TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES TSE

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TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES (TSE)

• Cast of characters

De Jonge Stier by Paulus Potter,
1647 (Maritshuis, Den Hague, Netherlands)
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TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES (TSE)

• Humans
• Creutzfeldt-Jakob Disease (CJD)
• Variant CJD (vCJD)
• Gertsmann-Straussler-Scheinker Syndrome (GSS)
• Kuru
• Fatal Familial Insomnia (FFI)

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TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES (TSE)

• Animals
• Bovine Spongiform Encephalopathy (BSE) cattle
• Scrapie sheep, goats
• Transmissible Mink Encephalopathy mink
• Chronic wasting disease (CWD) mule deer and elk
• Feline Spongiform Encephalopathy (FeSE) wild
  and domestic felids
• Hampster TSE

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TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES (TSE)

• Common characteristics
• prolonged incubation (months to years)
• progressive neurodegeneration/debilitation
• always fatal
• scrapie associated fibrils in brain tissue (human
  or animal) seen with EM studies
• pathology confined to CNS - vacuolation,
  astrocytosis, gliosis formation of amyloid
  plaques
• no apparent immune response elicited

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TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES (TSE)

• Causative agent history
• agent smaller than smallest known virus
• most accepted theory is that agent is a normal
  cell surface component called a prion protein,
  which is modified to a pathogenic form that is
  both less soluble and more resistant to enzyme
  degradation
• does not evoke immune/inflammatory response
• highly resistant to heat/normal sterilization

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TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES (TSE)

• Causative agent
• Stanley Prusiner, MD neurologist (US)
• obtain a highly purified infectious preparation
  known as prion (PrP)
• coined from proteinaceous infectious particles
• work began in 1972, published in 1982 (Science)
• containing no RNA or DNA
• considered heretical when first proposed (1982)
• awarded Nobel Prize for Medicine (1997)

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TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES (TSE)

• Causative agent
• PrP is a protein encoded by host gene, not a
  foreign invader (i.e., viral agent)
• Entire ORF (open reading frame) of all known
  mammalian and avian PrP genes resides within
  single exon
• a self-replicating protein not distinguished from
  other proteins by unique structural features
• in the low mid-range in size (35,000 Daltons)
• small proteinaceous infectious particle which
  resists inactivation by procedures that modify
  nucleic acids

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TSE phylogenetic tree
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TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES (TSE)

• Prion protein configuration

Left ?-helix PrP Right ?-sheet PrP
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TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES (TSE)

• Pathogenic mechanism
• protein changes configuration from ?-helix to
  ?-sheet (floppy soluble amyloid to stiff
  insoluble amyloid)
• most studies of experimental transmission by oral
  route indicate
• tonsils
• lymphatic tissue (intestinal)
• spleen
• CNS (via splanchnic nerve)

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TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES (TSE)

• Pathogenic mechanism
• familial form of CJD caused by more than 24
  insertion/point mutations in gene encoding PrP
• Mendelian form of inheritance
• all experimentally transmitted to laboratory
  animals
• increase likelihood of ?-helix to ?-sheet
  transformation
• altered protein deposited as extracellular
  amyloid or concentrated in synaptosomal region,
  both leading to accumulation of insoluble amyloid
  protein

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SCRAPIE

• Background
• Scrapie is the prototype of the TSE diseases
• recognized disease of sheep/goats (gt250 yrs)
• reported throughout the world first recognized
  in Great Britain and Western Europe
• not proven transmissible until 1936
• first diagnosed in US in 1947 in Michigan in
  sheep of British origin
• according to USDA-APHIS figures, has been
  diagnosed in gt900 flocks

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SCRAPIE

• Background
• in 1959, proposed by American veterinarian (WJ
  Hadlow, Lancet, 1959) to be analogous to newly
  described disease of humans known as Kuru which
  had been described earlier (Gajdusek and Zigas, N
  Eng J Med, 1957)

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SCRAPIE

• Clinical features
• wide range slowly developing
• early - behavioral changes
• scratching and rubbing
• incoordination
• weight loss with normal appetite
• biting, lip smacking
• gait abnormalities - goose-stepping, hopping,
  swaying
• upon stimulation - excessive movement, trembling,
  convulsion-like state

