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BSEvCJD: The European Ongoing Story


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Title: BSEvCJD: The European Ongoing Story

BSE/vCJD The European On-going Story
  • Prof J Ralph Blanchfield, MBE
  • Past President
  • Institute of Food Science Technology
  • President 2006-2008
  • International Academy of Food Science
  • IUFoST Governing Council Member 2003-2008
  • Food science, food technology and food law
  • E-mail Web
  • (updated 25 Sepember 2009)

BSE/vCJD The European On-going Story
  • ? Containment of risks of ?
  • a fatal cattle disease and
  • a fatal human disease
  • caused by entities which appear not to obey the
    rules of microbiology or toxicology
  • with incubation periods 3-5 years in cattle,
    6-30 years or more in humans
  • with clinical signs developing only very late in
    the incubation period and
  • no ante mortem test

Institute of Food Science Technology
  • the UK-based professional qualifying body of food
    scientists and technologists
  • democratic, not-for-profit, self-governing,
  • totally independent of government, of industry,
    and of any lobbying groups or special interest

Institute of Food Science Technology
  • professional members elected on academic
    qualifications and relevant experience, and
    signed undertaking to comply with the Institute's
    ethical Code of Professional Conduct

Institute of Food Science Technology
  • The first of its four purposes is
  • to serve the public interest by furthering the
    application of science and technology to all
    aspects of the supply of safe, wholesome,
    nutritious and attractive food, nationally and

Institute of Food Science TechnologyWhy
concerned with BSE?
  • New disease affecting major food source -
  • Rapid UK escalation to epidemic proportion
  • Not veterinarians, not neurologists, not
    pathologists, not geneticists, not molecular
    biologists, not epidemiologists, not BSE
  • Experts study small parts of picture close-up
  • Role of food scientists -
  • to stand back and observe whole picture.

  • Jigsaw many missing pieces
  • Every successive update of the IFST Information
    Statement on BSE has emphazised
  • "While that sums up the present state of
    knowledge, scientists always have to keep open
    minds. They have to act on existing knowledge
    while recognizing that further research will
    bring new information and knowledge, which may in
    turn lead to revised conclusions. We welcome the
    devotion of substantial extra resources to
    research in this field."

  • Origin?

Scrapie in rendered meat and bone meal (MBM) feed
becoming changed when passaged through
cows? Or Originating from a one-in-a-million case
of sporadic BSE infection in a cow which was
rendered and recycled? Or (latest speculation,
in Lancet 3 Sept 05) Originating from bonemeal
imported from Indian sub-continent containing
human remains
  • Origin?

Probably never be able to prove which. R
Capobianco et al (PLoS Pathogens, March 9 2007)
have proposed that the atypical form of
spongiform encephalopathy termed BASE, is caused
by a prion strain distinct from that of classical
BSE. They show that it converts into the
classical BSE strain on serial transmission to
inbred mouse lines. Accordingly BASE may be the
origin of BSE, following conversion of the causal
agent in an intermediate host. These findings may
have major implications with respect to the
origin of BSE . Role of contributory factors?
  • Whichever origin -
  • Rendered MBM was the vehicle.
  • Role of John Wilesmith
  • Wilesmith J W et al (1988) "Bovine spongiform
    encephalopathy epidemiological studies on the
    origin", Veterinary Record , 123, 638.
  • Wilesmith J W et al (1991) " Bovine spongiform
    encephalopathy epidemiological studies on the
    origin", Veterinary Record , 128, 200-203.

  • Rendered MBM was also the vector
  • Key factor in subsequent development of the
    epidemic was the use of MBM as cattle feed, as
    demonstrated when its prohibition led to
    successive year-by-year reductions in confirmed
    new cases.

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  • Incidence Rest of World
  • 2000 452 cases
  • 2001 987 cases (Detwiler Effect?)
  • 2002 864 cases
  • 2003 643 cases
  • 2004 524 cases
  • 2005 225 cases
  • 2006 139 cases
  • 2007 73 cases
  • 2008 57 cases
  • 2009 10 cases (to 7 Setember 2009)
  • Improved surveillance efforts
  • Passive versus active surveillance
  • 2001 increase due to active testing

  • Incidence Great Britain
  • Passive surveillance
  • 1988 to 2007 179,180 cases
  • 2000 1,311 cases
  • 2001 781 cases
  • 2002 445 cases
  • 2003 173 cases
  • 2004 82 cases
  • 2005 39 cases
  • 2006 15 cases
  • 2007 7 cases
  • Active surveillance 1,849
  • (2001 - 18 September 2009)

  • Active surveillance testing
  • Active surveillance has been possible on a large
    scale through development of rapid tests. So far
    a total of 12 rapid tests based on different
    detection principles are approved for BSE
    monitoring in cattle in Europe 1 Western blot,
    10 ELISA, and 1 Strip-based test. All are post
    mortem tests that use brain or spinal cord tissue
    of the slaughtered animal.

  • Occurrence of 17 cases in Canada
  • Between 20 May 2003 and 20 February 2009, there
    have been seventeen cases of BSE in Canada.
  • Completed investigation reports on the first
    eight are at http//

  • Occurrence of three cases in USA
  • On 23 December 2003 USDA reported its first BSE
    case in Washington State. By 6 January 2004
    investigation established by DNA that the cow,
    born in 1997, originated from a dairy farm in
    Alberta, Canada.
  • A second case,finally confirmed in June 2005 was
    a 12 year old cow born and bred in South-east
    Texas. A third case, a cow in Alabama, was
    confirmed on 13 March 2006.
  • Investigations are ongoing in both USA and
    Canada. Meanwhile USA is practising enhanced
    measures and controls. Full details and updates
    may be accessed at
  • http//
  • http//
  • http//

  • Prion protein
  • designated PrP or sometimes PrPC
  • small glycosylated protein molecule found mainly
    in the brain cell membrane
  • ?-helix structure

  • Infective agent Prion (Pree-on)
  • Misfolded protein molecule that causes normally
    folded prion protein molecules to misfold.
  • Separate lines of research provide strong
    evidence for the prion hypothesis.

