APPLIED
AND
ENVIRONMENTAL MICROBIOLOGY, Feb. 1979, p. 194-197
0099-2240/79/02-0194/04$02.00/0
Vol. 37, No.2
Clostridium botulinum in the Gulf of Thailand LEK TANASUGARN Section of Bacteriology, Veterinary College, Chulalongkorn University, Bangkok, Thailand Received for publication 6 October 1978
A survey was carried out to determine the incidence of Clostridium botulinum in samples of mud, sand, and fish from the Gulf of Thailand. Enrichment cultures from 762 samples of mud and sand from seven different areas around the Gulf were tested. C. botulinum type D was present in 10 samples, and type E was present in 2 samples taken from the west coast at Hua Hin. Enrichment cultures from 16,773 fish grouped into 2,151 samples yielded 10 filtrates containing C. botulinum type D and 5 containing type E. All of the toxic filtrates were obtained from samples of fish taken from the west coast of the Gulf of Thailand.
Clostridium botulinum is worldwide in its distribution (11), and its natural habitat is soil, even virgin and forest soil. A high degree of incidence of C. botulinum, especially types A and B, has also been reported under tropical conditions (15). In the United States, an extensive survey of C. botulinum type E in the fish of Lakes Erie, Huron, Superior, and Michigan was made after several type E botulism outbreaks in 1960 and 1963 (13). The organism was found in the digestive tracts of fish from all of the lakes, with incidences ranging from 1 to 9% (2). The association of C. botulinum type E with fishery products and the aquatic environment is now well established (1, 4, 9). The presence of type E in the deposits of lakes and soils of Hokkaido, Japan, (10, 12), British Columbia (6), and Sweden (8) has been reported. No survey of C. botulinum from Thailand has been reported previously nor have human cases of botulism been documented, but this does not necessarily mean that such food poisoning has not occurred. MATERIALS AND METHODS Collection of samples. Samples of mud and sand were collected from various freshwater sites of the Mekong River bed and from the Gulf of Thailand. Drop baskets with surface-controlled lid-sealing devices were used to collect samples of mud and sand at water depths of 1, 10, 20, and 30 m. Samples consisted of 2 kg of material, from which 100 g was aseptically separated for subsequent examination. Although the majority of fresh fish samples were purchased at various local markets where refrigeration is rather universally absent, some designated specimens were obtained at a 30-m water depth at Hua Hin (Fig. 1). Media. The media for the cultivation of C. botulinum used in this study were as follows: (i) sulfitereducing medium (15 g of tryptone, 10 g of yeast extract, 3 g of meat extract, 20 ml of 1% FeSO4, 10 ml of 5% Na2SO3, water added to 1,000 ml, pH 6.8); (ii) 194
beef heart infusion-cooked meat medium (10% fresh beef heart in cooked meat medium) (3); and (iii) egg yolk agar (reinforced Clostridium agar with 5% egg yolk emulsion). This last emulsion was prepared by aseptically removing the yolk of one egg and diluting with 15 ml of sterile normal saline. The emulsion was always prepared fresh. Method. A 10-g amount of each sample of mud or sand was inoculated into 80 ml of beef heart infusioncooked meat medium. In fish samples, the digestive tract was cultured for the presence of C. botulinum. For small fish, about eight intestines (10 to 15 g) were combined for one sample. Each sample of fish intestine was inoculated into 80 ml of beef heart infusion-cooked meat medium. After incubation at 25 to 300C for 7 days, the culture supernatant fluid was separated and centrifuged for 1 h at 6,000 rpm in a refrigerated centrifuge to eliminate the bacteria from the supernatant fluid. The clear supernatant fluid was again separated and kept in a freezer overnight. This frozen storage was used to reduce nonspecific death of mice. The routine use of a 1:5 dilution of the clear fluid of the culture in gelatin phosphate buffer (equal volumes of 0.4% gelatin solution and a pH 6.5 mixture of equal volumes of 0.2 M NaHPO4 and 0.2 M KH2PO4) to some extent also reduced the nonspecific death of mice. A 0.5-ml amount of a dilution of the clear, previously frozen fluid was injected intraperitoneally into mice (two mice for each culture). Mice were observed for symptoms of botulism, which resulted in death within 48 h. Injections of inactivated samples (heated for 10 min at 1000C) were made into mice as a control. If death of the animal was observed, the toxin was typed by using specific antitoxins (types A, B, C, D, and E) obtained from the Pasteur Institute, Paris. For confirmation, the original culture (the supernatant fluid of which caused death to the mice after inrtraperitoneal injection) was streaked onto the surface of egg yolk agar and incubated anaerobically in an anaerobic jar at 25 to 300C for 48 h. The lipase-positive zone was then picked and cultured in cooked meat medium at 25 to 300C. After 7 days of incubation, the supernatant fluid of the culture was typed by inoculating mice protected with specific antitoxins.
