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The effect of formic acid on BSE and scrapie infectivity in fixed and unfixed brain-tissue
veterinary microbiology ELSEVIER
Veterinary Microbiology 58 (1997) 167-174
The effect of formic acid on BSE and scrapie infectivity in fixed and unfixed brain-tissue D.M. Taylor *, J.M. Brown, K. Fernie, I. McConnell BBSRC and MRC Neuropathogenesis Unit, Institute for Animal Health, West Mains Road, Edinburgh EH9 3JF, UK
Received 15 May 1997; accepted 25 July 1997
Abstract Brain-tissue infected with scrapie-like agents remains infectious after histological fixation, and represents a source of occupational exposure. Infectivity titres in formol or paraformaldehydelysine-periodate (PLP)-fixed mouse-brains infected with the 301V strain of BSE agent were reduced by around six logs following treatment with formic acid. After PLP fixation and formic acid treatment, no infectivity was detectable in mouse-brain infected with the 87V strain of scrapie agent. Similarly-treated mouse-brain infected with the ME7 strain of scrapie agent showed a titre loss of ~ 5 logs. No infectivity was detectable in PLP-fixed, BSE-infected bovine brain after formic acid treatment, but this was an unreliable result. With unfixed brain homogenates, formic acid reduced the titre of ME7 by > 5.0 logs; technical problems limited the measured loss of BSE infectivity to > 1.0 logs. These studies confirm that formic acid treatment during fixation of brain-tissue significantly reduces the infectivity titres of scrapie-like agents, thus reducing the level of any occupational exposure. © 1997 Elsevier Science B.V. Keywords: Formic acid; BSE infectivity; Scrapie infectivity; Brain tissue
1. Introduction The u n c o n v e n t i o n a l but uncharacterised agents that cause transmissible d e g e n e r a t i v e e n c e p h a l o p a t h i e s ( T D E ) such as b o v i n e s p o n g i f o r m e n c e p h a l o p a t h y (BSE), scrapie in sheep, and C r e u t z f e l d t - J a k o b disease ( C J D ) in h u m a n s are r e m a r k a b l y resistant to inactivation ( K i m b e r l i n et al., 1983; B r o w n et al., 1986; T a y l o r et al., 1994). T D E agents
Corresponding author. BBSRC and MRC Neuropathogenesis Unit, Institute for Animal Health, West Mains Road, Edinburgh EH9 3JF, UK. Tel.: +44-131-667-5204; fax: +44-131-668-3872. 0378-1135/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 0 3 7 8 - 1 1 3 5 ( 9 7 ) 0 0 1 6 5 - X
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survive in formalin-fixed tissues (Fraser et al., 1992; Taylor, 1992) with little loss of titre (Brown et al., 1990a), and remain infective after full histological processing (Brown et al., 1982) thereby representing a potential source of occupational exposure. Scrapie agent is not considered to be a human pathogen, and there is no known risk to laboratory personnel who handle scrapie-infected materials (Advisory Committee on Dangerous Pathogens, 1994). In contrast, the same guidance document advises that CJD-infected materials should be handled under category 2 conditions. Although CJD had been observed in 24 healthcare workers (including two neurohistopathology technicians) by 1993, there was no evident link between their disease and previous occupational exposure to the causal agent (Berger and David, 1993). Nevertheless, scrapie infection can be established easily by introducing the causal agent through skin scarification in mice (Taylor et al., 1996), and the same may be true for CJD infection in humans. It was previously considered unlikely that the BSE agent would infect humans (Department of Health: Ministry of Agriculture Fisheries and Food, 1989; Taylor, 1989b) but because it is a relatively new disease, it was recommended that BSE agent should be handled under category 2 conditions (Advisory Committee on Dangerous Pathogens, 1994). However, it has been proposed more recently that the emergence of a new variant form of CJD in the UK may be linked to exposure of the human population to the BSE agent (Will et al., 1996). Adding sodium hypochlorite i or phenol (Kleinman, 1980; Brumback, 1988; Esiri, 1989) to formol saline are methods that have been recommended for reducing or eliminating infectivity in formol-fixed tissues but without any supporting experimental data. In the case of phenol the basis of the proposal was flawed (Taylor, 1989a), and phenolised formalin was shown subsequently to cause little inactivation (Brown et al., 1990b) and poor fixation (Brown et al., 1990b; Mackenzie and Fellowes, 1990). Another proposal was to autoclave formol-fixed brain (Masters et al., 1985a,b). However, although the microscopic architecture of autoclaved formol-fixed brain remains sufficiently good for routine diagnostic purposes (Taylor and McBride, 1987), substantial levels of infectivity survive (Taylor and McConnell, 1988; Brown et al., 1990a). In contrast, it has been demonstrated that the infectivity titres of scrapie and CJD agents in formol-fixed rodent brain were reduced from 101°2 and 108.5 to 1013 and 10 2.3 respectively by an exposure to > 96% formic acid for 1 h (Brown et al., 1990b). Furthermore, the tissue morphology was well preserved, and permitted meaningful subsequent microscopic examination. It has been reported previously that the degree of survival after formic acid treatment may be somewhat higher when brain infected with the mouse-passaged 301V strain of BSE agent has been fixed in paraformaldehyde-lysine-periodate (PLP) (Taylor, 1995). Although much of the experimental work on BSE and scrapie depends on the histopathological examination of brain-tissue fixed in formol saline, a significant amount of tissue is fixed in modified McLean's paraformaldehyde-lysine-periodate solution
i Cited in Ref. Titford and Bastian, 1989.