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SCRAPIE

• Clinical features
• most commonly spread from ewe to offspring and
  other lambs through direct contact with placenta
  and placental fluids
• incubation period normally 2 to 5 years
• survival after onset of signs 1-6 months
• dx based on signs and history
• no test available dx confirmed at necropsy
• DDx - rabies, external parasites, toxicosis,
  progressive pneumonia listeriosis

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SCRAPIE

• Histopathology of brain tissue from sheep

Vacuoles throughout with reactive astrocytes
(brown)
Cytoplasmic vacuoles push nucleus to outside of
cell
Courtesy UC-Davis SVM
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SCRAPIE

• Epidemiology
• 593 confirmed cases in UK in 1999 probably
  represents less than 15 of all cases
• Notably absent in Australia and New Zealand
• Most breeds affected
• Risk equal for males and females
• Age at greatest risk 3.5 yrs
• genetic variation may play a role
• laboratory can be transmitted to hamsters,
  rats, cattle, mink, some species of monkeys
• no evidence scrapie poses risk to humans

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SCRAPIE

• Control
• UK National Scrapie Plan (MAFF)
• Breeding genetic resistance into national flocks
• US - Voluntary Scrapie Flock Certification
  Program (1992)
• restriction of interstate movement from
  scrapie-infected and source flocks

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Bovine Spongiform Encephalopathy (BSE)

• Background
• 1986 - first identified in UK in indigenous
  cattle
• primarily in dairy herds
• source indicated to be meat and bone meal used in
  concentrated feeds which was banned in July 1988,
  reducing incidence but not completely eliminating
  disease
• still included in pig and poultry feed which may
  have caused cross-contamination of cattle feed
  during processing
• accidental exposure on farms

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BSE

• Background
• USDA instituted ban on mammalian-to-ruminant feed
  in 1997
• prohibits importation of live ruminants and most
  ruminant products from Europe

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BSE

• Clinical features
• progressive neurological disorder
• gt168,000 cases in Great Britain in over 34,000
  herds
• average age at onset 4-5 years
• three cardinal features
• nervousness
• heightened reactivity to external stimuli
• difficulty in movement, especially hind limbs
• no treatment all affected animals die
• 10 of newborns from infected dams will die

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BSE

• Epidemiology
• evidence in Great Britain suggests a
  common-source epidemic involving animal feed
  contaminated with animal protein
• causative agent suspected to be either from
  Scrapie-affected sheep or cattle with a
  previously unidentified TSE
• changes in rendering practices in early 1980s
  may have potentiated agents survival
• feeding of bovine meat and bone meal to young
  calves

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BSE

• Epidemiology
• occurs in UK, western Europe
• no cases in US according to USDA-APHIS report
  in 9/97, of 6,263 bovine brain specimens
  examined, none were positive
• one case in Canada in cow imported from Great
  Britain has now been slaughtered along with rest
  of herd
• recent (2001) quarantine of 2 herds in Texas
  preliminary findings negative for BSE agent

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BSE

• Epidemiology
• no evidence of horizontal transmission to other
  cattle or other species, but possibility of
  transmission to sheep is cause for recent alarm
  among officials in UK
• exotic ruminants in zoos have been affected from
  same contaminated feed source
• has not occurred in dogs, but outbreak in
  domestic cats (FeSE)
• maternal transmission may occur at low level (9
  increase in BSE among offspring of BSE-affected
  dams)

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BSE

• Epidemiology
• Support of sheep origin theory
• High incidence of scrapie in sheep in UK
• Large proportion of sheep in mix of carcases
  rendered for livestock feed
• Elimination of tallow extraction step in
  rendering process a few years prior to first
  case, allowing some scrapie infected tissue to
  survive process

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BSE

• Epidemiology
• Support of sheep origin theory
• Since scrapie does not infect humans, then why
  BSE?
• strain of TSE passing from one species to another
  may acquire altered host range
• Support
• Kuru or CJD (human) strains do not transmit to
  goats or ferrets unless first passaged through
  primates or cats
• BSE does not transmit to hamsters until passaged
  through mice