  • Infective agent Prion (Pree-on)
  • Concept J S Griffith (1967)
  • Formalized S B Prusiner (1982)
  • Does not obey conventional rules of microbiology
    or of toxicology
  • Replicates with no DNA or RNA
  • Misfolded prion protein molecule that causes
    normally folded prion protein molecules to
  • ?-helix ? ?sheet structure (PrPsc)
  • Largely protease-resistant (PrPres)
  • Resistant to heat, irradiation, most practicable
    chemical treatments

  • Infective agent Prion (Pree-on)
  • A study by Aguzzis group in Zurich now gives
    concrete evidence that the abnormal-normal prion
    interaction does in fact occur.
  • Meier, P et al. (2003). Misfolded soluble
    dimeric prion protein binds PrPSc in vivo and
    antagonizes prion disease. Cell, 113, 49 60.

  • Infective agent Prion (Pree-on)
  • A study by Prusinerss group in San Fransisco
    now provides compelling evidence that prions
  • are infectious proteins and demonstrates that
    artificially-produced prions can trigger
    development of a BSE-like neurological disorder
    in mice and damage brain tissue without the
    presence of viral DNA or RNA.
  • Legname G et al (2004), Synthetic Mammalian
    Prions, Science, 305, (5684), 673-676.

  • Infective agent Prion (Pree-on)
  • Collinges group (2004) have characterised two
    distinct prion strains derived from BSE
  • transmissions to inbred mice. These data indicate
    a crucial involvement of the host
  • genome in modulating prion strain selection and
    propagation in mice. It is possible that
  • multiple disease phenotypes may also be possible
    in BSE prion infection in humans and other
  • Lloyd SE et al, (2004), J Gen Virol, 85,

  • Infective agent Prion (Pree-on)
  • Studies by two independent research teams have
    shown that synthesised yeast protein Sup35
    clumped at different temperatures had structural
    differences and acted as different prion strains
    when put into yeast cells.
  • King C-H and Diaz-Avalos R (2004). Protein-only
    transmission of three yeast prion strains,
    Nature 428, 319 323.
  • Tanaka M et al (2004). Conformational variations
    in an infectious protein determine prion strain
    differences, Nature 428, 323 328.

  • Infective agent Prion (cont)
  • Five alternative treatments have been proposed
    for decontaminating prion infectivity on
    surfaces, including a phenolic disinfectant an
    alkaline cleaner the combination of an enzymatic
    cleaner and vaporised hydrogen peroxide (VHP) a
    mixture of two proteolytic enzmes and a
    detergent and radio-frequency gas plasma
  • The first two appear to be proprietary
    disinfectants already marketed by STERIS.

  • Infective agent Prion (cont)
  • Radio-frequency (RF) gas-plasma treatment was
    investigated as a method of removing both the
    protein debris and TSE infectivity.
    Stainless-steel spheres contaminated with the
    263K strain of scrapie and a variety of used
    surgical instruments, which had been cleaned by a
    hospital sterile-services department, were
    examined both before and after treatment by RF
    gas plasma.

  • Infective agent Prion (cont)
  • In vivo testing showed that RF gas-plasma
    treatment of scrapie-infected spheres eliminated
    transmission of infectivity. The infectivity of
    the TSE agent adsorbed on metal spheres could be
    removed effectively by gas-plasma cleaning with
    argon/oxygen mixtures. This treatment can
    effectively remove stubborn residual
    contamination on surgical instruments.
  • Baxter HC et al (2005), J Gen Virol 86,2393-2399

  • Infective agent Prion (cont)
  • Langeveld J et al (2003) Journal of Infectious
    Diseases, 1 December 2003 have shown that, when
    brain tissues from cows with BSE and sheep with
    scrapie are treated with a bacterial enzyme
    keratinase, the enzyme fully degraded the prion,
    rendering it undetectable.

  • Infective agent Prion (cont)
  • Prusiners group have reported in PNAS that they
    have genetically altered a strain of mice so that
    the animals made a mutated form of prion protein
    which prevented the animals from becoming ill
    when injected with infective prions. This raises
    the possibility of breeding cattle that are
    protected from BSE.

  • Infective agent Prion (cont)
  • In March 2005 LipidViro Tech, Inc. have announced
    pre-clinical research results demonstrating the
    ability to substantially inactivate infective
    prion proteins in bovine serum utilising patented
    equipment to deliver a precise, measured dose of
    ozone, allowing accurate targeting of the
    threshold which produces prion inactivation while
    maintaining the biological integrity of the
    treated fluid. Prion infectivity was reduced in
    bovine serum below the limit of detection in both
    cell and Western Blot assays. This ongoing
    research has been submitted for presentation at
    the Meeting of the International Union of
    Microbiological Societies, San Francisco, July
    2005. If confirmed these findings could be of
    value in treating bovine sera for pharmaceutical
    use and in treating blood.

Source SCI Biotechnology Group
  • PrPsc model structure based on electron
  • Source Wille et al (2002), PNAS, 99, 10, 1073

  • Infectivity of bovine materials?
  • Brain, spinal cord,
  • Retina (?)
  • Dorsal root and the trigeminal ganglia
  • Bone marrow slightly infectious
  • Distal ilium of calves (experimentally induced)
  • Muscle? Milk? Blood? Not detectable by current
  • BUT

  • Infectivity of bovine materials?
  • In January 2005 Aguzzis group has shown in mice
    suffering from any of five inflammatory diseases
    of kidney, pancreas or liver. that infective
    prions may spread further in the body to those
    tissues. In all cases,
  • chronic lymphocytic inflammation enabled prion
    accumulation in otherwise prion-free organs.
  • Heikenwalder et al., Chronic Lymphocytic
    Inflammation Specifies the Organ Tropism of
    Prions, Science 2005 0 11064601

  • Infectivity of bovine materials?
  • Then in October 2005 Aguzzis group found that
    scrapie-infected mice with kidney inflammation
    accumulated and replicated prions in their
    kidneys and excreted prions in their urine and
    these prions infected other mice with scrapie
    when injected. Further research will be carried
    out to investigate the possible implications for
  • Aguzzi et al, Science doi 101126/science.1117196

  • Infectivity of bovine materials?
  • No infectivity yet detected in blood of BSE
    infected cattle
  • but
  • BSE transmitted to one of six scrapie-free sheep
    by blood transfusion from apparently still
    healthy scrapie-free sheep fed orally with brain
    of BSE infected cattle.
  • Houston F et al (2000) Transmission of SE by
    blood transfusion in sheep, Lancet, 356,
  • Hunter, N et al (2002), Journal of General
    Virology 83, 2897-905.