VOL. 37, 1979
C. BOTULINUM IN GULF OF THAILAND
195
FIG. 1. Location of sampling sites.
TABLE 1. Incidence of C. botulinum in samples of mud and sand from the Gulf of Thailand Sampling site
Nature of sample
Paknam Bangpoo Bangsaen Gulf of Thailand Had Chao Sumran Hua Hin Klong Wan
Mud Mud Sand Mud and sand Sand Sand Sand
RESULTS A total of 300 samples of mud and sand were cultivated in sulfite-reducing medium, and no toxin of C. botulinum was ever observed in such cultures. When these same samples were cultivated in beef heart infusion-cooked meat medium, C. botulinum type D toxin was found in 10 samples. As a result, beef heart infusioncooked meat medium was used for propagation and toxin studies in the later part of this survey. I do not feel that sulfite-reducing medium gives
No. of sam- No. of cultures that ples cultured killed mice
20 100 25 43 47 475 52
C. botulinum serotype
10 Type D and 2 type E
12
TABLE 2. Incidence of C. botulinum in samples of mud and sand taken from various depths of the sea at Hua Hin No. of sam-Noofsm Depth of ofples of mud No. of sam-
septh
(i)
1 10
20 30
andsted 100 109 94 172
klled mice 2 10
C. botulinum
serotype 2 Type D 8 Type D and 2 type E
196
APPL. ENVIRON. MICROBIOL.
TANASUGARN
TABLE 3. Incidence of C. botulinum in samples of fish from the Gulf of Thailand
No. of fish
No. of composite sampecuculples tured
No. of culture filtrates ta that killed mice
Hua Hin
1,560
124
3
3 Type E
2 Squid and 1 Scalopsis
Petchburi Radchburi
1,985 704
361 88
7 3
5 Squid and 2 sea catfish
Nakorn-patom Mahachai Bangkok Paknam Cholburi
1,376 1,280 4,124 1,296 4,448
172 160 528 162 556
1
7 Type D 1 Type D and 2 type E Type D
1
Type D
Squid
Samplingsite
btli C.bulnmserotype
of fish sample giving Type positive results pstv
eut
dubiosus Squid Squid
a toxin level as high as that produced by beef mud and sand at Hua Hin, but only from samples taken from a 30-m depth; most of the C. heart infusion-cooked meat medium. Table 1 shows that C. botulinum types D and botulinum type D isolates were also found at E toxins were isolated from 12 of 475 samples of this depth. This indicated that the spores of C. mud and sand obtained at Hua Hin. C. botu- botulinum were present in deep water rather linum was not detected in enrichment cultures than in shallow water. C. botulinum type E is able to produce toxin of 287 samples of mud or sand from the other six coastal areas. Ten of the positive samples at 5°C (5, 7, 14). Hence, the presence in fish of from Hua Hin were collected at a 30-m depth, even small numbers of spores of C. botulinum and the other two were collected at a 20-m depth type E makes rigid temperature control imperative. The toxin is rapidly inactivated by heat(Table 2). Table 3 shows that C. botulinum toxin was ing, and raw, smoked, or fermented fish is a detected in enrichment cultures of 15 of 2,151 greater hazard than cooked fish. samples of fish obtained from different areas of ACKNOWLEDGMENTS the west coast of the Gulf of Thailand. Ten of I am indebted to T. A. Roberts, Meat Research Institute, the positive cultures were type D, and five were Langford, Bristol, England for his help in the initial stages of type E. this work and to the Department of Fisheries, Thailand for C. botulinum toxin was not detected in enrich- their help in the collection of samples of mud, sand, and fish. ment cultures of 44 samples of mud and sand This work was made possible by a generous grant from the taken from the Mekong River bed at various International Atomic Energy Agency, Vienna, Austria. sites. LITERATURE CITED
