focused on sheep carrying scrapie, a BSE-like disease that is widespread in flocks across Europe and North America.

Now scientists in France have stumbled across new evidence that adds weight to the campaigners' fears. To their complete surprise, the researchers found that one strain of scrapie causes the same brain damage in mice as sCJD.

"This means we cannot rule out that at least some sCJD may be caused by some strains of scrapie," says team member Jean-Philippe Deslys of the French Atomic Energy Commission's medical research laboratory in Fontenay-aux-Roses, south-west of Paris.

Hans Kretschmar of the University of Göttingen, who coordinates CJD surveillance in Germany, is so concerned by the findings that he now wants to trawl back through past sCJD cases to see if any might have been caused by eating infected mutton or lamb. ...

Tissue distribution. For atypical scrapie, what is PrPres and infectivity distribution within sheep of different genotypes, particularly with respect to SRM removal? For classical scrapie and experimental BSE in sheep, tissue distribution of infectivity is widespread. Thus, even with SRM controls in place, an infected sheep poses around 1000 times the risk to human health than does an infected cow22. Does the distribution depend on whether infection is by the oral or 21 Gubbins S. Prevalence of BSE in sheep: interpreting the results of retrospective and prospective testing of sheep TSE cases. SEAC 84 open meeting 22 paper presented to Food Standards Agency board on 9 December 2004.

Also see paper SEAC/84/2 Annex 2: McLean, A. Page 13 © SEAC 27 February 2006 intracerebral route? Are some VRQ sheep carriers with no neurological symptoms?




Diagnosis and Reporting of Creutzfeldt-Jakob Disease

Singeltary, Sr et al. JAMA.2001; 285: 733-734.

Diagnosis and Reporting of Creutzfeldt-Jakob Disease

Since this article does not have an abstract, we have provided the first 150 words of the full text and any section headings.

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. FREE FULL TEXT

APHIS-2006-0041-0006 TSE advisory committee for the meeting December 15, 2006

[Docket No. FSIS-2006-0011] FSIS Harvard Risk Assessment of Bovine Spongiform Encephalopathy (BSE)

[Docket No. 03-025IFA] FSIS Prohibition of the Use of Specified Risk Materials for Human Food and Requirement for the Disposition of Non-Ambulatory Disabled Cattle

Attachment to Singeltary comment

January 28, 2007

Greetings APHIS,

I would kindly like to submit the following to ;


[Federal Register: January 9, 2007 (Volume 72, Number 5)] [Proposed Rules] [Page 1101-1129] From the Federal Register Online via GPO Access [] [DOCID:fr09ja07-21]



Terry S. Singeltary Sr. P.O. Box 42 Bacliff, Texas USA 77518