  • Infectivity of bovine materials?
  • Further work resulted in a second transmission of
    BSE and four new cases of transmission of natural
    scrapie. Positive transmissions occurred with
    blood taken at pre-clinical and clinical stages
    of infection. These results confirmed the risks
    of TSE infection via blood products and suggest
    that the measures taken to restrict the use of
    blood in the UK have been fully justified.
  • Hunter, N et al (2002), Journal of General
    Virology 83, 2897-905.

  • The early UK containment controls (1988)
  • to slaughter and destroy animals clinically
    diagnosed on the farm or elsewhere
  • to prohibit the feeding of material containing
    animal protein derived from ruminants to cattle
    and other ruminants  
  • to prohibit Specified Bovine Offals (SBOs) from
    the food or feed chain

  • UK health controls since 1996 ?
  • Enhanced and strictly enforced ban on mammalian
    MBM for all farm animals, with recall of all
    existing stocks of MBM.
  • Exclusion of SRMs from the food or feed chain
    (SRM from 1995 includes vertebral column and
    dorsal root ganglia).
  • Over Thirty Months Scheme (OTMS) in which cattle
    aged over thirty months are slaughtered in
    designated abattoirs when they came to market and
    incinerated or rendered. To December 2001,
    5,410,716 cattle have been disposed of under the
  • Offspring cull, of offspring born after 1 August
    1996 to dams in which BSE was confirmed.
  • Compulsory Cattle Passport traceability system.

  • UK health controls since 1996 (cont)
  • On 2 December 2004 , following advice from SEAC
    and the Food Standards Agency, the UK Government
    agreed to phase out the OTMS, replacing it from
    2005 by a vigorous and extensive testing regime
    of all cattle.
  • On 15 August 2005, at the Food Standards Agency
    Board agreed its advice to Ministers that an
    effective system to test cattle aged over 30
    months (OTM) for BSE before they enter the food
    chain has now been developed. On 15 September
    2005, the UK Government announced that it is to
    replace the Over Thirty Months (OTM) Rule with
    BSE testing, Ministers also agreed to a number of
    pre-conditions set by the Food Standards Agency
    to ensure continued consumer protection during

  • Born after the real ban (BARB)
  • From 1 August 1996 to 31 December 2007
  • UK 178 cases
  • Rest of EU (since 1 Jan 1996) 298 cases
  • Possible causes
  • Inadequate practice/enforcement of controls?
  • Maternal/vertical transmission?
  • Contamination in calf feed?
  • Unknown cause?

  • Born after the real ban (BARB)
  • DEFRA asked Prof William Hill to review its work
    on cases of BSE in cattle born since 1 August
  • Prof Hill issued his report in June 2005. It can
    be found on the DEFRA Website http//

  • Born after the real ban (BARB)
  • Prof Hills principal conclusions are
  • a) There is no strong reason to believe that BSE
    in BARBs cases is a different disease from that
    in animals born before the reinforced feed ban.
  • b) The diagnostic tests used in active
    surveillance appear to be effective, but only
    when the animal is in the last few months of
    incubating the disease.
  • c) Obtaining hard evidence on the crucial
    hypothesis on the identity of BSE in BARBs and
    previous cases is highly desirable and the
    relevance of atypical molecular forms of BSE
    found by active surveillance in other countries
    needs to be resolved.
  • d) The efficacy and interpretation of the tests
    used in active surveillance of animals for BSE
    should be kept under review.

  • BSE controls across the EU
  • Essentially similar to the UK controls post-1996,
    plus post-mortem testing of
  • suspect cattle over 30 months from January 2001,
  • all over 30 months cattle, from 1 July 2001,
  • some categories of over 24 months cattle from 1
    July 2001.
  • tests mostly being carried out by the Prionics or
    Bio-Rad methods .

  • Vertical or maternal transmission?
  • Supposedly 10 during last six months of
    incubation, by a dubious 1996 assumption based on
    estimation from a flawed experiment started in
    1989 for another purpose.
  • More recently reported research (Wrathall et al,
    2002) appears to provide no support for vertical

  • BSE risk by countries or regions
  • EU Scientific Steering Committee (SSC) assessed
    countries in four Categories
  • Category I Highly unlikely to present a BSE risk
  • Category II Unlikely, but a BSE risk cannot be
  • Category III Likely to present a BSE risk, even
    if not confirmed, or presenting a low level of
    confirmed BSE risk
  • Category IV BSE risk confirmed at a high level
  • Note Assessment of risk of BSE being present,
  • NOT of level of risk to human health

  • BSE risk by countries or regions
  • Change proposed in BSE assessment 
  • On 22 November 2006,the European Food Safety
    Authoritys (EFSA) Panel on Biological Hazards
    (BIOHAZ) launched a public consultation on a
    revision of the methodology for Geographical
    BSE-Risk (GBR) assessment. The update takes
    account of new scientific knowledge on BSE and
    recent trends in BSE prevalence based on the most
    recent surveillance data. By allowing a more
    accurate assessment of geographical BSE risk, the
    revised methodology will assist risk managers in
    making decisions to protect consumers which are
    commensurate with the risk identified.

  • BSE risk by countries or regions
  • In December 2008 the EU provided updated lists of
  • Countries with negligible BSE risk
  • Countries with a controlled BSE risk
  • Commission Decision 2008/829/EC
  • http//

  • During 2004 a few atypical cases have been
    described in France, Italy, Belgium, Denmark, The
    Netherlands and Japan, showing prion
    immunopositive amyloid plaques, as opposed to the
    lack of amyloid deposition in typical BSE cases,
    and by a different pattern of regional
    distribution and topology of brain prion
  • This new form has been named bovine amyloidotic
    spongiform encephalopathy (BASE).