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because it exposes antigenic sites which can be exploited by immunocytochemical procedures (DeArmond et al., 1987). We have now carried out a quantitative study on formic acid inactivation of the mouse-passaged 301V strain of BSE agent in PLP-fixed and formol-fixed brain-tissue. Qualitative studies on formic acid inactivation were also carried out using PLP-fixed mouse-brain infected with the 87V or ME7 strains of scrapie agent, and PLP-fixed BSE-infected bovine brain. In addition, the effect of formic acid on infectivity in unfixed brain-tissue homogenates has been studied using mouse-brain infected with the ME7 strain of scrapie agent, and bovine brain-tissue infected with the BSE agent. The results of these various studies will be reported here.
2. Materials and methods
2.1. Quantitative assay for infectivity following formic acid treatment of 301V-infected mouse-brain during fixation in formalin or PLP A fresh brain was removed aseptically from a VM mouse terminally-affected with the 301V strain of mouse-passaged BSE agent; the infectivity titre in such brains is typically ~ 109-10 l° IDs0/g. The brain was bisected along the midline. Each half was then chopped into pieces, each measuring approximately 8 mm 3. The pieces from each half were immersed for 5 h in separate 10 ml quantities of freshly-prepared PLP. They were then transferred to 10 ml volumes of either sterile distilled water or Analar 98-100% formic acid (Merck, Glasgow) for 1 h. Each sample was then re-immersed in 10 ml of PLP for a further 5 h. The formic acid-treated sample was then subjected to three consecutive 4-h immersions in 100 ml volumes of sterile distilled water to leach out any residual formic acid so that the sample would be non-toxic for the recipient mice. The control (distilled water) sample was treated similarly. Following the removal of the tissues from the water, excess moisture was allowed to drain off before the samples were homogenised in sterile physiological saline (10% w / v ) using Griffiths tubes. Further dilutions were prepared in sterile physiological saline to obtain a range from 10 i up to a maximum of 10 -7. Various dilutions were injected intracerebrally into VM mice (20 /zl per mouse) as shown in Table 1. The mice were culled according to an established protocol when they displayed terminal clinical signs of neurological disease (Dickinson et al., 1968), or at 620 days after injection if they displayed no clinical signs. Brains from all mice were examined histologically for spongiform encephalopathy using tissue sections that were representative of all areas of the brain (Fraser and Dickinson, 1968). The ratios of positive and negative animals within each group were used to calculate the infectivity titres (Karber, 1931). A flesh brain from a further VM mouse terminally-affected with the 301V strain of mouse-passaged agent was processed similarly, except that the fixative used was 10% formol saline. The only difference from the above methodology was that each of the two periods of exposure to formol saline, before and after formic acid treatment, was for 24 h. The details of the dilution groups injected are shown in Table 1.
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Table 1 Quantitative assay of infectivity following exposure of PLP-fixed or formol-fixed 301V-infected brain-tissue to formic acid Treatment PLP/water
PLP/formic Formol/water
Formol/formic
Dilution 10 4 10-5 10-6 10-7 10-I 10-2 10-3 10-4 10 5 10 6 I0 7 10 i 10-2
No. mice infected/No, mice injected" 6/6 4/5 1/6 0/2 l/6 1/6 0/6 6/6 2/5 1/5
Infectivity titre (IDs0/g) 1072
< 1 0 2.5
106s
0/6
4/5 0/5
< 1030
aExcluding intercurrent deaths.
2.2. Qualitative assay for infectivi~" following formic acid treatment of 87V or ME7-infected mouse-brain, or BSE-infected bovine brain, during fixation in PLP The original titres of infectivity in the brain-tissues were ~ 108.o I D s 0 / g for 87V and ME7; the BSE titre was 1041 ID50/g. The methodology applied was mainly as described above for PLP-fixed brain-tissue but only 10 ~ dilutions of the treated materials were injected into mice. The only variation was with the BSE-infected bovine brain, where a sample equivalent in weight to an adult mouse-brain (375 mg) was removed from the brain-pool, and then processed as above. After processing, the samples were injected by the intracerebral (20 /xl) route. With the BSE-infected bovine brain, a further group of mice was injected by the combined intracerebral (20 /_tl) and intraperitoneal (100 ktl) routes because it is recognised that the greater efficiency of the intracerebral route for TDE agents passaged within a specific host-species does not apply with the BSE agent when crossing the bovine-to-mouse species-barrier (Taylor et al., 1994). The 87V material was injected into VM mice; ME7 into RIII and SV mice, and BSE into RIII mice. The experiments with the ME7 strain of agent were terminated at 496 days after injection; 87V at 456 days, and BSE at 656 days. The brains of all mice were examined microscopically for the presence of spongiform encephalopathy.
