NR ARXG
AU Weissmann,C.; Collinge,J.; Almond,J.W.; Dormont,D.; Kretzschmar,H.A.; Pocchiari,M.; Wierup,M.
TI Position paper by EC Group on BSE research
QU EC880928 report 1996 Oktober 5
PT Report
VT
POSITION PAPER BY EC GROUP ON BSE RESEARCH
(WEISSMANN REPORT)
OFFICIAL VERSION
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Position paper by EC Group on BSE research
I. EXECUTIVE SUMMARY
Introduction: Many questions regarding BSE are as yet unanswered and perhaps unasked. Because of the slow nature of the disease, it will take years to obtain answers and it may be countered that by the time the answers are known, the disease may have been eradicated. However, these predictions are based on assumptions which do not necessarily hold true, either in the UK or elsewhere, such as the immutability of the infectious agent and its mode of transmission. Because BSE could become endemic, as is scrapie, and because it might be transmissible to man, as scrapie is not, we consider it advisable to collect as much critical information as possible at the present time.
The following questions are addressed:
A. Questions related to practical aspects of BSE
1. Has BSE been transmitted to man perorally?
Epidemiological evidence has raised the suspicion that so-called variant Creutzfeldt-Jakob disease is linked to the consumption of products derived from BSE-infected cattle. Moreover, the BSE agent is orally transmissible to mice, mink, cats, cattle, sheep, goats and various other ruminants.
Proposals:
---> Compare agent strains recovered from vCJD patients with BSE and "normal" CJD, GSS and FFI strains.
---> Extend surveillance program for CJD, monitor for linkage to consumption of cattle and sheep products
---> Primate oral dose response experiment (see below).
2. Risk assessment
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Assuming that transmission of BSE from cattle to human has occurred, a number of as yet largely unknown parameters must be determined to allow the estimation of the risk of transmission.
Proposals:
---> a. Extend surveillance program for BSE to all of Europe.
---> b. Determine incidence of covert disease in cattle.
---> c. Determine infectivity titer of brain, spinal cord and other tissues of BSE-infected cattle before and after onset of clinical disease.
---> d. Determine contamination level of meat with brain/spinal cord after standard butchering procedures.
---> e. Determine oral and i.c. dose response to BSE agent in primates.
---> f. Determine whether multiple dosing is cumulative.
To estimate the past risk it is necessary to determine
---> g. The utilization of offal in different human food products prior to the ban and the consumption of such products by individual age groups and socio-economic criteria.
3. Is there infectivity in BSE-infected cattle-derived products other than SBO ?
Infectivity has been found in brain and spinal cord of diseased cattle, and in the intestinal wall of calves infected orally with BSE brain. Other tissue showed no infectivity, however the assays, carried out in the mouse, are very insensitive.
Proposals:
---> Implement sensitive assay, examine all products entering the human food chain (muscle, milk, cheese, blood etc.) and used in pharmaceutical products (collagen, gelatin, etc.).
4. Maternal risk factors for BSE in cattle
Recent data from the MAFF shows that offspring from BSE-sick dams have a higher incidence of BSE than controls (both were exposed to contaminated feed). The precise reason for this apparent transmission is not known.
Proposals:
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---> Determine infectivity in placenta, blood, colostrum, milk, excrements of BSE cattle using sensitive assay.
5. Did BSE originate in cattle and has it been transmitted to sheep in recent years?
It is possible that BSE originated as a (rare) spontaneous event in cattle and that the BSE agent is different from the scrapie agent. If so, then BSE may have been transmitted to sheep via bone and meat meal from cattle and BSE infected sheep could transmit the agent to man.
Proposals:
---> Extend surveillance of scrapie in sheep to the EU.
---> Examine the infectious agent derived from recent UK cases of sheep "scrapie" in regard to its strain properties.
---> Feed scrapie-infected brain from UK sheep to cattle and monitor for appearance of BSE-like disease.
6. Can BSE be transmitted orally to pigs ?
Pigs can be infected with BSE by intracerebral inoculation. Peroral transmissibility of BSE from BSE cattle brain to pigs is being tested by the MAFF in the UK. However, these experiments should be complemented to ascertain whether BSE derived from experimentally BSE-infected pigs can be transmitted to pigs:
---> Feed and inject brain extract of experimentally BSE-infected pig to pigs.
7. Diagnostic Research
There is still no reliable, sensitive method to detect CJD or BSE other than by analysis of CNS tissue by infectivity assays.
Proposals:
---> Develop a sensitive assay in transgenic mice carrying cattle transgenes or cattle/mouse chimeric PrP genes.
---> Explore surrogate markers for BSE.
---> Explore the presence of PrPsc in tonsils and spleen of man.
---> Sponsor a large-scale facility for assaying BSE infectivity in calves.
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---> Institute strain typing of human-derived prions (CJD, GSS, FFI, BSE-derived), correlate disease susceptibility with genetic markers.
8. Inactivation of the BSE agent under different conditions
There is a need for determining the efficiency of BSE agent inactivation by procedures used in food and pharmaceutical industry.
Proposals:
---> Determine inactivation kinetics of BSE infectivity under conditions used currently in industry and explore additional procedures.
9. Therapeutic Research
Possible approaches to therapy are specific inhibition of PrP synthesis, or prevention of the conversion of PrPc into the pathological form PrPsc.
Proposals:
---> Develop rapid throughput assay for inhibition of PrP synthesis and PrPc-->PrPsc conversion respectively; assay chemical libraries.
10. Predictive research
vCJD and other forms of prion disease occur very rarely in individuals heterozygous for the methionine-valine heterozygosity at position 129. A PCR test for the 129 haplotype could be used to assess the susceptibility of individuals for vCJD
---> Monitor haplotype of vCJD patients, develop routine PCR assay
11. Can BSE-resistant cattle and sheep be generated ?
Inactivation of the PrP gene has no major detrimental effect on the mouse and confers absolute protection against the mouse-adapted scrapie. The same effect might be achieved for sheep and cattle.