  • Biacabe et al have shown the existence of an
    atypical molecular phenotype among cattle
    diagnosed with BSE in France. Following western
    blot analysis, three cases showed unusual
    features of the electrophoretic profiles of the
    protease-resistant prion protein (PrPres)
    accumulating in the brain. The PrPres patterns
    were similar in these three atypical cases,
    showing a higher molecular mass of unglycosylated
    PrPres and strong labelling by P4 monoclonal
    antibody compared to 55 typical BSE cases. This
    finding suggests either some phenotypic
    modifications of PrPres following infection by
    the BSE agent or the existence of alternative
    origins of such diseases in cattle.
  • Biacabe A G et al (2004). Distinct molecular
    phenotypes in bovine prion diseases. EMBO Rep 5,

  • Similarly, C Casalone et al provide evidence of a
    second cattle TSE. The disorder was
    pathologically characterized by the presence of
    PrP-immunopositive amyloid plaques, as opposed to
    the lack of amyloid deposition in typical BSE
    cases, and by a different pattern of regional
    distribution and topology of brain PrPSc
    accumulation. In addition, Western blot analysis
    showed a PrPSc type with predominance of the low
    molecular mass glycoform and a protease-resistant
    fragment of lower molecular mass than BSE-PrPSc.
    Strikingly, the molecular signature of this
    previously undescribed bovine PrPSc was similar
    to that encountered in a distinct subtype of
    sporadic Creutzfeldt-Jakob disease.
  • Casalone C et al (2004). Identification of a
    second bovine amyloidotic spongiform
    encephalopathymolecular similarities with
    sporadic Creutzfeldt-Jakob disease.. Proc Natl
    Acad Sci USA, 101 306570.

  • Strikingly, the molecular signature of this
    previously undescribed bovine prion was similar
    to that encountered in a distinct subtype of
    sporadic Creutzfeldt-Jakob disease.
  • More recently, two of the US cases of BSE have
    been reported to have shown these atypical

  • Production of cattle lacking prion protein
  • In December 2006 Richt et al report the
    generation and characterization of PrPC-deficient
    cattle produced by a sequential gene-targeting
    system6. At over 20 months of age, the cattle are
    clinically, physiologically, histopathologically,
    immunologically and reproductively normal. Brain
    tissue homogenates are resistant to prion
    propagation in vitro as assessed by protein
    misfolding cyclic amplification. PrPC-deficient
    cattle may be a useful model for prion research
    and could provide industrial bovine products free
    of prion proteins.
  • Richt et al, Nature Biotechnology, published
    online 31 December 2006 doi10.1038/nbt1271
  • http//

  • BSE and sheep?
  • In sheep but masked by scrapie?
  • Serious concern since 1996 laboratory
  • August 1996 similar measures to protect human
    health against BSE in cattle were applied to
    sheep (and goats) in case BSE was present.
  • No evidence of presence in Europe BUT.
  • If present, far more tissues infected than in
  • If present and behaves in sheep like scrapie,
    both vertical and horizontal transmission,
    infection of pastures
  • Until 2005, no rapid test to distinguish BSE from
    scrapie in sheep
  • Full extent of scrapie in EU flocks is unknown

  • BSE and sheep? (cont)
  • From 1 April 2002, extensive testing for TSEs
    across the EU (560,000 a year, focussing on sheep
    aged over 18 months).
  • Extension of the current list of SRMs
  • New provisions on culling of scrapie-infected
  • More use of geno-typing of sheep breeds
  • Proposal on identification and traceability
  • September 2001 - UK Government contingency plan
    if BSE found in sheep. Worst case scenario"
    could include destruction of the whole 40 million
    national flock.
  • June 2002 UK Food Standards Agency proposal to
    ban sheep intestines.

  • BSE and sheep? (cont)
  • DEFRA scrapie surveillance survey showed that TSE
    status could not be determined for 28 out of
    29,201 abattoir sheep) due to inconclusive
    analytical results. The 28 sheep tested positive
    by the Bio-Rad Platelia assay but negative by
    immunohistochemistry (an OIE approved TSE test).
    An Expert Panel called by DEFRA and FSA reported
    its conclusions and research recommendations to
    SEAC, which were accepted.
  • (Report of SEAC 80th meeting, 26 November 2003)

  • BSE and sheep? (cont)
  • On 2 June 2004 DEFRA issues consultation document
    on Contingency plan for the emergence of
    naturally occurring BSE in sheep.
  • http//
  • In October 2005, Le Dur et al reported the
    discovery of a growing number of so-called
    atypical scrapie cases. A substantial proportion
    involved sheep with PrP genotypes known until now
    to confer natural resistance to conventional
    scrapie. Both Nor98 and discordant cases,
    including three sheep homozygous for the
    resistant PrPARR allele (A136R154R171),
    efficiently transmitted the disease to transgenic
    mice expressing ovine PrP, and they shared unique
    biological and biochemical features upon
    propagation in mice.

  • BSE and sheep? (cont)
  • On 9 March 2006 the EU Commission issued
    Questions and Answers on TSEs in Sheep
  • Through the required monitoring programme for
    small ruminants laid down in EU legislation,
    three sheep (two in France, one in Cyprus) were
    found positive for TSE. These required further
    tests to ensure that the TSE present is not BSE.
    In the case of these sheep, the initial
    rule-out tests identified unusual profiles and
    while some data suggested that the samples may
    not be BSE in sheep, there was insufficient
    evidence to definitively rule out the presence of
    BSE. This means that further rule-out tests are
    required. The definitive test is a mouse
    bio-assay, which the Community Reference
    Laboratory (CRL) will now carry out on tissues
    from the suspected sheep.
  • http//

  • BSE and sheep? (cont)
  • 4 April 2005 -The UK Veterinary Laboratories
    Agency (VLA) has developed a new Discriminatory
    Diagnostic Kit to distinguish between BSE and
    Scrapie in sheep, which has been authorised for
    use, and involves protein extraction and western
    blotting technology to differentiate between BSE
    and scrapie in sheep. It is a modified version of
    the Prionics - Check technique and provides a
    cleaner, more defined signal of the abnormal
    prion protein (PrPsc) profile, for analysis.
  • A homogenised sample of central nervous system
    tissue from cattle or sheep is exposed to the
    enzyme, proteinase K, and PrPsc can be
    distinguished from the normal prion protein
    (PrPc) by protease resistance and molecular size.
    This double antibody detection method has been
    validated to show there are different molecular
    weight fragment sizes, differential antibody
    affinity and glycoform ratio differences between
    BSE and scrapie in sheep.

