Follow Ups | Post Followup | Back to Discussion Board | VegSource
See spam or
inappropriate posts?
Please let us know.

From: TSS ()
Subject: Variant CJD (vCJD) and Bovine Spongiform Encephalopathy (BSE): 10 and 20 years on: part 1 & 2
Date: July 3, 2006 at 11:17 am PST

full text of the article:

Review article
Variant CJD (vCJD) and Bovine Spongiform Encephalopathy (BSE): 10 and 20 years on: part 1

Folia Neuropathol 2006; 44 (2): 93-101

authors: Ray Bradley, J. Gerald Collee, Paweł P. Liberski,

documents in PDF format:

> Variant CJD - part 1.pdf [0.10 MB]

Reports in The Lancet in 1997 summarised what was known about BSE and speculated on possible risks to human health [7,8]. At that time, the bovine epidemic had accounted for more than 165,000 cattle in British herds. In the present report, two decades after BSE was first recognised and one decade after the first cases of vCJD in human patients were announced in the UK, we re-examine important developments that may relate to human health.
The source of BSE
The true origin of BSE, first recognised in British cattle in 1985-86, may remain a mystery. It is virtually certain that the vehicle of infection was meat-and-bone-meal (MBM) fed as a dietary supplement predominantly to dairy calves [2,23]. MBM, and its associated by-product tallow, were derived mainly from the carcases of fallen stock and other animal and poultry material rejected or unwanted for human consumption. The starting materials were subjected to “rendering”, a cooking process in which water was extracted, fat was separated as tallow, and the remaining protein-rich material (“greaves”) was ground to make MBM for animal feed or agricultural and plant fertilizer. MBM was marketed primarily in the UK, but considerable amounts went abroad. The infective agent of BSE (see below) has remarkable resistance to physical and chemical destruction including dry heat, even up to 600°C [3]. Research has shown that some of the rendering procedures in use in the years leading up to the onset of BSE in the mid-1980s would not have reduced the titre of the causative agent to undetectable levels in the final MBM product. For one process the residual titre in the MBM was comparable to that of the untreated raw materials [32]. Residual BSE infectivity has not been detected in the tallow fraction after rendering, but the case against MBM as the vehicle of BSE in cows is convincing. The ineffective rendering processes have now been abandoned. There is just one approved world standard rendering process for use on ruminant materials that may carry a TSE risk and this demands exposure of particles ≤50 mm diameter to 133°C, at a pressure of 3 bar, for 20 min (often referred to as “pressure cooking”) [28].
Preventive measures
In late 1987, when MBM was identified as the likely vehicle of BSE [36], protective legislation was quickly introduced in the UK. The use of ruminant-derived protein in feed destined for ruminant animals was prohibited in July 1988 [2,14]. If this feed-ban had been effectively enforced and rigorously obeyed, it would have eliminated recycling of the agent in infected feed supplements, not only in cattle but also in various captive wild ruminants in zoos and wildlife parks. Sadly, it was not. Moreover, it did not restrict the use at that time of MBM in feed for pigs and poultry. In retrospect, we now see the many problems of cross-contamination that were to haunt us for many years to come. BSE mainly affects adult cattle in the 4-6 years age range, with a mean incubation period of 60 months. Accordingly, the entry of infected but clinically healthy animals into the food chain, and eventually into the animal feed chain, would occur both during and before the onset of the epidemic. Preclinical animals could not be recognized until appropriate tests were developed and even then only after death. Even with the ruminant feed ban in place, cattle could be carrying the infection for some years before going down. Therefore in 1989, in order to immediately protect public health it was decided to prohibit the use in human food of certain specified bovine offal (SBO) that, in an infected animal, might carry the BSE agent. The selection of the offal to be banned was based on existing knowledge on the pathogenesis of the natural analogous disease of sheep and goats known as scrapie. In 1990 a case of feline spongiform encephalopathy was reported in a domestic cat [38] and BSE was experimentally transmitted to a pig [34]. The SBO ban was therefore extended to protect all species of mammals and birds, and it was successively extended during the 1990s so that the whole head (except the tongue) of cattle more than six months old was banned, and sheep and goat heads were also removed from the human food chain. In time, the concept of specified animal materials that might carry a risk of BSE infection was developed. The full agreement of the EC in these matters was slowly achieved and a Specified Risk Material (SRM) ban became European law from 2000 [16], with subsequent amendment to take account of new knowledge and improvement in the general European BSE situation.
BSE in cows born after the ban
It was hoped that the feed ban of 1988 would have progressively reduced the number of infected cattle born after the ban (BAB) in the UK, which indeed it did, but it was not fully effective. Alternative mechanisms of infection such as maternal, horizontal or environmental transmission could not account for the extra cases. Sporadic BSE equivalent to sporadic CJD is not known to occur though a small number of cases of bovine amyloidotic spongiform encephalopathy (BASE) have been reported in Italy that may be different from BSE or merely reflect a different pathogenesis or phenotype [6]. No other sources were found, so it was concluded that continuing exposure of cattle to feed somehow contaminated with infected MBM must be the cause. By January 2006, more than 180,000 cases of BSE have been confirmed in British cattle, of which more than 44,000 were born after the 1988 feed ban [13]. This massive leakage is now attributed to cross-contamination of ruminant rations, mostly with those intended or prepared for pigs and poultry that were legally permitted to contain MBM until 29 Mar. 1996. A contribution was also made by inadequate separation and processing of SBO that permitted MBM prepared from SBO to enter non-ruminant feed and thus be a source for cross-contamination of cattle rations. To deal with contaminated feed ‘in the pipeline’ a feed recall scheme was introduced to remove and destroy it. The reinforced feed ban is regarded as effective from 1 Aug. 1996. When these deficits were appreciated and research determined that much less than 1g (now known to be about 1 mg) of BSE-infected brain was sufficient to provide an oral infecting dose for cattle, the feed ban was reinforced by testing feed for prohibited protein and it was more strenuously policed. Following a recommendation of the British Government’s Spongiform Encephalopathy Advisory Committee (SEAC), it was prohibited from 29 Mar 1996 to feed mammalian protein to any species of farmed food animal or bird, including horses and fish. This is known as the reinforced feed ban and is regarded as being fully effective from 1 Aug. 1996 due to rigorous enforcement following the feed recall period. Despite this, as at May 2006, 131 cattle born subsequently have been confirmed to have BSE (BARB cases), (DEFRA unpublished data). The first BARB cases were believed to result from feeding imported ruminant feed or feed ingredients contaminated with infected MBM. The contamination was suggested to have occurred during marine transport from the continent, e.g., if a vegetable feed cargo immediately followed a cargo of MBM. This is understandable because a ban prohibiting the feeding of processed animal protein to all farmed animals in the rest of the EU was not in place until 1 Jan. 2001. It is not even clear that this latter ban was totally effective from the date of its introduction; for example, ten new countries acceded to the EU in 2004 were not all operating completely to the new standard. A more recent detailed epidemiological investigation has shown that an important source of infection for BARB cases has been persistence of contamination in on-farm feed bins that had not been adequately cleaned and disinfected before the 1 Aug. 1996 deadline. A review by Professor William Hill of the evidence for the occurrence of BARB BSE cases in cattle has been published [10]. The BSE epidemic in Northern Ireland has followed a similar sequence of events and legislation as in GB. For example, of about 2150 cases of BSE there, almost 600 were BAB cases and 16 were BARB cases, as at August 2005.
The hazard for man
