Transmission of Scrapie by Steel-surface-bound Prions

Abstract

Background

Prions are unusually resistant to conventional disinfection procedures. An electrode used intracerebrally on a Creutzfeldt-Jakob disease (CJD) patient transmitted the disease to two patients in succession and finally to a chimpanzee, despite attempted disinfection. Concerns that surgical instruments may transmit variant CJD have been raised by the finding of PrP^Sc, a surrogate marker for infectivity, in various tissues other than brain.

Materials and Methods

Stainless steel wire was exposed to scrapie-infected brain or brain homogenate, washed exhaustively and inserted into the brain of indicator mice to measure infectivity.

Results

A contact time of 5 min with scrapie-infected mouse brain suffices to render steel wire highly infectious and insertion of infectious wire into the brain of an indicator mouse for 30 min suffices to cause disease. Infectivity bound to wires persists far longer in the brain than when injected as homogenate, which can explain the extraordinary efficiency of wire-mediated infection. No detectable amounts of PrP could be eluted with NaOH, however the presence of PrP on infectious wires was demonstrated by chemiluminescence. Several recommended sterilisation procedures inactivated wire-bound mouse prions, but exposure to 10% formaldehyde was insufficient.

Conclusions

Prions are readily and tightly bound to stainless steel surfaces and can transmit scrapie to recipient mice after short exposure times. This system mimics contaminated surgical instruments and will allow an assessment of sterilisation procedures.

Introduction

Prions are more resistant to inactivation than most conventional pathogens (1–4). An electrode used intracerebrally on a patient suffering from sporadic CJD (sCJD) transmitted the disease to two patients in succession and finally to a chimpanzee, despite exposure to benzene, 70% ethanol and formaldehyde vapour after each use (5,6). Concerns that surgical instruments may transmit variant CreutzfeldtJakob disease (vCJD) have been raised by the finding of PrP^Sc not only in nervous, but also in lymphatic tissue (7–10). We examined the ability of steel surfaces to bind scrapie prions by incubating steel wires overnight with scrapie-infected brain homogenates and inserting them permanently into the brain of indicator mice. This procedure resulted in efficient transmission of disease (11).

However, long-time exposure of steel wires to brain homogenate does not reflect conditions obtaining during surgical interventions. We now show that wires inserted into intact brain for as little as 5 min suffices to render the wires far more infectious than overnight exposure to brain homogenate and as infectious as 0.03 ml of 1% scrapie-infected brain homogenate injected directly into the brain. Furthermore, a contact time of 30 min was sufficient to elicit infection. Our experiments provide a model to assess the effectiveness of sterilisation procedures for steel bound prions and suggest a minimally invasive approach to assess infectivity in organs such as brain and tonsils.

Materials and Methods

Preparation of Infectious Wire

Stainless steel wire segments (diameter 0.15 mm; 5 mm length) were cut from “Stainless steel suture monofilament wire”, Art.Nr. 01614037, USP 4/0, B.Braun Melsungen AG, D-34209 Melsungen, Germany; batch 1/7502 or 1/8452). Gold wire segments (5 × 0.13 mm, Alfa Aesar Johnson Matthey GmbH Germany) were washed ultrasonically for 15 min in 2% Triton X-100, thoroughly rinsed in distilled water, dried at 37°C for 1 h as described (12). Brains were homogenized in 1 × Dulbecco’s phosphate-buffer saline (D-PBS; Gibco BRL, Glasgow, UK) by passing through 21G and 25G needles 8 times each, to give 10% (w/v) homogenates. These were centrifuged at 1,000 rpm (Eppendorf centrifuge 5415c, Hamburg, Germany) for 5 min at room temperature and the supernatants were recovered. We have recently determined that the centrifugation step result in the precipitation of about 80–90% of the PrP^Sc present in the sample (P. Klöhn, unpublished results) so that this step is better avoided. Wires were incubated with centrifuged 10% brain homogenate in PBS for 16 h and washed 5 times 10 min in 50 ml PBS, all at room temperature. The wires were air-dried, stored at room temperature for 1 day and inserted into brain of deeply anaesthetized indicator mice, using a 25-gauge injection needle as a trocar.

Chemiluminescence of Surface-bound PrP

Twenty stainless wire segments (0.15 × 5 mm) were inserted into one brain hemisphere for 5 minutes. The other hemisphere was homogenized and centrifuged as described above. Twenty stainless wire segments were incubated with 0.5 ml 10% centrifuged homogenate for 5 min at room temperature, washed five times for 10 min with 50 ml D-PBS, dried for 24 h and immediately assayed for PrP. Wires were incubated with 0.2 ml of D-PBS containing 5% non-fat dry milk (w/v; Marvel, Premier Brands UK Ltd., Wirral, Merseyside, U.K.) for 1 h with agitation. After removal of the blocking reagent, they were incubated for 1 h with 200 ng/ml of anti-PrP antibody (6H4; Prionics AG, Zürich, Switzerland) in D-PBS containing 1% non-fat dry milk and washed 3 times for 5 min with 0.2 ml of D-PBS, followed by incubation for 1 h with horseradish peroxidase-conjugated rabbit antimouse IgG1 (1: 5000 dilution; Zymed, South San Francisco, California, USA). After washing 5 times for 5 min with D-PBS, the wires were exposed to 0.2 ml of SuperSignal ELISA Femto Maximum Sensitivity Substrate (Pierce, Rockford, ILL, USA) according to the manufacturer’s instructions. Chemiluminescence was determined by luminometer (AutoLumat LB953; EG&G Berthold GmbH, Bad Wildbad, Germany).

Results

The ability of stainless steel surfaces to bind scrapie infectivity has been previously demonstrated by incubating steel wires (5 × 0.15 mm) for 16 h with 10% w/v brain homogenate of terminally scrapie-sick mice, referred to below as “standard conditions” (11). To model the exposure of surgical instruments to infected tissue more realistically, we inserted wire segments for 5, 30 or 120 min into brains of scrapieinoculated wild-type mice culled two months before the expected appearance of scrapie symptoms. These “transiently inserted” wires were washed, dried and assayed by permanent implantation into the brain of Tga20 indicator mice (13). Incubation times of the three groups lay between 65 ± 4 and 69 ± 5 days (Table 1, experiment 1), showing that even the shortest exposure to scrapie-infected brain rendered wires as infectious as intracerebral inoculation with 0.03 ml of 1% homogenate of the same brain homogenate (incubation time of 68 ± 8 days). Gold wires exposed to brain homogenate into brain also acquired infectivity (Table 1, experiment 2).

Table 1 Infectivity of steel or gold wires after exposure to intact brain or to brain homogenate of scrapie-infected mice

A second important question regards the length of time an infectious wire must contact brain tissue in order to initiate disease. Infectious wires were prepared by insertion for 5 min into the brain of an infected wild-type mouse culled one month before the expected onset of scrapie symptoms. After washing, the wires were inserted transiently into the brains of anaesthetised indicator mice. As shown in Table 2, all mice exposed to a wire for 30 min or 2 h developed symptoms after 94 ± 10 and 100 ± 18 days, respectively. The infectious wires, with or without subsequent exposure to brain tissue, were ultimately assayed in indicator mice and in all cases caused scrapie disease after about 70 days, showing that no detectable amounts of infectivity were lost by exposure to brain.

Table 2 Transient insertion of infectious wires into brains of indicator mice

Earlier experiments had shown that no detectable protein could be eluted with 2 M NaOH (<50 ng protein per wire) from wires exposed to 10% brain homogenate (11). To determine whether wires exposed to brain homogenate or to intact brain had surface-bound PrP, they were incubated with monoclonal PrP antibody 6H4 (14), followed by horseradish peroxidase-conjugated rabbit anti mouse IgG1 and chemiluminescence was measured in the presence of substrate. Fig. 1 shows that chemiluminescence of wires transiently inserted into infected brain of terminally sick indicator mice was about 5.5 times above reagent background. After background subtraction, the values were about 4 times higher than for wires exposed to infected brain homogenate and about 1.8 times higher than for those transiently inserted into uninfected brain. This experiment shows that PrP was bound to the wire surface; the higher chemiluminescence of the sample from infected brain is in keeping with the finding that total PrP content in terminally infected mouse brain is around 5 times higher than in uninfected controls (13,15), due to accumulation of PrP^Sc. We were not able to differentiate between PrP^C and PrP^Sc on wires because proteinase K treatment abolished immunofluorescence in all cases (data not shown). In an attempt to desorb PrP, we extracted 40 wire segments that had been transiently inserted into scrapie-infected brain with 0.05 ml 2 M NaOH for 1 h, neutralised the eluate with HCl and analysed half the sample by Western blot analysis. No PrP-specific immunoreactivity was detected under conditions where 0.3 ng purified glycosylated murine PrP, dissolved in NaOH and neutralised as described above, was clearly detectable (data not shown). Therefore, one wire released less than 15 pg PrP, that is less than 3 × 10⁸ molecules. Assuming that one log LD₅₀ unit of infectivity is associated with 10⁵ PrP^Sc molecules (16), one wire released less than 3000 LD50 units. Yet, the incubation time resulting from one wire is about the same as that following injection of 0.03 ml 1% brain homogenate, which corresponds to about 20,000 LD₅₀ units. This somewhat speculative calculation suggests that the amount of PrP that could have been released from the wire surface does not readily account for the wire’s infectivity, raising the question whether infectivity is due to irreversibly bound PrP^Sc (or PrP*) (17) rather than to desorbed prions.

Fig. 1

Chemiluminescence of surface-bound PrP on stainless steel wires exposed to brain or brain homogenates. Stainless steel wire segments were transiently inserted into brains (“dipped”) or incubated with 10% brain homogenates (homogenate) from PrP knockout mice (Prnp^0/0), uninfected (Tga20) and RML-infected, terminally sick Tga20 mice (RML-Tga20). Wires were washed, treated with anti-PrP antibody 6H4 and horseradish peroxidase-conjugated anti-mouse IgG1 antibody, and chemiluminescence was determined. The dotted line indicates the background level, given by reagents alone (reagent). Averages and standard deviations were calculated from three samples each. RLU, relative light units.

Why are wire-bound prions as infectious as concentrated homogenates? Upon intracerebral inoculation with brain homogenate, infectivity is rapidly distributed throughout the mouse (18) and after 4 days or less prions are no longer detectable in the brain (19). Perhaps wire-bound prions are more stable and can therefore act over a longer period of time. We assayed infectious wires directly or after leaving them for 1 or 5 days in brains of Prnp^+/+ or Prnp^0/0 mice. Table 3. shows that wires remained infectious even after residing in brain tissue for 5 days, albeit at a lower level, as evidenced by incubation times of about 90 days in indicator mice. Because wire-bound infectivity remains at a locally high concentration for 5 days or longer, it may result in a greater total exposure than injected homogenate.

Table 3 Infectivity of prion-coated wire after exposure to brain homogenate, PBS or brain of uninfected mice

The wire model will ultimately serve as model for the sterilisation of surgical instruments by recommended (1,3,20) or novel procedures. In a preliminary experiment, infectious wire segments were subjected to different treatments and assayed. Sodium hydroxide (1 M, 1 h) or guanidinium thiocyanate (4 M, 16 h) rendered the wires completely non-infectious to the limits of the bioassay (Table 4), however all 6 indicator mice challenged with formaldehyde-treated, prion-coated wires succumbed to scrapie after 92 ± 8 days.

Table 4 Infectivity of surface-bound mouse prions after various treatments

These preliminary decontamination studies should not be extrapolated to the level of instruments used in surgery. First, we used RML mouse prions, a mouse-adapted scrapie isolate (21) which is less heat stable than mouse-passaged BSE (301V) or the hamster strain 263K (3,22). It is clearly necessary to conduct sterilisation experiments of surface-bound infectivity using CJD, vCJD and BSE prions in an appropriately sensitive host. Second, the area of contact between wire surface and tissue is very small, compared with that of surgical instruments and it will ultimately be necessary to use scaled-up surfaces, such as could be provided by small steel beads introduced into larger indicator animals, to validate results obtained in the mouse.

Finally, it may become possible to use wires “dipped” for short times into brain or tonsils instead of biopsied tissue to determine the presence of PrP^Sc by chemiluminescence or infectivity in an appropriate indicator mouse or susceptible cultured cell line.