2.3. Qualitatit, e assay for infectivity following formic acid treatment of unfixed ME7-infected mouse-brain and BSE-infected bouine brain homogenates One gram samples of pooled macerated mouse-brains infected with the ME7 strain of scrapie agent, and pooled macerated bovine brains infected with BSE, were each homogenised in 10 ml of 9 8 - 1 0 0 % formic acid. The starting titres of infectivity in the homogenates was ~ 107 I D s 0 / m l for ME7, and 1031 I D s 0 / m l for BSE. After a period of 30 min at room temperature, the homogenates were brought to neutral pH by the
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addition of 1 M ammonium carbonate (105 ml in the case of ME7; 120 ml in the case of BSE). Two 5-ml aliquots were removed from each sample and dried over a period of 4 h using a Savant vacuum extractor to remove the neutralised, but still toxic, formic acid. Each dried aliquot was then re-homogenised in 0.5 ml of sterile distilled water, and the paired samples were pooled. At this stage the pH of the ME7 sample was slightly acidic, and was adjusted to neutral by the addition of a few drops of 1 M sodium hydroxide. To minimise toxicity problems in the recipient mice, the samples were diluted before injection (100-fold in the case of BSE: from 10- to 80-fold in the case of ME7). The BSE sample was injected by the combined intracerebral (20/zl) and intraperitoneal (500 /xl) routes into RIII mice; the ME7 sample was injected by the intracerebral (20 /zl) route into C57BL mice. The RIII mice were observed for the development of clinical neurological disease for up to 783 days post-injection; with the C57BL mice, this period was increased to 867 days because of their greater longevity. All brains were examined histopathologically for spongiform encephalopathy.
3. Results
The results from the quantitative bioassays of formol-fixed or PLP-fixed mouse-brain infected with the 301V strain of BSE agent, with and without formic acid treatment, are shown in Table 1. After formic acid treatment the PLP-fixed and formol-fixed tissues had < 10 2.5 and < 103° IDs0/g respectively of infectivity remaining. The infectivity titres of the fixed, water-treated controls w e r e 107.2 IDs0/g and 10 6.8 IDs0/g respectively. Table 2 shows the results of the qualitative assays for infectivity following formic acid treatment of PLP-fixed, 87V or ME7-infected mouse-brain, and BSE-infected bovine brain. All controls (PLP-fixed but water-treated) were positive, as were a number of the mice which received PLP-fixed, formic acid-treated, ME7-infected mouse-brain. However, no infectivity was detected in the 87V-infected mouse-brain or the BSE-infected bovine brain that had been PLP-fixed and treated with formic acid.
Table 2 Qualitative assay for infectivity following formic acid treatment of PLP-fixed 87V or ME7-infected mousebrain, and BSE-infected bovine brain Agent
Treatment
Injection route
No. mice infected/No, mice injected a
87V
PLP/water PLP/formic PLP/water PLP/formic PLP/water PLP/formic PLP/formic
ic b ic ic ic ic + ip c ic + ip ic
5//5 0/12 5/5 7/10 4/4 0/2 0/5
ME7 BSE
aExcluding intercurrent deaths. bIntracerebral. c Intraperitoneal.
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Table 3 The effect of formic acid on homogenates of ME7-infected mouse-brain and BSE-infected bovine brain Agent ME7
BSE
Dilution group 1:10 1:20 1:40 1:80 1:100
No. mice affected/No, mice injected" 0/4 0/4 0/4 0/15 0/10
Infectivity titre loss (IDs0/ml) > 105.0 > 1 0 4.7 > 10 ~4
>_ 10~ >~10I°
"Excluding intercurrent deaths. The effect of formic acid on homogenates of unfixed ME7-infected mouse-brain and BSE-infected bovine brain are shown in Table 3. None of the recipient mice developed spongiform encephalopathy but the sensitivity of the tests was reduced because the samples were diluted to avoid toxic reactions in the mice. Table 3 shows the maximum titre reductions that were achieved.
4. Discussion Table 1 shows that formic acid treatment of PLP-fixed brain-tissue infected with the 301V strain of BSE agent permitted the survival of < 102.5 I D s 0 / g of infectivity. This value was obtained by quantitative bioassay, and is lower than the titre of 103.2 I D s 0 / g which was estimated previously from data obtained by qualitative assay after a similar treatment of 301V (Taylor, 1995). This revised, more accurate, figure is not substantially different from the value of 102.3 I D s 0 / g previously obtained for formol-fixed, formic acid-treated hamster brain infected with the 263K strain of scrapie agent (Brown et al., 1990a) which has a similar starting titre to 301V. The titre of surviving infectivity in the 301 V-infected brain which had been formol-fixed and treated with formic acid (Table 1) was not significantly different from that in the PLP-fixed, formic acid-treated tissue. The qualitative studies on the effect of formic acid on infected, PLP-fixed brain-tissue show that there was no detectable infectivity after 87V-infected mouse-brain had been treated in this fashion (Table 2). In contrast, there was some survival of infectivity (estimated to be ~ 102.7 I D s 0 / g ) after a similar treatment of ME7-infected mouse-brain (Table 2). Given the comparable starting titres of these two strains of scrapie agent, this suggests that there is strain variation in terms of susceptibility to inactivation by formic acid. The fact that 87V appears to be more susceptible was surprising because, in contrast to ME7, it produces florid plaques of disease-specific PrP protein in the brains of infected mice, and it had been anticipated that these might have been more difficult to inactivate. With the BSE-infected bovine brain, the PLP-fixed, water-treated control material produced spongiform encephalopathy in all four recipient mice (Table 2) with an average incubation period of 336 days (standard error, _+ 5 days). However, only two of the mice injected with formic acid treated BSE agent survived long enough to regard them as being truly negative; these were culled at 561 and 656 days post-injection, and had no detectable spongiform encephalopathy. Therefore, this negative result cannot be considered to be reliable.
