1. Prion Diseases CMED 526/EPI 526 - May 6 th, 2009 Robert Harrington, DVM, PhD USDA – Agricultural Research Service UW – Dept. of Comparative Medicine rdhdvm@u.washington.edu

2. Transmissible Spongiform Encephalopathies~Prion Disease NORMALABNORMAL  -helix rich  -sheet rich Prusiner, et al. PrP c PrP d

3. PrP Conversion PrPc PrP d Heterodimer

5. TSE Pathogenesis Transmission occurs by oral route Transmission occurs by oral route PrP d localizes to regional lymphoid tissue PrP d localizes to regional lymphoid tissue –Transient in some species Migration to central nervous system Migration to central nervous system –Retrograde along nerves –Blood-borne transport Accumulation in brain with subsequent neurodegeneration Accumulation in brain with subsequent neurodegeneration

6. Diagnosis Postmortem Postmortem –Microscopic pathology –Immunohistochemistry –ELISA, Immunoblotting –Bioassay Antemortem Antemortem –Same techniques as above applied to: –Brain biopsy –Tonsil, lymph node, third eyelid biopsy, or rectal –? Blood test ?

7. Figure 3: Photomicrographs illustrating increased diameter and density of vacuoles in TME positive IC recipient (top row, a, b, c), as compared to CWD positive IC recipient, (middle row, d, e, f) and absence of lesions in CWD negative IC recipient (bottom, row, g, h, i). Left column = cerebral cortex, middle column = hippocampus, right column = thalamus. All sections stained with hematoxylin and eosin. Bar=100 µm. Histology

8. Immunohistochemistry

9. Western blot

10. Animal Prion Disease Scrapie - sheep, goats Scrapie - sheep, goats Chronic Wasting Disease (CWD) - deer, elk, moose Chronic Wasting Disease (CWD) - deer, elk, moose Bovine Spongiform Encephalopathy (BSE) - cattle Bovine Spongiform Encephalopathy (BSE) - cattle Transmissible mink encephalopathy (TME) - mink Transmissible mink encephalopathy (TME) - mink Feline spongiform encephalopathy - large & domestic cats Feline spongiform encephalopathy - large & domestic cats Spongiform encephalopathy of captive ungulates - exotic hoof-stock in zoological parks Spongiform encephalopathy of captive ungulates - exotic hoof-stock in zoological parks

11. Human Prion Disease Sporadic Sporadic –Creutzfeldt-Jakob disease (CJD) Familial (genetic) Familial (genetic) –Familial CJD –Gerstman-Straussler-Scheinker Syndrome (GSS) –Fatal Familial Insomnia (FFI) Acquired by transmission Acquired by transmission –Kuru –Iatrogenic CJD (neurosurgical instruments, dura mater grafts) –Variant CJD (vCJD)

12. Jackson, G S et al. Mol Pathol 2001;54:393-399 Human PrP Mutations

13. vertical and horizontal in utero, fetal fluids, fetal membranes Foodborne Direct only through bite wounds Foodborne, blood, tissue transplant, HGH, instruments Foodborne (MBM) No direct transmission from cow to cow horizontal Oral (urine, feces, or blood?) COMMON UNCOMMON Transmission Within Species

14. Species Barrier Concept Transmission within a species may occur readily Transmission within a species may occur readily Barrier between species limits transmission Barrier between species limits transmission –Inefficient transmission –Extended incubation times –Low or non-existent rate of disease Serial passage Serial passage –Required to overcome species barrier –Progressive reduction in incubation time –Increased rate of disease

15. BSESheepMinkDeer/ElkHumans IC; PO ICPO ScrapieCattleMinkDeer/ElkHumansICICIC TMECattleSheepDeer/ElkHumansICIC CWDCattleSheepMinkHumansICICIC SOURCE → → → → → → HOST ROUTE Transmission Between Species

16. Recognition of BSE Late 1985: Unusual neurologic disease in UK cattle Late 1985: Unusual neurologic disease in UK cattle Insidious onset Insidious onset –Irritabilty, agression –Motor system impairment (ataxia) –Difficulty in rising (e.g. “downer cow”) –Decreased milk production –Wasting –Death Predominantly dairy cattle Predominantly dairy cattle –Feeding practices –Relative herd age Neuropathology similar to Scrapie Neuropathology similar to Scrapie –Vacuolation, PrP d, astrocytosis, Scrapie associated fibrils

17. Cause of BSE Ruminant tissue in food chain Ruminant tissue in food chain –Meat and bone meal (MBM) –Scrapie –Sporadic BSE in cattle Alternative theories Alternative theories –Human tissue? –Toxin? –Environmental? –Other?