An early assumption that the agent of BSE in cows was a variant of the agent of scrapie in sheep led us to believe that transmission of BSE to man was unlikely. BSE might have come from cows or sheep [36]. We have no clear evidence of which, but we know that BSE has many puzzling characteristics and that strain typing studies indicated that the agent that causes BSE is biologically and molecularly different from the agents of scrapie and other transmissible encephalopathies except the vCJD agent [23] from which it is biologically indistinguishable [4]. Human cases of the disease, now called vCJD, began to be recognised in 1995 in the UK [37]. The most likely danger period for human ingestion of the BSE agent in meat and meat products is thought to be the years 1984-89 when public health protection was limited and before the SBO ban was in place to protect consumers. The “period of hazard” for humans could theoretically extend back into the 1970s if there has always been a low undetected level of BSE infection in cows. However, there is some evidence that a degree of protection was afforded to cattle until the late 1970s and early 1980s by the use of hydrocarbon solvents in the rendering process to extract tallow. During this period, the rendering industry progressively ceased to use these solvents. Whatever the reason, it is clear that a level of BSE agent sufficient to cause disease 5 years later was present in MBM fed to calves at least from 1980/81, and that a degree of recycling had occurred from 1984 (thus boosting the exposure) even before the first case of BSE in cattle was confirmed at the end of 1986. Thus, 1984 is taken as the start of the major hazard period for the consumption of contaminated beef and meat products in the UK. In view of the lack of adequate and effective control measures until 1995-6, the hazard period for possible human infection might extend forward until the use of mechanically recovered meat (MRM) and head meat was controlled. Some cheaper processed products such as burgers, pies, beef sausages and mince could have contained MRM and head meat that may have been contaminated with potentially infective tissues such as residues of bovine brain, spinal cord and dorsal root ganglia until controls were in place and the Meat Hygiene Service (MHS) was set up to monitor and enforce them in 1995. In regard to MRM, the Food Standards Agency reported a historical study on the uses of MRM in the period 1980-1995 [22]. Some 5,000 tonnes of bovine MRM was used annually, mostly in catering and retail economy foods such as burgers (40%), frozen and dried mince (40%), but not in burgers from major fast food outlets. Frozen mince was used in some hospitals and schools with the rest being exported or having minor uses. In regard to imported beef and sheep meat, the Food Standards authority records 144 breaches of the SRM regulations until Dec. 2005 [20]. The meat was almost entirely derived from BSE-infected countries in the EU [20]. The usual outcome has been destruction of the offending consignment [21]. There have been 17 breaches of the SRM regulations in the UK until Dec 2005, two of which were from imported animals or material [21]. SRM was removed and destroyed. In addition, there has been a small number of breaches of the regulation prohibiting the slaughter for human consumption of cattle over 30 months old. Most have related to slaughter (and usually consumption) of cattle over the prescribed age limit by just a few days, though a few were substantially older [21]. Any risk to the consumer from these collective events is regarded as low since either, the SRM was removed, or the whole consignment was destroyed. BSE worldwide BSE is known to have been spread widely, along cattle trade routes and where bovine products and by-products, including rendered animal proteins and compound animal feed containing MBM, are imported and marketed. Countries with cases reported to the Office International des Epizooties (OIE) are listed in Table I along with the date of the first report of BSE. The total number of cases outside the UK is currently around 5,400. Cases of vCJD have occurred in several countries with BSE and one that has not reported BSE. In a classification of perceived Geographical BSE Risk (GBR), the Scientific Steering Committee of the European Commission has proposed four categories ranging from I (BSE highly unlikely) to IV (BSE confirmed at a higher level) [29]. The UK and Portugal previously in Category IV, now satisfy the conditions for Category III and are currently so classified. Thus they align with all other EU Member States, Canada and the USA. The UK (since 3 May 2006) and Portugal now adopt the less stringent SRM regulations applying to EU Member States in Category III. They are also permitted to export live cattle and beef again, which in the UK was denied in 1996 by a world-wide ban, though a small volume of beef exports under the strict conditions of a date-based export scheme was subsequently introduced. A similar approach to GBR by the OIE specifies three levels of risk within its Code which gives recommendations for safe international trading in cattle and cattle products [28]. The OIE and EC rules are proposed to be aligned by 1 Jul. 2007 [19].
Preventive strategies
Such schemes depend heavily on reliable updating of information and upon the quality of animal identification, record keeping, notification and surveillance. Until 2001 in the EU, surveillance relied mainly on passive clinical detection and reporting of suspect cases for further investigation by the appropriate state veterinary services. In the latter years of the 1990s, various ‘rapid’ tests for BSE were developed for use on brain material and approved for use in the EU [24] and more have been approved since. These are applied rigorously throughout the EU and in some other countries, notably Japan [33] to detect the presence of PrPSc, which is an indicator of TSE infection and is the disease-specific structurally modified form of the normal host membrane protein PrPC (see below). The rapid tests were first applied to cattle and subsequently to sheep and goats (and a few other species), and they revealed many more unsuspected BSE cases because they could detect infected cattle 3-6 months before clinical onset. An Over Thirty Months (OTM) Rule was introduced in the UK in 1996 whereby cattle more than 30 months old were excluded from all food and feed chains and meat from them was prohibited for export. They were essentially dealt with as SRM and destroyed. This was based on the fact that the minimum age of occurrence of BSE was increasing and cases in animals less than 30 months old had been rare. In fact, none has been recorded in slaughter animals since 1996. In the rest of the EU from 2001, all cattle for human consumption >30 months old are compulsorily tested and if positive the whole carcase is destroyed, as well as closely proximate carcases on the slaughter line. All organs including hides and blood from these cattle are also destroyed. On 7 November 2005, the UK formally relinquished the OTM rule and adopted the same testing regimen as other Member States except that any cow born before 1 Aug. 1996 would forever be excluded for human consumption. Compulsory rapid testing was targeted not only at slaughter cattle >30 months of age but also “risk animals” >24 months of age. These include: fallen cattle stock (dead animals), casualty animals, animals found abnormal at ante mortem inspection, offspring of cases, feed cohorts of cases, and those slaughtered as part of a BSE elimination programme. Positive cases are confirmed by traditional methods. The diminishing number of cases confirmed each year in both the slaughter and risk populations and the continuously rising age at onset indicates good enforcement of the various measures [18,19]. The fallen stock and casualty slaughter populations are yielding the largest numbers and highest proportion of positives and this endorses the importance of targeting active surveillance. The proportion of cases detected by active surveillance using the rapid tests now exceeds that detected by passive surveillance. More than 10 million are completed each year in the EU. The cost is enormous and now probably out of proportion to the risk, especially in slaughter animals and in the 30-35 month age group and younger (Table II) [19]. It is important to note that in the early years of the epidemic and particularly before the SBO ban was introduced in GB in 1989 (and long before active surveillance became possible in the new millennium), pre-clinical and any sub-clinical cases of BSE that might have occurred could have been consumed undetected. The astonishing number of cattle, most of which were consumed in this way, has been estimated using modelling, to be between 1 and 3 millions [30]. The improving situation in the EU has resulted in the EC publishing a TSE Roadmap that lays out a proposed reduced programme for BSE control and elimination in the short, medium and long term [19]. The key piece of EC legislation to protect human and animal health from the hazard of any TSE was adopted on 22 May 2001 and is known as the TSE Regulation [15] laying down rules for the prevention, control and eradication of certain transmissible spongiform encephalopathies. The Roadmap acknowledges the progressively and continuously reducing numbers of confirmed BSE cases throughout the EU, coupled with an increasing age of peak occurrence, and proposes a reciprocal risk-based relaxation of some of the measures whilst assuring a high level of food safety and thus continuing protection of the consumer. An example, to show the start of the process, is that, as from 1 Jan. 2006, the age at which the vertebral column of cattle has to be removed as SRM in the whole EU is 24 months instead of 12 months. Since the vertebral column has not been removed for some years from UK cattle until 30 months of age as the BSE risk is so low, this is disappointing for the UK, but it is likely to be adopted on the grounds that rules should be harmonised. It is a pity that this rule applies to UK beef destined for domestic consumption as well as that for export, for there is no scientific justification for such action. However, now that the UK is a GBR Category III country, it benefits from the less strict SRM rules by not having to split bovine carcases from 6 to 12 months of age in order to remove the spinal cord, which previously was only exempt for cattle under 6 months of age. As at 1 January 2006 the UK still reports the largest annual number of cases of BSE, but the majority are cattle born before 1 Aug. 1996. None of these will ever enter the food or feed chain and the situation improves year on year. The peak age of onset of BSE in the UK cases is older (10 years) than in other countries, so that the UK (understandably) can look earlier towards the day of complete elimination of the disease particularly as an Older Cow Disposal Scheme for cattle born before 1 August 1996 was introduced by DEFRA in January 2006. This scheme will last three years with reducing compensation payments each year. This has the effect of making cattle over 13 years old on 1 January 2009 valueless and should stimulate their voluntary slaughter for destruction by this date [11]. From 2001 to 2004 the mean age at onset of BSE cases in slaughter animals in the 15 EU Member States increased from 76.2 to 95 months (say, 8 years). Ireland and Switzerland and then the other European countries follow the UK progress towards elimination. Outside of Europe the number of reported cases is low (Table I) but it is too early yet to be certain of progress towards universal elimination, as there is some worrying variation in the measures employed. At present, we can be sure that neither pigs nor poultry present a TSE risk to man, but sheep and goats might. One case of suspect BSE in a goat in France has been confirmed. The goat did not enter the food chain. The remainder of the herd was destroyed and tested and no goat was positive. No case has yet been reported in sheep and although maternal transmission of experimental BSE in sheep has recently been confirmed [1] it seems that no small ruminant species presents more than a negligible risk.
