Studies on the irradiation of toxins of Clostridium botulinum and Staphylococcus aureus
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Journal of Applied Bacteriology 1988,65,223-229 2643109187
Studies on the irradiation of toxins of Clostridium botulinum and Staphylococcus aureus
SALLY A. ROSE*, N.K. MODIS, H.S. T R A N T E R ~ , N.E. BAILEY*, M.F. STRINGER* & P. H A M B L E T O N ? *Campden Food and Drink Research Association, Chipping Campden, Gloucestershire, GL55 6LD, ?Centre for Applied Microbiological Research, Porton Down, Salisbury, Wiltshire SP4 ONG, and $Porton International plc, 100 Piccadilly, London WI V NFN, U K
Received I4 September 1987, revised 21 December 1987 and accepted 14 April 1988
ROSE, S.A., MODI, N.K., TRANTER, H.S., BAILEY, N.E., STRINGER, M.F. & HAMBLETON, P. 1988. Studies on the irradiation of toxins of Clostridium botu- h u m and Staphylococcus aureus. Journal of Applied Bacteriology 65,223-229.
The effects of irradiation of Clostridium botulinum neurotoxin type A (BNTA) and staphylococcal enterotoxin A (SEA) in gelatin phosphate buffer and cooked mince beef slurries were investigated. Estimation of toxins by immunoassays showed that in buffer, toxins were destroyed by irradiation at 8.0 kGy; in mince slumes however, 45% of BTNA and 27-34% of SEA remained after this level of irradiation. At 23.7 kGy, over twice the dose of irradiation proposed for legal acceptance in the UK, 15% of BNTA and 1626% of SEA still remained. Increasing concentrations of mince conferred increased protection against the effect of irradiation on both toxins. The biological activity of BNTA was more sensitive to irradiation than the immunological activity. Staphylococcal enterotoxin was more resistant to irradia- tion than BNTA. Irradiation should therefore only be used in conjunction with good manufacturing practices to prevent microbial proliferation and toxin pro- duction prior to irradiation.
At present, there is considerable interest in the potential use of ionizing radiation to preserve various types of food (Anon. 1986; Anon. 1987; Sonsino 1987). The process has become legally accepted in many countries, including France, The Netherlands, Belgium, Germany and South Africa, and others appear to be moving towards an acceptance of the process for specific applica- tions. In the United Kingdom, the sale of irradi- ated foods is currently forbidden under the Food (Control of Irradiation) Regulation 1967. In 1986, however, the Advisory Committee on Irradiated and Novel Foods (ACINF) endorsed the conclusion of the Joint FAO/IAEA/WHO Expert Committee on the Wholesomeness of Irradiated Foods (Anon. 1981) that an overall average dose of 10 kGy provides an efficacious food preservation treatment without affecting
$ Corresponding author.
the safety and wholesomeness of the food. Both committees recommended adequate microbio- logical evaluation during processing and ensuing storage to ensure food safety.
The effect of irradiation on bacterial vegeta- tive cells and spores is well documented in the literature. Irradiation at 10 kGy is capable of killing most common food poisoning micro- organisms (Erdman et al. 1961) but is unlikely to kill all bacterial spores unless the initial level of contamination is low (Urbain 1978). Informa- tion on the effects of irradiation on toxins in foods is restricted, however, particularly about staphylococcal enterotoxins which are known to be resistant to denaturation by heat (Tatini 1976). Although a number of D values (0.04- 0.06 Mrad) obtained by irradiating botulinum toxin have been reported (Wagenaar & Dack 1956; Wagenaar et al. 1959; Wagenaar & Dack 1960; Skulberg & Coleby 1960; Roberts et a[.
224 Sally A . Rose et al. 1965; Miura et a/. 1967; Licciardello et al. 1969), it is difficult to make a simple and valid com- parison of these values. The variety of complex menstrua and buffers used in these studies may vary in the protection against the effect of irra- diation on the toxin. Futhermore, prior to the availability of purified botulinum neurotoxins (mol. wt 150000) (Dasgupta et al. 1966; Moberg & Sugiyama 1978), complexes of the neurotoxin with other proteins, referred to as crystalline or 'purified' toxin, were used and these other pro- teins may also offer protection against denatur- ation by irradiation.