DISCUSSION It is interesting to note that C. botulinum type E was isolated from fish from only two places on the west coast of the Gulf of Thailand, Hua Hin and Radchburi. Fish sold in Radchburi were obtained from Hua Hin fish markets. C. botulinum type E was also found in samples of sand from Hua Hin from a 30-m depth, but was not isolated from samples of mud and sand collected from other areas. C. botulinum types D and E were found only in samples of mud, sand, and fish from the west coast of the gulf; C. botulinum was not isolated from any samples from the east coast. Geographically, the land along the west coast is much higher than that along the east coast. This might indicate that the spores of C. botulinum was washed into the sea from the west coast. C. botulinum type E was found in samples of
1. Ando, Y., and K. Inoue. 1957. Studies on growth and toxin production of Clostridium botulinum type E in fish products. Bull. Jpn. Soc. Sci. Fish. 23:8. 2. Bott, T. L., J. S. Deffner, E. McCoy, and E. M. Foster. 1966. Clostridium botulinum type E in fish from the Great Lakes. J. Bacteriol. 91:919-924. 3. Bott, T. L., J. Johnson, Jr., E. M. Foster, and H. Sugiyama. 1968. Possible origin of the high incidence of Clostridium botulinum type E in an inland bay (Green Bay of Lake Michigan). J. Bacteriol. 95: 1542-1547. 4. Cann, D. C., B. B. Wilson, G. Hobbs, J. M. Shewan, and A. Jopannsen. 1965. The incidence of Clostridium botulinum type E in fish and bottom deposits in the North Sea and off the coast of Scandinavia. J. Appl. Bacteriol. 28:462. 5. Dolman, C. E., H. Chang, D. V. Korr, and A. R. Shearer. 1950. Fish borne and type E botulism. Can. J. Public Health 46:135. 6. Dolman, C. E., and H. Iida. 1963. Type E botulism: its epidemiology, prevention and specific treatment. Can. J. Public Health 54:293-308. 7. Johannsen, A. 1961. Environmental condition for the growth and toxin formation of C. botulinum type E,
VOL. 37, 1979
8. 9.
10.
11.
with particular reference to the behaviour in vacuum packed foods. Report no. 100. Svenska Institute for Konservingsforskning, Gotenborg, Sweden. Johannsen, A. 1963. Clostridium botulinum in Sweden and adjacent waters. J. Appl. Bacteriol. 26:43-47. Johannsen, A. 1965. Clostridium botulinum type E in foods and the environment generally. J. Appl. Bacteriol. 28:90-94. Kanzawa, K. 1960. Ecological studies on Clostridium botulinum type E. Distribution of the organism in the soil of Hokkaido. Rep. Hokkaido Inst. Public Health 11:161-173. Meyer, K. F. 1956. The status of botulism as a world
C. BOTULINUM IN GULF OF THAILAND
197
health problem. Bull. W.H.O. 15:281-298. 12. Nakamura, Y., H. lida, S. Saeki, K. Kanzawa, and T. Karashimada. 1956. Type E botulism in Hokkaido. Jpn. J. Med. Sci. Biol. 9:45-59. 13. Osheroff, B. J., G. G. Slocum, and W. M. Decker. 1964. Status of botulism in the United States. Public Health. Rep. 79:871-878. 14. Schmidt, C. F., R. V. Lechowich, and J. F. Folinazzo. 1961. Growth and toxin production by type E Clostridium botulinum below 40'F. J. Food Sci. 26:626-630. 15. Wijewanta, E. A. 1962. Toxigenic strains of Clostridium botulinum in soils of Ceylon. Ceylon J. Med. Sci. 11:59.
