APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1979, p. 767-771 0099-2240/79/11-0767/05$02.00/0
Vol. 38, No. 5
Toxin Production by Clostridium botulinum in Grass S. NOTERMANS,'* S. KOZAKI,2 AND M. VAN SCHOTHORST' Laboratory for Zoonoses and Food Microbiology, National Institute of Public Health, Bilthoven, The Netherlands,1 and Department of Veterinary Science, College of Agriculture, University of Osaka Prefecture, Sakai-shi, Osaka, 591, Japan2 Received for publication 30 July 1979
Investigations on farms where botulism has occurred in cows showed that proteolytic Clostridium botulinum type B was present in newly made grass silages. Experiments were undertaken to study growth and toxin production of C. botulinum in grass. Of the strains tested only proteolytic strains of C. botulinum types A and B were able to produce toxin with grass as a substrate. Proteolytic strains of type B produced both medium (12S) and large (16S) toxin forms. The minimal water activity (a,,) for toxin production at pH 6.5 and 5.8 was 0.94. At pH 5.3, toxin was produced at an a,, of 0.985. These results indicate that proteolytic strains of C. botulinum (if present) may multiply and produce toxin in wilted grass silages.
Botulism in cattle has been reported from Africa (5, 19), South America (20), Australia (17), and Europe (4, 6, 8, 12, 18). The European outbreaks were caused by type C toxin, those in Africa and Australia by type D toxin, and those in South America by type C and D toxins. Most cases were due to the ingestion of toxin-containing carcass material or fodder contaminated with such carcass particles. Recent outbreaks of cattle botulism in The Netherlands (3, 9) were caused by feeding silage made from brewers' grains (malt) contaminated with high numbers (up to 106 g) of proteolytic Clostridium botulinum type B (14). Feces of cows fed contaminated fodder contained high numbers of these bacteria (up to 10' g) so pastures fertilized with the manure may be contaminated; grass silage prepared with grass from these pastures are probably contaminated also. Only a few reports describe botulism caused by grass silage (7, 10). Therefore, investigations were performed concerning growth of and toxin production by C. botulinum in grass. Grass silages from "botulism" farms were examined for C. botulinum, pH, and water activity (a.). Then the toxin production of various C. botulinum types and strains was determined, using grass as the growth medium. Finally, the effect of pH and a, on the toxin production of a selected strain was tested. MATERIALS AND METHODS Strains. The following C. botulinum cultures were used: type A strains 62A and t2k3; proteolytic type B strains SNB 77, Okra, CDI-3, and CDI-4; non-proteolytic type B strains CDI-1 and CDI-2; type C strains Ca and SNC 78; type E strain RIV 1. Type B strain 767
SNB 77 was isolated from malt which had caused botulism in cows. Type C strain Ca produced Cl toxin; strain SNC 78 produced only C2 toxin. Spore suspensions. A spore suspension was prepared by inoculating a liver broth culture incubated (30°C) overnight into a freshly prepared medium containing 2% proteose peptone no. 3 (Difco), 0.5% yeast extract (Difco), 0.5% glucose (Difco), and 0.1% cysteine-HCI at pH 7.0. For non-proteolytic type B and E strains, 0.1% ammonium sulfate was added after sterilization. After incubation at 30°C for 1 week, spores were collected by centrifugation and washed once with distilled water. The washed spores were suspended in distilled water and, after being heated at 70°C for 10 min, stored at -20°C until used. Grass media. Fresh grass medium consisted of 2 g of fresh grass, cut into pieces of 2 to 3 cm in length, and 20 ml of extract made by boiling 100 g of fresh grass for 1 h with 400 ml of distilled water. The medium was sterilized at 120°C for 15 min. Grass silage medium was made with grass silage (wilted type), which was obtained from a farm that had never been affected by botulism. The grass was cut into pieces of 2 to 3 cm in length, and 4 g was added to 18 ml of distilled water. The mixture was boiled for 30 min and used without further sterilization. Grass silage media of different a,, values were made by using different concentrations of glycerol instead of distilled water. The pH was adjusted with 0.01 N NaOH or 0.01 N HCI. Cultures of grass media were incubated anaerobically at 37°C, using McIntosh jars (Griffin, Wembley, Middlesex, England) which were evacuated three times and subsequently filled with 80% N2, 10% C02, and 10% H2. A disposable anaerobic indicator (GasPak, BBL Microbiology Systems, Cockeysville, Md.) was used. a. a,,. was determined at 20°C, using the a,, measurement device with a dew-point hygrometer described by Northolt et al. (13). The accuracy of mea-
surement was ±0.005. Detection of C. botulinum in silages. Samples
768
NOTERMANS, KOZAKI, AND
APPL. ENVIRON. MICROBIOL.
