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Observations on thermostable subpopulations of the unconventional agents that cause transmissible degenerative encephalopathies
Veterinary Microbiology 64 (1998) 33±38
Observations on thermostable subpopulations of the unconventional agents that cause transmissible degenerative encephalopathies D.M. Taylor*, K. Fernie, I. McConnell, P.J. Steele Neuropathogenesis Unit, Institute for Animal Health, West Mains Road, Edinburgh, EH9 3JF, UK Received 8 May 1998; accepted 18 September 1998
Abstract When scrapie agent is exposed to partially inactivating autoclave cycles, the fraction of infectivity that survives remains thermostable during relatively long periods of autoclaving. This resistant subpopulation can also be differentiated from the main population by its prolonged incubation periods in assay animals, compared with control material. Stabilisation of this subpopulation may occur through the smearing and drying of infected tissue that can occur prior to autoclaving, in which the disease-specific form of PrP protein (PrPSc) could become rapidly heatfixed. This may paradoxically be what protects this fraction of PrPSc from further inactivation during autoclaving. Data are presented showing that the thermostability acquired by the resistant subpopulation is a stable characteristic; autoclaving for a second time results in very little further loss of infectivity. These observations suggest that inactivation procedures that do not involve rapid and effective fixation of PrPSc may be better candidates for dealing effectively with scrapie-like agents. # 1998 Elsevier Science B.V. All rights reserved. Keywords: Transmissible degenerative encephalopathies; Scrapie agent; Thermostability
1. Introduction The transmissible degenerative encephalopathies (TDE) constitute a group of unusual and fatal neurological diseases of mammals that includes bovine spongiform encephalopathy (BSE), scrapie in sheep, and Creutzfeldt±Jacob disease of humans. While it is clear that the causal agents are unconventional, their precise nature has not yet * Corresponding author. Tel.: +44-131-667-5204; fax: +44-131-668-3872; e-mail: [email protected] 0378-1135/98/$ ± see front matter # 1998 Elsevier Science B.V. All rights reserved. PII S 0 3 7 8 - 1 1 3 5 ( 9 8 ) 0 0 2 5 7 - 0
34
D.M. Taylor et al. / Veterinary Microbiology 64 (1998) 33±38
Fig. 1. The inactivation curve for the 263 K strain of scrapie agent during autoclaving at 1348C showing the maximal and minimal effect between zero time and 18 min.
been elucidated. It is not known whether they consist entirely of PrPSc (a modified and disease-specific form of PrPc which is a normal host protein), or whether an as-yet unidentified informational molecule, such as a nucleic acid, is also required (Bruce et al., 1994). However, regardless of these uncertainties, it is known that TDE agents are relatively resistant to inactivation by procedures that are effective with conventional microorganisms (Taylor, 1992). When scrapie agent is completely inactivated by autoclaving, destruction of the agent over time proceeds in an exponential fashion (Rohwer, 1983). If the amounts of infectivity remaining after increasing exposure times, through to the time when complete inactivation is achieved, are plotted on a logarithmic scale, a straight line is obtained which shows that the death rate is constant. In marked contrast, when a heating procedure is only partially inactivating, the inactivation curve tends to show an initial decline and then flatten and persist with time (Rohwer, 1983). After autoclaving at 134±1388C for 18 min, it has been shown that the amount of BSE or scrapie infectivity that survives is relatively constant regardless of either the starting titre, or whether the agent is in bovine, hamster or mouse brain (Taylor, 1996a). An example of the tailing type of inactivation curve referred to above can be derived from data (Taylor et al., 1994) relating to the inactivation of the 263 K strain of scrapie agent by autoclaving (Fig. 1). In this experiment, 340 mg samples of a pooled macerate of infected hamster brains were autoclaved at 1348C for 18±60 min. Serial 10-fold dilutions of the autoclaved samples were injected intracerebrally into hamsters so that the infectivity titres could be measured (Karber, 1931). The mean incubation period for the recipients of the highest dilution of untreated brain macerate that contained detectable infectivity was 174 days. In contrast, the highest dilutions of autoclaved brain that were positive produced incubation periods of up to 391 days, demonstrating that autoclaving results in a modification of the dose± response curve (Taylor and Fernie, 1996). Unaffected animals were culled 788 days postinjection. The possibility that these differences were due to the autoclaving process