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In the e x p e r i m e n t s w h e r e i n f e c t e d b r a i n h o m o g e n a t e s w e r e t r e a t e d w i t h f o r m i c acid, the d e g r e e o f d i l u t i o n r e q u i r e d to r e n d e r the s a m p l e s n o n - t o x i c for m i c e r e d u c e d the s e n s i t i v i t y o f the assays. A l t h o u g h it w a s p o s s i b l e to s h o w a titre loss o f > 10 5.0 I D s 0 / m l for M E 7 , it w a s o n l y p o s s i b l e to c a l c u l a t e a loss o f > 10 l ° I D s 0 / m l for B S E i n f e c t i v i t y ( T a b l e 3). T h e studies o n the i n f e c t e d f o r m o l - f i x e d or P L P - f i x e d b r a i n - t i s s u e s h a v e d e m o n strated that f o r m i c acid t r e a t m e n t p r o d u c e d s i g n i f i c a n t r e d u c t i o n s in t h e i n f e c t i v i t y titres o f t h e 3 0 1 V strain o f m o u s e - p a s s a g e d B S E agent, a n d the M E 7 a n d 8 7 V strains o f m o u s e - p a s s a g e d scrapie agent. T h i s c o n f i r m s that the i n t r o d u c t i o n o f a f o r m i c acid step d u r i n g the f i x a t i o n o f b r a i n - t i s s u e w i t h f o r m o l s a l i n e or P L P is a u s e f u l m e a s u r e w h i c h r e d u c e s the level o f a n y o c c u p a t i o n a l e x p o s u r e o f l a b o r a t o r y w o r k e r s to T D E agents.
Acknowledgements T h i s s t u d y w a s s u p p o r t e d b y a g r a n t f r o m the M i n i s t r y o f A g r i c u l t u r e F i s h e r i e s a n d Food.
References Advisory Committee on Dangerous Pathogens 1994. Precautions for work with human and animal transmissible spongiform encephalopathies. HMSO, London. Berger, J.R., David, N.J., 1993. Creutzfeldt-Jakob disease in a physician: a review of the disorder in health care workers. Neurology 43, 205-206. Brown, P., Rohwer, R.G., Green, E.M., Gajdusek, D.C., 1982. Effects of chemicals, heat and histopathological processing on high-infectivity hamster-adapted scrapie virus. J. Infect. Dis. 145, 683-687. Brown, P., Rohwer, R.G., Gajdusek, D.C., 1986. Newer data on the inactivation of scrapie virus or Creutzfeldt-Jakob disease virus in brain tissue. J. Infect. Dis. 153, 1145-1148. Brown, P., Liberski, P.P., Wolff, A., Gajdusek, D.C., 1990a. Resistance of scrapie agent to steam autoclaving after formaldehyde fixation and limited survival after ashing at 360°C: practical and theoretical implications. J. Infect. Dis. 161,467-472. Brown, P., Wolff, A., Gajdusek, D.C., 1990b. A simple and effective method for inactivating virus infectivity in formalin-fixed tissue samples from patients with Creutzfeldt-Jakob disease. Neurology 40, 887-890. Brumback, R.A., 1988. Routine use of phenolised formalin in fixation of autopsy brain tissue to reduce risk of inadvertent transmission of Creutzfeldt-Jakob disease. New Engl. J. Med. 319, 654. DeArmond, S.J., Mobley, W.C., DeMott, D.L., Barry, R.A., Beckstead, J.H., Prusiner, S.B., 1987. Changes in the localization of brain prion proteins during scrapie infection. Neurology 37, 1271-1280. Department of Health: Ministry of Agriculture Fisheries and Food, 1989. Report of the working party on bovine spongiform encephalopathy. MAFF, London. Dickinson, A.G., Meikle, V.M.H., Fraser, H., 1968. Identification of a gene which controls the incubation period of some strains of scrapie in mice. J. Comp. Pathol. 78, 293-299. Esiri, M.M., 1989. Diagnostic Neuropathology. Blackwell, Oxford. Fraser, H., Dickinson, A.G., 1968. The sequential development of the brain lesions of scrapie in three strains of mice. J. Comp. Pathol. 78, 301-311. Fraser, H., Bruce, M.E., Chree, A., McConnell, I., Wells, G.A.H., 1992. Transmission of bovine spongiform encephalopathy and scrapie to mice. J. Gen. Virol., 1897. Karber, G., 1931. Beitrag zur kollectiven Behandlung pharmakologischer Reihenversuche. Arch. Exp. Pathol. 162, 480-483.
