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Bovine spongiform encephalopathy and biological products for human use
Biologicals
(1990) 18, 77-80
MEETING REPORT
Bovine Spongiform Encephalopathy and Biological Products for Human Use Report of an Informal Meeting Held at NIBSC on 16 May 1988 National
Philip Minor Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hefts EN6 3QG, U.K.
In 1987,l Wells et al. reported a novel syndrome in cattle characterized clinically by loss of condition, hyperaesthesia and neurolocomotor difficulties. Histopathological examination revealed a spongiform encephalopathy of the brain and extraction of brain by appropriate methods yielded fibrils very similar to those found in cases of scrapie (SAF). Neither the histopathology nor the fibrils could be found in brains of normal cows or of cows suffering from other disorders. The clinical signs, the histopathology and the fibrils are all consistent with the view that the syndrome bovine spongiform encephalopathy (BSE) is caused by an agent similar to those causing scrapie of sheep and kuru or CreutzfeldMakob disease of man. In so far as bovine products are used in the preparation of biological products for human use, the occurrence of such a syndrome raises safety considerations for medical products and a meeting was convened at NIBSC on 16 May 1988 to consider the relevant available information. The nature of scrapie-like agents is controversial and they are currently identified with certainty only by their ability to cause particular clinical signs and histopathology in infected animals. The incubation period is generally long, making rapid experimental studies difficult. Most of the current information on bovine spongiform encephalopathy thus derives from epidemiological studies. Analogies have also been drawn with scrapie and Creutzfeldt-Jakob disease where the properties of the agent are rather better defined. Epidemiological studies The international situation So far as is known cases of BSE have only been identified in the U.K. and the Republic of Ireland. 1045-1056190/010077+04
$03.00/O
Incidence in the U.K. The first case occurred in 1985.1,2 At the time of the meeting (mid 1988) 473 out of 45 000 dairy herds and 10 out of 54 000 beef suckler herds had confirmed cases. An epidemiological study of herds containing at least one histopathologically confirmed case was undertaken by Wilesmith of the Central Veterinary Laboratory. The conclusions of the study to date were firstly that BSE was not detected before 1985. While it may have occurred earlier the incidence prior to 1986 was very low compared with current levels. The incidence rose rapidly, but had levelled off at 60-70 newly diagnosed and confirmed cases per month; this incidence had been maintained since mid 1987. While this figure may be an underestimate due to underreporting and the occurrence of clinically suspected but unconfirmed cases, the rate of occurrence of new cases is now believed to be fairly constant. Distribution of cases Of the affected herds, 95% had three or fewer cases and 75% had only one case. The mean annual incidence within affected herds was 1.1% ) with the highest incidence being 5.5%. Affected herds were concentrated towards southern England. In Kent 6% of herds had at least one case, in Berkshire 5.3% and in southwest England which accounts for 25% of the U.K. population of cattle, Cornwall, Devon and Somerset all had confirmed cases in about 2% of herds. There is no breed or sex predisposition; one case had been confirmed in a bull and one was suspected, which is consistent with the population at risk. Cases were identified throughout the affected areas within a 3-4-month period. There is no evi@ 1990 The International Association ofBiological
Standardization
78
P. Minor
dence for spread of BSE from a single infected herd; furthermore a number of closed herds in which no animals were brought in are affected. Bovine spongiform encephalopathy is thus acting as an extended common source epidemic, where it appears that some factor induced the syndrome simultaneously in a number of locations. It is not an epidemic spreading from a single location. Infection
model
Cases have been confirmed in animals ranging from 2 years 9 months to 10 years of age, most being in the 3-to &year-old range. Based on a study of 113 adult herds containing 10 500 animals older than 2% years, the peak age specific incidence of cases in herds with confirmed cases was in 4-year-olds. The time between recognition of signs and slaughter ranged from 2 weeks to 14 months. Most animals were slaughtered 2-3 months after onset of symptoms. The incubation period of the disease is believed to be 3-8 years, and calves are about 30 times more likely to become infected than adults. Exposure is thought to have begun in 1982 and continued thereafter. The hypothesis The clinical and histopathological parallels between BSE and scrapie suggest that they might be directly related. The occurrence of the disease is temporally linked with a massive increase in the number of sheep in the U.K. in part due to imposed milk quotas, and it is postulated that the cattle have become infected from meat and bonemeal produced by rendering plants. In recent years, the rendering industry in the U.K. has become more rationalized, with many small units closing. Simultaneously the treatment of unfit carcasses, including those of sheep in the rendering process, has become less vigorous resulting in a more acceptable product. It may also allow scrapie-like agents to survive to a greater extent than with previous treatments. A model involving transmission by commercial feedstuffs supplements would be consistent with the higher incidence in dairy herds where such feedstuffs are used more extensively than in beef suckler herds. A number of other possible factors such as onset of lactation or stage of pregnancy and a range of management factors can be eliminated. There is as yet no evidence for transmission of BSE from cow to COW, or from mother to offspring. There is no epidemiological association of the disease with the presence of sheep on the farm.