Tissue infectivity in BSE
Research into the tissue distribution of infectivity in natural and experimental BSE in cattle is being extensively studied in the UK using conventional mice or cattle as the bioassay animals [35] and in Germany using highly BSE-sensitive transgenic mice [5]. The use of cattle to detect infectivity eliminates the species barrier to transmission and maximises test sensitivity. In studies of the natural bovine disease, infectivity in cattle tissues (using conventional mice and/or cattle for detection) was found only in the brain, spinal cord, the retina and the third eyelid (nictitating membrane). The experimental disease was induced by oral challenge of cattle with BSE-infected brain material followed by sequential killing of small groups of animals enabling the pathogenesis of the disease to be determined. In these studies infectivity has been found (using conventional mice and/or cattle) in the tonsil at 10 months post-challenge [35]. This resulted in a change in the legislation to include tonsil from all ages of cattle as specified risk material (SRM) for destruction instead of from those over 12 months of age [17] and modification of the method used to harvest tongues for human consumption. In addition, infectivity was found in the distal ileum from cattle groups killed at 6-18 months and from 36 months post-challenge. Dorsal root ganglia (DRG) were also infected in groups killed from 32 months post- challenge (about 3 months before clinical onset in the experiment, i.e., at 35 months post-challenge) [35]. Most recently, using transgenic mice, infectivity has been found in some peripheral nerves and in a single muscle from a single German cow with natural, clinically-advanced BSE, but at low titre compared with that in the brain [5]. These studies have also confirmed that the bovine lymphoreticular system (other than the tonsil and ileum, which are officially classed as SRM) is not significantly involved. This contrasts with the pathogenesis of scrapie in sheep and vCJD in man. Thus, to date, our most recently developed techniques confirm that BSE infectivity in slaughter cattle that have passed a rapid test is likely to be confined to the ileum and perhaps the tonsil. In more advanced cases of BSE, high levels of infectivity are likely to be confined for practical purposes to the CNS and its associated ganglia.
Concluding remarks
Careful collection and analysis of epidemiological data have enabled identification of the important factors responsible for the transmission and distribution of BSE. This led to the introduction, and later strengthening, of the measures introduced to reduce or eliminate the risk to cattle and other animals via feed. Effective research into the distribution of tissue infectivity in cattle with BSE, similarly has led to endorsement of the 1989 SBO ban and to later extensions on a wider geographic basis, thus providing protection for the consumer. The consequences of the transmission of the BSE agent to produce human cases of variant Creutzfeldt-Jakob disease (vCJD) will be considered in PART 2 of this Review.
1. Bellworthy SJ, Dexter G, Stack M, Chaplin M, Hawkins SAC, Simmons MM, Jeffrey M, Martin S, Gonazalez L, Hill P. Natural transmission of BSE between sheep within an experimental flock. Vet Rec 2005; 157: 206. 2. Bradley R, Wilesmith J. Epidemiology and control of bovine spongiform encephalopathy (BSE). Brit Med Bull 1993; 49: 932-959. 3. Brown P, Rau EH, Lemieux P, Johnson BK, Bacote AE, Gajdusek DC. Infectivity studies of both ash and air emissions from simulated incineration of scrapie-contaminated tissues. Environ Sci Technol 2004; 38: 6155-6160. 4. Bruce ME, Will, RG, Ironside JW, McConnell I, Drummond D, Suttle A, McCardle L, Chree A, Hope J, Birkett C, Cousens S, Fraser H, Bostock CJ. Transmissions to mice indicate that ‘new variant’ CJD is caused by the BSE agent. Nature 1997; 389: 498-501. 5. Buschmann A, Groschup MH. Highly bovine spongiform encephalopathy-sensitive transgenic mice confirm the essential restriction of infectivity to the nervous system in clinically diseased cattle. J Infect Dis 2005; 192: 934-942. 6. Casalone C, Zanusso G, Acutis P, Ferrari S, Capucci L, Tagliavini F, Monaco S, Caramelli M. Identification of a second bovine amyloidotic spongiform encephalopathy: molecular similarities with sporadic Creutzfeldt-Jakob disease. Proc Natl Acad Sci U S A 2004; 101: 3065-3070. 7. Collee JG, Bradley R. BSE: a decade on - part 1. Lancet 1997; 349: 636-641. 8. Collee JG, Bradley R. BSE: a decade on - part 2. Lancet 1997; 349: 715-721. 9. Department of Agriculture and Rural Development Northern Ireland. Website accessed Feb. 2006. file/dard0500.xls 10. Department of the Environment, Food and Rural Affairs (DEFRA). A review by Professor William Hill of the evidence for the occurrence of BARB BSE cases in cattle. Website accessed Feb. 2006. 11. Department of the Environment, Food and Rural Affairs (DEFRA). Older cow disposal scheme (OCDS) 23 Jan. 2006. Website accessed May 2006. livestock/strategy/otms/index.htm 12. Department of the Environment, Food and Rural Affairs (DEFRA). BSE statistics – confirmed cases of BSE reported worldwide as at 3 Jan. 2006 Website accessed Feb. 2006. http://www. 13. Department of the Environment, Food and Rural Affairs (DEFRA). Number of confirmed cases of BSE by year of birth where known. Website accessed Feb.2006. bse/statistics/bse/yrbirth.html 14. Department of the Environment, Food and Rural Affairs (DEFRA). Transmissible Spongiform Encephalopathies in Great Britain - A Progress Report. Dec. 2001. DEFRA, London 2002, pp. 152 15. European Commission (EC). Regulation (EC) No 999/2001 of the European Parliament and of the Council of 22 May 2001 laying down rules for the prevention, control and eradication of certain transmissible spongiform encephalopathies. Official J of the European Union 2001; L 147: 1-40. 16. European Commission (EC). Commission Decision 2000/418/EC of 29 June 2000 regulating the use of material presenting risks as regards transmissible spongiform encephalopathies and amending Decision 94/474/EC. Official J of the European Union 2000; L 158: 76-82. 17. European Commission (EC). Commission Regulation (EC) No 1974/2005 of 2 December 2005 amending Annexes X and X1 to regulation (EC) No 999/2001 of the European Parliament and of the Council as regards national reference laboratories and specified risk material. Official J of the European Union 2005; L317: 4-8. 18. European Commission (EC). Report on the monitoring and testing of ruminants for the presence of transmissible spongiform encephalopathy (TSE) in the EU in 2004. 31 May 2005. Health and Consumer Protection Directorate General. EC Brussels. Pp 93. 19. European Commission (EC). The TSE Roadmap. COM (2005) 322 FINAL. EC Brussels, 15 July 2005. EC website accessed Sep. 2005 20. Food Standards Agency. Website accessed Feb. 2006. 21. Food Standards Agency. Website accessed Feb. 2006. Facts and figures and BSE news. 22. Food Standards Agency. Website accessed Feb. 2006. 23. Horn G, Bobrow M, Bruce M, et al. Review of the origin of BSE. Report 5 July 2001. London: DEFRA, Pp 63. 24. Moynagh J, Schimmel H. Tests for BSE evaluated. Bovine spongiform encephalopathy. Nature 1999; 400: 105. 25. National Creutzfeldt-Jakob Disease Surveillance Unit. Website accessed Feb.2006. 26. Office International des Epizooties (OIE). Number of cases of BSE in the UK by year of restriction. Website accessed Feb. 2006. 27. Office International des Epizooties (OIE). Number of cases of BSE in farmed cattle worldwide excluding the UK by year of confirmation. Website accessed Feb. 2006. info/en_esbmonde.htm 28. Office International des Epizooties OIE). Terrestrial Animal Health Code, Chapter OIE, Paris, 2005. 29. Scientific Steering Committee (SSC). Opinion of the SSC on a method for assessing the geographical BSE-risk (GBR) of a country or region. Up-date Jan. 2000 EC Brussels Pp 9. 30. Smith PG, Bradley R. Bovine spongiform encephalopathy (BSE) and its epidemiology. Br Med Bull 2003; 66: 185-198. 31. Spongiform Encephalopathy Advisory Committee (SEAC). Draft minutes of 90th meeting held on 24 November 2005. Website accessed Feb. 2006. 32. Taylor DM, Woodgate SL, Atkinson MJ. Inactivation of the bovine spongiform encephalopathy agent by rendering procedures. Vet Rec 1995; 137: 605-610. 33. Tsutsui T, Yamane K, Shimura K. Preliminary evaluation of the prevalence of BSE in Japan. Vet Rec 2004; 154: 113-114. 34. Wells GAH, Hawkins SAC, Austin AR, Ryder SJ, Done SH, Green RB, Dexter I, Dawson M, Kimberlin RH. Studies of the transmissibility of the agent of bovine spongiform encephalopathy to pigs. J Gen Virol 2003; 84: 1021-1031. 35. Wells GAH, Spiropoulos J, Hawkins SAC, Ryder SJ. Pathogenesis of experimental bovine spongiform encephalopathy: preclinical infectivity in tonsil and observations on the distribution of lingual tonsil in slaughtered cattle. Vet Rec 2005; 156: 401-407. 36. Wilesmith J, Wells GAH, Cranwell M, Ryan JBM. Bovine spongiform encephalopathy: epidemiological studies. Vet Rec 1988; 123: 638-644. 37. Will RG, Ironside JW, Zeidler M, Cousens SN, Estibeiro K, Alperovitch A, Poser S, Pocchiari M, Hofman A, Smith PG. A new variant of Creutzfeldt-Jakob disease in the UK. Lancet 1996; 347: 921-925. 38. Wyatt JM, Pearson GR, Smerdon T, Gruffydd-Jones TJ, Wells GAH. Spongiform encephalopathy in a cat. Vet Rec 1990; 126: 513.