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Kimberlin, R.H., Walker, C.A., Millson, G.C., Taylor, D.M., Robertson, P.A., Tomlinson, A.H., Dickinson, A.G., 1983. Disinfection studies with two strains of mouse-passaged scrapie agent. J. Neurol. Sci. 59, 355-369. Kleinman, G.M., 1980. Case records of the Massachusetts General Hospital (case 45-1980). New Engl. J. Med. 303, 1162-1171. Mackenzie, J.M., Fellowes, W., 1990. Phenolized formalin may obscure early histological changes of Creutzfeldt-Jakob disease. Neuropathol. Appl. Neurobiol. 16, 255. Masters, C.L., Jacobsen, P.F., Kakulas, B.A., 1985a. Letter to the editor. Neuropathol. Appl. Neurobiol. 44, 304-307. Masters, C.L., Jacobsen, P.F., Kakulas, B.A., 1985b. Letter to the editor. Neuropathol. Appl. Neurobiol. 45, 760-776. Taylor, D.M., 1989a. Phenolized formalin may not inactivate Creutzfeldt-Jakob disease infectivity. Neuropathol. Appl. Neurobiol. 15, 585-586. Taylor, D.M., 1989b. Bovine spongiform encephalopathy and human health. Vet. Rec. 125, 413-415. Taylor, D.M. 1992. Inactivation of unconventional agents of the transmissible degenerative encephalopathies. In: Russell, A.D., Hugo, W.B., Ayliffe, G.A.J. (Eds.), Principles and Practice of Disinfection, Preservation and Sterilization. Blackwell, Oxford, pp. 171-179. Taylor, D.M., 1995. Survival of mouse-passaged bovine spongiform encephalopathy agent after exposure to paraformaldehyde-lysine-periodate and formic acid. Vet. Microbiol. 44, i I 1-112. Taylor, D.M., McBride, P.A., 1987. Autoclaved, formol-fixed scrapie brain is suitable for histopathological examination but may still be infective. Acta Neuropathol. 74, 194-196. 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., McConnell, I., Fraser, H., 1996. Scrapie infection can be established readily through skin scarification in immunocompetent but not immunodeficient mice. J. Gen. Virol. 77, 1595-1599. Titford, M., Bastian, F.L., 1989. Handling Creutzfeldt-Jakob disease tissues in the laboratory. J. Histotechnol. 12, 214-217. Will, R.G., Ironside, J.W., Zeidler, M., Cousens, S.N., Estibeiro, K., Alperovitch, A., Poser, S., Pocchiari, M., Hofman, A., Smith, P.G., 1996. A new variant form of Creutzfeldt-Jakob disease in the UK. Lancet 347, 921-925.
Veterinary Microbiology 58 (1997) 167-174
The effect of formic acid on BSE and scrapie infectivity in fixed and unfixed brain-tissue D.M. Taylor *, J.M. Brown, K. Fernie, I. McConnell BBSRC and MRC Neuropathogenesis Unit, Institute for Animal Health, West Mains Road, Edinburgh EH9 3JF, UK
Received 15 May 1997; accepted 25 July 1997
Abstract Brain-tissue infected with scrapie-like agents remains infectious after histological fixation, and represents a source of occupational exposure. Infectivity titres in formol or paraformaldehydelysine-periodate (PLP)-fixed mouse-brains infected with the 301V strain of BSE agent were reduced by around six logs following treatment with formic acid. After PLP fixation and formic acid treatment, no infectivity was detectable in mouse-brain infected with the 87V strain of scrapie agent. Similarly-treated mouse-brain infected with the ME7 strain of scrapie agent showed a titre loss of ~ 5 logs. No infectivity was detectable in PLP-fixed, BSE-infected bovine brain after formic acid treatment, but this was an unreliable result. With unfixed brain homogenates, formic acid reduced the titre of ME7 by > 5.0 logs; technical problems limited the measured loss of BSE infectivity to > 1.0 logs. These studies confirm that formic acid treatment during fixation of brain-tissue significantly reduces the infectivity titres of scrapie-like agents, thus reducing the level of any occupational exposure. © 1997 Elsevier Science B.V. Keywords: Formic acid; BSE infectivity; Scrapie infectivity; Brain tissue
1. Introduction The u n c o n v e n t i o n a l but uncharacterised agents that cause transmissible d e g e n e r a t i v e e n c e p h a l o p a t h i e s ( T D E ) such as b o v i n e s p o n g i f o r m e n c e p h a l o p a t h y (BSE), scrapie in sheep, and C r e u t z f e l d t - J a k o b disease ( C J D ) in h u m a n s are r e m a r k a b l y resistant to inactivation ( K i m b e r l i n et al., 1983; B r o w n et al., 1986; T a y l o r et al., 1994). T D E agents
Corresponding author. BBSRC and MRC Neuropathogenesis Unit, Institute for Animal Health, West Mains Road, Edinburgh EH9 3JF, UK. Tel.: +44-131-667-5204; fax: +44-131-668-3872. 0378-1135/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 0 3 7 8 - 1 1 3 5 ( 9 7 ) 0 0 1 6 5 - X
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survive in formalin-fixed tissues (Fraser et al., 1992; Taylor, 1992) with little loss of titre (Brown et al., 1990a), and remain infective after full histological processing (Brown et al., 1982) thereby representing a potential source of occupational exposure. Scrapie agent is not considered to be a human pathogen, and there is no known risk to laboratory personnel who handle scrapie-infected materials (Advisory Committee on Dangerous Pathogens, 1994). In contrast, the same guidance document advises that CJD-infected materials should be handled under category 2 conditions. Although CJD had been observed in 24 healthcare workers (including two neurohistopathology technicians) by 1993, there was no evident link between their disease and previous occupational exposure to the causal agent (Berger and David, 1993). Nevertheless, scrapie infection can be established easily by introducing the causal agent through skin scarification in mice (Taylor et al., 