D.M. Taylor et al. / Veterinary Microbiology 64 (1998) 33±38
35
selecting a thermostable strain, from what might have been a mixture of strains, was excluded by studying the brain±lesion profiles in the animals receiving untreated, compared with autoclaved, material. These were identical. As insufficient data were available to show precisely the shape of the curve between zero time and 18 min, the two possible extremes have been plotted. These show that if, as is often the case with conventional microorganisms, the initial steep decline in infectivity is used to predict the time that it will take to achieve complete inactivation, this would be quite incorrect for scrapie agent, regardless of which of the two curves between zero time and 18 min are used. This reinforces the viewpoint that, although such estimates may often be useful (and sometimes accurate), there is no substitute for establishing a full inactivation curve (Greene, 1992). Tailing inactivation curves are not uncommon for conventional microorganisms; these may result from protection of some of the organisms through clumping, or be due to population heterogeneity where differing straight-line inactivation curves for two subpopulations combine to produce a tailing curve. Where there is no population heterogeneity, the same sort of tailing curve is still usually obtained when the surviving organisms are cultured and re-tested (Gardner and Peel, 1991). One explanation for the presence of a heat- or chemical-resistant (Taylor, 1991) subpopulation of scrapie agent might be the protective effect of aggregation which could occur in homogenates of infected tissue but not in undiluted tissue. However, Fig. 1 shows that even in undiluted tissue, small fractions of infectivity can survive heating for extended periods; this cannot be due to protective aggregation. Although more than seven logs of infectivity were lost, two logs of the 263 K strain of scrapie agent in 340 mg samples of macerated hamster brain survived autoclaving at 1348C for 1 h. Surprisingly, similarly sized samples of infected brain tissue are completely inactivated within 18 min by autoclaving if the brain tissue is undisrupted, as opposed to macerated (Taylor and McConnell, 1988; Taylor et al., 1997; unpublished data). The lesser efficiency of inactivating macerates may result from the fact that some smearing and drying before autoclaving occurs with this type of sample. Asher et al. (1986, 1987) found that scrapieinfected tissue is more difficult to inactivate by autoclaving when it has become dried onto surfaces. We have now shown that a relatively heat-resistant subpopulation of scrapie agent retains its thermostability when re-heated, suggesting that this is an acquired but stable characteristic of this subpopulation that differentiates it from the main population. Evidence for the intrinsic and fundamental difference of this subpopulation comes from the fact that, at its limiting dilution, it produces an average incubation period in recipient animals that is well beyond that for unheated material at its limiting dilution (Taylor and Fernie, 1996). 2. Methods From a pool of macerated hamster brain infected with the 263 K strain of scrapie agent, two 340 mg aliquots were placed on the grinding surfaces of two Griffiths tubes. These were subjected to porous-load autoclaving at 1348C for 18 min, after which one of the samples was homogenised in sterile physiological saline and a series of 10-fold dilutions was prepared. Each dilution was injected intracerebrally into groups of Syrian hamsters
10/10 (148) 3/5 (176)
10
ÿ1
9/12 (171) 7/12 (196)
10
ÿ2
9/12 (207) 0/6
10
ÿ3
ÿ4
10
No. infected hamsters in each dilution groupa
Mean incubation periods (in days) are shown in brackets. a Excluding intercurrent deaths.