full text of the article:

Review article
Variant CJD (vCJD) and Bovine Spongiform Encephalopathy (BSE): 10 and 20 years on: part 2

Folia Neuropathol 2006; 44 (2): 102-110

authors: J. Gerald Collee, Ray Bradley, Paweł P. Liberski,

documents in PDF format:

> Variant CJD - part 2.pdf [0.10 MB]

In our preceding paper [3], we summarised current issues in relation to BSE in cattle and the possible further impact on public health. Here, we consider uncertainties in regard to the vCJD epidemic that was initiated by the consumption of food produced from BSE-infected cattle and now threatens to be extended by human-to-human transmission.
Transmission of BSE to man
Is the link between bovine BSE and human vCJD clearly established? Biological and molecular strain typing studies have demonstrated clearly that for practical purposes the BSE agent and the vCJD agent are one and the same, or at least have a similar pathogenicity for man and for macaque monkeys to which both agents have been transmitted. The association between BSE and vCJD is further supported by the epidemic curves, the presumed response to the various bans, the time-scales of the two diseases and their geographical occurrence (Fig. 1, Table I). Following a recommendation from the Southwood Working Party on BSE, from 8 Aug 1988 all cattle suspected to have BSE were removed from all food and feed chains and destroyed. Subsequently, a specified bovine offal (SBO) and finally a specified risk material (SRM) ban was introduced and controls were put in place on the use of the vertebral column and restrictions on the manufacture and sale of mechanically recovered meat (MRM) (Fig. 1). Epidemiological enquiry into vCJD has revealed no association with occupation, medicines, immunisation, gelatine, surgery (including the use of catgut derived from cattle intestine) or exposure to bovine materials or by-products by routes other than orally. Even brain consumption (as brain) has not been incriminated though this last cannot be completely ruled out because brain might have been included in processed food products before the 1989 SBO ban was in place or complete. A theoretical source that has caused great concern (to the extent that it is now completely banned from use in the EU) is mechanically recovered (or separated) meat (MRM) from ruminant animal bones. Historically, skulls were not used to prepare MRM because the teeth were destructive to the machinery. The specific risk would be from the vertebral column because it is virtually impossible to remove all vestiges of spinal cord, dorsal root ganglia (DRG) and associated autonomic and spinal nerves. Thus, there is a global restriction recommended by the OIE that from countries with a controlled or undetermined BSE risk, MRM from skulls and vertebral column from cattle >30 months old should not be traded. As European law prohibits any use or trade of ruminant MRM, any risk from this source is now eliminated completely in the EU. All of these considerations generate confidence that new exposure of humans born and resident in the EU and Switzerland is most unlikely from cattle. No new challenges for the European population are apparent from cattle even though a variant form of BSE (bovine amyloidotic spongiform encephalopathy, (BASE)) has been detected in a small number of animals in Italy [6]. However, scrapie is presently receiving considerable attention in the EC. Although it is apparently not a risk for humans and has not been shown to be transmitted even to sheep and goats via feed, there are several plans to eradicate it. This is partly because there is no easy way to distinguish the various scrapie agents from the BSE agent in a rapid and convincing way, especially in the live animal, and partly because the presence of scrapie severely restricts international trade in sheep and goats and some of their products. One case of BSE in a goat in France has been reported [3].
Incubation periods for vCJD
In regard to the primary transmission from cattle to man, if the most likely period for transmission of BSE to humans was indeed 1984-89, the incubation periods for the early cases of human BSE infection (vCJD) seem to be of the order of 10 years. If the danger period is extended forward to 1996, shorter human incubation periods are a possibility. On the other hand, if the risk of human infection extended back before the 1980s, much longer incubation periods might be postulated. Since the date of exposure for no individual is known, the actual length of the incubation period is also unknown, though analysis of the epidemiological data enables a reasoned judgment to be made at least for patients with the PRNP MM polymorphism. By contrast, and in regard to secondary transmission from man to man as a result of blood transfusion, for two patients (see below) the incubation period is known with a greater degree of precision, namely 6.5 years [31] and 8 years [10]. Assuming transfusion to be the cause in these cases, there would be no species barrier. Furthermore, the intravenous route of exposure is orders of magnitude more efficient than the oral route and the amount of blood transfused is likely to be greater than the amount of infected cattle product consumed at one meal. These considerations seem to be consistent with the (presently unproven) assumption that a shorter incubation period is likely in secondary (man-to-man) transfused infections than in primary transmission from cattle by the oral route (c.10 years or so). Caution should be exercised however, because we do not know the titre of infectivity in human blood from an infected donor (estimated to be 0-60 i/v ID50/g) [15]. This is likely to be orders of magnitude less than in brain material from affected cattle (106 cattle i/c ID50/g) [5] or humans (estimated to be 108 i/c ID50/g [15]) and bearing in mind that the i/v route is about 10 times less efficient than the i/c route [15]. Longer incubation periods could arise in patients with codon 129 MV and VV polymorphism (see below). Parallels have been drawn between vCJD and kuru, a TSE associated with endocannibalism and funeral rites in the eastern highlands of Papua New Guinea where the range of incubation periods extends from 4 to more than 40 years. Thus, it has been postulated [9] that some early cases of vCJD seen in the UK may have received their infections earlier than the mid-1980s and could represent infection acquired before we were even aware of BSE in cows. This is worrying, but a note of caution is justified on at least two counts. It seems that a factor, or several factors, operated when BSE transmitted to human patients. Perhaps this is related to the size of the challenge dose, or to repeated (cumulative) challenges, though evidence for the latter is unconvincing [16,21,28]. It may be that a coincident infection acted to make a victim susceptible to the BSE (vCJD) agent at a particular time (sometimes perhaps even early in childhood, in the case of teenagers who have died). Indeed, various models indicate that vCJD infection seems to be preferentially acquired by young people, and older subjects may be more resistant [11,41]. We do not yet know if they resist infection or become infected but do not develop clinical disease, either at all, or after extended incubation. In contrast to the situation with BSE in cattle [3], since 1996 the average age of vCJD patients has not changed significantly (median age at onset 26 and at death 28 years) [32] despite the fact that the major exposures from food were probably confined to a relatively short period. As noted above, teenagers and young adults might be more susceptible to infection and develop clinical signs more readily [11,41], such as may have occurred in the kuru epidemic where some young children succumbed and some adults exposed around the same time had very long incubations indeed. However, it is important to note that the agent that causes vCJD is unique among human TSE agents and is biologically indistinguishable from the BSE agent [3], so kuru might not be a good model to guide us. Another consideration is that, in the UK, young people may have selectively consumed food containing mechanically recovered meat (MRM) with a potentially high TSE-risk [3].
BSE/vCJD agent, PrP and its associated gene
Prusiner and colleagues suggested that the infectious agent in BSE, and thus in vCJD, is a prion - a small proteinaceous infectious particle that resists inactivation by procedures which modify nucleic acids and thus may be devoid of detectable DNA [37]. To date, the prion is known to be very small, passing through small-pore filters, and to be remarkably heat-stable and resistant to a wide range of antimicrobial chemicals including formaldehyde when tested in crude mixtures of infective brain homogenates. Despite this, chemical and physical methods have been devised that are effective and widely used in hospitals, autopsy rooms and industrial suites to secure safety [40]. The importance of thorough cleaning is stressed, with careful removal of all traces of protein from surfaces before disinfection and from surgical instruments prior to sterilization. The particle has not yet been visualized as such, but some structures called scrapie-associated fibrils and prion rods, seen by electron-microscopy after chemical extraction and treatment of infective material, may be aggregates of prions. The terminology has evolved in a confusing manner. A host-encoded glycoprotein that is a normal constituent of cell membranes in man and animals is designated PrPC. The letter C denotes the normal form of the protein expressed in many cell types and notably in the brain. The abnormal form PrPSc was so called because it was first associated with scrapie (Sc) in sheep but is commonly used to relate to any TSE. (Various other symbols are used to differentiate between the normal and abnormal forms of PrP.) Attention has been drawn to codon 129 of the gene that codes for PrPC in man, the prion protein (PRNP) gene. Homozygous individuals may have two methionine alleles (MM) or two valine (VV) alleles. Heterozygous individuals have one of each allele (MV) at codon 129. All of the patients who have died of vCJD and have been genetically investigated so far are homozygous for methionine (MM). A possible, but uncertain, exception was a heterozygous (MV) patient apparently infected by blood transfusion from a donor who subsequently developed vCJD [36]. The heterozygote recipient developed no neurological symptoms prior to death and showed no evidence of vCJD-related neuropathology or PrPSc accumulation in the central nervous system. This case is therefore provisionally considered to be an asymptomatic vCJD infection that may (preclinical case) or may not (subclinical case) have gone on to be expressed as vCJD. Present evidence indicates that MM homozygosity at codon 129 is associated with susceptibility to vCJD. Alternatively, it may be that MM is a factor that predisposes to a shorter incubation period, whilst MV heterozygosity or VV homozygosity may be considered as factors conferring relative resistance or longer incubation periods. If this is the case, then we might expect to see another wave or waves of cases of vCJD representing infections that were incubated in MV or VV patients. Yet another worrying possibility is that the disease produced in the latter patients (if any) might differ clinically from vCJD in patients with the MM allele. These points are part of the reason for caution when the experts are pressed to predict the future course of events in this difficult field. Studies of the PrP gene of cows (which, like most other animal species, are homozygous for methionine at the equivalent codon) have revealed polymorphisms in the octa-repeat region but these show no association with disease occurrence. It is believed that cattle are uniformly susceptible to BSE. By contrast, the sheep PrP gene is polymorphic at several codons and notably at codons 136, 154 and 171. Some genotypes are highly susceptible to scrapie, others are much more resistant and some are of intermediate ‘resistance’[20]. Use has been made of this variability by breeding rams homozygous for ‘resistance’ to increase the desirable alleles in the national flock in various European and other countries [12].