Proposals:
---> Determine whether sheep devoid of PrP are viable and resistant to scrapie.
---> Develop methods for generating cattle devoid of PrP.
B. QUESTIONS RELATED TO BASIC RESEARCH ON PRION DISEASE
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1. Nature of the infectious agent.
Proposals:
---> Purify infectious agent from BSE-infected cattle brain and other sources and analyze.
---> 3 D structure of complete PrPc (PrP-sens) from different sources.
---> 3 D structure of PrPsc.
2. Multiplication of the agent.
Proposals:
---> Determine whether PrPc can be converted into the infectious agent (rather than only to PrPsc) in vitro.
---> Determine which other components are required for propagation of the infectious agent.
3. Pathogenesis
Proposals:
---> Determine mechanism of pathogenesis.
4. Transport of infectious agent
Proposals:
---> Determine how the agent moves from periphery to CNS and vice-versa.
5. Strains
- Proposals:
---> Elucidate the molecular feature of the infectious agent that determines strain specificity.
6. Susceptibility of the host to the infectious agent
Proposals:
---> Determine which genetic factors contribute to susceptibility ?
---> Determine which factors other than the sequence of PrP determines the species barrier ?
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II. DETAILED DISCUSSION
A. Questions related to practical aspects of BSE
1. Has BSE been transmitted to man perorally?
Arguments in favor :
(a) Epidemiological evidence: The occurrence since 1994 of nine vCJD cases at ages 34 years or less in the UK and one in France) and none between 1990 and 1993 (despite intensive surveillance) is statistically highly significant on the basis of age (the odds are less than 1:10^7 reference D.Morrison]). vCJD shows a characteristic histopathology, in particular high incidence of PrP positive plaques. No archival cases of vCJD as characterized by histopathology have been found in recent searches 1.
Because BSE originated in the UK in the mid-eighties and has an incidence about 2 orders of magnitude higher than in any other country, a causal connection between BSE and vCJD is suspected.
(b) Experimental evidence: The BSE agent is experimentally perorally transmissible to mice, mink, cattle, goats and sheep. Strain specific properties of BSE agent are retained after passaging through a variety of animals such as sheep, pig, goats or mouse and differ from those of passaged scrapie agent. An agent indistinguishable from BSE agent has been isolated from cases of "naturally" occurring TSEs in domestic cats, kudu and nyala which are attributed to feeding of contaminated bone and meat meal. Experimental transmission of BSE to primates by intracerebral injection has been reported (Baker et al., 1993a: Lasmézas et al., 1996) and the appearance of vCJD-like neuropathology in macaques was noted (Lasmézas et al., 1996). However, there have been no experimental feeding experiments.
Scrapie agent experimentally transmitted from U.S. sheep to cattle caused a disease which is unlike BSE; strain typing is in progress (R.B., K.B., pers. commun.). If scrapie prions and BSE prions represent distinctly different strains, the argument cannot be upheld that the BSE agent is
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1 Reported at the meeting of the EU Concerted Action on Neuropathology of Prion Diseases, Vienna, May 1996)
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not transmissible to man because it is identical with the scrapie agent (believed not to be transmissible to man on the basis of epidemiological data).
Arguments against:
(a) Mice containing human PrP transgenes as well as the mouse PrP wild-type background, when inoculated with BSE came down with scrapie-like disease but only PrPsc of murine and not of human origin was detected, suggesting that human PrPc was not being converted to PrPsc (Collinge, 1995 #3681). This experiment was not conclusive because there may be competition between human and murine PrPc in the conversion process (Telling et al., 1994). Moreover, the human transgene used had valine at position 129; the methionine allele should also be tested (underway in Collinge's lab).
Mice transgenic for the human PrP gene on a mouse PrP knockout background were inoculated with BSE agent (30 µl of 1% medulla homogenate) and have not come down with prion disease 500 days after inoculation; inoculation with human CJD agent leads to prion disease after 200 days. This experiment is more persuasive than the first one if the mice remain free of prion disease permanently. However, differences in the PrP gene of humans may affect their susceptibility to BSE prions. Initiation of BSE disease in mouse may require more than just the presence of "compatible" PrPc. Moreover, it has to be considered that the nature of the cattle prions may vary with the PrP gene sequence of the donor (for example, in regard to the number of octa repeats) and hence differ in their transmissibility to humans.
What experiments can cast more light on this question?
---> Determination of the strain-specific properties of agent from the variant human CJD cases and the vCJD-like disease elicited in macaques (Lasmézas et al., 1996) after transmission to mouse. Such experiments are underway in the laboratory of John Collinge (with mice carrying the human PrP transgene) and in NPU (with normal mice).
---> Determine whether infectious agent from vCJD or sporadic CJD cause BSE-like disease in cattle.
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---> Extend surveillance program for CJD, monitor for linkage to beef and mutton consumption.
---> Experimental peroral transmission of BSE to primates (see below).
2. Assessment of risk associated with consumption of cattle-derived products
One of the most important questions to be resolved is assessment of risk for the transmission of BSE to man as a function of BSE incidence in the cattle population and of consumption of cattle-derived products. The parameters required for assessment are:
a. The frequency of cattle suffering from overt BSE.
b. The frequency of clinically healthy cattle carrying BSE agent .
c. The infectivity titer in BSE-sick cattle of brain and spinal cord (these data are available of clinically ill animals 2) and of other tissues (these data are in part available but should be determined at higher sensitivity).
d. The decontamination level of meat with brain/spinal cord after standard butchering procedure.
e. The minimal infectious BSE dose for man; the best estimate will be from a primate oral dose response experiment. In this context we recommend elucidating whether peroral administration of brain from sheep experimentally infected with BSE causes disease.
f. The effect of repeated dosing with BSE agent.