  • BSE and sheep? (cont)
  • Scientists from the Veterinary Laboratories
    Agency have revealed that two ewes fed 5mg of
    BSE-infected material had lambs that died of BSE
    after showing signs of infection in their
    tonsils, 546 days after birth. Their mothers had
    shown no outward signs of the disease at lambing,
    one showing them 73 days after lambing, and the
    other 198 days after.
  • It is still not certain that the lambs were
    infected while in the uterus, or shortly before
    or after lambing. The disease may have spread
    through the birthing fluids or in some other way.
    The evidence so far suggests this is far more
    likely than the lambs catching the disease from
    other apparently unaffected sheep.
  • Bellworthy SJ et al (2005), Natural transmission
    of BSE between sheep within an experimental
    flock,Vet Record, 157. 206.

  • BSE and sheep? (cont)
  • Statement of the Scientific Panel on Biological
    Hazards related to report of the EU TSE Community
    Reference Laboratory on the recent TSE cases in
  • Last updated 17 March 2006
  • The result does not fit the criteria for
    BSE-like by this test, nor does it match
    those for classical scrapie isolates. It also
    does not share the properties of atypical
    scrapie, as defined in the EFSA Opinion.

  • BSE and sheep? (cont)
  • Atypical scrapie has now been identified in the
    British sheep flock. On 15 June 2006, the UK Food
    Standards Agency updated its advice to the public
    because it did not know whether atypical scrapie
    could affect humans. While it is not advising
    people to stop eating sheep or goat meat, or
    their dairy products, it says that consumers can
    reduce the risk by avoiding meat from older sheep
    (mutton) but points out to the EU that new
    labelling provisions are needed for the public to
    be able to identify products containing mutton.

  • BSE and sheep? (cont)
  • On 17 November 2006 a UK Defra Information
    Bulletin stated that atypical scrapie had been
    detected in a sheep from a research flock all the
    founder animals of which were imported from New
    Zealand, a country considered to be free of TSEs.
    The flock is managed under strict bio-security
    conditions to ensure that the animals do not come
    into contact with other sheep. All animals that
    die or are culled in the flock are routinely
    tested for the presence of TSEs and as result of
    this routine testing one of the animals has
    tested positive for atypical scrapie. As the
    origin is not clear, independent scientific
    investigation will include ensuring that the
    bio-security on the farm was not compromised and
    that there was no possibility of cross
    contamination of the sample.

  • BSE and goats?
  • On 12 November 2004 the French General
    Directorate for Food (DGAL) reported that a goat
    was found, when slaughtered in 2002, to exhibit a
    TSE molecularly and biologically compatible with
    BSE. On 28 January 2005 BSE was confirmed. The
    goat was born before the Europe wide ban on MBM.
  • Over 140,000 goats have been tested for BSE
    throughout the EU, with only this one case
    discovered. The rest of the infected animal's
    herd of 600 was tested, all with negative
    results. The EU Commission intends increased
    testing of goats.
  • EFSA has advised that based on current
    scientific knowledge, goat milk and derived
    products are unlikely to present any risk of TSE
    contamination if the milk comes from healthy
    animals and is assessing whether restrictions
    are necessary on French goat meat.

  • BSE and goats?
  • In the UK, all sheep and goat TSE positive cases
    are tested by molecular methods that are capable
    of differentiating between classical scrapie,
    atypical scrapie and experimental BSE. Cases
    where the presence of BSE is ruled out and which
    are confirmed as classical or atypical scrapie
    are not routinely subjected to mouse bioassay. If
    the presence of BSE cannot be excluded, the
    sample is subjected to mouse bioassay.
  • In each of the six cases of TSE detected in goats
    in the UK in the past three years, both BSE and
    atypical scrapie have been ruled out they have
    all been confirmed as classical scrapie.

  • Transmissible to humans?
  • March 1996 UK Government announcement of assumed
    causal connection.
  • October 1996 First scientific evidence
    consistent with transmissibility of BSE
    infectivity to at least some humans (possibly to
    all humans but at varying incubation rates).
  • Subsequent findings increased likelihood of
    causal connection.

  • Transmissible to humans?
  • In a paper given at the Prion 2008 Conference in
    Madrid in October 2008, Baron V et al reported
    the ransmission of atypical BE to non-human
    primates (lemurs). Only one of the four lemurs
    challenged with (H-type) BSE died without
    clinical signs after 19 months post inoculation
    (pmi), wheras all four lemurs inoculated with
    L-type BSE died at 19mpi (2 males) and 22mpi (2
    females), have shown blindness, tremor. abnormal
    posure, uncoordinated movements and balance loss.
  • http//

  • Transmissible to humans?
  • In December 2008 a study showed the transmission
    of atypical bovine prions to mice transgenic for
    human prion protein, with no significant
    transmission barrier. Extrapolation of the data
    raises the theoretical possibility that the
    zoonotic risk associated with BSE-L prions might
    be higher than that associated with classical
  • Béringue V et al, Emerg. Infect. Dis., 2008 Dec
    Epub ahead of print

  • Current UK incidence of vCJD
  • As at 7 September 2009 UK - Deaths
  • From definite vCJD (confirmed) 116
  • From probable vCJD (without 7neuropathological
    confirmation) 49
  • From probable vCJD (neuropathological
    confirmation pending) 0
  • Total deaths from definite or probable vCJD
    (as above) 165
  • Alive probable vCJD cases still alive 4
  • Total number of definite or probable vCJD
    (dead and alive) 169

  • Current UK incidence of vCJD?
  • (per million population aged 10 yrs )

  • Current incidence of vCJD
  • As at 25 September 2009
  • Deaths outside UK
  • France 24
  • Ireland 4
  • Italy 1
  • Hong Kong 1
  • USA (1 British born, 1 Saudi born) 3
  • Canada (spent some time in the UK)
  • Italy (alive, confirmed by brain scan / tonsil
    biopsy) 1
  • Spain 4
  • Japan (visited UK during 1989) 1
  • The Netherlands 2
  • Portugal 1
  • Saudi Arabia 1

  • The role of MRM?
  • Attention focused on MRM as a possible source of
    high titre infectivity in the food chain,
    particularly prior to 1995 when the use of spinal
    column for MRM was banned. Current EU measures
    prohibit MRM from any bovine or ovine bones.