Nil Autoclave 1 Autoclave 2
Procedure
6/6 (71)
10
ÿ5
4/6 (109)
10
ÿ6
3/6 (149)
10
ÿ7
4/12 (174)
10
ÿ8
0/12
10
ÿ9
Table 1 Titration of 263 K infectivity in hamster brain macerate before autoclaving, and after autoclaving once or twice at 1348C for 18 min
108.3 105.0 103.3
Infectivity titre (ID50 g)
36 D.M. Taylor et al. / Veterinary Microbiology 64 (1998) 33±38
D.M. Taylor et al. / Veterinary Microbiology 64 (1998) 33±38
37
(0.05 ml per animal). The second tube was autoclaved again at the same temperature and for the same time. This sample was then diluted and injected in the same fashion as the first sample. The hamsters were also injected with serial 10-fold dilutions of the untreated brain macerate. Animals were culled when they displayed clinical signs of neurological disease. Hamsters that remained healthy were kept for 18 months before they were culled. All brains were examined histologically for spongiform encephalopathy to confirm whether or not the animals had developed scrapie. In addition, to confirm that the strain of agent present after autoclaving was the same as that present in the untreated brain pool, the lesion profiles in these two groups of hamsters were compared. Titres of infectivity in the samples were calculated by the method of Karber (1931). The results are shown in Table 1. 3. Results Table 1 shows that, after one autoclaving cycle, the titre of infectivity was reduced by 3.3 logs. However, the titre was reduced by only a further 1.3 logs after autoclaving for a second time. Given the high calorific value of steam at 1348C, there can be no question of inadequate heat penetration into the samples because the Griffiths tubes were unstoppered. Also, thermocouple readings have shown that when a temperature of 1348C is achieved inside the autoclave chamber, the delay before there is complete heat penetration into 375 mg samples of brain tissue is only around 1 min (Taylor, unpublished data). There were no differences in the brain lesion profiles of the animals receiving untreated, compared with autoclaved, material. 4. Discussion The lesser efficiency of inactivating partially smeared and dried macerated tissue, compared with intact undisrupted tissue, may relate to the very rapid heating that would occur in the film of dried material (compared with the bulk of the sample) and the consequently very rapid fixation of PrPSc in the dried film. Protection by fixation has been shown to occur during the inactivation of poliovirus by formalin (Gard and Maaloe, 1959); prior fixation in ethanol (Taylor, 1996b) or formalin (Taylor and McConnell, 1988) has been shown to enhance considerably the thermostability of scrapie agent. Also, it has been observed that the amount of scrapie infectivity inactivated after 4 h under vacuum at 728C is greater than that achieved over the same timescale at atmospheric pressure when an end temperature of 1208C is achieved (Taylor et al., 1997). Given that the molecular nature of TDE agents has not yet been characterised (Coles, 1997) it is difficult to know with any certainty what accounts for the characteristics of the subpopulation of scrapie agent that is intrinsically more thermostable than the main population. The fact that the brain lesion profiles were the same in the hamsters that received untreated, compared with autoclaved, material shows that this is not due to the presence of a minor (but thermostable) contaminating strain of agent. Although the complete molecular structure of TDE agents has still to be determined, it is likely that
38
D.M. Taylor et al. / Veterinary Microbiology 64 (1998) 33±38
PrPSc is an important component. Overall, the data presented or discussed suggest that procedures which produce rapid and/or extremely effective fixation of protein result in enhanced resistance of TDE agents. Thus, there seems to be a potential for developing effective inactivation procedures by the use of procedures that avoid rapid and/or efficient protein fixation. References Asher, D.M., Pomeroy, K.L., Murphy, L., Rohwer, R.G., Gibbs, C.J., Gajdusek, D.C., 1986. Practical inactivation of scrapie agent on surfaces, In: Abstr. IXth Int. Congress of Infectious and Parasitic Diseases, Munich, 20±26 July. Asher, D.M., Pomeroy, K.L., Murphy, L., Gibbs, C.J., Gajdusek, D.C., 1987. Attempts to disinfect surfaces contaminated with etiological agents of the spongiform encephalopathies, In: Abstr. VIIth Int. Congress of Virology, Edmonton, 9±14 August, p. 147. Bruce, M.E., Chree, A., McConnell, I., Foster, J., Pearson, G., Fraser, H., 1994. Transmission of bovine spongiform encephalopathy and scrapie to mice; strain variation and the species barrier. Philosophical Transactions of the Royal Society B 343, 405±411. Coles, H., 1997. Nobel panel