To estimate the past risk it is necessary to determine:
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2 Infectivity titer of BSE-infected brain and spinal cord (data: Wells, GAH, CVL, unpublished; CVO report for 1995):
BSE brain: 10^6 to 1 0^7 infectious units/g as assayed intracerebrally in calves
10-100 infectious units/g as assayed orally
3-4 logs less when assayed intracerebrally in mouse
(0.02 g insufficient to infect mouse perorally).
Oral administration is usually 10^5 times less effective than i.c. administration (for calves), however in transspecies infection the relative efficiency of oral administration is increased (Fraser and Foster, 1994).
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g. The utilization of offal in different human food products prior to the ban and the consumption of such products by individuals of different age and socio-economic status.
To answer these questions, the following actions need to be implemented:
---> a. Extend surveillance program for BSE.
Institute EC-wide reliable, standardized criteria for BSE. Include search for possible occurrence of "spontaneous" BSE. Search for possible cofactors 3. Determine whether the apparent incidence of BSE in European countries other than the UK is lower than expected on the basis of cattle and feed imported from the UK 4.
---> b. Determine incidence of covert disease in cattle
It has been shown in the mouse that high titers of scrapie infectivity and PrPsc can be measured months before any clinical symptoms are evident, particularly when PrP is expressed at a low level (Büeler et al., 1994). The same is likely true for cattle 5. It is thus important to determine in several herds where there have been one or more cases of BSE (and if possible also herds, where no animals have fallen sick), the number of animals that show infectious agent in their CNS. To this purpose, brain stem homogenates should be analyzed for their content of infectivity at several dilutions and of PrPsc (by different assays: Western, ELISA or whatever is available at the time). In this connection the importance of establishing a reliable, sensitive and rapid assay for BSE should be reiterated.
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3 It has been proposed that nematode infection promotes scrapie in sheep (Paris Meeting 1996) and that mites can carry scrapie agent (Wisniewski et al., 1996).
4 It is said that in GB about 30% of "suspected BSE" cases turn out to be due to metabolic or other causes. However, in some other countries not even any "suspected" cases are reported, suggesting that surveillance could be improved.
5 Narang has claimed in a recent review (Narang, 1996) that 29% of cattle brain (8 of 27 brains from an abattoir examined at random) were positive by his "SAF touch assay"; this assay has been checked by Stack et al.(Stack et al., 1995) as mentioned in a report from Dr.Bradley ("Advice to the Minister") and is said to state that there were no false positives among 5 healthy cattle and that 2 (or perhaps 3) of 5 BSE cattle were correctly diagnosed.
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The cattle (clinically healthy, from affected and non-affected herds) to be targeted:
Animals < 30 months,
Targeted cull animals,
Old dairy cows > 30 months,
Random animals coming to slaughter.
---> c. Determine infectivity titer of brain, spinal cord and other tissues of BSE-infected cattle before and after onset of clinical disease.
The data for clinically ill animals are available1 and will become available for experimentally infected cattle at various times after infection (MAFF). Infectivity measurements of other tissues of BSE-infected cattle are in part available but should be determined at higher sensitivity (See below, section 3).
The titer of a BSE agent preparation administered i.c. and orally should be compared in a variety of non-primate species.
---> d. Determine contamination level of meat with brain/spinal cord after standard butchering procedures.
To what extent can BSE agent derived from brain and spinal cord contaminate meat, in particular "head meat", during processing? Carcasses are decapitated and split longitudinally, opening the spinal canal; does this result in significant contamination? It should be possible to estimate the extent of contamination by "mock-up" experiments in which the spinal cord and brain are stained by a fluorescent dye 6 and the amount of fluorescent material deposited on meat cuts following standard butchering procedure is determined. Moreover, it has been claimed (Garland et al., 1996) that slaughtering with use of bolts may lead to brain emboli in the lung; could microemboli make their way to the periphery? What is the
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6 for example the fluorescent lipid probe rhodamine phosphatidyl ethanolamine. Radioactive probes may be less sensitive and more difficult to use and measure.
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effect of stunning and pithing ? Devise a test for these conditions of slaughter.
---> e. Determine oral and i.c. dose response to BSE agent in primates 7
Although experimentation on primates may meet with considerable opposition 8 on both ethical and economic grounds, and despite the objection that oral susceptibility data obtained with primates may not reflect susceptibility of humans, we believe this is the best approach to obtaining these data, which will be of greatest importance if more vCJD cases were to appear and if BSE were to become endemic. If and when this were to happen, it may well be considered a serious omission if no effort bad been made to collect the best possible data.
So far, it has been shown that marmosets inoculated with BSE brain homogenate (i.p. and i.c. simultaneously) came down with disease after 4 years (Baker et al., 1993b). Lasmézas et al. inoculated 3 cynomoulgus macaques with BSE brain homogenate i.c.; the macaques showed clinical disease after 4 years and presented histopathology similar to that of variant CJD (Lasmézas et al., 1996).
The experiments should comprise the following parts:
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7 There are at least six primate centers in which the work could in principle be done:
TNO (Holland)
Porton Down; 100 monkeys, 20 macaques under P3 conditions
Göttingen: 18 animals under P3, 36 under P2 conditions
Rome: 24-48 squirrel monkeys under P3 conditions
Lyon
Zürich
Porton Downs facilities could be upgraded to P3. Perhaps the experiment would have to be spilt between several centers.
Estimated cost: purchase of monkey ca. Sfr. 3000,-
upkeep: 365x12= Sfr. 4'400/yr
Assuming 100 monkeys have to be kept for 10 years:
10 x 4'400 x 100 = Sfr 4.4 million
8 Three consultants from the UK considered primate feeding experiments without sufficient merit to warrant the expense and effort of implementation.
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(i) Determination of the titer of a BSE agent preparation 9 by end point dilution in mice. This should be done early on.
(ii) The dose response to BSE cattle brain homogenate administered by intracerebral injection (about 40 monkeys) and peroral administration10 is determined using about 6 different doses, 6 monkeys each 11. As controls, guaranteed healthy brain extracts (10 monkeys) are to be used at the highest dosage.
