- Original Articles
- Open Access
Infectivity of Scrapie Prions Bound to a Stainless Steel Surface
Molecular Medicine volume 5, pages240–243(1999)
The transmissible agent of Creutzfeldt-Jakob disease (CJD) is not readily destroyed by conventional sterilization and transmissions by surgical instruments have been reported. Decontamination studies have been carried out thus far on solutions or suspensions of the agent and may not reflect the behavior of surface-bound infectivity.
Materials and Methods
As a model for contaminated surgical instruments, thin stainless-steel wire segments were exposed to scrapie agent, washed exhaustively with or without treatment with 10% formaldehyde, and implanted into the brains of indicator mice. Infectivity was estimated from the time elapsing to terminal disease.
Stainless steel wire (0.15 × 5 mm) exposed to scrapie-infected mouse brain homogenate and washed extensively with PBS retained the equivalent of about 105 LD50 units per segment. Treatment with 10% formaldehyde for 1 hr reduced this value by only about 30-fold.
The model system we have devised confirms the anecdotal reports that steel instruments can retain CJD infectivity even after formaldehyde treatment. It lends itself to a systematic study of the conditions required to effectively inactivate CJD, bovine spongiform encephalopathy, and scrapie agent adsorbed to stainless steel surfaces such as those of surgical instruments.
The agent responsible for transmissible spongiform encephalopathies, the prion, is far more resistant to physical and chemical inactivation than conventional pathogens. These properties are reflected in the difficulties encountered in sterilizing prion-containing material by conventional procedures, in particular heat sterilization and formaldehyde treatment (1–5). More than 100 cases of proven or suspected iatrogenic transmissions to humans have been catalogued (6).
A particularly well-documented and disturbing report concerns an electrode that had been inserted into the cortex of a patient with unrecognized Creutzfeldt-Jakob disease (CJD) and, following a decontamination procedure involving treatment with benzene, 70% ethanol, and formaldehyde vapor after each use, was employed in succession on two additional patients who subsequently came down with CJD. Following these events, the tip of the electrode was implanted into the brain of a chimpanzee where it again caused lethal spongiform encephalopathy (7,8). The electrodes in question had a complex structure: a steel shaft of about 6 mm in diameter, with multiple silver contacts separated by rings of insulating plastic, allowing for the existence of crevices into which infectious material might have penetrated and become inaccessible to mechanical cleansing.
Instruments used for tonsillectomy or appendectomy on unrecognized nvCJD sufferers could become contaminated with the agent (9,10), so the question of how surgical instruments could be sterilized effectively without being damaged has assumed increased importance. In view of the report that scrapie-infected tissue dried onto surfaces is more resistant to inactivation than a suspension of the same material (quoted in ref. 4), extrapolation from available data on sterilization of suspensions may be unreliable. It thus became of interest to generate a model system for the sterilization of stainless steel instruments. We here show that mouse-adapted scrapie prions can firmly bind to stainless steel wire and give rise to infection when implanted into the brain of indicator mice, even after treatment with 10% formaldehyde.
Materials and Methods
Scrapie Infection and Diagnosis
RML is a mouse-adapted scrapie isolate (11) that was passaged in Swiss CD-1 mice (Charles River Laboratories, Wilmington, MA). Inoculum stock was prepared as a 10% (w/v) homogenate of RML scrapie-infected CD-1 mouse brains in 0.32 M sucrose; RML4.1 had a titer of 8.7 log LD50 units/ml and 20 mg/ml total protein. Inoculated mice were checked for the development of scrapie symptoms every other day and, once they developed disease, every day (12). Apparent prion titers were estimated from incubation times (13) using the parameters derived for Tga20 indicator mice (14).
Scrapie Agent-Exposed Wire
Stainless steel wire segments (diameter, 0.15 mm; length, 5 mm; stainless steel suture monofilament wire, Art. Nr. 01614037, USP 4/0, B. Braun Melsungen AG, Melsungen, Germany; batch 1/7502) were incubated with 10% homogenate of RML scrapie-infected CD-1 mouse brain in phosphate-buffered saline (PBS) for 16 hr, washed five times for 10 min in 50 ml PBS, all at room temperature. The wire segments were air dried and stored at room temperature for 1 day, and one segment was inserted into the brain of each of four Tga20 mice, while in deep anesthesia, by pushing it through a 25-gauge injection needle.
To determine the amount of protein bound to a wire segment, ten 5-mm wire segments were treated with brain homogenate, washed and dried as above. They were then incubated in 0.1 ml 2 M NaOH for 1 hr at 20°C and the eluate was diluted with 0.3 ml water. Protein was determined by the Micro BCA Protein assay (Pierce, Rockford, IL), using bovine serum albumin (BSA) dilutions as standards.
We exposed segments of thin stainless-steel wire to a 10% homogenate of freshly prepared scrapie-infected mouse brain, washed them exhaustively with only PBS or with PBS followed by exposure to 10% formaldehyde for 1 hr, and inserted them into the brains of indicator mice. Aliquots (30 µl) of a standard 1% brain homogenate (RML4.1) and of the last PBS wash of the wires were inoculated intracerebrally. The effective infectious dose was estimated by the incubation time method (13) using the parameters determined for Tga20 indicator mice (14).
As shown in Table 1, prion-exposed wire segments that had been washed exhaustively with PBS caused terminal disease after 72 ± 3 days; this is equivalent to a dose of about 5.2 ± 0.4 log LD50 units administered as brain homogenate. After formaldehyde treatment, terminal disease occurred after 87 ± 9 days (equivalent to a dose of about 3.5 ± 1 log LD50 units). Thirty microliters of a 1% brain homogenate (RML4.1) produced disease after 65 ± 1 days (a dose of 6 ± 0.1 log LD50 units) whereas 30 µl of the fifth PBS wash did not cause disease.
Ten segments of 0.5-cm wire segment, exposed to scrapie-infected brain homogenate and exhaustively washed with PBS, were eluted with 0.1 ml 2 M NaOH. No protein was detected in the eluate; the limit of detection was about 50 ng per segment.
Stainless steel wire exposed to scrapie-infected brain homogenate and washed exhaustively with PBS retained a high level of infectivity, which was diminished about 30-fold but not abolished by formaldehyde treatment.
Our experiments show that the anecdotal clinical findings of Bernoulli et al. (7) could be reproduced under defined laboratory settings, both in regard to the tight binding of prion infectivity to a stainless steel surface and to the considerable resistance of the bound infectivity to formaldehyde treatment. Failure to elute detectable amounts of protein from the exposed wires means that <50 ng per wire segment was bound and/or that the binding is partly or entirely irreversible under our experimental conditions.
The approach we report here enables the performance of sterilization experiments that more closely mimic real-life circumstances than experiments with solutions, suspensions, and homogenates. It is advisable that not only scrapie but also BSE and CJD prions be used in such tests, for which appropriate indicator mice exist (15–17).
Our findings also raise the interesting question as to whether the wire-bound prions desorb from the wire when inserted into the brain or whether they initiate infection from the bound state.
Taylor DM, Fraser H, McConnell I, et al. (1994) Decontamination studies with the agents of bovine spongiform encephalopathy and scrapie. Arch. Virol. 139: 313–326.
Brown P, Gibbs CJ Jr, Amyx HL, et al. (1982) Chemical disinfection of Creutzfeldt-Jakob disease virus. N. Engl. J. Med. 306: 1279–1282.
Ernst DR, Race RE. (1993) Comparative analysis of scrapie agent inactivation methods. J. Virol. Methods 41: 193–201.
Taylor DM. (1996) Exposure to, and inactivation of, the unconventional agents that cause transmissible degenerative encephalopathies. In: Baker H, Ridley RM (eds). Prion Diseases. Humana Press, Totowa, NJ, pp. 105–118.
Taylor DM. (1993) Inactivation of SE agents. Br. Med. Bull. 49: 810–821.
Brown P. (1996) Environmental causes of human spongiform encephalopathy. In: Baker H, Ridley RM (eds). Prion Diseases. Humana Press, Totowa, NJ, pp. 139–154.
Bernoulli C, Siegfried J, Baumgartner G, et al. (1977) Danger of accidental person-to-person transmission of Creutzfeldt-Jakob disease by surgery. Lancet 1: 478–479.
Gibbs CJ Jr, Asher DM, Kobrine A, Amyx HL, Sulima MP, Gajdusek DC. (1994) Transmission of Creutzfeldt-Jakob disease to a chimpanzee by electrodes contaminated during neurosurgery. J. Neurol. Neurosurg. Psychiatry 57: 757–758.
Collinge J, Rossor MN, Thomas D, Frosh A, Tolley N. (1998) Diagnosis of Creutzfeldt-Jakob disease by measurement of S100 protein in serum. Tonsil biopsy helps diagnose new variant Creutzfeldt-Jakob disease. BMJ 317: 472–473.
Hilton DA, Fathers E, Edwards P, Ironside JW, Zajicek J. (1998) Prion immunoreactivity in appendix before clinical onset of variant Creutzfeldt-Jakob disease. Lancet 352: 703–704.
Chandler RL. (1961) Encephalopathy in mice produced by inoculation with scrapie brain material. Lancet 1: 1378–1379.
Büeler H, Aguzzi A, Sailer A, et al. (1993) Mice devoid of PrP are resistant to scrapie. Cell 73: 1339–1347.
Prusiner SB, Cochran SP, Groth DF, Downey DE, Bowman KA, Martinez HM. (1982) Measurement of the scrapie agent using an incubation time interval assay. Ann. Neurol. 11: 353–358.
Brandner S, Isenmann S, Raeber A, et al. (1996) Normal host prion protein necessary for scrapie-induced neurotoxicity. Nature 379: 339–343.
Scott MR, Safar J, Telling G, et al. (1997) Identification of a prion protein epitope modulating transmission of bovine spongiform encephalopathy prions to transgenic mice. Proc. Natl. Acad. Sci. U.S.A. 94: 14279–14284.
Telling GC, Scott M, Hsiao KK, et al. (1994) Transmission of Creutzfeldt-Jakob disease from humans to transgenic mice expressing chimeric human-mouse prion protein. Proc. Natl. Acad. Sci. U.S.A. 91: 9936–9940.
Collinge J, Palmer MS, Sidle KC, et al. (1995) Unaltered susceptibility to BSE in transgenic mice expressing human prion protein. Nature 378: 779–783.
We thank Dr. Michael A. Klein for RML4.1 and Mr. Josef Ecsoedi for care and surveillance of the mice. This work was supported by the Kanton of Zürich and by grants of the Schweizerischer Nationalfonds and the European Union to C.W.
Communicated by C. Weissmann.
About this article
Cite this article
Zobeley, E., Flechsig, E., Cozzio, A. et al. Infectivity of Scrapie Prions Bound to a Stainless Steel Surface. Mol Med 5, 240–243 (1999). https://doi.org/10.1007/BF03402121
- Stainless Steel Surface
- Wire Segments
- Formaldehyde Treatment
- Injured Mice
- Creutzfeldt-Jakob Disease