18. BSE Epidemic ~180,000 cumulative cases in UK ~180,000 cumulative cases in UK Peaked at 37,000 cases per annum in 1992 Peaked at 37,000 cases per annum in 1992 Recycling of ruminant tissue in food chain implicated Recycling of ruminant tissue in food chain implicated Progressive decline with introduction of feed bans Progressive decline with introduction of feed bans

19. Transmissible Spongiform Encephalopathy as a Zoonotic Disease, Brown, P., et. al. ILSI, March 2003 (mammals)

20. Spread of BSE Epidemic 1990: Domestic BSE detected in Switzerland, imported cases in Portugal 1990: Domestic BSE detected in Switzerland, imported cases in Portugal 1999: 7 other EU countries with domestic BSE 1999: 7 other EU countries with domestic BSE Jan 2000 to Oct 2002: 11 additional EU countries Jan 2000 to Oct 2002: 11 additional EU countries 2001: BSE detected in Japan 2001: BSE detected in Japan 2002: BSE detected in Israel 2002: BSE detected in Israel 2003: BSE in Canadian cow 2003: BSE in Canadian cow

22. 30-40 million cattle slaughtered/year 30-40 million cattle slaughtered/year 1997: ban on feeding US cattle meat-and- bone meal 1997: ban on feeding US cattle meat-and- bone meal 3 cases to date - RARE! 3 cases to date - RARE! –2003: 6.5yo dairy cow imported from Canada –2004: 12yo beef cow born and raised in Texas –2006: 10yo beef cow in Alabama uncertain origin –Were there previously unrecognized cases? BSE in the United States

23. 2003: Additional measures post WA BSE case 2003: Additional measures post WA BSE case –“downer” cattle excluded from human consumption –Ban on SRM from animals >30 months of age from human consumption –Ban on mechanically-separated meat

24. New variant CJD Unusual form of neurologic disease in teenagers and young adults Unusual form of neurologic disease in teenagers and young adults Spongiform encephalopathy Spongiform encephalopathy Neuropathology not consistent with sporadic forms of CJD Neuropathology not consistent with sporadic forms of CJD Stimulus for US National Prion Disease Center Stimulus for US National Prion Disease Center

25. Clinical Feature or Procedures Classical sCJD vs. vCJD Average / Median age at clinical onset → 63 yr / 68 yr 29 yr / 28 yr Survival from date of clinical onset → 4 mo 14 mo Early psychiatric symptoms → Unusual early, Dementia later Common (psychosis, depression, anxiety, apathy, withdrawal, delusions) EEG → Bi- or triphasic periodic complexes Nonspecific, slow MRI → Increased signal in basal ganglia, caudate nucleus, and putamen Hyper-intense signal in pulvinar region of the thalamus Genetics (PRNP codon 129) → MM, MV, or VV MM CSF 14-3-3 protein → Usually elevated Not usually elevated Histopathology of brain tissue → No amyloid plaques 100% florid plaques PrP immunohistochemical staining pattern of brain tissue → Punctate pattern Widespread plaque staining pattern Immunohistochemical staining of tonsil or appendix tissue → Negative PrP d present in tissue, especially toward late-stage disease PrP d isotype by Western blot → Type 1A Type 2B or 4