(iii) Dose response as above using brain of New Zealand sheep (in which scrapie seems not to occur or UK sheep with a scrapie-resistant genotype infected with BSE (about 20 monkeys). Inoculation of sheep with BSE, preferably orally, must be initiated early an (some sheep have been inoculated at MAFF: are they sufficient?).
-Choice of primate: The primate should be known to be susceptible to BSE by parenteral inoculation (CJD has been transmitted to squirrel monkey, macaque, chimpanzees and others), have a PrP sequence as close to the human as possible (although the importance of this parameter is not clear; only a few residues may be critical for the species barrier, and other, as yet unknown factors may be just as
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9 A homogenate of BSE-infected brain stem (50 g each; titer: 10^3 to 10^5 LD5O bovine units/g). It is important that sufficient BSE brain stems be collected, pooled and aliquoted as soon as possible, because this invaluable material will become scarce as the epidemic subsides (inquiry has been addressed to Mr. Meldrum at MAFF and Mr.Schapps at the EC). The samples should be sterile: it should be ascertained early on whether heating at 80C causes significant loss of infectivity.
10 There is some question as to whether the homogenate should be administered by stomach tube or by first starving the monkeys and then letting them feed autonomously (J.Gibbs has stated that with CJD or Kuru transmission occurred only in the latter case [pers.commun.]). Although it would seem from the experiment on cattle that orally administered BSE agent entered through the gut, it is not clear whether this only occurs generally or only under special circumstances, such as lesions in the Gl tract (see report that nematodes increase incidence of scrapie in sheep; Paris 1996).
11 There will be no systematic multiple dosing experiments at this time; only where very large doses are to be administered, the material can be given in two or more sequential sessions.
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important), but availability, ease of maintenance and monitoring and cost will be important considerations 12.
It is to be discussed whether the experiment should (and could) be done with bath squirrel monkeys and macaques; the inclusion of a small number of chimpanzees should be considered.
The PrP genes of the experimental animals should be genotyped.
-Monitoring:
Inspection for ataxia, EEG. Infectivity in tonsil biopsy.
Assay for surrogate markers in CSF (14-3-3 protein) (Hsieh et al., 1996).
Sacrifice 2 animals of each group after 5 years and remaining animals after 10 years and determine infectivity in brain, spinal cord, spleen.
Determine whether BSE strain specificity is retained.
---> f. Determine whether multiple dosing is cumulative.
In order to examine this important question, the end point titer of an agent preparation in a particular host must be known; therefore, the experiment cannot be carried out on primates at this time. However, it can and should be carried out on the mouse or hamster.
---> g. Determine the utilization of offal in different human food products prior to the ban.
A survey of the industry in regard to the utilization in Europe and the UK in particular may be difficult because of fear of liability claims.
A survey in Australia or New Zealand may be more likely to yield information.
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12 Tamarins (squirrel monkeys, marmosets), 200-500g, life expectancy 10-15 y, very sensitive, difficult to handle. Macaques (rhesus mulatta, cynomolgus fascicularis), 2-8 kg, life expectancy 20-25 y, robust.
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The consumption of offal-containing products by individual age groups and socio-economic criteria for .the period 1985-1990 and 1990 - present should be ascertained as accurately as possible.
3. Is there infectivity in BSE-infected cattle-derived products other than SBO ?
Infectivity has been found in brain, eye and spinal cord of diseased cattle, and in the intestinal wall of calves experimentally infected perorally with brain from BSE-infected cattle.
Other tissues, in particular muscle, spleen, lymph nodes, peripheral nervous system showed no infectivity, however the assays were carried out in the mouse. Moreover, the tissue was obtained from cattle sacrificed by barbiturate injection and not by the usual slaughtering procedures, which might result in microemboli of brain tissue in the periphery. In late-stage scrapie-infected sheep, peripheral organs had titers 10-100 times lower than brain.
If infectivity determination is carried out in the mouse, the volume that can be injected into the brain is small (about 0.025 ml). If for example milk contained 100 infectious units per liter, about 1800 mice would have to be injected intracerebrally in order to have a 95% probability of finding one sick mouse. There is an ongoing epidemiological analysis of suckled offspring from BSE-infected dams however the results are not yet available.
A similar question may be raised in the case of blood, nerve tissue, or in fact any tissue. It may be argued that if the infectivity is so low, then it is also of no concern for human health. On the basis of current knowledge this sounds reasonable, but this assessment lacks a scientific basis without an estimate of the LD50 for man.
---> The most sensitive assays available, i.e. intracerebral administration into calves, should be implemented for bovine products used for human consumption 13. Possibly intraperitoneal
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13 A facility appropriate for such assays may become available in the Padua region (pers.commun. M.Pocchiari).
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injection may prove to be more sensitive 14. Simultaneous i.c. and i.p. has been used by MAFF.
Products from BSE-infected cattle to be assayed:
-mechanically recovered meat
-Colostrum, cells concentrated from milk and colostrum
-Blood, cells concentrated from blood
-Skeletal and heart muscle, heart valves
-Rennet
-Collagen, gelatin
-lung
4. Maternal risk factors for BSE in cattle
Recent data from the MAFF demonstrated a maternal risk factor in cattle. It is not clear whether one is dealing with actual agent transmission, as proposed, or with genetically based susceptibility to, for example, bone and meat meal. The experimental data suggested a risk of 10% in the cattle studied, where all calves were born within 13 months of clinical onset of BSE in the dams. It was extrapolated that the risk is 1% for herds in general and that this factor alone will not lead to endemic BSE in the UK. This might not be true if there is transmission from dams with subclinical disease, if the agent mutates to a more easily transmissible form or if there are or will be modes of transmission we are currently unaware of.
Placenta as assayed in the mouse is negative. This may not have been sufficiently sensitive to exclude transmission in utero.
---> Assay placenta, colostrum, milk in an i.c. calf assay. Can there be transmission through saliva (the agent is found in salivary gland of mouse, mink and goat) or through excrements?
---> Determine infectivity in saliva?