rewards prion theory after years of heated debate. Nature 389, 529. Gard, S., Maaloe, O., 1959. Inactivation of viruses, In: Burnet, F.M., Stanley, W.M. (Eds.), The Viruses, vol. 1, Academic Press, New York, pp. 359±427. Gardner, J.F., Peel, M.M., 1991. Introduction to Sterilization, Disinfection and Infection Control, Churchill Livingstone, Edinburgh. Greene, V.W., 1992. Sterility assurance: Concepts, methods and problems, In: Russell, A.D., Hugo, W.B., Aycliffe, G.A.J. (Eds.), Principles and Practice of Disinfection, Preservation and Sterilization, Blackwell, Oxford, pp. 605±624. Karber, G., 1931. Beitrag zur kollectiven Behandlung pharmakologischer Reihenversuche. Arch. Exp. Path. 162, 480±483. Rohwer, R.G., 1983. Scrapie inactivation kinetics ± an explanation for scrapie's apparent resistance to inactivation ± a re-evaluation of estimates of its small size, In: Court, L.A., Cathala, F. (Eds.), Virus non Conventionnels et Affections du Systeme Nerveux Central, Masson, Paris, pp. 84±113. Taylor, D.M., 1991. Resistance of the ME7 scrapie agent to peracetic acid. Vet. Microbiol. 27, 19±24. Taylor, D.M., 1992. Inactivation of unconventional agents of the transmissible degenerative encephalopathies, In: Russell, A.D., Hugo, W.B., Aycliffe, G.A.J. (Eds.), Principles and Practice of Disinfection, Preservation and Sterilisation, Blackwell, Oxford, pp. 605±624. Taylor, D.M., 1996a. Creutzfeldt±Jacob disease, Lancet, 347, 1333. Taylor, D.M., 1996b. Transmissible subacute spongiform encephalopathies: Practical aspects of agent inactivation, In: Court, L., Dodet, D. (Eds.), Transmissible Subacute Spongiform Encephalopathies: Prion Diseases, IIIrd Int. Symp. Subacute Spongiform Encephalopathies, 18±20 March 1996, Paris, pp. 479±482. Taylor, D.M., Fernie, K., 1996. Exposure to autoclaving or sodium hydroxide extends the dose response curve of the 263K strain of scrapie agent in hamsters. J. Gen. Virol. 77, 811±813. Taylor, D.M., McConnell, I., 1988. Autoclaving does not decontaminate formol-fixed scrapie tissues, Lancet i, 1463±1464. Taylor, D.M., Fraser, H., McConnell, I., Brown, D.A., Brown, K.L., Lamza, K.A., Smith, G.R.A., 1994. Decontamination studies with the agents of bovine spongiform encephalopathy and scrapie. Arch. Virol. 139, 313±326. Taylor, D.M., Woodgate, S.L., Fleetwood, A.J., Cawthorne, R.J.G., 1997. The effect of rendering procedures on the scrapie agent. Vet. Rec. 141, 643±649.
Observations on thermostable subpopulations of the unconventional agents that cause transmissible degenerative encephalopathies D.M. Taylor*, K. Fernie, I. McConnell, P.J. Steele Neuropathogenesis Unit, Institute for Animal Health, West Mains Road, Edinburgh, EH9 3JF, UK Received 8 May 1998; accepted 18 September 1998
Abstract When scrapie agent is exposed to partially inactivating autoclave cycles, the fraction of infectivity that survives remains thermostable during relatively long periods of autoclaving. This resistant subpopulation can also be differentiated from the main population by its prolonged incubation periods in assay animals, compared with control material. Stabilisation of this subpopulation may occur through the smearing and drying of infected tissue that can occur prior to autoclaving, in which the disease-specific form of PrP protein (PrPSc) could become rapidly heatfixed. This may paradoxically be what protects this fraction of PrPSc from further inactivation during autoclaving. Data are presented showing that the thermostability acquired by the resistant subpopulation is a stable characteristic; autoclaving for a second time results in very little further loss of infectivity. These observations suggest that inactivation procedures that do not involve rapid and effective fixation of PrPSc may be better candidates for dealing effectively with scrapie-like agents. # 1998 Elsevier Science B.V. All rights reserved. Keywords: Transmissible degenerative encephalopathies; Scrapie agent; Thermostability
1. Introduction The transmissible degenerative encephalopathies (TDE) constitute a group of unusual and fatal neurological diseases of mammals that includes bovine spongiform encephalopathy (BSE), scrapie in sheep, and Creutzfeldt±Jacob disease of humans. While it is clear that the causal agents are unconventional, their precise nature has not yet * Corresponding author. Tel.: +44-131-667-5204; fax: +44-131-668-3872; e-mail: [email protected] 0378-1135/98/$ ± see front matter # 1998 Elsevier Science B.V. All rights reserved. PII S 0 3 7 8 - 1 1 3 5 ( 9 8 ) 0 0 2 5 7 - 0
34
D.M. Taylor et al. / Veterinary Microbiology 64 (1998) 33±38
Fig. 1. The inactivation curve for the 263 K strain of scrapie agent during autoclaving at 1348C showing the maximal and minimal effect between zero time and 18 min.