Studies of infection
The epidemiology and the properties of the BSE suggest that it is caused by a scrapie-like agent. Transmission experiments involving hamsters, mice, cattle and marmosets have been set up and transmission to mice has now been demonstrated. All species have received intracerebral injection of crude brain homogenate. Four separate sources of homogenate have been used. The hamsters and mice have also received material intraperitoneally and the cattle have received it intravenously. No attempts to transmit the disease by feeding have yet been reported. Where transmission to mice has been demonstrated the latent period was at least several months, comparable to that of scrapie-like agents. It is as yet unknown whether the putative causative agent of BSE can be transmitted to non-bovine species in general and humans in particular. No studies have yet been reported to identify specific tissues as sources of infection. Scrapie and Creutzfeldt-Jakob disease It has been demonstrated3 that fibrils from the brains of cows affected by BSE contain scrapie-associated protein. Even so the question of possible detection methods, the stability of the agent and its partition in purification processes remain difficult to address directly. A number of predictions can be made, however, based on the assumption that BSE is caused by an infectious agent comparable to the causative agents of scrapie and CreutzfeldtrJakob disease. The nature of scrapie-like agents is controversial.* They may be viruses, in which the protein is encoded by a viral nucleic acid virinos in which there is a sequence of RNA with a regulatory non-coding function enclosed in a host-coded protein, or prions, which are regarded as self-replicating proteins. Experimental
transmission
to other species
Intracerebral inoculation of concentrated brain homogenates from wild cases of scrapie in sheep will transmit the disease to mice in most cases. Passage in a new host species is usually slow and inefficient but the agent can also be transmitted to primates, hamsters and sheep. In all cases the typical latent period is 2 years although incubation periods of 60 days in hamsters and 120 days in mice can be observed in adapted strains. Comparable findings are made with CreutzfeldtJakob disease. By analogy BSE may be transmissible to humans although this may be difficult because of species differences. Scrapie can be transmitted orally,
Meeting report
although used.
this is not the normal
experimental
route
Sources of infection
Creutzfeldtiakob disease has been reported to be transmitted to humans by cornea1 transplant, dura mater transplant in the course of constructing an artificial eardrum, intracortical electrodes in neurosurgery and by growth hormone obtained from human pituitaries and administered intramuscularly or subcutaneously. 5s6Cases have not been reported as a result of general surgery, organ transplantation or blood transfusion. Iatrogenic infection of humans has thus been associated with material derived from nervous tissue. However, 7,8 there are reports of transmission of CJD to animals by intracerebral injection of buffy coat cells from the blood of infected animals and patients. Similarly central nervous tissue is the best source of infectious scrapie agent, and while it may also be obtained from organs of the lympho-reticular system, such as the spleen, the titres are at least ten-fold lower. This is probably also true of CreutzfeldtrJakob disease. It is sometimes possible to transmit scrapie by injecting blood of an infected mouse but infectivity titres are much lower than in the lymphoreticular system. Detection of the agent
The only method for detecting the agents of scrapielike disease is by their infectivity. There is no specific immunological response. The long incubation period and the insensitivity of the infectivity assays make it impractical to screen materials for wild-type scrapielike agents. It may be possible to reduce the chance of contaminating bovine products with BSE by selecting appropriate animals from regions where the incidence is low and examining the herds for clinical signs. Routine examination of all animals for histological lesions is unlikely to be practicable. If the route of infection is established as being from feed supplements it may be possible to eliminate BSE by appropriate treatment of such material. It is unlikely to be practical to establish that a particular product is free of scrapie-like agents by examining the product alone. Removal of infectivity
Several common disinfectants have no effect on the scrapie agent. It is resistant to ionizing radiation although reductions in titre can be effected. It is destroyed by autoclaving at 30 lbs psi (134°C) for 18 min (current DHSS recommendations for Creutz-
79
feldt-Jakob disease agent). At least one strain of scrapie is resistant to exposure to 121°C for 1 h. Infectivity is partially lost by treatment with phenolic disinfectants while concentrated hypochlorite solution is more effective. Organic solvents such as hexane may have some effect. Disinfection is therefore difficult in the context of a biological product. Scrapie is an adherent moiety; it may be removed by precipitation. It may also be excluded by column chromatography or ultrafiltration with an exclusion limit of 100 K daltons. This may reflect its adherent character. While it cannot be readily destroyed, therefore, it may be possible to remove it by purification methods. Conclusions Bovine spongiform encephalopathy presents features suggesting that it is caused by a scrapie-like agent. The incidence varies geographically with a lower incidence in the north. In the absence of data on transmission the properties of the agent are expected to be similar to those of the causative agents of scrapie and Creutzfeldt-Jakob disease, which are, in practical terms, undetectable by existing technology and cannot be selectively destroyed, although they may be removed. If BSE is held to be a problem, the only option is to ensure that bovine materials for manufacture of biological medicinal products are derived from cattle in areas free of the disease. Transmission to humans may not readily occur. This statement is based on experience with scrapie which is unlikely to cause a disease in humans under natural conditions, and in particular has been shown to be epidemiologically unlinked to CreutzfeldtJakob disease. There is no evidence for transmission of scrapie to occupational groups such as shepherds and veterinarians which have a high exposure to sheep and scrapie-infected sheep. This is true for research workers and abattoir workers or butchers, where there may be exposure to brain tissues. The tissue distribution of infectious agents may also act against ease of transmission. In particular, serum is a poor source of infectivity in animals affected by scrapie. Vertical transmission of acquired Creutzfeldt-Jakob disease, kuru or BSE has not been demonstrated. Transmission of scrapie from infected ewes to their lambs occurs with relatively high frequency, however, implying possible transplacental spread or transmission via milk. It is thus possible that BSE poses no real threat to human health provided the main exposures are either as a contaminant of food stuffs, which will be minimized by inspection of
80
P. Minor
animals, or from products that are not contaminated with nervous tissue. The information on which assessment of the risk is based is, however? extremely sparse. Postscript BSE was made notifiable on 21 June 1988, under the Bovine Spongiform Encephalopathy Order 1988. This Order requires affected animals to be restricted to the premises until they go for slaughter, under licence. Heads of suspected animals will then be surrendered to MAFF for histopathological examination of the brain. In addition, from 18 July to 31 December 1988, the Order prohibits the inclusion of animalderived protein from ruminants, including imported material, in feed rations for ruminants. A committee chaired by Sir Richard Southwood
investigated the available reported in 1988.
information
on BSE and
References 1. Wells GAH, Scott AC, Johnson CT, Gunning RF, Hancock RD, Jeffrey M, Dawson M, Bradley R. Vet Ret 1987; 121: 419. Anon. Vet Ret 1988; 122: 477. 32: Hope J, Reekie LJD, Hunter N, Multhaup G, Beyreuther K, White, Scott AC, Stack MJ, Dawson M, Wells GAH. Nature 1988; 336: 390. 4. Carp RI, Merz PA, Karcsak RJ, Merz GS, Wisniewski HM. J Gen Viro11985; 66: 1357-1368. 5. Powell-Jackson J, Kennedy P, Whitcombe EM, Weller RO, Preece MA, Newson Davies J. Lancet 1985; ii: 244. 6. Koch, Berg BO, de Armond SJ, Gravina RF. New Eng J Med 1985; 313: 731. 7. Manuelidis, Kim JH, Mericargas JR, Manuelidis L. Lancet 1985; ii: 896-897. 8. Tateishi J. Lancet 1985; ii: 1074.
(1990) 18, 77-80
MEETING REPORT
Bovine Spongiform Encephalopathy and Biological Products for Human Use Report of an Informal Meeting Held at NIBSC on 16 May 1988 National
Philip Minor Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hefts EN6 3QG, U.K.
In 1987,l Wells et al. reported a novel syndrome in cattle characterized clinically by loss of condition, hyperaesthesia and neurolocomotor difficulties. Histopathological examination revealed a spongiform encephalopathy of the brain and extraction of brain by appropriate methods yielded fibrils very similar to those found in cases of scrapie (SAF). Neither the histopathology nor the fibrils could be found in brains of normal cows or of cows suffering from other disorders. The clinical signs, the histopathology and the fibrils are all consistent with the view that the syndrome bovine spongiform encephalopathy (BSE) is caused by an agent similar to those causing scrapie of sheep and kuru or CreutzfeldMakob disease of man. In so far as bovine products are used in the preparation of biological products for human use, the occurrence of such a syndrome raises safety considerations for medical products and a meeting was convened at NIBSC on 16 May 1988 to consider the relevant available information. The nature of scrapie-like agents is controversial and they are currently identified with certainty only by their ability to cause particular clinical signs and histopathology in infected animals. The incubation period is generally long, making rapid experimental studies difficult. Most of the current information on bovine spongiform encephalopathy thus derives from epidemiological studies. Analogies have also been drawn with scrapie and Creutzfeldt-Jakob disease where the properties of the agent are rather better defined. Epidemiological studies The international situation So far as is known cases of BSE have only been identified in the U.K. and the Republic of Ireland. 1045-1056190/010077+04
$03.00/O
Incidence in the U.K. The first case occurred in 1985.1,2 At the time of the meeting (mid 1988) 473 out of 45 000 dairy herds and 10 out of 54 000 beef suckler herds had confirmed cases. An epidemiological study of herds containing at least one histopathologically confirmed case was undertaken by Wilesmith of the Central Veterinary Laboratory. The conclusions of the study to date were firstly that BSE was not detected before 1985. While it may have occurred earlier the incidence prior to 1986 was very low compared with current levels. The incidence rose rapidly, but had levelled off at 60-70 newly diagnosed and confirmed cases per month; this incidence had been maintained since mid 1987. While this figure may be an underestimate due to underreporting and the occurrence of clinically suspected but unconfirmed cases, the rate of occurrence of new cases is now believed to be fairly constant. Distribution of cases Of the affected herds, 95% had three or fewer cases and 75% had only one case. The mean annual incidence within affected herds was 1.1% ) with the highest incidence being 5.5%. Affected herds were concentrated towards southern England. In Kent 6% of herds had at least one case, in Berkshire 5.3% and in southwest England which accounts for 25% of the U.K. population of cattle, Cornwall, Devon and Somerset all had confirmed cases in about 2% of herds. There is no breed or sex predisposition; one case had been confirmed in a bull and one was suspected, which is consistent with the population at risk. Cases were identified throughout the affected areas within a 3-4-month period. There is no evi@ 1990 The International Association ofBiological
Standardization
78
P. Minor
dence for spread of BSE from a single infected herd; furthermore a number of closed herds in which no animals were brought in are affected. Bovine spongiform encephalopathy is thus acting as an extended common source epidemic, where it appears that some factor induced the syndrome simultaneously in a number of locations. It is not an epidemic spreading from a single location. Infection
model
Cases have been confirmed in animals ranging from 2 years 9 months to 10 years of age, most being in the 3-to &year-old range. Based on a study of 113 adult herds containing 10 500 animals older than 2% years, the peak age specific incidence of cases in herds with confirmed cases was in 4-year-olds. The time between recognition of signs and slaughter ranged from 2 weeks to 14 months. Most animals were slaughtered 2-3 months after onset of symptoms. The incubation period of the disease is believed to be 3-8 years, and calves are about 30 times more likely to become infected than adults. Exposure is thought to have begun in 1982 and continued thereafter. The hypothesis The clinical and histopathological parallels between BSE and scrapie suggest that they might be directly related. The occurrence of the disease is temporally linked with a massive increase in the number of sheep in the U.K. in part due to imposed milk quotas, and it is postulated that the cattle have become infected from meat and bonemeal produced by rendering plants. In recent years, the rendering industry in the U.K. has become more rationalized, with many small units closing. Simultaneously the treatment of unfit carcasses, including those of sheep in the rendering process, has become less vigorous resulting in a more acceptable product. It may also allow scrapie-like agents to survive to a greater extent than with previous treatments. A model involving transmission by commercial feedstuffs supplements would be consistent with the higher incidence in dairy herds where such feedstuffs are used more extensively than in beef suckler herds. A number of other possible factors such as onset of lactation or stage of pregnancy and a range of management factors can be eliminated. There is as yet no evidence for transmission of BSE from cow to COW, or from mother to offspring. There is no epidemiological association of the disease with the presence of sheep on the farm.