VAN SCHOTHORST
were drawn from various grass silages from eight farms with a history of botulism. These silages had been prepared with wilted grass. The stability of this silage results from its low oxygen content, low a,,, and a low pH from lactic acid fermentation (21). Each sample was macerated by using a Waring blender. Serial 10fold dilutions of macerate were made, and 1 ml of a dilution was transferred into two tubes containing 30 ml of freshly prepared, fortified egg meat medium (150 g of egg meat [Difco], 10 g of glucose, and 10 g of ammonium sulfate per liter, sterilized at 120°C for 15 min). One tube of the pair was heated at 70°C for 20 min. The heated and the unheated tubes were incubated anaerobically at 30°C. After 5 days of incubation, the culture fluids were examined for botulinal toxin. Molecular size of toxin in grass medium. Tubes of fresh grass medium were incubated at 37°C for 5 and 14 days and then centrifuged. Supernatant fluid, 0.2 ml, was loaded on tubes containing 5 ml of a sucrose gradient (5 to 20% in 0.05 M acetate buffer, pH 6.0). Centrifugation was at 189,000 x g for 5 h at 4°C with an SW 50 rotor and a Beckman L-2 65B centrifuge. Purified type B-L and B-M toxins (11) were used as references. Toxicity. To determine botulinal toxins in culture fluids, 2-ml portions were diluted with 6 ml of sterile 0.05 M phosphate buffer, pH 6.0, containing 2 g of gelatin (Difco), 1 mg of streptomycin, and 10' U of penicillin per liter. Activation of the diluted samples was performed by adding trypsin (Sigma type III, Sigma Chemical Co., St. Louis, Mo.) at a final concentration of 0.2 mg/ml and incubating at 37°C for 30 min. Samples, 2 ml, of the diluted and trypsinized fluids were mixed with 0.5 ml of antibotulinal serum (Institut Pasteur, Paris) of types homologous to the toxin expected. Two mice (18 to 20 g) were injected intraperitoneally with 0.5 ml of each preparation and observed for 4 days. Toxin assay by the time-to-death method was as described by Boroff and Fleck (2) and by Sakaguchi et al. (16). Two mice (18 to 20 g) were injected intravenously with 0.1 ml of each preparation, and the time between injection and death was noted.
RESULTS a. and pH of silages and presence of C.
botulinum. Samples of 11 silages from 8 farms at which cow botulism occurred in 1977 were investigated. In four silages C. botulinum type B was detected (Table 1). In two silages C. botulinum type B was present in 0.01-g samples. An isolated culture of these bacteria was proteolytic. Botulinal toxin could not be detected in the silages. There was no correlation between the presence of C. botulinum and pH or a,,, of the silages. Toxin production of different C. botulinum strains in grass. A spore suspension or a culture developed by overnight incubation in liver broth was inoculated into grass silage medium. One of the two type A strains and three of
the five type B strains produced toxin during incubation for 5 days at 37°C (Table 2). The strains producing toxin were proteolytic. In a fresh grass medium prepared with grass harvested during spring, the toxicity of strain SNB 77 after incubation at 37°C for 4 days reached 3.6 x 106 50% lethal doses (LD60) per ml, which was six times higher than in grass silage medium. The level of toxicity remained constant during 4 weeks of incubation. Molecular size of toxin produced in fresh grass medium. To examine the molecular size TABLE 1. pH and a,,, ofgrass silages and occurrence of C. botulinum No. of
Farmer silages samA
C. botulinum (type) in: pH
au, 1
1 5.77 Nja 1 6.35 0.931 2 6.26 0.937 2 5.40 0.979 3 5.30 0.958 B 1 5.23 0.958 C 1 5.79 0.854 D 1 5.38 0.965 E 1 6.42 0.932 F 1 5.80 0.929 G 1 6.14 0.948 2 5.91 0.966 H 2 5.78 0.899 NI, Not investigated. b-, Not detected.