vCJD: Concepts and trends
The change from normal PrPC to misfolded PrPSc is post-translational. The misfolding results in the conversion of an a helix-rich protein to a form rich in b sheet, for example by a process of dimerisation in a type of chain reaction. The abnormal form is partially protease-resistant whereas PrPC is denatured by proteases and this enables a distinction to be made between the two. Results of experimental studies in rodents suggest that, in natural human infection by the oral route, it is likely that the agent is initially taken up by migrating intestinal dendritic cells. These transport it to follicular dendritic cells (FDC) resident in lymphoreticular sites in the gut and elsewhere such as in Peyer’s patches of the jejunum and ileum and lymphoid tissue of the appendix and large intestine. FDC maturation is assisted by lymphocytes, suggesting an important role for these cells in TSE, including increasing the risk of infection in chronically inflamed tissues [30]. Accumulation and/or replication of infectivity in the FDC is a necessary prelude to neuroinvasion effected through peripheral nerves of the autonomic nervous system to the spinal cord and then the brain [1,8,38]. PrPSc accumulates in the brain, infectivity titres rise and there is progressive neurodegeneration leading to death. Patients with symptoms and signs of the disease that we now recognise as vCJD were first observed in the UK in 1995. The number of definite or probable deaths from vCJD in the UK and worldwide are given in table I and the UK data are presented graphically in figure 1. Annual numbers of deaths from vCJD rose thereafter to a peak of 28 in 2000 and have then fallen progressively (at least, up to Feb. 2006). Once again, however, a note of caution is needed. The number of onsets of vCJD (new cases each year) increased in 2004 (9 cases) compared with 2003 (5 cases). This may be a more reliable indication of the current trend and must moderate our optimism.
vCJD: The uncertain future
The ominous interpretation suggested by Collinge [9] is that the graph of the UK figures might include some cases of infection acquired before the overt infection in British cattle, so that an unknown number of human cases relating to infection acquired in the 1980s and later have still to be accounted for. It is tempting to draw premature but more reassuring conclusions from the shape of the graph of these mortality figures. If the patients with vCJD seen in the last decade were not attributable to infections acquired from 1984 or later (when the BSE outbreak started), they would be part of a cohort of patients who had long incubation times of 12-22 years. If such cases were going on to represent the extremes of long incubation, as in kuru, their numbers should not be so strikingly affected by the controls that were introduced from 1988-1996. Perhaps, however, there might be a strange biphasic character in their presentation, and a possible explanation could rest on the influence on incubation times of MV heterozygosity or VV homozygosity at the polymorphic codon 129. Even without this influence of host genetics on possible susceptibility, Cooper and Bird [11] have noted that subjects in the birth cohorts studied by them had different dietary exposure intensities to BSE that prevailed before and after significant preventive actions in 1989. Their calculations indicate that, for presently unknown reasons, significant numbers of later (primary) cases of vCJD may yet be seen in the UK in the present decade as a second wave of patients who presumably ingested the agent in cattle meat products containing or contaminated with SRM. The shape of the graph of vCJD incidence in the UK in the last decade seems to be compatible with the view that we are experiencing the effects of infection transmitted to human patients when the BSE outbreak occurred in cows in the mid-1980s or 1990s, or just a few years before this, say, in the early 1980s. In the light of the worst predictions on this premise, we have been fortunate so far. Most of these early extrapolations were based on the assumption that all of the deaths from vCJD recorded to date in the UK were attributable to infections resulting from the overt outbreak of BSE in cattle. This is a reasonable basis for prediction, though some of the earlier published estimates were alarming and present estimates are more reassuring (but see below). In the present paper, we have taken account of alternative suggestions that there may be another (later) human sequel to the bovine outbreak. We also bear in mind that BSE and cases of vCJD in other countries may have longer courses to run. Most of the literature on BSE and vCJD to date implies that vCJD in man is acquired as a primary disease from cattle. The probable (secondary) human-to-human transmission of vCJD infection via transfusion of infected blood now appears to be almost certain, but doubt remains (as noted above) about the patient originally reported by Peden et al.[36], who may be an example of an asymptomatic infection in an MV heterozygote recipient. A death has certainly been reported in a methionine homozygote recipient who received blood from a donor who was healthy at the time but who developed and died from vCJD subsequently [31]. The most recent report in Feb. 2006 is of another patient who is symptomatic but presently alive [10]. There is much concern that further cases of vCJD may arise in this way. The worry extends beyond the UK to other countries across the world and it has led to major alerts and product withdrawal, with restrictions on donors of blood, organs and tissues. There are on-going urgent reviews of the safety of human blood and plasma and derived products, and similar reconsiderations of the safety of human tissues and medical and surgical instruments that may have become contaminated with the vCJD agent. In the context of transfusion, the use of leucodepletion is a recognised step to safeguard blood for transfusion, but it is not considered to be an absolute protection as tests (with blood from scrapie-infected hamsters) showed that about half of the potential infectivity may remain after the treatment [22].
There is another area of concern relating to the widespread distribution of PrPSc that is known to occur in lymphatic tissues of vCJD patients [26] when tests are done based on immunohistochemical (IHC) techniques and/or sensitive Western Blot technology [26,29]. There is evidence that, at least in the spleen and the tonsil, infectivity can also be demonstrated [4]. A worrying development has been the finding of PrPSc in an appendix removed from an otherwise healthy patient who went on to develop vCJD 8 months later [23]. In 2004, a report mentioned another incident where appendix tissue removed at appendicectomy was positive for PrPSc two years before the symptoms and signs of vCJD appeared and four years before death [25]. Appendix tissue removed from a third patient at appendicectomy 10 years before vCJD was diagnosed, was negative for PrPSc [4]. Three anonymous surveys have been done to determine the prevalence of PrPSc in appendices removed at routine appendicectomy. In the first, no positives were found in 3075 samples [27]. In the second survey, 1 positive appendix was found out of 8318 specimens examined by IHC [24] and the distribution of PrPSc was similar to that found in the two preclinical samples noted above. In the third study, 3 out of 12,674 appendix samples were positive for PrPSc by IHC [25]. The latter figure gives an estimated prevalence of 237 vCJD infections per million in the UK (95% CI 49-692 per million) [25]. However, although the immunocytochemical method performed to detect PrPSc in this study appears to be specific, it is unlikely to be fully sensitive for all phases of the (unknown) incubation period, and this causes further uncertainty in the estimation of the numbers of vCJD infections that have occurred. Similar anonymous studies have been done on tonsils removed at tonsillectomy. In the first of these studies [27], all 95 tonsils examined by IHC were negative for PrPSc; and, in the second tonsil survey, no positives were found in 2,000 tonsils examined [18]. The positive findings with appendicectomy specimens have led to the speculation that pre-clinical or sub-clinical vCJD may be present in a proportion of the population. There is an understandable call for large-scale prospective screening to provide evidence for estimations of prevalence and to increase awareness of possible secondary transmission in surgery, particularly in relation to young people who may have been exposed to BSE before it was brought under control, though the magnitude of any hidden pre/subclinical population is likely to be underestimated [29]. All of these considerations increase the urgent need for tests that would reliably and speedily determine whether a patient may be in danger of developing vCJD, ideally by a minimally invasive procedure. Promising results are being reported, for example by Castilla, Saá and Sato [7] who have employed protein misfolding cyclic amplification (PMCA) technology to amplify very small amounts of PrPSc (that might be present in blood) by over 6,500 times and thus substantially increase the sensitivity of the test. A number of other potentially useful tests for detecting tiny amounts of the misfolded protein are under development and are being presented at recent and future meetings on TSE and blood. For a review of diagnostic methods in animals see Gavier-Widén et al. [19]. Technology and research is also progressing in the elimination of TSE infectivity and decontamination and the evaluation and control of instrument- and device-borne prion infection [2,17].