Studies of infection
The epidemiology and the properties of the BSE suggest that it is caused by a scrapie-like agent. Transmission experiments involving hamsters, mice, cattle and marmosets have been set up and transmission to mice has now been demonstrated. All species have received intracerebral injection of crude brain homogenate. Four separate sources of homogenate have been used. The hamsters and mice have also received material intraperitoneally and the cattle have received it intravenously. No attempts to transmit the disease by feeding have yet been reported. Where transmission to mice has been demonstrated the latent period was at least several months, comparable to that of scrapie-like agents. It is as yet unknown whether the putative causative agent of BSE can be transmitted to non-bovine species in general and humans in particular. No studies have yet been reported to identify specific tissues as sources of infection. Scrapie and Creutzfeldt-Jakob disease It has been demonstrated3 that fibrils from the brains of cows affected by BSE contain scrapie-associated protein. Even so the question of possible detection methods, the stability of the agent and its partition in purification processes remain difficult to address directly. A number of predictions can be made, however, based on the assumption that BSE is caused by an infectious agent comparable to the causative agents of scrapie and CreutzfeldtrJakob disease. The nature of scrapie-like agents is controversial.* They may be viruses, in which the protein is encoded by a viral nucleic acid virinos in which there is a sequence of RNA with a regulatory non-coding function enclosed in a host-coded protein, or prions, which are regarded as self-replicating proteins. Experimental
transmission
to other species
Intracerebral inoculation of concentrated brain homogenates from wild cases of scrapie in sheep will transmit the disease to mice in most cases. Passage in a new host species is usually slow and inefficient but the agent can also be transmitted to primates, hamsters and sheep. In all cases the typical latent period is 2 years although incubation periods of 60 days in hamsters and 120 days in mice can be observed in adapted strains. Comparable findings are made with CreutzfeldtJakob disease. By analogy BSE may be transmissible to humans although this may be difficult because of species differences. Scrapie can be transmitted orally,
Meeting report
although used.
this is not the normal
experimental
route
Sources of infection
Creutzfeldtiakob disease has been reported to be transmitted to humans by cornea1 transplant, dura mater transplant in the course of constructing an artificial eardrum, intracortical electrodes in neurosurgery and by growth hormone obtained from human pituitaries and administered intramuscularly or subcutaneously. 5s6Cases have not been reported as a result of general surgery, organ transplantation or blood transfusion. Iatrogenic infection of humans has thus been associated with material derived from nervous tissue. However, 7,8 there are reports of transmission of CJD to animals by intracerebral injection of buffy coat cells from the blood of infected animals and patients. Similarly central nervous tissue is the best source of infectious scrapie agent, and while it may also be obtained from organs of the lympho-reticular system, such as the spleen, the titres are at least ten-fold lower. This is probably also true of CreutzfeldtrJakob disease. It is sometimes possible to transmit scrapie by injecting blood of an infected mouse but infectivity titres are much lower than in the lymphoreticular system. Detection of the agent
The only method for detecting the agents of scrapielike disease is by their infectivity. There is no specific immunological response. The long incubation period and the insensitivity of the infectivity assays make it impractical to screen materials for wild-type scrapielike agents. It may be possible to reduce the chance of contaminating bovine products with BSE by selecting appropriate animals from regions where the incidence is low and examining the herds for clinical signs. Routine examination of all animals for histological lesions is unlikely to be practicable. If the route of infection is established as being from feed supplements it may be possible to eliminate BSE by appropriate treatment of such material. It is unlikely to be practical to establish that a particular product is free of scrapie-like agents by examining the product alone. Removal of infectivity