- in excrements 15
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14 Although intraperitoneal injection is reportedly 2 orders of magnitude less efficient than intracerebral injection, it may be possible to administer 3 orders of magnitude larger volumes (500 ml or more in repeated injections i.p. versus 0.5ml i.c.).
15 Sterilize by radiation, filter through 0.2-µm Millipore filters which do not retain scrapie agent (pers.comm. Dr.Pocchiari), inject intraperitoneally.
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5. Did BSE originate in cattle and has it been transmitted to sheep in recent years?
Three possibilities for the origin of BSE may be considered. (a) scrapie-infected sheep, (b) a (rare) spontaneous (sporadic) event in cattle (Chastel, 1996) akin to CJD in humans, or (c) a change of strain of an endemic, symptomless form of BSE. In all cases spread of the disease is attributed to introduction of contaminated tissue into cattle feed, perhaps in connection with a change in the rendering procedure. Spontaneous occurrence of spongiform encephalopathy (Creutzfeldt Jakob disease) in man is of course well documented, and it has been proposed that kuru, an epidemic spongiform encephalopathy that occurred in Papua New Guinea, may have originated from a spontaneous case of CJD and was spread by the consumption or handling of brain tissue of deceased family members. A spontaneous case of spongiform encephalopathy in a rhesus monkey has been reported (Bons et al., 1996), however infection by feeding is not excluded.
The arguments in favor of origin in cattle are (1) the unique properties of the BSE-derived agent, namely: (a) speed of transmission to mice and other animals, (b) lesion pattern in mice, (c) incubation times in 4 strains of mice, and (2) failure to generate BSE-like disease by feeding/ inoculating cattle with scrapie-infected sheep brain (but there was spongiform encephalopathy) (Cutlip et al., 1994), whereas feeding with BSE-infected cattle brain causes typical BSE in cattle.
In favor of origin from scrapie-infected sheep the following arguments have been advanced (1) epidemiological: the disease appeared more or less simultaneously in various distant sites in the UK while feed was not widely distributed (Wilesmith, 1992b); (2) appearance of BSE occurred a few years after a change in the procedure for processing offal was instituted (Wilesmith, 1992a).
If BSE originated in cattle (or even if it is a mutated form of an agent that originally came from sheep) and is transmissible to man, its transmission to sheep might well generate a disease different from scrapie, possibly with the transmission properties of BSE, i.e. possibly transmissible to man. Cheviot sheep infected with BSE show scrapie-like symptoms; the emerging agent has BSE-like properties when tested in mouse.
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Because cattle-derived animal feed has been fed to sheep in the past (the practice is forbidden in the UK since 1988 but cross-contamination of ruminant rations has been likely until April 1996), transmission may already have occurred; if so, it might be maintained within the sheep population as is scrapie.
---> Surveillance of scrapie in sheep of the UK and EU; offer compensation for reported cases in EU; correlate susceptibility to scrapie and to BSE with genotype (natural scrapie is almost always associated with the sA/sA genotype (Goldmann et al., 1994)).
---> Examine the infectious agent derived from recent UK cases of sheep "scrapie" in regard to its strain properties. Because sheep may be slaughtered to early for clinical symptoms to be observed, It may be necessary to monitor a large sampling of sheep brain for PrPsc by an immunoassay and test infectivity in positive cases. In order to reduce the large number of samples that may have to be investigated, sheep with a genotype that renders them non-susceptible to natural scrapie should be preferentially examined.
---> Feed scrapie-infect brain from UK sheep to cattle and monitor for appearance of BSE-like disease.
6. Can BSE be transmitted orally to pigs and chickens?
Pigs inoculated with BSE agent i.c., i.p. and i.v. (simultaneously) acquire disease, with infectivity (as detected by the mouse i.c. assay) in brain, jejunum, distal ileum; marginally in stomach and pancreas. Oral administration of BSE brain to pigs has not given rise to disease; no infectivity was found in ileum and spleen after 2 years (G.A.H. Wells, as communicated by R. Bradley). Inoculation of chickens with BSE i.c. has so far not given signs of disease. It would seem that no immediate action is required on these issues, but it is of interest to determine whether pig-to-pig transmission occurs, in particular orally, because if a pig at same time became infected with BSE, the disease might be further transmitted by feeding of offal.
---> Feed and inject brain extract of experimentally BSE-infected pig to pigs.
7. Diagnostic Research
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There is still no rapid, specific and sensitive method to detect CJD or "BSE" in man. Histopathological and immunohistochemical analysis of CNS tissue is usually carried out post mortem. The most sensitive but very slow method is determination of infectivity. In spongiform encephalopathies of man, transmission to primates has been achieved with spinal cord, CSF, eye, lung, liver, kidney, spleen and lymph nodes with varying degrees of success (Brown et al., 1994). In the late stages of disease, appearance of protein 14-3-3 in cerebrospinal fluid may provide an approach (Hsieh et al., 1996). A polarographic method for urine analysis is underway. The specificity of these surrogate markers need to be determined carefully.
---> Develop a sensitive assay in transgenic mice carrying cattle transgenes or cattle/mouse chimeric PrP genes; such experiments are ongoing in S.Prusiner's lab, so far without positive results.
---> Explore surrogate markers mentioned above. A surrogate marker would be extremely valuable. Search for additional surrogate markers.
---> PrPsc is found in the CNS which, however, is not readily accessible in vivo, and in some species in spleen or tonsils at lower levels (Schreuder et al., 1996). Explore the presence of PrPsc in tonsils but also spleen of man. (In cattle, the BSE agent is not found in spleen and lymph nodes; no corresponding data for CJD in man is available).
---> In the absence of a reliable surrogate marker, infectivity assays have to be performed by i.c. inoculation in calves, currently the most sensitive assay for BSE agent. This is very costly and time consuming; it is being implemented on a larger scale by MAFF. The EC may want to sponsor a large-scale facility for assaying BSE in calves independently 12
---> Strain typing of human-derived prions (CJD, GSS, FFI, BSE-derived), correlation of disease susceptibility with genetic markers. Differentiation of BSE and scrapie agent 16.