Concluding remarks
Our analysis of the currently available data allows us to be optimistic in foreseeing the elimination of BSE in the EU and countries that follow the EU plan. The outlook is necessarily less assured for the control of BSE in other countries adopting less stringent remedies. In consequence, in regard to vCJD in ‘Europe’, we are content that there has been effective and virtually complete elimination of food-borne BSE from cattle to man, but we are more cautious for some countries elsewhere. The current trends in the human epidemic of vCJD in the UK are reassuring, but we note that the present reduction in numbers of these cases stems from measures adopted in the veterinary, rather than the medical field. We are presently much more uncertain about the future trends in the human epidemic that might result from a currently concealed, unknown number of infected people in the European population, due either to their PRNP genotype (MV or VV) or to existing pre-clinical or sub-clinical infection. If such concealed populations exist, are their numbers too small to permit maintenance of the epidemic, or are they already considerable and being added to by current practices? There is a need for continuing surveillance for BSE and all forms of CJD in the UK, EU and indeed globally, in view of the uncertainty of the occurrence and extent of BSE in many countries of the world, the absence of fully effective measures and their enforcement, and the uncertainty about future trends in the human epidemic of vCJD in the UK despite effective controls on BSE. The hazard of vCJD has greatly complicated the careful vetting of blood donors and there is an urgent requirement for a test that might detect infected persons. We are convinced that blood transfusion is a means of transmission of vCJD but note that the recent UK infections occurred before leucodepletion of blood was in place. Currently we know of no certain human-to-human transmissions by other routes or mechanisms, but it is probably too early to say that they will not occur. Whilst we are strongly encouraged by the actions taken to research and to reduce infectivity in blood and to determine effective ways to clean and decontaminate instruments and surfaces, we are aware that many problems remain to be solved if we are to deal effectively with this daunting challenge.
We thank Professor JW Ironside for valuable comments and much generous help with this review. We gratefully acknowledge guidance from Mr DE Bradbury on the presentation of data in the charts and Dr PW Brown for guidance on presenting vCJD case data. And we thank DEFRA, SEAC, OIE, EC, EFSA and the National CJDSU at Edinburgh for access to and regular updating of information.
1. Aguzzi A. Neuro-immune connection in spread of prions in the body? Lancet 1997; 349: 742-743. 2. Baxter HC, Campbell GA, Whittaker AG, Jones AC, Aitken A, Simpson AH, Casey M, Bountiff L, Gibbard L, Baxter RL. Elimination of transmissible spongiform encephalopathy infectivity and decontamination of surgical instruments by using radio-frequency gas-plasma treatment. J Gen Virol 2005; 86: 2393-2399. 3. Bradley R, Collee JG, Liberski PP. Variant CJD (vCJD) and bovine spongiform encephalopathy (BSE): 10 and 20 years on – part 1. Folia Neuropathol 2006; 44: 93-101. 4. Bruce ME, McConnell I, Will RG, Ironside JW. Detection of variant Creutzfeldt- Jakob disease. Infectivity in extraneural tissues. Lancet 2001; 358: 208-209. 5. Buschmann A, Groschup MH. Highly bovine spongiform encephalopathy-sensitive transgenic mice confirm the essential restriction of infectivity to the nervous system in clinically diseased cattle. J Infect Dis 2005; 192: 934-942. 6. Casalone C, Zanusso G, Acutis P, Ferrari S, Capucci L, Tagliavini F, Monaco S, Caramelli M. Identification of a second bovine amyloidotic spongiform encephalopathy: molecular similarities with sporadic Creutzfeldt-Jakob disease. Proc Natl Acad Sci U S A 2004; 101: 3065-3070. 7. Castilla J, Saá P, Soto C. Detection of prions in blood. Nat Med 2005; 11: 982-985. 8. Collee JG. Transmissible Spongiform Encephalopathies. In: The Microbiological Safety and Quality of Food, Barbara M Lund, TC Baird-Parker, GW Gould eds, vol. II, Aspen, Maryland, USA 2000. Ch 57, p. 1618. 9. Collinge J. Variant Creutzfeldt-Jakob disease. Lancet 1999; 354: 317-323. 10. Communicable Disease Report Weekly. New case of transfusion-associated variant-CJD. CDR Weekly 2006; 16: 9 Feb. 2006. 11. Cooper JD, Bird SM. Predicting incidence of variant Creutzfeldt-Jakob disease from UK dietary exposure to bovine spongiform encephalopathy for the 1940 to 1969 and post-1969 birth cohorts. Int J Epidemiol 2003; 32: 784-791. 12. Dawson M, Hoinville LJ, Hosie BD, Hunter N. Guidance on the use of PrP genotyping as an aid to the control of clinical scrapie. Vet Rec 1998; 142: 623-625. 13. Department of the Environment, Food and Rural Affairs (DEFRA). BSE statistics - confirmed cases of BSE reported worldwide as at 3 Jan. 2006 Website accessed Feb. 2006. animalh/bse/statistics/bse/worldwide.htm 14. Department of the Environment, Food and Rural Affairs (DEFRA). Transmissible Spongiform Encephalopathies in Great Britain - A Progress Report, Dec. 2001. DEFRA, London, 2002, p. 152. 15. Det Norske Veritas. Assessment of the risk of exposure to vCJD infectivity in blood and blood products. Appendix II: Risk assessment of vCJD infectivity in blood. Report for the department of Health, Revision D. Feb. 2003. DNV Consulting London. Pp 55. Website accessed Feb. 2006. AppII_tcm23-74416.pdf 16. Diringer H, Roehmel J, Beekes M. Effect of repeated oral infection of hamsters with scrapie. J Gen Virol 1998; 79: 609-612. 17. Fichet G, Comoy E, Duval C, Antloga K, Dehen C, Charbonnier A, McDonnell G, Brown P, Lasmezas CI, Deslys JP. Novel methods for disinfection of prion-contaminated medical devices. Lancet 2004; 364: 521-526. 18. Frosh A, Smith LC, Jackson CJ, Linehan JM, Brandner S, Wadsworth JD, Collinge J. Analysis of 2000 consecutive UK tonsillectomy specimens for disease-related protein. Lancet 2004; 364: 1260-1262. 19. Gavier-Widén D, Stack MJ, Baron T. Diagnosis of transmissible spongiform encephalopathies in animals: a review. J Vet Diagn Invest 2005; 17: 509-527. 20. Goldmann W, Baylis M, Chihota C, Stevenson E, Hunter N. Frequencies of PrP gene haplotypes in British sheep flocks and the implications for breeding programmes. J Appl Microbiol 2005; 98: 1294-1302. 21. Gravenor MB, Stallard N, Curnow R, McLean AR. Repeated challenge with prion disease: the risk of infection and impact on incubation period. Proc Natl Acad Sci U S A 2003; 100: 10960-10965. 22. Gregori L, McCombie N, Palmer D, Birch P, Sowemimo-Coker SO, Giulivi A, Rohwer RG. Effectiveness of leucoreduction for removal of infectivity of transmissible spongiform encephalopathies from blood. Lancet 2004; 364: 529-531. 23. Hilton DA, Fathers E, Edwards P, Ironside JW, Zajicek J. Prion immunoreactivity in appendix before clinical onset of variant Creutzfeldt-Jakob disease. Lancet 1998; 352: 703-704. 24. Hilton DA, Ghani AC, Conyers L, Edwards P, McCardle L, Penney M, Ritchie D, Ironside JW. Accumulation of prion protein in tonsil and appendix: review of tissue samples. BMJ 2002; 325: 633-634. 25. Hilton DA, Ghani AC, Conyers L, Edwards P, McCardle L, Ritchie D, Penney M, Hegazy D, Ironside JW. Prevalence of lymphoreticular prion protein accumulation in UK tissue samples. J Pathol 2004; 203: 733-739. 26. Hilton DA, Sutak J, Smith MEF, Penney M, Conyers L, Edwards P, McCardle L, Ritchie D, Head MW, Wiley CA, Ironside JW. Specificity of lymphoreticular accumulation of prion protein for variant Creutzfeldt-Jakob disease. J Clin Pathol 2004; 57: 300-302. 27. Ironside JW, Hilton DA, Ghani A, Johnston N, Conyers L, McCardle LM, Best D. Retrospective study of prion-protein accumulation in tonsil and appendix tissues. Lancet 2000; 255: 1693-1694. 28. Jacquemot C, Cuche C, Dormont D, Lazarini F. High incidence of scrapie induced by repeated injections of subinfectious prion doses. J Virol 2005; 79: 8904-8908. 29. Joiner S, Linehan J, Brandner S, Wadsworth JDF, Collinge J. Irregular presence of abnormal prion protein in appendix in variant Creutzfeldt-Jakob disease. J Neurol Neurosurg Psychiatry 2002; 73: 597-603. 30. Ligios C, Sigurdson CJ, Santucciu C, Carcassola G, Manco G, Basagni M, Maestrale C, Cancedda MG, Madau L, Aguzzi A. PrPSc in mammary glands of sheep affected by scrapie and mastitis. Nat Med 2005; 11: 1137-1138. 31. Llewelyn CA, Hewitt PE, Knight RS, Amar K, Cousens S, Mackenzie J, Will RG. Possible transmission of variant Creutzfeldt-Jakob disease by blood transfusion. Lancet 2004; 363: 417-421. 32. National CJD Surveillance Unit. Thirteenth Annual Report for 2004, 2005. Website accessed Feb. 2006. report13.pdf 33. National Creutzfeldt-Jakob Disease Surveillance Unit. Website accessed Feb.2006. 34. Office International des Epizooties (OIE). Website accessed Feb. 2006. 35. Office International des Epizooties (OIE). Website accessed Feb. 2006. 36. Peden AH, Head MW, Ritchie DL, Bell JE, Ironside JW. Preclinical vCJD after blood transfusion in a PRNP codon 129 heterozygous patient. Lancet 2004; 364: 527-529. 37. Prusiner SB. Novel proteinaceous particles cause scrapie. Science 1982; 216: 136-144. 38. Shmakov AN, Ghosh S. Prion proteins and the gut: une liaison dangereuse? Gut 2001; 48: 443-447. 39. Spongiform Encephalopathy Advisory Committee (SEAC). Draft minutes of 90th meeting held on 24 November 2005. Website accessed Feb. 2006. 40. Taylor DM. Resistance of TSE agents to decontamination. In: Rabenau HF, Cinatl J, Doerr HW, eds. Prions. A challenge for science, medicine and the public health system. Contrib Microb. Basel Karger 2004; 11: 136-145. 41. Valleron AJ, Boelle PY, Will RG, Cesbron JY. Estimation of epidemic size and incubation time based on age characteristics of vCJD in the United Kingdom. Science 2001; 294: 1726-1728.
Note added in Proof (June 2006)
The PRNP genotype of two of the three appendix tissue samples that tested positively for PrPSc in the large retrospective prevalence study referred to in this paper has now been determined [1]. The genotype in both is confirmed as homozygous for the valine allele (VV) at codon 129 of the PRNP gene. There was insufficient material to test the third sample. This is the first indication that valine homozygotes may be susceptible to vCJD infection and shows now that all PRNP codon 129 genotypes may be susceptible to vCJD infection, which was not known before.
1. Ironside JW, Bishop MT, Connolly K, Hegazy D, Lowrie S, Le Grice M, Ritchie D, McCardle LM, Hilton DA. Variant Creutzfeldt-Jakob Disease: a prion protein genotype analysis of positive appendix tissue samples from a retrospective prevalence study. BMJ 2006; 332: 1186-1188.