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BSE in the UK, 1986-2000
Brown P, Will RG et al, EID (2001) 7(1) 6-16.
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Source Department of Environment Food and Rural
Affairs (DEFRA), UK
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Source DEFRA, UK
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Chronic Wasting Disease (CWD)
1960s recognized in captive deer 1977 CWD
classified as a TSE 1981 recognized in
free-ranging cervids 1996 recognized in farmed
elk
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Chronic Wasting Disease (CWD)
Agent prion (?) Origin unknown Hosts deer,
elk Signs emaciation, abnormal
behavior Epidemiology lateral transmission,
prolonged incubation, environmental persistence
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CWD in deer and elk
Mule deer (also white-tailed deer elk) Started
gt20 years ago Apparently self-sustaining Slow
geographic spread (natural movements)
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Source USDA-APHIS
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CWD in endemic areas
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CWD in Farmed Elk (1996-2000)
Started gt11 years ago Apparently
self-sustaining Wide geographic spread (market
movements)
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CWD in Farmed Elk, 2001-2002
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CWD Control Measures

• Goal - ? prevalence ? incidence and spread
• Increase surveillance
• Reduce or depopulate
• Colorado
• western Slope depopulate game farm and all
  deer/elk within 5 mile radius
• endemic area - depopulate 5000 deer
• Wisconsin
• survey, reduce population

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CWD Public Health Risk

• Data is limited
• No evidence of transmission to humans
• No association between deer/elk consumption and
  CJD
• Strain typing in mice
• CWD is different from BSE and Scrapie

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Can CWD cause vCJD in U.S.?

• Stable rates for classical CJD last 20 years
• 12 cases of CJD in U.S. lt39 years old
• 8 received pituitary growth hormone from cadaver
  all ruled out for vCJD by neuropath
• 3 recent cases of CJD investigated by CDC
• young onset (28 - 30) game consumption
• Clinically and pathologically similar to Classic
  CJD
• 2 CO cases - high game consumption (endemic area)
• no evidence of vCJD

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Transmission (species barrier)

• 250 brains from downer cows in endemic area
  negative for spongiform encephalopathies
• 3 ongoing studies gt 4 years
• CWD intracerebral to cattle
• 3/13 died with spongiform changes
• J Vet Diagn Invest 2001 Jan13(1)91-6
• CWD orally to cattle
• CWD deer penned with cattle

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In vitro Study - Species Barrier Cell-free
conversion of purified prion proteins EMBO J 2000
Sep 1 19 (17) 4425-30

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CWD Summary

• No evidence of BSE/vCJD in U.S.
• CWD transmission to humans/other species
• low risk
• high uncertainty
• More research needed
• Risk assessments needed
• Continued surveillance

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Creutzfeld-Jakob Disease (CJD)

• Background
• first described in the early 1920s by two German
  neurologists
• a rapidly fatal dementing illness
• reported worldwide with familial clusters in
  certain regions
• similar pathology to Kuru as reported in 1959
• transmitted to chimpanzee (Science, 1968)

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CJD

• Background
• Underlying etiologic mechanism
• 90 occur sporadically
• 10 genetic
• lt1 iatrogenic

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Inherited Prion Diseases

• Genetic Etiology
• autosomal dominant mode of inheritance
• mutation on Chromosome 20 that encodes precursor
  protein (PrP) gene
• Phenotypes
• Fatal Familial Insomnia (FFI)
• Gerstmann-Sträussler-Scheinker Disease (GSS)

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Inherited Prion Diseases

• Gerstmann-Sträussler-Scheinker Disease (GSS)
• Codon 102 proline (P) -gt leucine (L) mutation
• Thymine (T) -gt Cytosine (C) substitution
• Many families in numerous countries
• Familial CJD (fCJD)
• 144 bp containing 6 octarepeats at codon 53
• 4 families residing in Southern England
• Single point mutation at Codon 200 Glu -gt Lys
  substitution
• Israeli Jews of Libyan or Tunisian origin
• Slovaks originating from Orava
• Cluster of familial cases in Chile
• Large German family living in US
• 100 penetrance