Several common disinfectants have no effect on the scrapie agent. It is resistant to ionizing radiation although reductions in titre can be effected. It is destroyed by autoclaving at 30 lbs psi (134°C) for 18 min (current DHSS recommendations for Creutz-
79
feldt-Jakob disease agent). At least one strain of scrapie is resistant to exposure to 121°C for 1 h. Infectivity is partially lost by treatment with phenolic disinfectants while concentrated hypochlorite solution is more effective. Organic solvents such as hexane may have some effect. Disinfection is therefore difficult in the context of a biological product. Scrapie is an adherent moiety; it may be removed by precipitation. It may also be excluded by column chromatography or ultrafiltration with an exclusion limit of 100 K daltons. This may reflect its adherent character. While it cannot be readily destroyed, therefore, it may be possible to remove it by purification methods. Conclusions Bovine spongiform encephalopathy presents features suggesting that it is caused by a scrapie-like agent. The incidence varies geographically with a lower incidence in the north. In the absence of data on transmission the properties of the agent are expected to be similar to those of the causative agents of scrapie and Creutzfeldt-Jakob disease, which are, in practical terms, undetectable by existing technology and cannot be selectively destroyed, although they may be removed. If BSE is held to be a problem, the only option is to ensure that bovine materials for manufacture of biological medicinal products are derived from cattle in areas free of the disease. Transmission to humans may not readily occur. This statement is based on experience with scrapie which is unlikely to cause a disease in humans under natural conditions, and in particular has been shown to be epidemiologically unlinked to CreutzfeldtJakob disease. There is no evidence for transmission of scrapie to occupational groups such as shepherds and veterinarians which have a high exposure to sheep and scrapie-infected sheep. This is true for research workers and abattoir workers or butchers, where there may be exposure to brain tissues. The tissue distribution of infectious agents may also act against ease of transmission. In particular, serum is a poor source of infectivity in animals affected by scrapie. Vertical transmission of acquired Creutzfeldt-Jakob disease, kuru or BSE has not been demonstrated. Transmission of scrapie from infected ewes to their lambs occurs with relatively high frequency, however, implying possible transplacental spread or transmission via milk. It is thus possible that BSE poses no real threat to human health provided the main exposures are either as a contaminant of food stuffs, which will be minimized by inspection of
80
P. Minor
animals, or from products that are not contaminated with nervous tissue. The information on which assessment of the risk is based is, however? extremely sparse. Postscript BSE was made notifiable on 21 June 1988, under the Bovine Spongiform Encephalopathy Order 1988. This Order requires affected animals to be restricted to the premises until they go for slaughter, under licence. Heads of suspected animals will then be surrendered to MAFF for histopathological examination of the brain. In addition, from 18 July to 31 December 1988, the Order prohibits the inclusion of animalderived protein from ruminants, including imported material, in feed rations for ruminants. A committee chaired by Sir Richard Southwood
investigated the available reported in 1988.
information
on BSE and
References 1. Wells GAH, Scott AC, Johnson CT, Gunning RF, Hancock RD, Jeffrey M, Dawson M, Bradley R. Vet Ret 1987; 121: 419. Anon. Vet Ret 1988; 122: 477. 32: Hope J, Reekie LJD, Hunter N, Multhaup G, Beyreuther K, White, Scott AC, Stack MJ, Dawson M, Wells GAH. Nature 1988; 336: 390. 4. Carp RI, Merz PA, Karcsak RJ, Merz GS, Wisniewski HM. J Gen Viro11985; 66: 1357-1368. 5. Powell-Jackson J, Kennedy P, Whitcombe EM, Weller RO, Preece MA, Newson Davies J. Lancet 1985; ii: 244. 6. Koch, Berg BO, de Armond SJ, Gravina RF. New Eng J Med 1985; 313: 731. 7. Manuelidis, Kim JH, Mericargas JR, Manuelidis L. Lancet 1985; ii: 896-897. 8. Tateishi J. Lancet 1985; ii: 1074.