  • The role of MRM?
  • A research report prepared for the Food Standards
    Agency, issued 10 October 2002, on Sources of
    bse infectivity attempted retrospectively to
    estimate the uses of MRM in various meat products
    prior to 1996.
  • http//

  • Susceptibility to vCJD
  • Assuming causal link between consumption of BSE
    infectivity and vCJD, and millions exposed from
    1985 to 1996, why still only ca 170 cases by
    early 2005?
  • In vitro research Raymond et al (1997) found
    that host prion protein that is
    methionine/methionine homozygous at codon 129 is
    converted to prions more rapidly than host prion
    protein valine/valine homozygous at codon 129 (no
    evidence about heterozygous met/val). All of the
    victims reported prior to July 2004 have been
    met/met homozygous at codon 129, i.e. suggesting
    that met/met has a shorter incubation period,
    than val/val or met/val.

  • Susceptibility to vCJD (cont)
  • Wadsworth et al have reported that in transgenic
    mice expressing human PrP, human PrP 129 valine
    appears not to be a compatible substrate for the
    type of prion (type 4) seen in vCJD. These
    animal models suggest that human infection with
    BSE-derived prions may not be restricted to a
    single disease phenotype, but may result in
    sporadic CJD-like or novel phenotypes in addition
    to vCJD, with the type of disease experienced
    depending on the genotype of the host source of
    the infection, and the genotype of the recipient.
  • (Wadsworth JD et al (2004). Science 2004 Nov 11

  • Asante et al have shown that transgenic mice
    expressing human PrP methionine 129, inoculated
    with either BSE or vCJD prions, may develop the
    neuropathological and molecular phenotype of
    vCJD, consistent with these diseases being caused
    by the same prion strain. Surprisingly, however,
    BSE transmission to these transgenic mice, in
    addition to producing a vCJD-like phenotype, can
    also result in a distinct molecular phenotype
    that is indistinguishable from that of sporadic
    CJD with PrPSc type 2. These data suggest that
    more than one SE-derived prion strain might
    infect humans it is therefore possible that some
    patients with a phenotype consistent with
    sporadic CJD may have a disease arising from BSE
  • Asante et al (2002). EMBO Journal, 21 ( 23),

  • First CJD case of valine homozygosity at codon
  • In a paper describing the histopathologic and
    molecular investigation in a young British woman
    with atypical sporadic CJD and valine
    homozygosity at PRNP codon 129. autopsy findings
    were atypical of sporadic CJD, with marked gray
    and white matter degeneration and widespread
    prion protein (PrP) deposition. Lymphoreticular
    tissue was not available for analysis. Molecular
    analysis of PrPSc from cerebellar tissue
    demonstrated a novel PrPSc type similar to that
    seen in vCJD (PrPSc type 4). Further studies will
    be required to characterize the prion strain seen
    in this patient and to investigate its etiologic
    relationship with BSE.
  • Mead s et al Arch Neurol. 200764(12)1780-1784.

  • Aguzzis group in Zurich studied 114 brain
    samples from 70 patients with sporadic CJD and
    three patients with variant CJD. Every patient
    classified as CJD type 2, and all variant CJD
    patients, showed POM2/POM12 reactivity in the
    cerebellum and other PrPSc-rich brain areas, with
    a typical PrPSc type 1 migration pattern.
  • Interpretation
  • The regular coexistence of multiple PrPSc types
    in patients with CJD casts doubts on the validity
    of electrophoretic PrPSc mobilities as surrogates
    for prion strains, and questions the rational
    basis of current CJD classifications.
  • Polymenidou et al (2005), Coexistence of
    multiple PrPSc types in individuals with
    Creutzfeldt-Jakob disease, Lancet Neurology,

  • Susceptibility to vCJD (cont)
  • However, codon 129 may not be the only
    significant location. Collinge et al (2001) have
    shown that other genes are likely to play an
    important role in susceptibility to infection.
    Two different strains of mice with known
    differences in incubation periods for disease
    were used in the study and the mice given a
    thorough analysis of their whole genome, which
    allowed the three genes to be located that are
    involved in susceptibility to prion disease in
    mice. As the mouse and human genomes are so
    similar it is almost certain that corresponding
    genes in humans will be found which have the same
    role to play,

  • Susceptibility to vCJD (cont)
  • A genome-wide association study of the risk of
    vCJD with follow-up analyses of the genetic
    control of the clinical phenotype of prion
    disease and analysis candidate gene expression in
    a mouse cellular model of prion infection. Showed
    that although the main contribution to disease
    risk was conferred by PRNP polymorphic codon 129,
    another nearby SNP conferred increased risk of
    vCJD. In addition to PRNP, one technically
    validated SNP association upstream of RARB (the
    gene that encodes retinoic acid receptor beta)
    had nominal genome-wide significance.
  • Mead S et al (2009), The Lancet Neurology, (8)1,
    57 66
  • Possible conclusion?

  • Susceptibility to vCJD (cont)
  • Possible conclusion?
  • There could up to three variably-susceptible
    genetic groups and at least three sub-groups in
    each. All the reported victims to early July
    2004, all met/met at codon 129, could be in the
    most susceptible genetic sub-group of the most
    susceptible group.
  • This wave shows signs of declining but there
    may be several further waves to come.

vCJDJanuary 1994 December 2004
  • Incidence of vCJD?

  • Incidence of vCJD?
  • (age vs calendar year)

  • Estimated influence of age on a) risk for
    infection with the variant Creutzfeldt-Jakob
    disease (vCJD) agent and b) risk for death from
    vCJD after infection
  • (Barchetti, P (2003). EID serial online 9 (12))

vCJDJanuary 1994 June 2006
  • Susceptibility to vCJD (cont)
  • Following a case in December 2003 of vCJD
    possibly acquired by a 1999 blood transfusion, a
    second case of possible transmission of vCJD from
    person to person via a 1999 blood tranfusion was
    reported Peden, AH et al, (2004), The Lancet,
    264, 527-29. A patient in the UK received a
  • blood transfusion in 1999 from a donor who later
    went on to develop vCJD. The patient died of
    causes unrelated to vCJD but a post mortem
  • revealed the presence of vCJD infectivity in the
    patient's spleen. This was the first instance of
    vCJD in a person in the codon 129 heterozygous
    (met/val) group.

  • Susceptibility to vCJD (cont)
  • On 18 December 2008 it was reported that an
    individual with methionine/valine heterozygosity
    had been diagnosed on a clinical basis as vCJD.
    Prof Higgins, Chairman of SEAC, estimates that
    the number of met/val victims could be probably
    between 50 and 350.