1996), and the same may be true for CJD infection in humans. It was previously considered unlikely that the BSE agent would infect humans (Department of Health: Ministry of Agriculture Fisheries and Food, 1989; Taylor, 1989b) but because it is a relatively new disease, it was recommended that BSE agent should be handled under category 2 conditions (Advisory Committee on Dangerous Pathogens, 1994). However, it has been proposed more recently that the emergence of a new variant form of CJD in the UK may be linked to exposure of the human population to the BSE agent (Will et al., 1996). Adding sodium hypochlorite i or phenol (Kleinman, 1980; Brumback, 1988; Esiri, 1989) to formol saline are methods that have been recommended for reducing or eliminating infectivity in formol-fixed tissues but without any supporting experimental data. In the case of phenol the basis of the proposal was flawed (Taylor, 1989a), and phenolised formalin was shown subsequently to cause little inactivation (Brown et al., 1990b) and poor fixation (Brown et al., 1990b; Mackenzie and Fellowes, 1990). Another proposal was to autoclave formol-fixed brain (Masters et al., 1985a,b). However, although the microscopic architecture of autoclaved formol-fixed brain remains sufficiently good for routine diagnostic purposes (Taylor and McBride, 1987), substantial levels of infectivity survive (Taylor and McConnell, 1988; Brown et al., 1990a). In contrast, it has been demonstrated that the infectivity titres of scrapie and CJD agents in formol-fixed rodent brain were reduced from 101°2 and 108.5 to 1013 and 10 2.3 respectively by an exposure to > 96% formic acid for 1 h (Brown et al., 1990b). Furthermore, the tissue morphology was well preserved, and permitted meaningful subsequent microscopic examination. It has been reported previously that the degree of survival after formic acid treatment may be somewhat higher when brain infected with the mouse-passaged 301V strain of BSE agent has been fixed in paraformaldehyde-lysine-periodate (PLP) (Taylor, 1995). Although much of the experimental work on BSE and scrapie depends on the histopathological examination of brain-tissue fixed in formol saline, a significant amount of tissue is fixed in modified McLean's paraformaldehyde-lysine-periodate solution
i Cited in Ref. Titford and Bastian, 1989.
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because it exposes antigenic sites which can be exploited by immunocytochemical procedures (DeArmond et al., 1987). We have now carried out a quantitative study on formic acid inactivation of the mouse-passaged 301V strain of BSE agent in PLP-fixed and formol-fixed brain-tissue. Qualitative studies on formic acid inactivation were also carried out using PLP-fixed mouse-brain infected with the 87V or ME7 strains of scrapie agent, and PLP-fixed BSE-infected bovine brain. In addition, the effect of formic acid on infectivity in unfixed brain-tissue homogenates has been studied using mouse-brain infected with the ME7 strain of scrapie agent, and bovine brain-tissue infected with the BSE agent. The results of these various studies will be reported here.
2. Materials and methods
2.1. Quantitative assay for infectivity following formic acid treatment of 301V-infected mouse-brain during fixation in formalin or PLP A fresh brain was removed aseptically from a VM mouse terminally-affected with the 301V strain of mouse-passaged BSE agent; the infectivity titre in such brains is typically ~ 109-10 l° IDs0/g. The brain was bisected along the midline. Each half was then chopped into pieces, each measuring approximately 8 mm 3. The pieces from each half were immersed for 5 h in separate 10 ml quantities of freshly-prepared PLP. They were then transferred to 10 ml volumes of either sterile distilled water or Analar 98-100% formic acid (Merck, Glasgow) for 1 h. Each sample was then re-immersed in 10 ml of PLP for a further 5 h. The formic acid-treated sample was then subjected to three consecutive 4-h immersions in 100 ml volumes of sterile distilled water to leach out any residual formic acid so that the sample would be non-toxic for the recipient mice. The control (distilled water) sample was treated similarly. Following the removal of the tissues from the water, excess moisture was allowed to drain off before the samples were homogenised in sterile physiological saline (10% w / v ) using Griffiths tubes. Further dilutions were prepared in sterile physiological saline to obtain a range from 10 i up to a maximum of 10 -7. Various dilutions were injected intracerebrally into VM mice (20 /zl per mouse) as shown in Table 1. The mice were culled according to an established protocol when they displayed terminal clinical signs of neurological disease (Dickinson et al., 1968), or at 620 days after injection if they displayed no clinical signs. Brains from all mice were examined histologically for spongiform encephalopathy using tissue sections that were representative of all areas of the brain (Fraser and Dickinson, 1968). The ratios of positive and negative animals within each group were used to calculate the infectivity titres (Karber, 1931). A flesh brain from a further VM mouse terminally-affected with the 301V strain of mouse-passaged agent was processed similarly, except that the fixative used was 10% formol saline. The only difference from the above methodology was that each of the two periods of exposure to formol saline, before and after formic acid treatment, was for 24 h. The details of the dilution groups injected are shown in Table 1.