Bradley et al state ;

> No new challenges for the European population are

> apparent from cattle even though a variant form of

> BSE (bovine amyloidotic spongiform encephalopathy,

> (BASE)) has been detected in a small number of

> animals in Italy [6].

THIS is an absurd statement, with gross negligence, in my opinion, if you consider the rest of the story. what Bradley et al fail to mention with the study of BASE, is a most important factor to this equation ;

BASE in cattle in Italy of Identification of a second bovine amyloidotic spongiform encephalopathy: Molecular
similarities with sporadic Creutzfeldt-Jakob disease

Medical Sciences
Identification of a second bovine amyloidotic spongiform encephalopathy: Molecular similarities with sporadic Creutzfeldt-Jakob disease

Cristina Casalone *, Gianluigi Zanusso , Pierluigi Acutis *, Sergio Ferrari , Lorenzo Capucci , Fabrizio Tagliavini ¶, Salvatore Monaco ||, and Maria Caramelli *
*Centro di Referenza Nazionale per le Encefalopatie Animali, Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta, Via Bologna, 148, 10195 Turin, Italy; Department of Neurological and Visual Science, Section of Clinical Neurology, Policlinico G.B. Rossi, Piazzale L.A. Scuro, 10, 37134 Verona, Italy; Istituto Zooprofilattico Sperimentale della Lombardia ed Emilia Romagna, Via Bianchi, 9, 25124 Brescia, Italy; and ¶Istituto Nazionale Neurologico "Carlo Besta," Via Celoria 11, 20133 Milan, Italy

Edited by Stanley B. Prusiner, University of California, San Francisco, CA, and approved December 23, 2003 (received for review September 9, 2003)

Transmissible spongiform encephalopathies (TSEs), or prion diseases, are mammalian neurodegenerative disorders characterized by a posttranslational conversion and brain accumulation of an insoluble, protease-resistant isoform (PrPSc) of the host-encoded cellular prion protein (PrPC). Human and animal TSE agents exist as different phenotypes that can be biochemically differentiated on the basis of the molecular mass of the protease-resistant PrPSc fragments and the degree of glycosylation. Epidemiological, molecular, and transmission studies strongly suggest that the single strain of agent responsible for bovine spongiform encephalopathy (BSE) has infected humans, causing variant Creutzfeldt-Jakob disease. The unprecedented biological properties of the BSE agent, which circumvents the so-called "species barrier" between cattle and humans and adapts to different mammalian species, has raised considerable concern for human health. To date, it is unknown whether more than one strain might be responsible for cattle TSE or whether the BSE agent undergoes phenotypic variation after natural transmission. Here we 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.