+ + + + + -
g
0.1
g
0.01
(B) + (B) (B) + (B) + (B) + -
(B) _b (B) + (B) (B) (B) + (B) -
TABLE 2. Toxin production of different C. botulinum types in grass silage medium C. botulinum Toxi.i. Inoculum
g
ToxLclty Strain(Lo/) A 62A b Spores (2.7 x 107)a Culturec 1.2 x 106 t2K3 Culture B SNB 77 Spores (2.5 x 105) 5.6 x 105 SNB 77 Culture 4.6 x 105 CDI-1 Culture CDI-2 Culture CDI-3 Spores (2.5 x 106) Culture 2.2 x 105 CDI-4 Spores (1.4 x 106) 5.4 x 104 Culture 1.4 x 105 C Cad Culture SNC 78d Culture E RIV-1 Culture ab Number of spores added. -, Toxin not detected. c 0.5 ml of an overnight liver broth culture was used for inoculation. d Ca strain produces Cl toxin, and SNC 78 strain produces C2 toxin. Type
TOXIN PRODUCTION BY C. BOTULINUM IN GRASS
VOL. 38, 1979
of toxin produced in fresh grass medium type B proteolytic strains SNB 77 and Okra, isolated from cow botulism and human botulism, respectively, were used. A tube of fresh grass medium was inoculated with 104 spores of each culture and incubated in anaerobic jars at 37°C. Culture supernatants of the two strains showed two toxin peaks (Fig. 1). Culture supernatant of strain SBN 77 contained more M toxin than L toxin, whereas the amounts of L and M toxins in the Okra culture supernatant were nearly the same. Prolonged incubations hardly influenced the proportion of L and M toxins and the amount present in the culture supernatant (Fig. 1). Effect of pH and a, on toxin production
., I6
769
in grass silage medium. Tubes of freshly prepared grass silage media with different pH and a,,. values were inoculated with 104 spores of C. botulinum strain SNB 77. At pH 5.8, which was the pH of the grass without adjustment, toxin production occurred down to a,,. 0.944. Toxin production was less than 10i LDro per ml when the a,, was below 0.951, whereas with an a,,. of >0.955, toxin production was more than 4 x 104 LD,o per ml. The minimal au. for toxin production at pH 6.5 was the same as that at pH 5.8. However, toxin production at pH 5.4 was inhibited at fairly high at,, levels; the minimal a,, for toxin production was interpolated to be 0.98 (Table 3).
tS
A
0
l\ I'
I'
5
10
15
25 20 fraction No (10 drops)
B O0; 4 0 1-
Ln
0 c:
2
'ib -P
0
5
10
15
20
25 fraction No(10 drops)
FIG. 1. Sucrose density gradient centrifugation of culture supernatant of C. botulinum strains SNB 77 and Okra, grown in grass medium. (A) Strain SNB 77: 5-day culture contained 6.3 x 104 LD5o; 14-day culture contained 9.3 x 104 LD5o. (B) Strain Okra: 5-day culture contained 1.8 x 104 LD5o; 14-day culture contained 2.7 x 104 LD5o. Symbols: 0, 7-day culture; x, 14-day culture. L and M indicate the sedimentation positions of B-L (16S) and B-M (12S) toxins, respectively. i.p., Intraperitoneal.
770
NOTERMANS, KOZAKI, AND VAN SCHOTHORST
TABLE 3. Effect of a, and pH on toxin production by C. botulinum type B, strain SNB 77, in grass silage medium pH au, Toxicity (LDWo/ml)' 6.5
5.8
5.3
0.995 0.968 0.962 0.957 0.943 0.940
1.5 x 105 1.5 x 105 1.5 x 105
0.993 0.963 0.955
8.7 x 104 7.5 x 105 4.3 x I05
0.951
102_103b
0.944 0.935
ND
0.995 0.985
102_103b
Vol. 38, No. 5
Toxin Production by Clostridium botulinum in Grass S. NOTERMANS,'* S. KOZAKI,2 AND M. VAN SCHOTHORST' Laboratory for Zoonoses and Food Microbiology, National Institute of Public Health, Bilthoven, The Netherlands,1 and Department of Veterinary Science, College of Agriculture, University of Osaka Prefecture, Sakai-shi, Osaka, 591, Japan2 Received for publication 30 July 1979
Investigations on farms where botulism has occurred in cows showed that proteolytic Clostridium botulinum type B was present in newly made grass silages. Experiments were undertaken to study growth and toxin production of C. botulinum in grass. Of the strains tested only proteolytic strains of C. botulinum types A and B were able to produce toxin with grass as a substrate. Proteolytic strains of type B produced both medium (12S) and large (16S) toxin forms. The minimal water activity (a,,) for toxin production at pH 6.5 and 5.8 was 0.94. At pH 5.3, toxin was produced at an a,, of 0.985. These results indicate that proteolytic strains of C. botulinum (if present) may multiply and produce toxin in wilted grass silages.
Botulism in cattle has been reported from Africa (5, 19), South America (20), Australia (17), and Europe (4, 6, 8, 12, 18). The European outbreaks were caused by type C toxin, those in Africa and Australia by type D toxin, and those in South America by type C and D toxins. Most cases were due to the ingestion of toxin-containing carcass material or fodder contaminated with such carcass particles. Recent outbreaks of cattle botulism in The Netherlands (3, 9) were caused by feeding silage made from brewers' grains (malt) contaminated with high numbers (up to 106 g) of proteolytic Clostridium botulinum type B (14). Feces of cows fed contaminated fodder contained high numbers of these bacteria (up to 10' g) so pastures fertilized with the manure may be contaminated; grass silage prepared with grass from these pastures are probably contaminated also. Only a few reports describe botulism caused by grass silage (7, 10). Therefore, investigations were performed concerning growth of and toxin production by C. botulinum in grass. Grass silages from "botulism" farms were examined for C. botulinum, pH, and water activity (a.). Then the toxin production of various C. botulinum types and strains was determined, using grass as the growth medium. Finally, the effect of pH and a, on the toxin production of a selected strain was tested. MATERIALS AND METHODS Strains. The following C. botulinum cultures were used: type A strains 62A and t2k3; proteolytic type B strains SNB 77, Okra, CDI-3, and CDI-4; non-proteolytic type B strains CDI-1 and CDI-2; type C strains Ca and SNC 78; type E strain RIV 1. Type B strain 767
SNB 77 was isolated from malt which had caused botulism in cows. Type C strain Ca produced Cl toxin; strain SNC 78 produced only C2 toxin. Spore suspensions. A spore suspension was prepared by inoculating a liver broth culture incubated (30°C) overnight into a freshly prepared medium containing 2% proteose peptone no. 3 (Difco), 0.5% yeast extract (Difco), 0.5% glucose (Difco), and 0.1% cysteine-HCI at pH 7.0. For non-proteolytic type B and E strains, 0.1% ammonium sulfate was added after sterilization. After incubation at 30°C for 1 week, spores were collected by centrifugation and washed once with distilled water. The washed spores were suspended in distilled water and, after being heated at 70°C for 10 min, stored at -20°C until used. Grass media. Fresh grass medium consisted of 2 g of fresh grass, cut into pieces of 2 to 3 cm in length, and 20 ml of extract made by boiling 100 g of fresh grass for 1 h with 400 ml of distilled water. The medium was sterilized at 120°C for 15 min. Grass silage medium was made with grass silage (wilted type), which was obtained from a farm that had never been affected by botulism. The grass was cut into pieces of 2 to 3 cm in length, and 4 g was added to 18 ml of distilled water. The mixture was boiled for 30 min and used without further sterilization. Grass silage media of different a,, values were made by using different concentrations of glycerol instead of distilled water. The pH was adjusted with 0.01 N NaOH or 0.01 N HCI. Cultures of grass media were incubated anaerobically at 37°C, using McIntosh jars (Griffin, Wembley, Middlesex, England) which were evacuated three times and subsequently filled with 80% N2, 10% C02, and 10% H2. A disposable anaerobic indicator (GasPak, BBL Microbiology Systems, Cockeysville, Md.) was used. a. a,,. was determined at 20°C, using the a,, measurement device with a dew-point hygrometer described by Northolt et al. (13). The accuracy of mea-
surement was ±0.005. Detection of C. botulinum in silages. Samples
768
NOTERMANS, KOZAKI, AND
APPL. ENVIRON. MICROBIOL.
VAN SCHOTHORST
were drawn from various grass silages from eight farms with a history of botulism. These silages had been prepared with wilted grass. The stability of this silage results from its low oxygen content, low a,,, and a low pH from lactic acid fermentation (21). Each sample was macerated by using a Waring blender. Serial 10fold dilutions of macerate were made, and 1 ml of a dilution was transferred into two tubes containing 30 ml of freshly prepared, fortified egg meat medium (150 g of egg meat [Difco], 10 g of glucose, and 10 g of ammonium sulfate per liter, sterilized at 120°C for 15 min). One tube of the pair was heated at 70°C for 20 min. The heated and the unheated tubes were incubated anaerobically at 30°C. After 5 days of incubation, the culture fluids were examined for botulinal toxin. Molecular size of toxin in grass medium. Tubes of fresh grass medium were incubated at 37°C for 5 and 14 days and then centrifuged. Supernatant fluid, 0.2 ml, was loaded on tubes containing 5 ml of a sucrose gradient (5 to 20% in 0.05 M acetate buffer, pH 6.0). Centrifugation was at 189,000 x g for 5 h at 4°C with an SW 50 rotor and a Beckman L-2 65B centrifuge. Purified type B-L and B-M toxins (11) were used as references. Toxicity. To determine botulinal toxins in culture fluids, 2-ml portions were diluted with 6 ml of sterile 0.05 M phosphate buffer, pH 6.0, containing 2 g of gelatin (Difco), 1 mg of streptomycin, and 10' U of penicillin per liter. Activation of the diluted samples was performed by adding trypsin (Sigma type III, Sigma Chemical Co., St. Louis, Mo.) at a final concentration of 0.2 mg/ml and incubating at 37°C for 30 min. Samples, 2 ml, of the diluted and trypsinized fluids were mixed with 0.5 ml of antibotulinal serum (Institut Pasteur, Paris) of types homologous to the toxin expected. Two mice (18 to 20 g) were injected intraperitoneally with 0.5 ml of each preparation and observed for 4 days. Toxin assay by the time-to-death method was as described by Boroff and Fleck (2) and by Sakaguchi et al. (16). Two mice (18 to 20 g) were injected intravenously with 0.1 ml of each preparation, and the time between injection and death was noted.
RESULTS a. and pH of silages and presence of C.
botulinum. Samples of 11 silages from 8 farms at which cow botulism occurred in 1977 were investigated. In four silages C. botulinum type B was detected (Table 1). In two silages C. botulinum type B was present in 0.01-g samples. An isolated culture of these bacteria was proteolytic. Botulinal toxin could not be detected in the silages. There was no correlation between the presence of C. botulinum and pH or a,,, of the silages. Toxin production of different C. botulinum strains in grass. A spore suspension or a culture developed by overnight incubation in liver broth was inoculated into grass silage medium. One of the two type A strains and three of
the five type B strains produced toxin during incubation for 5 days at 37°C (Table 2). The strains producing toxin were proteolytic. In a fresh grass medium prepared with grass harvested during spring, the toxicity of strain SNB 77 after incubation at 37°C for 4 days reached 3.6 x 106 50% lethal doses (LD60) per ml, which was six times higher than in grass silage medium. The level of toxicity remained constant during 4 weeks of incubation. Molecular size of toxin produced in fresh grass medium. To examine the molecular size TABLE 1. pH and a,,, ofgrass silages and occurrence of C. botulinum No. of
Farmer silages samA
C. botulinum (type) in: pH
au, 1
1 5.77 Nja 1 6.35 0.931 2 6.26 0.937 2 5.40 0.979 3 5.30 0.958 B 1 5.23 0.958 C 1 5.79 0.854 D 1 5.38 0.965 E 1 6.42 0.932 F 1 5.80 0.929 G 1 6.14 0.948 2 5.91 0.966 H 2 5.78 0.899 NI, Not investigated. b-, Not detected.
+ + + + + -
g
0.1
g
0.01
(B) + (B) (B) + (B) + (B) + -
(B) _b (B) + (B) (B) (B) + (B) -
TABLE 2. Toxin production of different C. botulinum types in grass silage medium C. botulinum Toxi.i. Inoculum
g
ToxLclty Strain(Lo/) A 62A b Spores (2.7 x 107)a Culturec 1.2 x 106 t2K3 Culture B SNB 77 Spores (2.5 x 105) 5.6 x 105 SNB 77 Culture 4.6 x 105 CDI-1 Culture CDI-2 Culture CDI-3 Spores (2.5 x 106) Culture 2.2 x 105 CDI-4 Spores (1.4 x 106) 5.4 x 104 Culture 1.4 x 105 C Cad Culture SNC 78d Culture E RIV-1 Culture ab Number of spores added. -, Toxin not detected. c 0.5 ml of an overnight liver broth culture was used for inoculation. d Ca strain produces Cl toxin, and SNC 78 strain produces C2 toxin. Type
TOXIN PRODUCTION BY C. BOTULINUM IN GRASS
VOL. 38, 1979
of toxin produced in fresh grass medium type B proteolytic strains SNB 77 and Okra, isolated from cow botulism and human botulism, respectively, were used. A tube of fresh grass medium was inoculated with 104 spores of each culture and incubated in anaerobic jars at 37°C. Culture supernatants of the two strains showed two toxin peaks (Fig. 1). Culture supernatant of strain SBN 77 contained more M toxin than L toxin, whereas the amounts of L and M toxins in the Okra culture supernatant were nearly the same. Prolonged incubations hardly influenced the proportion of L and M toxins and the amount present in the culture supernatant (Fig. 1). Effect of pH and a, on toxin production
., I6
769
in grass silage medium. Tubes of freshly prepared grass silage media with different pH and a,,. values were inoculated with 104 spores of C. botulinum strain SNB 77. At pH 5.8, which was the pH of the grass without adjustment, toxin production occurred down to a,,. 0.944. Toxin production was less than 10i LDro per ml when the a,, was below 0.951, whereas with an a,,. of >0.955, toxin production was more than 4 x 104 LD,o per ml. The minimal au. for toxin production at pH 6.5 was the same as that at pH 5.8. However, toxin production at pH 5.4 was inhibited at fairly high at,, levels; the minimal a,, for toxin production was interpolated to be 0.98 (Table 3).
tS
A
0
l\ I'
I'
5
10
15
25 20 fraction No (10 drops)
B O0; 4 0 1-
Ln
0 c:
2
'ib -P
0
5
10
15
20
25 fraction No(10 drops)
FIG. 1. Sucrose density gradient centrifugation of culture supernatant of C. botulinum strains SNB 77 and Okra, grown in grass medium. (A) Strain SNB 77: 5-day culture contained 6.3 x 104 LD5o; 14-day culture contained 9.3 x 104 LD5o. (B) Strain Okra: 5-day culture contained 1.8 x 104 LD5o; 14-day culture contained 2.7 x 104 LD5o. Symbols: 0, 7-day culture; x, 14-day culture. L and M indicate the sedimentation positions of B-L (16S) and B-M (12S) toxins, respectively. i.p., Intraperitoneal.
770
NOTERMANS, KOZAKI, AND VAN SCHOTHORST
TABLE 3. Effect of a, and pH on toxin production by C. botulinum type B, strain SNB 77, in grass silage medium pH au, Toxicity (LDWo/ml)' 6.5
5.8
5.3
0.995 0.968 0.962 0.957 0.943 0.940
1.5 x 105 1.5 x 105 1.5 x 105
0.993 0.963 0.955
8.7 x 104 7.5 x 105 4.3 x I05
0.951
102_103b
0.944 0.935
ND
0.995 0.985
102_103b