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Inherited Prion Diseases

• Fatal Familial Insomnia (FFI)
• Codon 178 mutation resulting in Asn for Asp with
  a Met encoded at the polymorphic codon 129
• Produces progressive sleep disorder and death
  within 1 year of onset
• In contrast, same D178N mutation with a Val
  encoded at position 129 produces fCJD

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Inherited Prion Diseases

• Human PrP gene polymorphisms
• Codon 129 polymorphism for M -gt V
• May influence prion disease expression on all
  forms of CJD (inherited, sporadic, iatrogenic)
• Race/ethnicity differences?
• Caucasian
• Patients with sCJD are homozygous for Met or Val
  at codon 129
• general population is 12 V/V, 37 M/M, 51 M/V
• Japanese population
• Patients with sCJD more frequently heterozygous
  (18)
• General population is 0 V/V, 92 M/M, 8 M/V
• Codon 219 polymorphism for E -gt K
• Reported in Japan
• K allele frequency 12

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CJD

• Background
• Multiple strains (isolates) of prions have
  distinct biological properties
• Diversity encoded by differences in PrP
  conformation
• Molecular techniques have identified 4 main types
  of CJD
• Sporadic and inatrogenic PrPSC types 1-3
• Variant CJD PrPSC type 4

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CJD

• Epidemiology
• estimated incidence 1/1,000,000
• from 1979 - 1994, highest mortality rate in US
  among 65-74 year age group (gt5/1,000,000)
• estimated that 1 in 10,000 individuals are
  infected with CJD at the time of death

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CJD

• Pathogenic mechanism
• familial form of CJD caused by more than 24
  insertion/point mutations in gene encoding PrP
• Mendelian form of inheritance
• all experimentally transmitted to laboratory
  animals
• increase likelihood of ?-helix to ?-sheet
  transformation
• altered protein deposited as extracellular
  amyloid or concentrated in synaptosomal region,
  both leading to accumulation of insoluble amyloid
  protein

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CJD

• Clinical features
• insidious onset of confusion, progressive
  dementia, and ataxia
• normally occurs in individuals gt35 years of age
• no fever, CSF normal
• EEG shows typical periodic high-voltage complexes
• rapid progression with death in 3-12 months

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CJD

• Clinical features
• mode of transmission unknown
• de novo spontaneous generation of
  self-replicating protein (prion protein)
• iatrogenic
• corneal transplant
• cortical electrodes
• dura mater grafts
• gonadotropic hormone injections (pituitary
  extracts) from CJD-affected patients

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CJD

• Histopathology Sporadic CJD

Spongiform (vacuolar) change in cortex
Prion protein positive amyloid
plaques (immunochemical stain)
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Variant CJD (vCJD)

• There is no conceivable risk of BSE being
  transmitted from cows to people. Minister of
  Health, 1995
• No conceivable risk, beef is safe. John Major,
  Parliament, 1996
• British beef is the safest in the world. Prime
  Minister and other high ranking officials, 1996
• Yes, we could face a major epidemic... Yes, it
  is possible that very many people will die...
  Yes, it could theoretically be hundreds of
  thousands. Professor John Pattison, Chairman of
  SEAC, 1996

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vCJD

• Background
• 1994-1995, an unusual cluster of CJD in young
  patients (lt 36 years, now found to be 16-52 years
  of age)
• in 1997, this entity was confirmed to be Human
  BSE by subsequent experimentation

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vCJD

• Background
• atypical clinical features
• early symptoms often psychiatric (behavioral
  changes, dysesthesia) followed within weeks or
  months by cerebellar syndrome, dementia, and
  myoclonus (late stages)
• longer duration (up to 14 months)
• non-characteristic EEG findings

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vCJD

• Epidemiology
• Occurrence (figures as of 4/1/01)
• UK 90 confirmed deaths 7 living (suspected)
• France 1 confirmed death 2 living (suspected)
• Ireland 1 living (suspected)
• Single-dose vs cumulative low-dose exposure
• Incubation period
• Variable - 8-10 yrs or longer

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CJD in the UK, 1991-2003
(ProMED, 01/6/2004)
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vCJD

• Age at onset vCJD vs CJD

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vCJD

• Epidemiology
• Risk
• No history of dietary or occupational exposure
• Possible co-factors (genetic, etc) affecting
  sensitivity, shorter incubation
• All patients PRNP codon 129 methionine
  homozygotes as are cattle with disease however
  40 of normal caucasian population have this
  genotype
• Mouse studies suggest other genetic loci affect
  incubation period

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vCJD

• Histopathology vCJD

Spongiform changes in cerebral cortex
with plaques (arrow)
Halo vacuolation surrounding plaques
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CJD Diagnosis

• Lumbar puncture
• not possible to detect prion protein in CSF
• a test for a protein in the CSF known as '14-3-3'
  protein supports the diagnosis of CJD
• EEG
• there are characteristic EEG abnormalities that
  are often seen in sCJD
• not seen in vCJD
• MRI scan
• may show heightened signal in the caudate and
  putamen regions of the basal ganglia in sCJD
• high signal in the putamen in vCJD
• presence supports a diagnosis of CJD

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CJD Diagnosis

• Tonsil biopsy
• lymphoreticular tissues from patients with
  neuropathologically confirmed vCJD positive for
  the abnormal protein associated with prion
  diseases
• Brain biopsy
• only way to diagnose CJD with certainty is by
  neuro-pathological examination
• brain biopsy carried out in some cases, but it is
  an invasive procedure and may give a negative
  result if an unaffected part of the brain is
  biopsied
• Genetic testing
• genes responsible for familial types of CJD have
  been identified and are detectable on blood
  testing

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Diagnosis - vCJD
Protease resistant prion protein (red-brown
stain) in tonsillar tissue from patient with vCJD
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TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES (TSE)

• Chronology of discovery
• 1732 Scrapie in sheep in UK
• 1920 CJD in Germany
• 1947 TME in US
• 1950 Kuru in Papua New Guinea
• 1965 transmission of Kuru to chimpanzees
• 1967 CWD in deer and elk in US
• 1968 transmission of CJD to primates
• 1986 BSE in cattle in UK
• 1990 FSE in wild and domestic felids in UK
• 1994 vCJD in humans in UK

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TSE - Chronology

• Sheep (scrapie)
• Cattle (BSE)
• Humans (vCJD)

1750
Year initial cases
Year exposed
1986
1994
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TSE species origin and transmission
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BSE in the US Chronology

• 12/09/2003
• Non-ambulatory cow slaughtered in Moses Lake, WA
• 12/22/2003
• Preliminary tests positive for BSE
• 12/23/2003
• Recall of meat (10,410 lbs) from the 20 animals
  slaughtered on 12/09/2003 herd quarantined
  traceback initiated
• 12/30/2003
• New safeguards/regulations announced by USDA
• 01/20/2004
• 170 animals identified from facilities/herd for
  testing
• 02/06/2004
• Update report that 29 of 81 animals from original
  herd in Canada had been located

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BSE in the US USDA Proposed Surveillance
Would allow for detection of 1 in 10,000,000
cattle with 99 confidence level
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BSE in the US USDA Proposed Surveillance

• Seven labs approved for high throughput rapid
  testing
• California Animal Health and Food Safety Lab
  System, University of California-Davis
• Colorado State University Veterinary Diagnostic
  Laboratory
• Texas Veterinary Medical Diagnostic Laboratory
  (College Station)
• Wisconsin Animal Health Laboratory (Madison)
• Washington State University Animal Disease
  Diagnostic Laboratory
• Athens Diagnostic Laboratory, College of
  Veterinary Medicine, University of Georgia
• NY State College of Veterinary Medicine,
  Veterinary Diagnostic Laboratory, Cornell
  University
• Confirmatory testing at USDA National Veterinary
  Services Laboratory (Ames)

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BSE in the US Environmental Issues - Disposal

• Incineration
• 1,000C (gt1800F)
• Costly, limited volume
• 75 per lb
• Alkaline Hydrolysis
• Costly, limited volume
• 25 per lb
• Landfill
• Cost effective but is it safe?
• Hydrophobic nature would indicate leaching into
  soil unlikely

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BSE in the US Biodigester
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How now mad cow?

• Absence of evidence should not be taken as
  evidence of absence.
• Sherlock Holmes

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How now mad cow?