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18 Differences in mobility of PrPres after digestion by a variety of proteases should be explored.
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8. Inactivation of the BSE agent under different conditions
There is a need for determining the efficiency of BSE agent inactivation by procedures used in food and pharmaceutical industry. This should not be considered a substitute for using BSE-free sources
---> Determine inactivation kinetics of BSE infectivity under conditions used currently in industry and explore additional procedures.
9. Therapeutic research
One could consider specific inhibition of PrP synthesis, because disease progression is clearly linked to PrPc content, or search for compounds preventing the postulated conversion of PrPc into the pathological form; such an effect has been reported for sulfated glycosaminoglycans, Congo Red, 4'-iodo-4'-deoxydoxorubicin, amphotericin B and some of its derivatives.
---> Develop rapid throughput assay for PrP synthesis and PrPc-->PrPsc conversion; assay chemical libraries, antisense oligonucleotides.
10. Predictive research
It has been shown that vCJD and other forms of prion disease occur very rarely in individuals heterozygous for the methionine-valine heterozygosity at position 129. The monitoring for this connection should be maintained. Linkage to other genetic markers should be sought for if there is an increase in vCJD. A PCR test for the 129 haplotype could be used to assess the susceptibility of individuals for vCJD, however the implications of the availability of such a test must be examined from the ethical, economic, insurance and legal point of view.
---> Monitor haplotype of vCJD patients, develop routine PCR assay
11. Can BSE-resistant, cattle and sheep be generated and would these be of practical use ?
It has been shown that inactivation of the PrP gene has no major detrimental effect on the mouse and that it confers absolute protection against the mouse-adapted scrapie (BSE is being tested in Dormont's lab) agent. The tools for gene disruption, namely omnipotent ES cell lines, have recently become available for sheep (Campbell et al., 1996),
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however not yet for cattle. It is therefore now possible to generate and test PrP knockout sheep for resistance to scrapie and BSE.
(Whether it is practical to envisage replacing existing flocks by absolutely prion-resistant ones remains to be determined, in view of the fact that many different breeds are required and that production of the required number of animals poses problems.
In the case of cattle, the preparation of ES cell lines and successful generation of calves derived from such lines has not yet been reported, however efforts are underway in ( several labs and biotech companies. Herds of prion-resistant cattle would be very desirable for the production of medicinal products; a general replacement of existing herds would be a long-term project. It may be desirable to establish proof of principle and decide wider implementation if and when the necessity arises. If BSE can arise spontaneously, as does CJD, then no country or continent may ultimately be "guaranteed" free of BSE.
---> Determine whether sheep devoid of PrP are viable and resistant to scrapie.
---> Develop methods for generating cattle devoid of PrP.
B. QUESTIONS RELATED TO BASIC RESEARCH ON PRION DISEASE
We have not considered this issue as being within the remit of this group, however some comments may be useful.
1. Nature of the infectious agent.
No informational nucleic acid has been found in highly purified preparations and the major protein component has been identified as PrPsc (PrPres) but other components may be present and important. Purification is very difficult because infectivity is associated with aggregated, insoluble material (mostly PrPsc). As far as we know, the only source used more or less successfully for purification is scrapie-infected hamster brain; efforts with bovine BSE brain seem not to have been successful.
---> Purify infectious agent from BSE-infected cattle brain and other sources and analyze.
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---> 3 D structure of PrPc (PrP-sens) from different sources (the structure of the carboxyterminal half of recombinant mouse PrPc produced in E.coli (i.e. unglycosylated) has been published.
---> 3 D structure of PrPsc should be established- very difficult because purification yields insoluble, heterogeneous material.
2. Multiplication of the agent.
The "protein only" hypothesis proposes that the infectious agent is PrPsc and that it multiplies by converting PrPc into PrPsc. The two forms are believed to differ only in their conformation.
---> Determine whether PrPc can be converted into the infectious agent (rather than only to PrPsc) in vitro.
Not all cells that produce PrPc allow multiplication of the infectious agent. Prusiner has proposed a "protein X" as requirement.
---> Determine which other components are required for propagation of the infectious agent (receptors? chaperones?).
3. Pathogenesis
It is still not understood how vacuolization and neuronal death come about.
---> Determine mechanism of pathogenesis. Depletion of PrPc? Toxicity of PrPsc ? Other ?
---> Which cell types and tissues can replicate the infectious agent? What are the biochemical requirements?
4. Transport of infectious agent
It is very likely that the agent can enter the organism via the digestive tract (although entry through nasal mucosa has been cited as an alternative possibility).
---> Determine where and how the agent enters the organism after ingestion.
---> Determine how the agent moves within the periphery, from periphery to CNS and vice-versa.
5. Strains
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it has been convincingly shown that there are different strains of TSE agent which can be passaged in inbred mice homozygous for a particular PrP allele. It is not evident how an agent consisting of protein only could encode species specific characteristics, however two explanations have been offered, the conformational hypothesis and the target cell theory.
---> What is the molecular feature of the infectious agent that determines strain specificity?
6. Susceptibility of the host to the infectious agent
At least in the case of the mouse, susceptibility of the host and incubation time in response to a given agent strain depend not only on the sequence of the host PrP gene but also on other genetic factors of the host.
---> Which genetic factors contribute to susceptibility and determine incubation time?
---> Which factors other than the sequence of PrP determines the species barrier ?
7. Natural function of PrP
PrP knockout mice generated by the Weissmann and Manson laboratories develop normally, show only minor behavioral deviations (modification of the circadian sleep rhythm. which remain to be confirmed) and mild electrophysiological abnormalities 17. What is the natural function of PrP? Why does ablation of a conserved protein, expressed early in development in many organs not show any notable phenotype? Can the developing organism compensate for absence of putative PrP function?
---> Detailed analysis of the PrP knockout mice.