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Table 1 Quantitative assay of infectivity following exposure of PLP-fixed or formol-fixed 301V-infected brain-tissue to formic acid Treatment PLP/water
PLP/formic Formol/water
Formol/formic
Dilution 10 4 10-5 10-6 10-7 10-I 10-2 10-3 10-4 10 5 10 6 I0 7 10 i 10-2
No. mice infected/No, mice injected" 6/6 4/5 1/6 0/2 l/6 1/6 0/6 6/6 2/5 1/5
Infectivity titre (IDs0/g) 1072
< 1 0 2.5
106s
0/6
4/5 0/5
< 1030
aExcluding intercurrent deaths.
2.2. Qualitative assay for infectivi~" following formic acid treatment of 87V or ME7-infected mouse-brain, or BSE-infected bovine brain, during fixation in PLP The original titres of infectivity in the brain-tissues were ~ 108.o I D s 0 / g for 87V and ME7; the BSE titre was 1041 ID50/g. The methodology applied was mainly as described above for PLP-fixed brain-tissue but only 10 ~ dilutions of the treated materials were injected into mice. The only variation was with the BSE-infected bovine brain, where a sample equivalent in weight to an adult mouse-brain (375 mg) was removed from the brain-pool, and then processed as above. After processing, the samples were injected by the intracerebral (20 /xl) route. With the BSE-infected bovine brain, a further group of mice was injected by the combined intracerebral (20 /_tl) and intraperitoneal (100 ktl) routes because it is recognised that the greater efficiency of the intracerebral route for TDE agents passaged within a specific host-species does not apply with the BSE agent when crossing the bovine-to-mouse species-barrier (Taylor et al., 1994). The 87V material was injected into VM mice; ME7 into RIII and SV mice, and BSE into RIII mice. The experiments with the ME7 strain of agent were terminated at 496 days after injection; 87V at 456 days, and BSE at 656 days. The brains of all mice were examined microscopically for the presence of spongiform encephalopathy.
2.3. Qualitatit, e assay for infectivity following formic acid treatment of unfixed ME7-infected mouse-brain and BSE-infected bouine brain homogenates One gram samples of pooled macerated mouse-brains infected with the ME7 strain of scrapie agent, and pooled macerated bovine brains infected with BSE, were each homogenised in 10 ml of 9 8 - 1 0 0 % formic acid. The starting titres of infectivity in the homogenates was ~ 107 I D s 0 / m l for ME7, and 1031 I D s 0 / m l for BSE. After a period of 30 min at room temperature, the homogenates were brought to neutral pH by the
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addition of 1 M ammonium carbonate (105 ml in the case of ME7; 120 ml in the case of BSE). Two 5-ml aliquots were removed from each sample and dried over a period of 4 h using a Savant vacuum extractor to remove the neutralised, but still toxic, formic acid. Each dried aliquot was then re-homogenised in 0.5 ml of sterile distilled water, and the paired samples were pooled. At this stage the pH of the ME7 sample was slightly acidic, and was adjusted to neutral by the addition of a few drops of 1 M sodium hydroxide. To minimise toxicity problems in the recipient mice, the samples were diluted before injection (100-fold in the case of BSE: from 10- to 80-fold in the case of ME7). The BSE sample was injected by the combined intracerebral (20/zl) and intraperitoneal (500 /xl) routes into RIII mice; the ME7 sample was injected by the intracerebral (20 /zl) route into C57BL mice. The RIII mice were observed for the development of clinical neurological disease for up to 783 days post-injection; with the C57BL mice, this period was increased to 867 days because of their greater longevity. All brains were examined histopathologically for spongiform encephalopathy.
3. Results
The results from the quantitative bioassays of formol-fixed or PLP-fixed mouse-brain infected with the 301V strain of BSE agent, with and without formic acid treatment, are shown in Table 1. After formic acid treatment the PLP-fixed and formol-fixed tissues had < 10 2.5 and < 103° IDs0/g respectively of infectivity remaining. The infectivity titres of the fixed, water-treated controls w e r e 107.2 IDs0/g and 10 6.8 IDs0/g respectively. Table 2 shows the results of the qualitative assays for infectivity following formic acid treatment of PLP-fixed, 87V or ME7-infected mouse-brain, and BSE-infected bovine brain. All controls (PLP-fixed but water-treated) were positive, as were a number of the mice which received PLP-fixed, formic acid-treated, ME7-infected mouse-brain. However, no infectivity was detected in the 87V-infected mouse-brain or the BSE-infected bovine brain that had been PLP-fixed and treated with formic acid.