  • Susceptibility to vCJD (cont)
  • This could possibly signal the beginning of a new
    wave of heterozygous met/val cases.
  • It also highlights the possibilities of
    iatrogenic person-to-person transmission of vCJD
    from persons silently incubating the disease to
    others via earlier blood transfusions or surgical

  • Susceptibility to vCJD (cont)
  • In February 2006 a third case was reported. The
    patient developed symptoms of vCJD about 8 years
    after receiving a blood transfusion from a donor
    who developed symptoms of vCJD about 20 months
    after donating this blood. The patient who was at
    the at the National Prion Clinic and has now died
    was one of 25 living persons known to have
    received a blood transfusion in the UK from a
    donor who later developed vCJD. All have
    previously been informed of their potential
    exposure to vCJD and asked to take certain
    precautions to reduce the chance of passing on
    vCJD on to other people via healthcare
    procedures, such as surgery.

  • Susceptibility to vCJD (cont)
  • This was confirmed by a case study published
  • in December 2006 The patient, who has since
    passed away, is the first to have been diagnosed
    whilst still alive. At the age of 23, the patient
    was given a blood transfusion from a donor who
    later developed vCJD. Seven and a half years
    later he was referred to the NHS National Prion
  • where his symptoms were confirmed to be caused by
  • The Lancet 2006 3682061-2067
  • http//

  • Susceptibility to vCJD (cont)
  • Following a statement by the UK Secretary of
    State for Health on 9 September 2004, to reduce
    the risk of onward patient-to-patient
    transmission of vCJD, warning letters were sent
    during the week commencing 21 September 2004 to
    selected groups of patients (and their healthcare
    professionals) about the results of the risk
    assessment, informing them that, because they
    have received certain batches of plasma products
    in the past, which were derived from blood
    donated from someone who has later gone on to
    develop vCJD, they could be at a small increased
    risk of carrying the vCJD agent.

  • Susceptibility to vCJD (cont)
  • People who may be affected are
  • Some people with haemophilia and other bleeding
    disorders. All of these people (around 6,000)
    will receive letters about the background to this
    exercise. The number who may be affected directly
    is estimated to be around 4,000 people.
  • A small group of people suffering from primary
    immunodeficiency, estimated to number around 50
  • A small number of people who have been treated
    with large quantities of particular plasma
    products for a range of conditions (e.g.
    secondary immunodeficiency).

  • Transmission by blood
  • The first two reported cases could have acquired
    infectivity by other means but statistical
    analysis suggests it is more probable it was
    acquired via (non-leucodepleted) blood donated in
    1997 by a person who subsequently developed vCJD
    (there are 13 other persons known to have
    received blood from that donor).

  • Transmission by blood
  • In February 2009 evidence of abnormal prion
    protein infection has been found at post mortem
    in the spleen of a person with haemophilia. The
    patient, who was over 70 years old, died of a
    condition unrelated to vCJD and had shown no
    symptoms of vCJD or any other neurological
    condition prior to his death. Patients with
    bleeding disorders are being made aware of this
    preliminary information which is being further
    investigated. It is known that the patient had
    been treated with several batches of UK sourced
    clotting factors before 1999, when measures to
    improve the safety of blood in relation to vCJD
    were introduced. The patient's treatment had
    included one batch of Factor VIII that was
    manufactured using plasma from a donor who went
    on to develop symptoms of vCJD six months after
    donating the plasma in 1996.

  • Transmission by blood
  • Nov 1997 - evidence that pathogenesis of vCJD may
    involve lymphoreticular tissues possibly
    involving circulating lymphocytes, led to UK
    leucodepletion of blood for transfusions and
    purchase of blood supplies from USA.

  • Transmission by blood
  • However, in August 2004 research was published
    showing that leucodepletion removed only 42 of
    the initial TSE infectivity from whole blood.
    While further work was needed to identify the
    location of the residual infectivity, it was
    presumed that it is plasma associated.
  • Gregori L, (2004). Effectiveness of
    leucoreduction for removal of infectivity of
    transmissible spongiform encephalopathies from
    blood,The Lancet, 264, 529-31.

  • Transmission by blood
  • On 17 October 2005 research studies demonstrating
    the effectiveness of prion filtration technology
    to safeguard the blood supply were presented at
    the American Association of Blood Banks Annual
    Meeting in Seattle. 99.9 prion removal
    efficiency was claimed for the Pall Leukotrap
    Affinity Prion Reduction Filter System. A new
    study conducted by the American Red Cross and the
    Eastern Virginia Medical School, Norfolk Va.,, found that red blood cells filtered
    through the system retain their therapeutic value
    and quality after 42 days of storage.

  • Transmission by blood
  • In December 2006, Rohwer et al reported
    identifying a resin, called L13, that binds to
    both the normal and disease-causing forms of
    prion protein. They tested the resin using
    hamster blood spiked with scrapie. They then used
    this blood to transfuse hamsters. Around half of
    the animals injected with untreated blood
    developed disease, but the nearly 200 hamsters
    injected with blood filtered with L13 remained
  • Nature News, published on-line, 22 December 2006

  • Iatrogenic transmission
  • Bishop et al (2006) have modeled in mice
    iatrogenic spread to enable a comparison of
    transmission efficiencies of vCJD and BSE and an
    assessment of the effect of the codon-129
  • polymorphism on human susceptibility.
  • BSE was transmitted to the bovine line but did
    not transmit to the human lines. By contrast,
    vCJD was transmitted to all three human lines
    with different pathological characteristics for
    each genotype and a gradation of transmission
    efficiency from MM to MV to VV.
  • Lancet Neurology, DOI10.1016/S1474-4422(06)
  • .

  • How many future cases of vCJD?
  • Short-term forecast by CJDSU
  • At July 2006, extrapolating the best fitting
    CJDSU model (the quadratic model) gives an
    estimate of one or 2 deaths in the following 12
    months (95 prediction interval 0 to 3), however
    with 5 cases currently alive a prediction of one
    or 2 deaths is likely to be a slight
    underestimate. It is important to note that
    although a peak has been passed, it is possible
    that there will be future peaks, possibly in
    other genetic groups.  There is also the
    possibility of ongoing person to person spread.