---> Generate mice in which PrP expression is experimentally controllable.
References
-----------------------------------------------
17 A PrP knockout mouse line generated by Sakaguchi et al. (Sakaguchi et al., 1996) shows Purkinje cell degeneration and ataxia in aging animals. This may be concomitant with the deletion of the open reading frame.
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Baker, H. F., Ridley, R. M. and Wells, G. A. (1993a). Experimental transmission of BSE and scrapie to the common marmoset. Vet Rec. 132, 403-406.
Baker, H. F., Ridley, R. M. and Wells, G. A. H. (1993b). Experimental transmission of BSE and scrapie to the common marmoset. Vet.Rec. 132, 403-406.
Bons, N., Mestre-Francés, N., Charnay, Y. and Tagliavini, F. (1996). Spontaneous spongiform encephalopathy in a young adult rhesus monkey. Lancet 348, 55.
Brown, P.. Gibbs, C. J. J., Rodgers, J. P., Asher, D. M., Sulima, M. P., Bacote, A., Goldfarb, L. G. and Gajdusek, D. C. (1994). Human spongiform encephalopathy: the National Institutes of Health series of 300 cases of experimentally transmitted disease. Ann Neurol 35, 513-29.
Büeler, H., Raeber, A., Sailer, A., Fischer, M., Aguzzi, A. and Weissmann, C. (1994). High prion and PrPsc levels but delayed onset of disease in scrapie-inoculated mice heterozygous for a disrupted PrP gene. Molecular Medicine 1, 19-30.
Campbell, K. H., McWhir, J., Ritchie, W. A. and Wilmut, I. (1996). Sheep cloned by nuclear transfer from a cultured cell line. Nature 380, 64-66.
Chastel, C. E. (1996). ,BSE a specific bovine disease? Nature 381, 360.
Cutlip, R. C., Miller, J. M., Race, R. E., Jenny, A. L., Katz, J. B., Lehmkuhl, H. D., DeBey, B. M. and Robinson, M. M. (1994). Intracerebral transmission of scrapie to cattle. J. infect. Dis. 169, 814-20.
Fraser, H. and Foster, J. D. (1994). Transmission to mice, sheep and goats and bioassay of bovine tissues. In Transmissible Spongiform Encephalopathies, R. Bradley and B. Marchant, eds.: Brussels: Working document for the European Commission Ref.F.II.3-JC/0003), pp. 145-159.
Garland, T., Bauer, N. and Bailey Jr. , M. (1996). Brain emboli. Lancet, 610.
Goldmann, W., Hunter, N., Smith,G., Foster, J. and Hope, J. (1994). PrP genotypes and the Sip gene in Cheviot sheep form the basis for scrapie strain typing in sheep. Ann. N. Y. Acad. Sci. 724, 296-299.
Hsieh, G., Kenney, K., Gibbs, J., C.J., Lee, K. H. and Harrington, M. G. (1998). The 14-3-3 brain protein in cerebrospinal fluid as a marker for transmissible spongiform encephalopathies. N. Engl. J.Med. 335, 924-930.
Lasmézas, C.I., Deslys, J. P., Demaimay, R., Adjou, K. T., Lamoury, F., Dormont, D., Robain. O., Ironside, J. and Hauw, J. J. (1996). BSE transmission to macaques. Nature 381, 743-744.
Narang, H. (1996). Origin and implications of bovine spongiform encephalopathy. Proc.Soc.Exp.Biol.Med. 211, 306-22.
Sakaguchi, S., Katamine, S., Nishida, N., Moriuchi, R., Shigematsu, K., Sugimoto, T., Nakatani, A., Kataoka,Y., Houtani, T., Shirabe, S., Okada, H., Hasegawa, S., Miyamoto, T.
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and Noda, T. (1996). Loss of cerebellar Purkinje cells in aged mice homozygous for a disrupted PrP gene. Nature 380, 528-531.
Schreuder, B. E. C., van Keulen, L. J. M., Vromans, M. E. W., Langeveld, J. P. M. and Smits, M. A. (1996) . Preclinical test for prion disease. Nature 381, 563.
Stack, M. J., Aldrich, A. M., Kitching, A. D. and Scott, A. C. (1995). Comparative study of electron microscopical techniques for the detection of scrapie-associated fibrils. Res.Vet.Sci. 59, 247-254.
Telling, G.C., Scott, M., Hsiao, K.K., Foster, D., Yang, S. L., Torchia, M., Sidle, K. C., Coillnge, J., DeArmond, S. J. and Prusiner, S. B. (1994). Transmission of Creutzfeldt-Jakob disease from humans to transgenic mice expressing chimeric human-mouse prion protein. Proc. Natl. Acad. Sci. USA 91, 9938-40.
Wilesmith, J. W. (1992a). Bovine spongiform encephalopathy: a brief epidemiography, 1985-1991. in Prion diseases of humans and animals, S. Prusiner, 11. Collinge, J. Powell and B. Anderton, eds. (New York: Ellis Horwood), pp. 243-255.
Wilesmith, J. W. (1992b). Epidemiology of bovine spongiform encephalopathy. Semin. Virol. 2, 239-245.
Wisniewski, H. M., Sigurdson, S., Rubenstein, R., Kascsak, R. J. and Carp, R. I. (1996). Mites as vectors for scrapie. Lancet 347,1114.
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Glossary and Abbreviations
BSE: Bovine spongiform encephalopathy.
CJD: Creutzfeldt-Jakob disease.
CNS: Central nervous system.
EEG: Electroencephalogram. ES cells: Embryonal stem cells.
FFI: Fatal familial insomnia.
GSS: Gerstmann-Sträussler-Scheinker disease.
i.c.: intracerebral.
i.p.: intraperitoneal.
i.v.: intravenous.
knockout mouse: A mouse in which a particular gene has been disrupted and inactivated.
MAFF: Ministry of Agriculture, Fisheries and Food.
NPU: Neuropathogenesis Unit (Edinburgh).