AND
ENVIRONMENTAL MICROBIOLOGY, Feb. 1979, p. 194-197
0099-2240/79/02-0194/04$02.00/0
Vol. 37, No.2
Clostridium botulinum in the Gulf of Thailand LEK TANASUGARN Section of Bacteriology, Veterinary College, Chulalongkorn University, Bangkok, Thailand Received for publication 6 October 1978
A survey was carried out to determine the incidence of Clostridium botulinum in samples of mud, sand, and fish from the Gulf of Thailand. Enrichment cultures from 762 samples of mud and sand from seven different areas around the Gulf were tested. C. botulinum type D was present in 10 samples, and type E was present in 2 samples taken from the west coast at Hua Hin. Enrichment cultures from 16,773 fish grouped into 2,151 samples yielded 10 filtrates containing C. botulinum type D and 5 containing type E. All of the toxic filtrates were obtained from samples of fish taken from the west coast of the Gulf of Thailand.
Clostridium botulinum is worldwide in its distribution (11), and its natural habitat is soil, even virgin and forest soil. A high degree of incidence of C. botulinum, especially types A and B, has also been reported under tropical conditions (15). In the United States, an extensive survey of C. botulinum type E in the fish of Lakes Erie, Huron, Superior, and Michigan was made after several type E botulism outbreaks in 1960 and 1963 (13). The organism was found in the digestive tracts of fish from all of the lakes, with incidences ranging from 1 to 9% (2). The association of C. botulinum type E with fishery products and the aquatic environment is now well established (1, 4, 9). The presence of type E in the deposits of lakes and soils of Hokkaido, Japan, (10, 12), British Columbia (6), and Sweden (8) has been reported. No survey of C. botulinum from Thailand has been reported previously nor have human cases of botulism been documented, but this does not necessarily mean that such food poisoning has not occurred. MATERIALS AND METHODS Collection of samples. Samples of mud and sand were collected from various freshwater sites of the Mekong River bed and from the Gulf of Thailand. Drop baskets with surface-controlled lid-sealing devices were used to collect samples of mud and sand at water depths of 1, 10, 20, and 30 m. Samples consisted of 2 kg of material, from which 100 g was aseptically separated for subsequent examination. Although the majority of fresh fish samples were purchased at various local markets where refrigeration is rather universally absent, some designated specimens were obtained at a 30-m water depth at Hua Hin (Fig. 1). Media. The media for the cultivation of C. botulinum used in this study were as follows: (i) sulfitereducing medium (15 g of tryptone, 10 g of yeast extract, 3 g of meat extract, 20 ml of 1% FeSO4, 10 ml of 5% Na2SO3, water added to 1,000 ml, pH 6.8); (ii) 194
beef heart infusion-cooked meat medium (10% fresh beef heart in cooked meat medium) (3); and (iii) egg yolk agar (reinforced Clostridium agar with 5% egg yolk emulsion). This last emulsion was prepared by aseptically removing the yolk of one egg and diluting with 15 ml of sterile normal saline. The emulsion was always prepared fresh. Method. A 10-g amount of each sample of mud or sand was inoculated into 80 ml of beef heart infusioncooked meat medium. In fish samples, the digestive tract was cultured for the presence of C. botulinum. For small fish, about eight intestines (10 to 15 g) were combined for one sample. Each sample of fish intestine was inoculated into 80 ml of beef heart infusion-cooked meat medium. After incubation at 25 to 300C for 7 days, the culture supernatant fluid was separated and centrifuged for 1 h at 6,000 rpm in a refrigerated centrifuge to eliminate the bacteria from the supernatant fluid. The clear supernatant fluid was again separated and kept in a freezer overnight. This frozen storage was used to reduce nonspecific death of mice. The routine use of a 1:5 dilution of the clear fluid of the culture in gelatin phosphate buffer (equal volumes of 0.4% gelatin solution and a pH 6.5 mixture of equal volumes of 0.2 M NaHPO4 and 0.2 M KH2PO4) to some extent also reduced the nonspecific death of mice. A 0.5-ml amount of a dilution of the clear, previously frozen fluid was injected intraperitoneally into mice (two mice for each culture). Mice were observed for symptoms of botulism, which resulted in death within 48 h. Injections of inactivated samples (heated for 10 min at 1000C) were made into mice as a control. If death of the animal was observed, the toxin was typed by using specific antitoxins (types A, B, C, D, and E) obtained from the Pasteur Institute, Paris. For confirmation, the original culture (the supernatant fluid of which caused death to the mice after inrtraperitoneal injection) was streaked onto the surface of egg yolk agar and incubated anaerobically in an anaerobic jar at 25 to 300C for 48 h. The lipase-positive zone was then picked and cultured in cooked meat medium at 25 to 300C. After 7 days of incubation, the supernatant fluid of the culture was typed by inoculating mice protected with specific antitoxins.
VOL. 37, 1979
C. BOTULINUM IN GULF OF THAILAND
195
FIG. 1. Location of sampling sites.
TABLE 1. Incidence of C. botulinum in samples of mud and sand from the Gulf of Thailand Sampling site
Nature of sample
Paknam Bangpoo Bangsaen Gulf of Thailand Had Chao Sumran Hua Hin Klong Wan
Mud Mud Sand Mud and sand Sand Sand Sand
RESULTS A total of 300 samples of mud and sand were cultivated in sulfite-reducing medium, and no toxin of C. botulinum was ever observed in such cultures. When these same samples were cultivated in beef heart infusion-cooked meat medium, C. botulinum type D toxin was found in 10 samples. As a result, beef heart infusioncooked meat medium was used for propagation and toxin studies in the later part of this survey. I do not feel that sulfite-reducing medium gives
No. of sam- No. of cultures that ples cultured killed mice
20 100 25 43 47 475 52
C. botulinum serotype
10 Type D and 2 type E
12
TABLE 2. Incidence of C. botulinum in samples of mud and sand taken from various depths of the sea at Hua Hin No. of sam-Noofsm Depth of ofples of mud No. of sam-
septh
(i)
1 10
20 30
andsted 100 109 94 172
klled mice 2 10
C. botulinum
serotype 2 Type D 8 Type D and 2 type E
196
APPL. ENVIRON. MICROBIOL.
TANASUGARN
TABLE 3. Incidence of C. botulinum in samples of fish from the Gulf of Thailand
No. of fish
No. of composite sampecuculples tured
No. of culture filtrates ta that killed mice
Hua Hin
1,560
124
3
3 Type E
2 Squid and 1 Scalopsis
Petchburi Radchburi
1,985 704
361 88
7 3
5 Squid and 2 sea catfish
Nakorn-patom Mahachai Bangkok Paknam Cholburi
1,376 1,280 4,124 1,296 4,448
172 160 528 162 556
1
7 Type D 1 Type D and 2 type E Type D
1
Type D
Squid
Samplingsite
btli C.bulnmserotype
of fish sample giving Type positive results pstv
eut
dubiosus Squid Squid
a toxin level as high as that produced by beef mud and sand at Hua Hin, but only from samples taken from a 30-m depth; most of the C. heart infusion-cooked meat medium. Table 1 shows that C. botulinum types D and botulinum type D isolates were also found at E toxins were isolated from 12 of 475 samples of this depth. This indicated that the spores of C. mud and sand obtained at Hua Hin. C. botu- botulinum were present in deep water rather linum was not detected in enrichment cultures than in shallow water. C. botulinum type E is able to produce toxin of 287 samples of mud or sand from the other six coastal areas. Ten of the positive samples at 5°C (5, 7, 14). Hence, the presence in fish of from Hua Hin were collected at a 30-m depth, even small numbers of spores of C. botulinum and the other two were collected at a 20-m depth type E makes rigid temperature control imperative. The toxin is rapidly inactivated by heat(Table 2). Table 3 shows that C. botulinum toxin was ing, and raw, smoked, or fermented fish is a detected in enrichment cultures of 15 of 2,151 greater hazard than cooked fish. samples of fish obtained from different areas of ACKNOWLEDGMENTS the west coast of the Gulf of Thailand. Ten of I am indebted to T. A. Roberts, Meat Research Institute, the positive cultures were type D, and five were Langford, Bristol, England for his help in the initial stages of type E. this work and to the Department of Fisheries, Thailand for C. botulinum toxin was not detected in enrich- their help in the collection of samples of mud, sand, and fish. ment cultures of 44 samples of mud and sand This work was made possible by a generous grant from the taken from the Mekong River bed at various International Atomic Energy Agency, Vienna, Austria. sites. LITERATURE CITED
DISCUSSION It is interesting to note that C. botulinum type E was isolated from fish from only two places on the west coast of the Gulf of Thailand, Hua Hin and Radchburi. Fish sold in Radchburi were obtained from Hua Hin fish markets. C. botulinum type E was also found in samples of sand from Hua Hin from a 30-m depth, but was not isolated from samples of mud and sand collected from other areas. C. botulinum types D and E were found only in samples of mud, sand, and fish from the west coast of the gulf; C. botulinum was not isolated from any samples from the east coast. Geographically, the land along the west coast is much higher than that along the east coast. This might indicate that the spores of C. botulinum was washed into the sea from the west coast. C. botulinum type E was found in samples of
1. Ando, Y., and K. Inoue. 1957. Studies on growth and toxin production of Clostridium botulinum type E in fish products. Bull. Jpn. Soc. Sci. Fish. 23:8. 2. Bott, T. L., J. S. Deffner, E. McCoy, and E. M. Foster. 1966. Clostridium botulinum type E in fish from the Great Lakes. J. Bacteriol. 91:919-924. 3. Bott, T. L., J. Johnson, Jr., E. M. Foster, and H. Sugiyama. 1968. Possible origin of the high incidence of Clostridium botulinum type E in an inland bay (Green Bay of Lake Michigan). J. Bacteriol. 95: 1542-1547. 4. Cann, D. C., B. B. Wilson, G. Hobbs, J. M. Shewan, and A. Jopannsen. 1965. The incidence of Clostridium botulinum type E in fish and bottom deposits in the North Sea and off the coast of Scandinavia. J. Appl. Bacteriol. 28:462. 5. Dolman, C. E., H. Chang, D. V. Korr, and A. R. Shearer. 1950. Fish borne and type E botulism. Can. J. Public Health 46:135. 6. Dolman, C. E., and H. Iida. 1963. Type E botulism: its epidemiology, prevention and specific treatment. Can. J. Public Health 54:293-308. 7. Johannsen, A. 1961. Environmental condition for the growth and toxin formation of C. botulinum type E,
VOL. 37, 1979
8. 9.
10.
11.
with particular reference to the behaviour in vacuum packed foods. Report no. 100. Svenska Institute for Konservingsforskning, Gotenborg, Sweden. Johannsen, A. 1963. Clostridium botulinum in Sweden and adjacent waters. J. Appl. Bacteriol. 26:43-47. Johannsen, A. 1965. Clostridium botulinum type E in foods and the environment generally. J. Appl. Bacteriol. 28:90-94. Kanzawa, K. 1960. Ecological studies on Clostridium botulinum type E. Distribution of the organism in the soil of Hokkaido. Rep. Hokkaido Inst. Public Health 11:161-173. Meyer, K. F. 1956. The status of botulism as a world
C. BOTULINUM IN GULF OF THAILAND
197
health problem. Bull. W.H.O. 15:281-298. 12. Nakamura, Y., H. lida, S. Saeki, K. Kanzawa, and T. Karashimada. 1956. Type E botulism in Hokkaido. Jpn. J. Med. Sci. Biol. 9:45-59. 13. Osheroff, B. J., G. G. Slocum, and W. M. Decker. 1964. Status of botulism in the United States. Public Health. Rep. 79:871-878. 14. Schmidt, C. F., R. V. Lechowich, and J. F. Folinazzo. 1961. Growth and toxin production by type E Clostridium botulinum below 40'F. J. Food Sci. 26:626-630. 15. Wijewanta, E. A. 1962. Toxigenic strains of Clostridium botulinum in soils of Ceylon. Ceylon J. Med. Sci. 11:59.