been elucidated. It is not known whether they consist entirely of PrPSc (a modified and disease-specific form of PrPc which is a normal host protein), or whether an as-yet unidentified informational molecule, such as a nucleic acid, is also required (Bruce et al., 1994). However, regardless of these uncertainties, it is known that TDE agents are relatively resistant to inactivation by procedures that are effective with conventional microorganisms (Taylor, 1992). When scrapie agent is completely inactivated by autoclaving, destruction of the agent over time proceeds in an exponential fashion (Rohwer, 1983). If the amounts of infectivity remaining after increasing exposure times, through to the time when complete inactivation is achieved, are plotted on a logarithmic scale, a straight line is obtained which shows that the death rate is constant. In marked contrast, when a heating procedure is only partially inactivating, the inactivation curve tends to show an initial decline and then flatten and persist with time (Rohwer, 1983). After autoclaving at 134±1388C for 18 min, it has been shown that the amount of BSE or scrapie infectivity that survives is relatively constant regardless of either the starting titre, or whether the agent is in bovine, hamster or mouse brain (Taylor, 1996a). An example of the tailing type of inactivation curve referred to above can be derived from data (Taylor et al., 1994) relating to the inactivation of the 263 K strain of scrapie agent by autoclaving (Fig. 1). In this experiment, 340 mg samples of a pooled macerate of infected hamster brains were autoclaved at 1348C for 18±60 min. Serial 10-fold dilutions of the autoclaved samples were injected intracerebrally into hamsters so that the infectivity titres could be measured (Karber, 1931). The mean incubation period for the recipients of the highest dilution of untreated brain macerate that contained detectable infectivity was 174 days. In contrast, the highest dilutions of autoclaved brain that were positive produced incubation periods of up to 391 days, demonstrating that autoclaving results in a modification of the dose± response curve (Taylor and Fernie, 1996). Unaffected animals were culled 788 days postinjection. The possibility that these differences were due to the autoclaving process
D.M. Taylor et al. / Veterinary Microbiology 64 (1998) 33±38
35
selecting a thermostable strain, from what might have been a mixture of strains, was excluded by studying the brain±lesion profiles in the animals receiving untreated, compared with autoclaved, material. These were identical. As insufficient data were available to show precisely the shape of the curve between zero time and 18 min, the two possible extremes have been plotted. These show that if, as is often the case with conventional microorganisms, the initial steep decline in infectivity is used to predict the time that it will take to achieve complete inactivation, this would be quite incorrect for scrapie agent, regardless of which of the two curves between zero time and 18 min are used. This reinforces the viewpoint that, although such estimates may often be useful (and sometimes accurate), there is no substitute for establishing a full inactivation curve (Greene, 1992). Tailing inactivation curves are not uncommon for conventional microorganisms; these may result from protection of some of the organisms through clumping, or be due to population heterogeneity where differing straight-line inactivation curves for two subpopulations combine to produce a tailing curve. Where there is no population heterogeneity, the same sort of tailing curve is still usually obtained when the surviving organisms are cultured and re-tested (Gardner and Peel, 1991). One explanation for the presence of a heat- or chemical-resistant (Taylor, 1991) subpopulation of scrapie agent might be the protective effect of aggregation which could occur in homogenates of infected tissue but not in undiluted tissue. However, Fig. 1 shows that even in undiluted tissue, small fractions of infectivity can survive heating for extended periods; this cannot be due to protective aggregation. Although more than seven logs of infectivity were lost, two logs of the 263 K strain of scrapie agent in 340 mg samples of macerated hamster brain survived autoclaving at 1348C for 1 h. Surprisingly, similarly sized samples of infected brain tissue are completely inactivated within 18 min by autoclaving if the brain tissue is undisrupted, as opposed to macerated (Taylor and McConnell, 1988; Taylor et al., 1997; unpublished data). The lesser efficiency of inactivating macerates may result from the fact that some smearing and drying before autoclaving occurs with this type of sample. Asher et al. (1986, 1987) found that scrapieinfected tissue is more difficult to inactivate by autoclaving when it has become dried onto surfaces. We have now shown that a relatively heat-resistant subpopulation of scrapie agent retains its thermostability when re-heated, suggesting that this is an acquired but stable characteristic of this subpopulation that differentiates it from the main population. Evidence for the intrinsic and fundamental difference of this subpopulation comes from the fact that, at its limiting dilution, it produces an average incubation period in recipient animals that is well beyond that for unheated material at its limiting dilution (Taylor and Fernie, 1996). 2. Methods From a pool of macerated hamster brain infected with the 263 K strain of scrapie agent, two 340 mg aliquots were placed on the grinding surfaces of two Griffiths tubes. These were subjected to porous-load autoclaving at 1348C for 18 min, after which one of the samples was homogenised in sterile physiological saline and a series of 10-fold dilutions was prepared. Each dilution was injected intracerebrally into groups of Syrian hamsters
10/10 (148) 3/5 (176)
10
ÿ1
9/12 (171) 7/12 (196)
10
ÿ2
9/12 (207) 0/6
10
ÿ3
ÿ4
10
No. infected hamsters in each dilution groupa
Mean incubation periods (in days) are shown in brackets. a Excluding intercurrent deaths.
Nil Autoclave 1 Autoclave 2
Procedure
6/6 (71)
10
ÿ5
4/6 (109)
10
ÿ6
3/6 (149)
10
ÿ7
4/12 (174)
10
ÿ8
0/12
10
ÿ9
Table 1 Titration of 263 K infectivity in hamster brain macerate before autoclaving, and after autoclaving once or twice at 1348C for 18 min
108.3 105.0 103.3
Infectivity titre (ID50 g)
36 D.M. Taylor et al. / Veterinary Microbiology 64 (1998) 33±38
D.M. Taylor et al. / Veterinary Microbiology 64 (1998) 33±38
37
(0.05 ml per animal). The second tube was autoclaved again at the same temperature and for the same time. This sample was then diluted and injected in the same fashion as the first sample. The hamsters were also injected with serial 10-fold dilutions of the untreated brain macerate. Animals were culled when they displayed clinical signs of neurological disease. Hamsters that remained healthy were kept for 18 months before they were culled. All brains were examined histologically for spongiform encephalopathy to confirm whether or not the animals had developed scrapie. In addition, to confirm that the strain of agent present after autoclaving was the same as that present in the untreated brain pool, the lesion profiles in these two groups of hamsters were compared. Titres of infectivity in the samples were calculated by the method of Karber (1931). The results are shown in Table 1. 3. Results Table 1 shows that, after one autoclaving cycle, the titre of infectivity was reduced by 3.3 logs. However, the titre was reduced by only a further 1.3 logs after autoclaving for a second time. Given the high calorific value of steam at 1348C, there can be no question of inadequate heat penetration into the samples because the Griffiths tubes were unstoppered. Also, thermocouple readings have shown that when a temperature of 1348C is achieved inside the autoclave chamber, the delay before there is complete heat penetration into 375 mg samples of brain tissue is only around 1 min (Taylor, unpublished data). There were no differences in the brain lesion profiles of the animals receiving untreated, compared with autoclaved, material. 4. Discussion The lesser efficiency of inactivating partially smeared and dried macerated tissue, compared with intact undisrupted tissue, may relate to the very rapid heating that would occur in the film of dried material (compared with the bulk of the sample) and the consequently very rapid fixation of PrPSc in the dried film. Protection by fixation has been shown to occur during the inactivation of poliovirus by formalin (Gard and Maaloe, 1959); prior fixation in ethanol (Taylor, 1996b) or formalin (Taylor and McConnell, 1988) has been shown to enhance considerably the thermostability of scrapie agent. Also, it has been observed that the amount of scrapie infectivity inactivated after 4 h under vacuum at 728C is greater than that achieved over the same timescale at atmospheric pressure when an end temperature of 1208C is achieved (Taylor et al., 1997). Given that the molecular nature of TDE agents has not yet been characterised (Coles, 1997) it is difficult to know with any certainty what accounts for the characteristics of the subpopulation of scrapie agent that is intrinsically more thermostable than the main population. The fact that the brain lesion profiles were the same in the hamsters that received untreated, compared with autoclaved, material shows that this is not due to the presence of a minor (but thermostable) contaminating strain of agent. Although the complete molecular structure of TDE agents has still to be determined, it is likely that
38
D.M. Taylor et al. / Veterinary Microbiology 64 (1998) 33±38
PrPSc is an important component. Overall, the data presented or discussed suggest that procedures which produce rapid and/or extremely effective fixation of protein result in enhanced resistance of TDE agents. Thus, there seems to be a potential for developing effective inactivation procedures by the use of procedures that avoid rapid and/or efficient protein fixation. References Asher, D.M., Pomeroy, K.L., Murphy, L., Rohwer, R.G., Gibbs, C.J., Gajdusek, D.C., 1986. Practical inactivation of scrapie agent on surfaces, In: Abstr. IXth Int. Congress of Infectious and Parasitic Diseases, Munich, 20±26 July. Asher, D.M., Pomeroy, K.L., Murphy, L., Gibbs, C.J., Gajdusek, D.C., 1987. Attempts to disinfect surfaces contaminated with etiological agents of the spongiform encephalopathies, In: Abstr. VIIth Int. Congress of Virology, Edmonton, 9±14 August, p. 147. Bruce, M.E., Chree, A., McConnell, I., Foster, J., Pearson, G., Fraser, H., 1994. Transmission of bovine spongiform encephalopathy and scrapie to mice; strain variation and the species barrier. Philosophical Transactions of the Royal Society B 343, 405±411. Coles, H., 1997. Nobel panel rewards prion theory after years of heated debate. Nature 389, 529. Gard, S., Maaloe, O., 1959. Inactivation of viruses, In: Burnet, F.M., Stanley, W.M. (Eds.), The Viruses, vol. 1, Academic Press, New York, pp. 359±427. Gardner, J.F., Peel, M.M., 1991. Introduction to Sterilization, Disinfection and Infection Control, Churchill Livingstone, Edinburgh. Greene, V.W., 1992. Sterility assurance: Concepts, methods and problems, In: Russell, A.D., Hugo, W.B., Aycliffe, G.A.J. (Eds.), Principles and Practice of Disinfection, Preservation and Sterilization, Blackwell, Oxford, pp. 605±624. Karber, G., 1931. Beitrag zur kollectiven Behandlung pharmakologischer Reihenversuche. Arch. Exp. Path. 162, 480±483. Rohwer, R.G., 1983. Scrapie inactivation kinetics ± an explanation for scrapie's apparent resistance to inactivation ± a re-evaluation of estimates of its small size, In: Court, L.A., Cathala, F. (Eds.), Virus non Conventionnels et Affections du Systeme Nerveux Central, Masson, Paris, pp. 84±113. Taylor, D.M., 1991. Resistance of the ME7 scrapie agent to peracetic acid. Vet. Microbiol. 27, 19±24. Taylor, D.M., 1992. Inactivation of unconventional agents of the transmissible degenerative encephalopathies, In: Russell, A.D., Hugo, W.B., Aycliffe, G.A.J. (Eds.), Principles and Practice of Disinfection, Preservation and Sterilisation, Blackwell, Oxford, pp. 605±624. Taylor, D.M., 1996a. Creutzfeldt±Jacob disease, Lancet, 347, 1333. Taylor, D.M., 1996b. Transmissible subacute spongiform encephalopathies: Practical aspects of agent inactivation, In: Court, L., Dodet, D. (Eds.), Transmissible Subacute Spongiform Encephalopathies: Prion Diseases, IIIrd Int. Symp. Subacute Spongiform Encephalopathies, 18±20 March 1996, Paris, pp. 479±482. Taylor, D.M., Fernie, K., 1996. Exposure to autoclaving or sodium hydroxide extends the dose response curve of the 263K strain of scrapie agent in hamsters. J. Gen. Virol. 77, 811±813. Taylor, D.M., McConnell, I., 1988. Autoclaving does not decontaminate formol-fixed scrapie tissues, Lancet i, 1463±1464. Taylor, D.M., Fraser, H., McConnell, I., Brown, D.A., Brown, K.L., Lamza, K.A., Smith, G.R.A., 1994. Decontamination studies with the agents of bovine spongiform encephalopathy and scrapie. Arch. Virol. 139, 313±326. Taylor, D.M., Woodgate, S.L., Fleetwood, A.J., Cawthorne, R.J.G., 1997. The effect of rendering procedures on the scrapie agent. Vet. Rec. 141, 643±649.