Prion: Agent of transmissible spongiform encephalopathy, with unconventional properties. The term does not have structural implications other than that a protein is an essential component.
"Protein only" hypothesis: Maintains that the prion is devoid of informational nucleic acid and consists of protein (or glycoprotein) as essential pathogenic component. Genetic evidence strongly suggests that the protein is an ,abnormal form of PrP (PrP* or perhaps specifically PrPsc). The association with other "non-informational" molecules (for example lipids or glycosamino glycans) is not excluded.
PrPc or PrP-sen: The naturally occurring form of the mature Prnp gene product. It is glycosylated and anchored to the plasma membrane by a glycosyl phosphatidylinositol residue. Its presence in a given cell type is necessary but not sufficient for the replication of the prion.
PrPsc or PrPres: An "abnormal" form of the mature Prnp gene product found in tissue of TSE sufferers, operationally defined as being partly resistant to proteinase K digestion under defined reaction conditions. It is believed to differ from PrPc only (or mainly ) conformationally and is rich in ß sheet structure. Within the framework of the "protein only" hypothesis it is often considered to be the transmissible agent or prion (however, see PrP*).
PrP*: Within the framework of the "protein only" hypothesis PrP* is defined as the form of PrP constituting the essential (or only) component of the prion. It may but need not be identical with PrPsc.
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This concept was introduced (Weissmann, 1991) because the ratio of infectivity to PrPsc is 1:100'000 or less and the determination of chemical or physical characteristics of purified infectious preparations consisting mainly of PrPsc does not necessarily reflect the characteristics of the infectious component.
SBO: Specified bovine offal.
TSE: Transmissible spongiform encephalopathy.
Virino hypothesis: Maintains that the TSE agent (prion) consists of an agent-specific nucleic acid enveloped in or associated with a host protein, perhaps PrPsc. Such a nucleic acid has not been identified in highly purified preparations of infectious agent.
"Unconventional virus" hypothesis: Maintains that the TSE agent is a virus however with unusual properties. The requirement for PrP in the host to allow replication and pathogenesis is explained by assuming that PrP forms part or all of a receptor for the virus. As mentioned above, no nucleic acid specific for TSEs has been isolated.
vCJD: Variant Creutzfeldt-Jakob disease, a new variant of CJD associated with a particular pathology and occurring in young individuals.
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The Committee
Permanent members:
C.Weissmann, University of Zürich (chairman)
J.Collinge, Imperial College School of Medicine, London (deputy chairman)
J.Almond, University of Reading
D.Dormont, Commissariat à l'Energie Atomique, Fontenay aux Roses, France
H.Kretzschmar, Georg-August-Universität, Göttingen
M.Pocchiari, Istituto Superiore di Sanita, Roma
M.Wierup, Swedish Animal Health Service, Johannashov, Sweden
Consultants:
A.Aguzzi, University of Zürich
L.K.Borysiewicz, University of Wales, Cardiff
C.Bostock, Compton Laboratory, Compton, UK
K.Brown, Ministry of Agriculture, Fisheries und Food, London
R.Bradley, Central Veterinary Laboratory, Weybridge, UK
H.Diringer, Robert Koch Institut, Berlin
M.Flacher, University of Zürich
G.Hunsmann, Deutsches Primatenzentrum, Göttingen
R.H.Kimberlin, SARDAS, Edinburgh
B.Oesch, University of Zürich
J.R.Pattison, University College London
A.Raeber, University of Zürich
R.M.Ridley, University of Cambridge
J.Wilesmith, Central Veterinary Laboratory, Weybridge, UK
IN
Die Autoren behaupten leichtfertig, Scrapie sei nicht auf Menschen übertragbar. Um herauszufinden ob und gegebenenfalls mit welcher Dosis-Wirkungs-Relation BSE auf Menschen übertragen wurde, schlagen die Autoren ebenso sinnlose wie ausgedehnte Tierversuche mit höheren Affen vor. Sie halten es für möglich, dass BSE nicht durch einen Übersprung von Scrapie, sondern einfach durch eine Ausbreitung eines spontan entstandenen BSE-Falles entstand. Sie wollen diese gewagte These durch Verfütterung von Scrapie-Infektiosität an Rinder überprüfen. Sie behaupten im Widerspruch zu ihrer These, BSE sei Mitte der achtziger Jahre entstanden. Wiederum durch einen überflüssigen Tierversuch solch untersucht werden, ob intrazerebral mit BSE infizierte Schweine für Schweine infektiös sind. Die Autoren regen die Entwicklung eines Massen-PCR-Tests für die Ermittlung des BSE-Risikos einzelner Menschen aufgrund ihrer genetischen Ausstattung am Codon 129 an. Ich frage mich, wem dies nutzen soll. Die Autoren haben offenbar die SEAC-Interpretation der Weybridge-Zahlen nicht nachgerechnet. Sie glauben der dümmlichen Rechnung und bezweifeln lediglich ihre Verallgemeinerbarkeit. Natürlich ohne einen Beweis dafür liefern zu können behaupten die Autoren, die spontane Entstehung der Creutzfeldt-Jakob-Krankheit sei gut belegt.
Offene Fragen:
Effekt von BSE auf die Milchleistung
Creutzfeldt-Jakob-Inkubationszeiten
Eine kleine Anzahl oral infizierter Schweine erlaubt nur bei postivem Ergebnis eine Aussage. Es hätte nicht nur eine infizierte Malzeit geben dürfen.zumindest müssen die Tiere bis zu ihrem natürlichen Tod beobachtet werden.
Es muß endlich aufhören, dass Geheimexperimente durchgeführt werden, über die einige Beamte, jedoch nur sehr wenige Wissenschaftler informiert sind.
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AD Prof. Charles Weissmann, Universität Zürich, Institut für Molekularbiologie, Abteilung I, Hönggerberg, 8093 Zürich, Schweiz, Postfach, Tel. 633 24 91, Fax 371 72 05
SP englisch
OR Prion-Krankheiten 8