26. Clinical Feature or Procedures Classical sCJD vCJD Average / Median age at clinical onset 63 yr / 68 yr 29 yr / 28 yr Length of survival from date of clinical onset 4 mo 14 mo Early psychiatric symptoms Unusual early, Dementia later Common (psychosis, depression, anxiety, apathy, withdrawal, delusions) EEG Bi- or triphasic periodic complexes Nonspecific, slow MRI Increased signal in basal ganglia, caudate nucleus, and putamen Hyper-intense signal in pulvinar region of the thalamus Genetics (PRNP codon 129) MM, MV, or VV MM CSF 14-3-3 protein levels usually elevated 14-3-3 protein levels not usually elevated Histopathology of brain tissue No amyloid plaques 100% florid plaques PrP immunohistochemical staining pattern of brain tissue Punctate pattern Widespread plaque staining pattern Immunohistochemical staining of tonsil or appendix tissue Negative PrP d present in tissue, especially toward late-stage disease PrP d isotype by Western blot Type 1A Type 2B or 4

27. Percent distribution of non-iatrogenic # UK vCJD and US CJD deaths, by age group, 1995-2005 # Excludes blood transfusion-associated vCJD and pituitary hormone- or dural graft-associated CJD * UK vCJD deaths, including UK-related nonresident cases, 1995-2003 (Will, RG; personal communication, 2004) ** US CJD deaths, 1995- 2001. +

28. Jackson, G S et al. Mol Pathol 2001;54:393-399 Western Blots

29. BSE-vCJD Link New variant disease that differs from classical CJD New variant disease that differs from classical CJD –Similarities to BSE –Geographically related to areas of BSE –Hypothesis: consumption of contaminated beef products Epidemiologic curve Epidemiologic curve Animal challenge studies Animal challenge studies Molecular biology Molecular biology

30. BSE and vCJD Hilton, 2006. J of Pathology, 208:134

31. Goldfarb, L. G. Microbes and Infection 4 (2002) 875-882

32. Animal Challenge Studies BSE → primates ≈ vCJD → primates BSE → primates ≈ vCJD → primates –Similar lesions and biochemistry Transgenic mice Transgenic mice –BSE → humanized mice –vCJD → bovinized mice –Lesions, biochemistry of BSE ≈ vCJD regardless of mouse type –Both differ from sCJD

33. Jackson, G S et al. Mol Pathol 2001;54:393-399 Western Blots

34. Continuing US Cattle Surveillance USDA National Veterinary Services Laboratory USDA National Veterinary Services Laboratory –AAVLD certified labs, refer positives to NVSL If 1 case per 1 million slaughter then 95% CI requires: If 1 case per 1 million slaughter then 95% CI requires: –All slaughters = 2,995,731 –Suspect cattle = 40,000 (neuro signs, fallen, “downers”) –45,803 samples in 2007 –Meets OIE “controlled risk” classification National animal ID system National animal ID system

35. Continuing US Human Surveillance National Prion Disease Center National Prion Disease Center –Established 1996 Monitoring for unusual trends in mortality data Monitoring for unusual trends in mortality data ↑ # of autopsies in US, ↑ # of referrals ↑ # of autopsies in US, ↑ # of referrals CJD monitoring in CWD endemic areas CJD monitoring in CWD endemic areas

37. CWD to Humans? CJD flatline in endemic areas CJD flatline in endemic areas Challenge studies indicate natural transmission is unlikely Challenge studies indicate natural transmission is unlikely –Cattle –Mink –Humanized Tg mice

38. CWD risk reduction? Hunting Hunting –Avoid endemic areas –Test animals in effected areas Consumption Consumption –Don’t eat brain, nerves, spleen, lymph nodes, or eyes –Avoid composite foods (sausage, head cheese)

39. Conclusion Prion diseases vary by species, distinct differences Prion diseases vary by species, distinct differences TSE transmission usually limited to within a species TSE transmission usually limited to within a species Only Scrapie and CWD are readily transmissible Only Scrapie and CWD are readily transmissible Species barrier limits transmission between species Species barrier limits transmission between species

40. Questions?

41. Do you think this study supports the association of beef consumption as a primary risk factor for development of vCJD?

42. Did the study authors take adequate measures to address the limitations to the study design? What other measures could they have considered?

43. Are there other ways that the question of risk factors for vCJD may be addressed?