In recent years, sources of highly purified botulinal neurotoxins and staphylococcal enterotoxins have become available and major advances have been made in the development of techniques for the detection of these toxins in foods. For example, enzyme-linked immuno- sorbent assay (ELISA) techniques for the detec- tion of staphylococcal enterotoxins (Freed et al. 1982; Fey et al. 1984) are significantly more sen- sitive than the immunodiffusion technique used by Read & Bradshaw (1967). In addition, an ELISA method for the detection of BNTA using highly specific antisera raised against purified neurotoxin (Modi et af. 1988) has been devel- oped. Such improved techniques facilitate the examination of toxins in foods, particularly those containing levels of toxin commensurate with food poisoning and the aim of the present study was to apply these techniques to investi- gate the effect of irradiation on the purified toxins of Clostridium botulinum and Staphylo- coccus aureus.
Materials and Methods
Staphylococcal enterotoxin A (SEA) was a kind gift from Mr D. Reynolds, Vaccine Research and Production Laboratory, PHLS Centre for Applied Microbiology and Research, Porton Down, Salisbury. The toxin was purified (Reynolds 1987) from culture supernatant fluids of Staph. aureus strain FRI-722 which was obtained from Professor M.S. Bergdoll, Food Research Institute, University of Wisconsin- Madison, Wisconsin. Clostridium botulinum type A neurotoxin (BNTA) was purified (Hambleton et al. 1981) from culture supernatant fluids of Cl. botulinum type A strain NCTC 2916. The
purified toxin had a specific toxicity of 2 x lo8 LD,,/mg protein.
Phosphate-buffered saline, pH 7.4 (PBS) con- tained (g,']): NaCI, 8.0; KCI, 0 2 ; Na,HPO,, 1.15; NaH,PO,. 2H,O; and was autoclaved at 121C for 15 min.
Gelatin phosphate buffer, pH 6.5 (GPB) con- tained (&): gelatin, 2-0; Na,HPO,, 9.94 and was autoclaved at 121C for 15 min.
Tris-gelatin-salt buffer, pH 7.0 (TGS) con- tained (g/l): NaCI, 8.77; gelatin, 2.0; Tris, 2.4, and was autoclaved at 121C for 15 min.
P R E P A R A T I O N OF MEAT SAMPLES
Mince slurries (YO, w/v) were prepared by homogenizing the appropriate weight of fresh lean minced beef in GPB for 5-10 min in an electric blender and autoclaving at 121C for 15 min. The mince was re-homogenized and 18 ml amounts aseptically transferred into sterilized 25 ml screw-capped bottles.
Neat ( l0O0/0) mince samples were prepared by adding 18 g of minced beef to a 25 ml screw- capped bottle, autoclaving, then aseptically mixed using a sterile glass rod.
Mince slurries and gelatin phosphate buffer (18 ml) were innocuiated with either BNTA (1 x lo6 mouse LD,,/ml or SEA (100 pg/ml) to give a final concentration in the mince of 1 x lo4 mouse LD,,/ml or 111.1 ng/ml respec- tively. The inoculated slurries were mixed for 10 min using a bottle roller (Multimix MM1, Luckham) and maintained at 4C until irradia- tion within 12 h of inoculation.
I R R A D I A T I O N
Two cobalt-60 sources were used for sample irradiation, One source had a strength of approximately 40000 Ci with a dose rate of 9.4 kGy/h while the other had a strength of approx- imately 50000 Ci at a dose rate of 12.2 kGy/h. Actual dosage received by the samples was assessed with red 4034 ( 5 5 0 kGy) or amber 3042 (1-30 kGy) perspex dosimeters (AERE, Harwell), irradiated in screw-capped bottles in parallel with the test samples, and the dose- induced absorbance was measured spectropho- tometrically after irradiation (Whittaker 1970).
Irradiation of bacterial toxins 225 The temperature during irradiation was main- tained at 2&25C and non-irradiated control samples were kept at ambient temperatures during the period of irradiation.
E X T R A C T I O N P R O C E D U R E
Mince slurries were centrifuged (3000 g for 30 min at 4C) and the supernatant fluid collected. The residual mince was resuspended in 9 ml of GPB, mixed for 10 min using a bottle roller and recentrifuged. The supernatant fractions were combined, centrifuged (20000 g) for 30 min at 4C and the total volume of supernatant fluid recorded.
T O X I N ASSSAYS
Mouse Lethality Test: Groups of four Porton mice weighing 15-20 g were each injected intra- peritoneally (0.5 ml/mouse) with test samples serially diluted in GPB and the mouse lethal dose 50 (MLD,,) estimated (Reed & Muench 1938).
ELISA: NUNC-Immuno 1 plates were coated with guinea-pig IgG anti-BNTA (20 pg/ml PBS, 100 pl/well) overnight at 4C. Blanking, toxin binding, conjugate addition and washing steps were as described by Shone et al. (1985). After incubation for 1 h at room temperature with test samples and alkaline phosphatase conju- gate, the plates were washed six times and p- nitrophenyl phosphate (Sigma 104) added at 100 pl/well. The plate was shakn for 1 h at room temperature, the reaction stopped by adding 0.5 mol/l NaOH (50 pl/well) and the absorbence measured at 405 nm using an ELISA plate reader (Dynatech MR 580). Concentrations of BNTA in the samples were calculated from a calibration curve prepared using pure BNTA (2-1000 LD,,/ml) in TGS, on the same plate as the samples.
The detection of staphylococcal enterotoxin A was carried out using two different ELISA systems.
ELISA method I: Dynatech microtiter plates were coated with guinea-pig IgG anti-SEA (20 pg/ml PBS, 100 pl/well) overnight at 4C. The plates were washed three times with PBS con- taining 0.05% Tween 20. The excess binding sites were blocked by adding (100 pl/well) RPMI 1640 medium (Imperial Labs) containing 10% (v/v) fetal calf serum and 1% (w/v) bovine serum albumin and shaking the plate for 30 min at room temperature, followed by incubation, at 37C for If h. The plate was washed three times, samples (suitably diluted in GPB) added (50 pl/well) and the plate shaken at room tem- perature for 2 h. After washing the plate, rabbit IgG anti-SEA conjugated to horseradish peroxi- dase (Avrameas et al. 1978) diluted 1 : 200 in the blocking reagent described above, was added at 100 pl/well. The plate was washed four times and 100 p1 3, 3, 5, 5-tetramethylbenzidine (TMB) solution (0.1 mg/ml) added per well. The plates were shaken at room temperature for 10-15 min, the reaction stopped by adding 2 mol/l H,SO, (50 pl/well) and the absorbence measured at 450 nm using an MR580 ELISA plate reader. The toxin concentrations in the samples were calculated from a calibration curve (0.1-1OOO ng/ml) constructed using puri- fied SEA diluted in GPB.
ELISA method 2 : The SET-EIA kit (available from Labor Dr W. Bommeli, Langass-Strasse 7, CH-3012 Bern, Switzerland) developed by Fey et af. (1984) was used, according to the manufac- turers instruction. After the substrate reaction had been stopped by addition of 100 pl 2 mol/l NaOH to 1.0 ml of reaction mixture, four 0.2 ml amounts of the solution were transferred to a microtitration plate and the A,os read on a Dynatech MR 600 plate reader. The toxin con- centration of samples was estimated from a standard curve (0.1-1-6 ng/ml) of SEA diluted in GPB.
The effect of irradiation dosage on the toxins is shown in Table 1. An increase in the irradiation resulted in increased losses of both SEA and BNTA.
Although the biological activity of BNTA in gelatin phosphate buffer was destroyed at all doses of irradiation, some immunological activ- ity was still detectable after irradiation at a dose
226 Sally A . Rose et al. Table 1. Effect of varying doses of irradiation on Clostridium botulinum type A neurotoxin and
staphylococcal enterotoxin A
Gelatin phosphate buffer
Mince slurry (15"/0)
BNTA-MLT BNTA-ELISA* SEA-ELISAl* SEA-ELISA2* BNTA-MLT BNTA-ELISA* SEA-ELISAI' SEA-ELISA2*
0 1.9 5.2 7.5 8.0 23.7
111.0 ND ND ND ND ND 82.0 15.7 ND ND N D ND 53.2 14.0 2.7 2.2 ND ND 87.2 4.5 0.2 ND ND ND
161.3 78.0 43.7 49.8 22.0 2.0 71.0 59.0 51.3 47.3 45.0 15.3 41.9 29.5 24.5 30.4 33.6 26.5 42.7 32.4 31.4 31.5 27.1 16.0
D value W Y ) < 0-4
2.65 5.32 2.01
* Mean of triplicate results. ND not detected. Figures in the table refer to the amounts of toxin detected after irradiation expressed as a percent-
age of the initial toxin added (1.8 x lo' mouse LD,, BNTA and 2.0 pg SEA).
of 1.9 kGy. The greater sensitivity of the bio- logical compared with the immunological activ- ity to irradiation was also seen in experiments using different concentrations of mince slurry irradiated at 23.7 kGy (Table 2). Detection of residual toxin was consistently lower with the MLT than with the ELISA (Table 2). Although the two ELISAs used for the detection of SEA showed different absolute values, the protective effect of mince on the toxin was evident in both cases.
Comparison of results for the immunological detection of the two toxins irradiated in buffer
(Table 1) showed that denaturation of SEA required a higher dose of irradiation than for BNTA, and that D values were greater for SEA than BNTA. The greater susceptibility of BNTA was also seen in experiments on irradiation of different concentrations of mince slurry (Table 2).
Results showing the effect of the concentra- tion of mince slurry on irradiation of toxins are given in Table 2. Recovery of toxin from non- irradiated samples decreased as the concentra- tion of mince in the sample increased. If results are expressed as the ratio of recoveries from
Table 2. Effect of composition of mince slurry on recovery of Clostridium botulinum type A neurotoxin and staphylococcal enterotoxin A after irradiation
Concentration of slurry (% w/v)
Assay 0 3 15 30 50 100
Non-irradiated samples BNTA-MLT BNTA-ELISA" SEA-ELISAl* SEA-ELISA2*
Irradiated samplest BNTA-MLT BNTA-ELISA' SEA-ELISA 1 * SEA-ELISA2*
100.0 102.6 120.3 110.0
ND N D N D N D
150.9 161.3 102.6 76.7 95.9 103.3 70.7 57.3 67.8 85.3 123.1 41.9 57.4 72.3 66.2 112.7 42.7 71.1 79.9 73.5
ND 2.0 5.7 6.4 28.5 ND 15.3 18.0 27.5 34.0 1.2 26.5 33.5 25.8 53.8 0.9 16.0 51.5 50.6 77.0
* Mean of triplicate results. 7 Samples were irradiated at an average dose of 23.4 ( 5 0.76) kGy. ND not detected. Figures in the table refer to the amounts of toxin detected in the extracts
expressed as a percentage of the initial toxin added (1.8 x to5 mouse LD,, BNTA, 2.0 pg SEA).
Irradiation of bacterial toxins 221 irradiated to non-irradiated samples, to over- come the problem of the inconsistency of toxin extraction from the slurries, they show that as the concentrations of mince in the slurry increased so did the recovery of both BNTA and SEA. In neat mince approximately 30% of both the biological and immunological activity of BNTA and over 50% of the immunological activity of SEA remained after irradiation at 23.8 kGy.