BSE/vCJD: The European On-going Story

1. 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 & Technology IUFoST Governing Council Member 2003-2008 Food science, food technology and food law consultant E-mail: jralphb@easynet.co.uk Web: www.jralphb.co.uk (updated 25 Sepember 2009)

2. 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

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

4. 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

5. 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 internationally.

6. 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 researchers Experts study small parts of picture close-up Role of food scientists - to stand back and observe whole picture.

7. BSE 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."

8. 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 BSE Origin?

9. 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? BSE Origin?

10. 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. BSE Whichever origin -

11. BSE 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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19. BSE 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

20. BSE 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)

21. BSE 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.

22. BSE 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://www.inspection.gc.ca/english/anima/heasan/disemala/bseesb/comenqe.shtml

23. BSE 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://www.aphis.usda.gov/lpa/issues/bse/bse.html http://www.fda.gov/oc/opacom/hottopics/bse.html http://www.cdc.gov/ncidod/diseases/cjd/cjd.htm

24. BSE Prion protein designated PrP or sometimes PrPC small glycosylated protein molecule found mainly in the brain cell membrane -helix structure

25. BSE 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.

26. BSE 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 misfold -helix  –sheetstructure (PrPsc) Largely protease-resistant (PrPres) Resistant to heat, irradiation, most practicable chemical treatments

27. BSE Infective agent – Prion (“Pree-on”) A study by Aguzzi’s 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].

28. BSE Infective agent – Prion (“Pree-on”) A study by Prusiner’s’s 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].

29. BSE Infective agent – Prion (“Pree-on”) Collinge’s 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 animals. [Lloyd SE et al, (2004), J Gen Virol, 85, 2471-2478].

30. BSE 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”, Nature428, 319 – 323. Tanaka M et al (2004). “Conformational variations in an infectious protein determine prion strain differences”, Nature428, 323 – 328.

31. BSE 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 treatment. The first two appear to be proprietary disinfectants already marketed by STERIS.

32. BSE 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.

33. BSE 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]

34. BSE 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.

35. BSE Infective agent – Prion (cont) Prusiner’s 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.

36. 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.

37. BSE Source: SCI Biotechnology Group

38. BSE PrPsc model structure based on electron crystallography Source: Wille et al (2002), PNAS, 99, 10, 1073

39. BSE 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 methods. BUT —

40. BSE Infectivity of bovine materials? In January 2005 Aguzzi’s grouphas 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

41. BSE Infectivity of bovine materials? Then in October 2005 Aguzzi’s groupfound 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 humans. Aguzzi et al, Science doi: 101126/science.1117196

42. BSE 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, 999-1000. Hunter, N et al (2002), Journal of General Virology83, 2897-905.

43. BSE 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 Virology83, 2897-905.

44. BSE 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

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46. BSE 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 OTMS. Offspring cull, of offspring born after 1 August 1996 to dams in which BSE was confirmed. Compulsory Cattle Passport traceability system.

47. BSE 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 implementation.

48. BSE 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?

49. BSE 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 1996. Prof Hill issued his report in June 2005. It can be found on the DEFRA Website http://www.defra.gov.uk/animalh/bse/index.html

50. BSE Born after the “real” ban (BARB) Prof Hill’s 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.