Table 2 Qualitative assay for infectivity following formic acid treatment of PLP-fixed 87V or ME7-infected mousebrain, and BSE-infected bovine brain Agent
Treatment
Injection route
No. mice infected/No, mice injected a
87V
PLP/water PLP/formic PLP/water PLP/formic PLP/water PLP/formic PLP/formic
ic b ic ic ic ic + ip c ic + ip ic
5//5 0/12 5/5 7/10 4/4 0/2 0/5
ME7 BSE
aExcluding intercurrent deaths. bIntracerebral. c Intraperitoneal.
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Table 3 The effect of formic acid on homogenates of ME7-infected mouse-brain and BSE-infected bovine brain Agent ME7
BSE
Dilution group 1:10 1:20 1:40 1:80 1:100
No. mice affected/No, mice injected" 0/4 0/4 0/4 0/15 0/10
Infectivity titre loss (IDs0/ml) > 105.0 > 1 0 4.7 > 10 ~4
>_ 10~ >~10I°
"Excluding intercurrent deaths. The effect of formic acid on homogenates of unfixed ME7-infected mouse-brain and BSE-infected bovine brain are shown in Table 3. None of the recipient mice developed spongiform encephalopathy but the sensitivity of the tests was reduced because the samples were diluted to avoid toxic reactions in the mice. Table 3 shows the maximum titre reductions that were achieved.
4. Discussion Table 1 shows that formic acid treatment of PLP-fixed brain-tissue infected with the 301V strain of BSE agent permitted the survival of < 102.5 I D s 0 / g of infectivity. This value was obtained by quantitative bioassay, and is lower than the titre of 103.2 I D s 0 / g which was estimated previously from data obtained by qualitative assay after a similar treatment of 301V (Taylor, 1995). This revised, more accurate, figure is not substantially different from the value of 102.3 I D s 0 / g previously obtained for formol-fixed, formic acid-treated hamster brain infected with the 263K strain of scrapie agent (Brown et al., 1990a) which has a similar starting titre to 301V. The titre of surviving infectivity in the 301 V-infected brain which had been formol-fixed and treated with formic acid (Table 1) was not significantly different from that in the PLP-fixed, formic acid-treated tissue. The qualitative studies on the effect of formic acid on infected, PLP-fixed brain-tissue show that there was no detectable infectivity after 87V-infected mouse-brain had been treated in this fashion (Table 2). In contrast, there was some survival of infectivity (estimated to be ~ 102.7 I D s 0 / g ) after a similar treatment of ME7-infected mouse-brain (Table 2). Given the comparable starting titres of these two strains of scrapie agent, this suggests that there is strain variation in terms of susceptibility to inactivation by formic acid. The fact that 87V appears to be more susceptible was surprising because, in contrast to ME7, it produces florid plaques of disease-specific PrP protein in the brains of infected mice, and it had been anticipated that these might have been more difficult to inactivate. With the BSE-infected bovine brain, the PLP-fixed, water-treated control material produced spongiform encephalopathy in all four recipient mice (Table 2) with an average incubation period of 336 days (standard error, _+ 5 days). However, only two of the mice injected with formic acid treated BSE agent survived long enough to regard them as being truly negative; these were culled at 561 and 656 days post-injection, and had no detectable spongiform encephalopathy. Therefore, this negative result cannot be considered to be reliable.
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In the e x p e r i m e n t s w h e r e i n f e c t e d b r a i n h o m o g e n a t e s w e r e t r e a t e d w i t h f o r m i c acid, the d e g r e e o f d i l u t i o n r e q u i r e d to r e n d e r the s a m p l e s n o n - t o x i c for m i c e r e d u c e d the s e n s i t i v i t y o f the assays. A l t h o u g h it w a s p o s s i b l e to s h o w a titre loss o f > 10 5.0 I D s 0 / m l for M E 7 , it w a s o n l y p o s s i b l e to c a l c u l a t e a loss o f > 10 l ° I D s 0 / m l for B S E i n f e c t i v i t y ( T a b l e 3). T h e studies o n the i n f e c t e d f o r m o l - f i x e d or P L P - f i x e d b r a i n - t i s s u e s h a v e d e m o n strated that f o r m i c acid t r e a t m e n t p r o d u c e d s i g n i f i c a n t r e d u c t i o n s in t h e i n f e c t i v i t y titres o f t h e 3 0 1 V strain o f m o u s e - p a s s a g e d B S E agent, a n d the M E 7 a n d 8 7 V strains o f m o u s e - p a s s a g e d scrapie agent. T h i s c o n f i r m s that the i n t r o d u c t i o n o f a f o r m i c acid step d u r i n g the f i x a t i o n o f b r a i n - t i s s u e w i t h f o r m o l s a l i n e or P L P is a u s e f u l m e a s u r e w h i c h r e d u c e s the level o f a n y o c c u p a t i o n a l e x p o s u r e o f l a b o r a t o r y w o r k e r s to T D E agents.
Acknowledgements T h i s s t u d y w a s s u p p o r t e d b y a g r a n t f r o m the M i n i s t r y o f A g r i c u l t u r e F i s h e r i e s a n d Food.
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