  • How many future cases of vCJD?
  • No such thing as the incubation period
  • Unknown dates of infection of individuals.
  • Age-based susceptibility?
  • Dose-response relationship?
  • Three main populations (codon 129) with
    differing rates.
  • Unknown number of possible genetic
  • Secondary transmission via blood, surgical
  • Long-term forecast ?

  • Hilton et al (2004) tested samples from 16 703
    patients (14 964 appendectomies, 1739
    tonsillectomies), approximately 60 of whom were
    from the age group 20-29 years at operation (25
    of samples containing inadequate amounts of
    lymphoid tissue were excluded from final
    analyses), suggests a prevalence of vCJD among UK
    people aged 20-29  of 237 per million.. Three
    appendicectomy samples showed lymphoreticular
    accumulation of prion protein, giving a
    estimated prevalence of 3/12,674 or 237 per
    million (95 CI 49-692 per million). The pattern
    of lymphoreticular accumulation in two of these
    samples was dissimilar from that seen in known
    cases of vCJD. The margin of error for this
    figure is high and the authors stress the need
    for large scale screening of tonsil tissue to
    obtain precise data.
  • Hilton DA et al (2004) Prevalence of
    lymphoreticular prion protein
  • accumulation in UK tissue samples,Journal of
    Pathology, 202.

  • Comment
  • Extrapolation from 3 (of which 2 are problematic)
    in 12,674 to the whole population is an
    unwarranted assumption.
  • Moreover, we do not know at what stage in the
    long but variable incubation period, infective
    prions become detectable in the tonsils or
  • Tonsils removed from 100,000 patients, mostly
    children or teenagers, over the next three years
    may provide a better basis.

  • In April 2008, SEAC noted the progress of the
    National Anonymous Tonsil Archive (NATA) and of
    discussions around a proposed post mortem tissue
    archive. These would provide data to estimate the
    prevalence of subclinical vCJD (vCJD infections
    that have yet to develop, or may never develop,
    into clinical disease). Approximately 55,000 NATA
    samples had been screened by the end of March
    2008. Although none was positive for abnormal
    prion protein (PrPvCJD), some testing remains to
    be done on some samples.

  • Aguzzi's group in Zurich engineered mice to
    produce a soluble protein that would stick to the
    scrapie prion, so the team could retrieve the
    prion protein for testing. The engineered mice
    turned out to take twice as long as normal to
    develop the disease. Aguzzi now hopes to
    mass-produce the protein so he can test it on
    macaque monkeys that have been exposed to BSE.
  • (presentation at European Life Science
    Organisation conference reported in New
    Scientist, 4 October 2003)

  • On 8 January 2003 the EU issued Questions and
    Answers on BSE What is the current state of play
    on BSE in the EU?
  • http//

  • O Koperek et al have demonstrated
    disease-associated prion protein deposits in
    intracranial vessel walls, in sporadic and
    variant Creutzfeldt-Jakob disease. They conclude
    that mobile cells in vessel walls like dendritic
    and monocyte/macrophage lineage cells may be
    involved in spread of disease-associated prion
    protein and possibly also of infectivity.
  • O Koperek et al (2002). Disease-associated prion
    protein in vessel walls, American Journal of
    Pathology. 161,1979-1984)

  • G Zanusso et al have demonstrated that PrPSc is
    deposited in the neuroepithelium of the olfactory
    mucosa in patients with sporadic CJD, indicating
    that olfactory biopsy may provide diagnostic
    information in living patients. They conclude
    that the olfactory pathway may represent a route
    of infection and a means of spreading prions.
  • (NB This has not yet been shown with vCJD)
  • G Zanusso et al (2003)NEJM, 348(8),711-719.

  • G Mallucci and J Collinge suggest that new
    insights into the mechanisms of neurotoxicity in
    prion diseases support the concept that PrPSc
    itself is not directly neurotoxic. They suggest
    that neuronal prion propagation results in the
    production of a toxic intermediate or depletion
    of a key constituent. Prevention of the formation
    of such a species rather than PrPSc
    accumulation itself is a clear target for prion
  • Mallucci, G and Collinge, J (2004) Update on
    Creutzfeldt-Jakob disease. Current Opinion in
    Neurology, 17(6),641-647, Dec 2004.

  • H Yull et al (NCJDSU) have shown that the PrPSc
    that accumulates in the brain in vCJD also
    contains a minority type 1 component. This
    minority type 1 PrPSc was found in all 21 cases
    of vCJD tested, irrespective of brain region
    examined, and was also present in the vCJD
  • The quantitative balance between PrPSc types was
    maintained when vCJD was transmitted to wild-type
    mice and was also found in BSE cattle brain,
  • that the agent rather than the host specifies
  • relative representation. These results indicate
    that PrPSc
  • molecular typing is based on quantitative rather
  • qualitative phenomena and point to a complex
  • between prion protein biochemistry, disease
  • and agent strain.
  • (Am J Pathol 2006, 168151-157 DOI

  • What makes a good prion?
  • The Cold Spring Harbor Laboratory/Wellcome Trust
    Conference on Prion Biology took place between 7
    and 11 September 2005 in Hinxton, UK, and was
    organized by A. Aguzzi, B. Chesebro, M. Tuite and
    R. Wickner.
  • EMBO reports 7, 3, 254258 (2006)
  • doi10.1038/sj.embor.7400642
  • AOP Published online 17 February 2006
  • http//
  • (see next slide for I ndividual References)

  • References
  • Aguzzi A , Polymenidou M (2004) Mammalian prion
    biology one century of evolving concepts. Cell
    116 313327 Article PubMed ISI ChemPort
  • Chien P , Weissman JS , DePace AH (2004) Emerging
    principles of conformation-based prion
    inheritance. Ann Rev Biochem 73 617656
    Article PubMed ISI ChemPort
  • Dobson CM (2004) In the footsteps of alchemists.
    Science 304 12591262 Article PubMed ISI
  • Shorter J , Lindquist S (2005) Prions as adaptive
    conduits of memory and inheritance. Nat Rev Genet
    6 435450 Article PubMed ISI ChemPort
  • Weissmann C (2005) Birth of a prion spontaneous
    generation revisited. Cell 122 165168 Article
    PubMed ISI ChemPort

  • Main knowledge gaps?
  • There is no treatment or cure for BSE or vCJD