C.C. and G.Z. contributed equally to this work.

||To whom correspondence should be addressed.


THIS _is_ the title to the study and Bradley et al removed it and stated ;

> No new challenges for the European population are

> apparent from cattle

AGAIN, this was an absurd statement with gross negligence, with severe ramifications for public health, in my opinion, deliberately done to obscure the truth, and or, the rest of the story.

NEXT, just look at the dramatic rate of sporadic CJD in Italy from 27 in 1993 to 95 in 2005, the highest documented country for sCJD that year ;

FURTHERMORE, Bradley et al fail to be concerned with this study by Collinge, Asante, et al ;

BSE prions propagate as either variant CJD-like or

sporadic CJD-like prion strains in transgenic mice

expressing human prion protein

The EMBO Journal Vol. 21 No. 23 pp. 6358±6366, 2002

Emmanuel A.Asante, Jacqueline M.Linehan,

Melanie Desbruslais, Susan Joiner,

Ian Gowland, Andrew L.Wood, Julie Welch,

Andrew F.Hill, Sarah E.Lloyd,

Jonathan D.F.Wadsworth and

John Collinge1

MRC Prion Unit and Department of Neurodegenerative Disease,

Institute of Neurology, University College, Queen Square,

London WC1N 3BG, UK

1Corresponding author


Variant Creutzfeldt±Jakob disease (vCJD) has been

recognized to date only in individuals homozygous for

methionine at PRNP codon 129. Here we show that

transgenic mice expressing human PrP methionine

129, inoculated with either bovine spongiform

encephalopathy (BSE) or variant CJD 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

from that of sporadic CJD with PrPSc

type 2. These data suggest that more than one BSEderived

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 exposure. ...

The EMBO Journal Vol. 21 No. 23 pp. 6358±6366, 2002

6358 ãEuropean Molecular Biology Organization

WE have atypical BSE showing up in other countries besides Italy. The cow in Texas and Alabama we now know were atypical BSE. Japan and Belgium also have atypical BSE in cattle. WE also have atypical scrapie showing up in countries, with Stanley Prusiner warning ;

> These observations support the view that a truly infectious TSE agent, unrecognized until recently, infects sheep and goat flocks and may have important implications

> in terms of scrapie control and public health.

Published online before print October 20, 2005

Proc. Natl. Acad. Sci. USA, 10.1073/pnas.0502296102
Medical Sciences

A newly identified type of scrapie agent can naturally infect sheep with resistant PrP genotypes

( sheep prion | transgenic mice )

Annick Le Dur *, Vincent Béringue *, Olivier Andréoletti , Fabienne Reine *, Thanh Lan Laï *, Thierry Baron , Bjørn Bratberg ¶, Jean-Luc Vilotte ||, Pierre Sarradin **, Sylvie L. Benestad ¶, and Hubert Laude *
*Virologie Immunologie Moléculaires and ||Génétique Biochimique et Cytogénétique, Institut National de la Recherche Agronomique, 78350 Jouy-en-Josas, France; Unité Mixte de Recherche, Institut National de la Recherche Agronomique-Ecole Nationale Vétérinaire de Toulouse, Interactions Hôte Agent Pathogène, 31066 Toulouse, France; Agence Française de Sécurité Sanitaire des Aliments, Unité Agents Transmissibles Non Conventionnels, 69364 Lyon, France; **Pathologie Infectieuse et Immunologie, Institut National de la Recherche Agronomique, 37380 Nouzilly, France; and ¶Department of Pathology, National Veterinary Institute, 0033 Oslo, Norway

Edited by Stanley B. Prusiner, University of California, San Francisco, CA, and approved September 12, 2005 (received for review March 21, 2005)

Scrapie in small ruminants belongs to transmissible spongiform encephalopathies (TSEs), or prion diseases, a family of fatal neurodegenerative disorders that affect humans and animals and can transmit within and between species by ingestion or inoculation. Conversion of the host-encoded prion protein (PrP), normal cellular PrP (PrPc), into a misfolded form, abnormal PrP (PrPSc), plays a key role in TSE transmission and pathogenesis. The intensified surveillance of scrapie in the European Union, together with the improvement of PrPSc detection techniques, has led to the discovery of a growing number of so-called atypical scrapie cases. These include clinical Nor98 cases first identified in Norwegian sheep on the basis of unusual pathological and PrPSc molecular features and "cases" that produced discordant responses in the rapid tests currently applied to the large-scale random screening of slaughtered or fallen animals. Worryingly, a substantial proportion of such cases involved sheep with PrP genotypes known until now to confer natural resistance to conventional scrapie. Here we report that 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 that they shared unique biological and biochemical features upon propagation in mice. These observations support the view that a truly infectious TSE agent, unrecognized until recently, infects sheep and goat flocks and may have important implications in terms of scrapie control and public health.


Author contributions: H.L. designed research; A.L.D., V.B., O.A., F.R., T.L.L., J.-L.V., and H.L. performed research; T.B., B.B., P.S., and S.L.B. contributed new reagents/analytic tools; V.B., O.A., and H.L. analyzed data; and H.L. wrote the paper.

A.L.D. and V.B. contributed equally to this work.

To whom correspondence should be addressed.

Hubert Laude, E-mail:


Full Text
Diagnosis and Reporting of Creutzfeldt-Jakob Disease
Singeltary, Sr et al.
JAMA.2001; 285: 733-734

HUMAN and ANIMAL TSE Classifications i.e. mad cow
disease and the UKBSEnvCJD only theory

TSEs have been rampant in the USA for decades in many
species, and they all have been rendered and fed back
to animals for human/animal consumption. I propose that
the current diagnostic criteria for human TSEs only
enhances and helps the spreading of human TSE from the
continued belief of the UKBSEnvCJD only theory in 2005.
With all the science to date refuting it, to continue
to validate this myth, will only spread this TSE agent
through a multitude of potential routes and sources
i.e. consumption, surgical, blood, medical, cosmetics
etc. I propose as with Aguzzi, Asante, Collinge,
Caughey, Deslys, Dormont, Gibbs, Ironside, Manuelidis,
Marsh, et al and many more, that the world of TSE
Tranmissible Spongiform Encephalopathy is far from an
exact science, but there is enough proven science to
date that this myth should be put to rest once and for
all, and that we move forward with a new classification
for human and animal TSE that would properly identify
the infected species, the source species, and then the
route. This would further have to be broken down to
strain of species and then the route of transmission
would further have to be broken down. Accumulation and
Transmission are key to the threshold from subclinical
to clinical disease. However key to all
this, is to stop the amplification and transmission of
this agent, the spreading of, no matter what strain.
BUT, to continue with this myth that the U.K. strain of
BSE one strain in cows, and the nv/v CJD, one strain in
humans, and that all the rest of human TSE is one
single strain i.e. sporadic CJD (when to date there are
6 different phenotypes of sCJD), and that no other
animal TSE transmits to humans, to continue with this
masquerade will only continue to spread, expose, and
kill, who knows how many more in the years and decades
to come. ONE was enough for me, My Mom, hvCJD, DOD
12/14/97 confirmed, which is nothing more than another
mans name added to CJD, like CJD itself, Jakob and
Creutzfeldt, or Gerstmann-Straussler-Scheinker
syndrome, just another CJD or human TSE, named after
another human. WE are only kidding ourselves with the
current diagnostic criteria for human and animal TSE,
especially differentiating between the nvCJD vs the
sporadic CJD strains and then the GSS strains and also
the FFI fatal familial insomnia strains or the ones
that mimics one or the other of those TSE? Tissue
infectivity and strain typing of the many variants of
the human and animal TSEs are paramount in all variants
of all TSE. There must be a proper classification that
will differentiate between all these human TSE in order
to do this. With the CDI and other more sensitive
testing coming about, I only hope that my proposal will
some day be taken seriously.

My name is Terry S. Singeltary Sr. and I am no
scientist, no doctor and have no PhDs, but have been
independently researching human and animal TSEs since
the death of my Mother to the Heidenhain Variant of
Creutzfeldt Jakob Disease on December 14, 1997
'confirmed'. ...TSS

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

Follow Ups:

Post a Followup

E-mail: (optional)


Optional Link URL:
Link Title:
Optional Image URL: