Microbiol.
Immunol.
Vol. 36 (6), 583-591, 1992
Prevalence of Clostridium botulinum Type E and Coexistence of C. botulinum Nonproteolytic Type B
in the River Soil of Japan Kiyotaka YAMAKAWA, and Shinichi NAKAMURA* Department of Bacteriology,Schoolof Medicine, Kanazawa University, Kanazawa,
Ishikawa 920, Japan
(Accepted for publication, March 9, 1992)
Abstract Soil samples from 98 sites in the whole systems of four rivers in Japan were examined for the presence of Clostridium botulinum. Type E organism was prevalently shown throughout the whole river systems including upper part ; detection rates of type E toxin in soil culture ranged from 33 to 82% . This type was also detected in soil of adjacent mountainous district . Type B and C toxins were detected at 7% and 9% of the sites examined , respectively. C, botulinum type E and nonproteolytic type B strains were isolated from enrichment cultures of soil samples. These results suggest that the terrestrial origin of type E organism would be considered as one of the reasons for the high incidence of this organism in the sea areas, and prove that C. botulinumnonproteolytic type B exists in the soil of Japan .
Clostridiumbotulinumtype E is an important type in human botulism as well as types A and B. C. botulinumtype E is disseminated in aquatic land of the northern high-latitude areas (19). In Japan, since type E outbreak caused by a foodstuff "izushi" was recognized in Hokkaido, the northern island of the country , in 1951 (15), many investigations have been performed to know occurrence of the bacterium in soil. In these investigations soil of sea shore , lakes and rivers were extensively surveyed and a wide distribution of type E throughout Japan was demonstrated (7, 9, 11,20). In rivers, however, the surveys have been mainly restricted to lower reaches closely related to human behaviors . From a standpoint of the hypothesis that the marine areas are catchment basins in which are concentrated the type E organisms that have multiplied on land and are carried in by the drainage water (4, 10), it is expected that type E organism is present in the upper regions of rivers. In the present study, we performed a survey of C. botulinumtype E in soil of the whole systems of four rivers in Japan to evaluate a distributional state of upper regions. Furthermore, a preliminary survey on mountainous district was conducted . We also report the presence of C. botulinumnonproteolytic type B in the river soil. MATERIALS
AND METHODS
Soil samples. Soil samples were collected from 98 sites of four rivers in Japan 583
584
K.
YAMAKAWA
AND S. NAKAMURA
during 1986 and 1990: 38 sites 1 to 80 km from the mouth of a river (0 to 1,200 m above sea level) in the Tedori River (August 1986 and August 1987), 18 sites 66 to 160 km (50 to 750 m) in the Nagara River (November 1989), 22 sites 1 to 245 km (0 to 800 m) in the Tenryu River (July 1990) and 20 sites 123 to 245 km (200 to 1,200 m) in the Chikuma River (July 1990). These rivers are located in the central mountainous part of Honshu, the major island of the country (Fig. 1). Soil specimens were collected at some distance of 1 to 20 km. At each site, a pair of soil samples was taken; one from waterside and the other from several meters apart from the waterside. Soil samples were also collected from 15 sites on a mountainous district of Mt. Hakusan in August 1988. The height of these sites was between 1,000 and 2,700 m (near the peak) above sea level. Specimens were collected at some distance of 0.5 to 3 km.
Fig. 1. Location of four rivers examined in Japan. 1, The Tedori River; 2, The Nagara River; 3, The Tenryu River; 4, The Chikuma River; 5, Mt. Hakusan.
C. BOTULINUM
IN
RIVER
SOIL
585
About 100 g specimen was sampled from soil at a depth of 10 to 15 cm and stored at 4 C until use. Detectionof botulinumtoxin. Presence of C. botulinumin soil was demonstrated according to the method of Yamakawa et al (20) except cultivation condition. Five 1-g portions of each soil sample (ten 1-g portions per each site) were inoculated into tubes containing chopped meat-glucose (CMG) medium (8) and incubated anaerobically under CO2 gas at 25 C for 7 days. The culture was frozen, thawed, and then each supernatant fluid from the culture was examined for botulinum toxins with and without trypsinization. When at least one culture of a site showed botulinum toxicity, the site was expressed as positive for the corresponding type of C. botulinum. Isolation of C. botulinum typeE and nonproteolytictypeB. Isolation of C. botulinum from soil was performed using the samples in which botulinum toxin was detected in the original experiment. Culture of additive 1-g portions of the soil sample in CMG or CMG containing D-cycloserine (Sigma Chemical Co., St. Louis, Mo., U.S.A.) (250 pg/ml) was done for isolation of type E or nonproteolytic type B, respectively, under the same conditions as mentioned above. A toxic culture was then diluted with pre-reduced diluent (8), and 0.1 ml of 10-5- or 10-6-fold diluted culture was spread on 5% egg yolk-containing nutrient agar (EYNA) stored anaerobically overnight. The plates were placed in anaerobe jars with some amount of silica gel and cold catalyst pellets, and incubated at 25 C for 4 to 6 days under an atmosphere of 75% H2 and 25% CO2 gas. Colonies of nonproteolytic C. botulinumon EYNA plate were 1 to 5 mm in size, circular to irregular and white or milky, and surrounded with a wide (about 1 cm or more) opaque zone and pearly layer of similar size. Suspected colonies with these characters were fished into liver broth and incubated at 25 C for 1 to 2 days. Cultures showing marked gas production in liver broth were checked on purity by re-cultivation on EYNA plates and subjected to a test of toxigenicity in CMG medium. Biochemicalproperties of the isolates. Tests for biochemical properties were performed according to the description of Nakamura et al (14). Gas-chromatographic analysis of fermentation products in PYG medium was performed according to the method of Holdeman et al (8). RESULTS
Surveyof Four Rivers and the Related MountainousDistrict A wide distribution of C. botulinumtype E was demonstrated in the soil of the Tedori and the Nagara (Fig. 2). Type E toxin was detected in the lower to upper reaches of these rivers, and even in mountainous branches. Detection rates of type E toxin were 39% (15 sites) of examined sites in the Tedori and 33% (6 sites) in the Nagara. The most upper site yielding the toxic culture was 75 km from the mouth of a river (1,000 m above sea level) in the Tedori, and 160 km (750 m) in the Nagara. Type B toxin was detected at 3 (8%) and 4 (22%) sites in the Tedori and the Nagara, respectively, mainly in the upper reaches. Samples from two of
586
K. YAMAKAWA
Fig.2.
Distribution
positive
sites;○,type
positive
site;C2,
of C.
botulinum E
C2
in
AND S. NAKAMURA
the
rivers
toxin-negative
toxin-positive
of
sites;B, site;*,type
Tedori
type B
B
isolation
and
Nagara.●,type
toxin-positive sites;★,type
E
sites;C1, E
C isolation
toxinI
toxinsites.
the 7 type B-positive sites, one in the Tedori and the other in the Nagara, contained both types B and E. In these 7 sites, a total of 10 cultures were type B toxin-positive. Trysinization was effective on detecting type B toxin in soil cultures. In 8 cultures the toxicity was not detected before trypsinization and increased to 4 to 40 MLD/ml after trypsinization. In other two cultures the toxicity was 4 MLD/ml before the treatment, and 80 and 400 MLD/ml after the treatment. A mountainous district, a common source of these rivers, was surveyed. The type E toxin was detected at 3 (20%) sites. These sites were not directly related to a river stream and their heights were 1,000, 1,000 and 1,700 m above sea level, respectively. In comparison with the results of the Tedori and the Nagara, the other two rivers showed extremely high incidence of type E. Detection rates of type E toxin
C. BOTULINUM
IN RIVER
SOIL
587
were 82% (18 sites) in the Tenryu and 80% (16 sites) in the Chikuma (Fig. 3). The toxin-yielding sites were distributed throughout the river systems. The most upper site yielding toxic culture was 245 km from the mouth of a river (800 m above sea level) in the Tenryu, and 330 km (1,200 m) in the Chikuma. Unlike the Tedori and the Nagara, no type B toxin was found in these rivers. Detection rates of type C (Ci or C2 toxin) were 3% (1 site) in the Tedori, 6% (1 site) in the Nagara, 14% (3 sites) in the Tenryu and 20% (4 sites) in the Chikuma. Isolationof C. botulinum TypesB and E from River Soil Type E organisms could be isolated from soil by the method of enrichment culture using CMG at 4 sites (refer to Fig. 2) in the Tedori in 1987. Five to 90% of the suspected colonies on EYNA plates were demonstrated to be type E in each
Fig.3.
Distribution
positive
sites;○,
posltlve
slte.
of C.botulinum type
E
in
toxin-negative
the
rivers
of
sites;C1,
Tenryu
and
Chikuma.●,type
C I toxin-positive
sites;C2,
E
toxin.
C2
toxin-
588
K. Table
YAMAKAWA 1.
Properties
AND S. NAKAMURA of C. botulinum
isolates
site.
Cultural and biochemical properties of these isolates are given in Table 1. Isolation of type B organisms from soil taken at a site (refer to Fig. 2) in the Tedori in 1987 was unsuccessful by the method for type E. Type B organisms could. be isolated by the modified method of enrichment culture using D-cycloserinecontaining CMG from soil taken in 1989 at a site of the Nagara (refer to Fig. 2), culture of which exhibited 40 MLD/ml of type B toxicity. A total of 128 isolates from
the culture,
which
showed
colonial
characters
of nonproteolytic
C. botulinum,
were examined for the toxigenicity and 4 isolates produced type B toxin. In July 1990 soil was taken at the same site of the Tedori stated above and isolation was attempted again by the modified method. Type B toxicity was detected at 400 MLD/ml in this soil culture and 5 toxigenic isolates were obtained out of 36 suspected isolates tested. Cultural and biochemical properties of the toxigenic isolates showed nonproteolytic nature and extremely high similarity to those of the type E isolates (Table 1).
C. BO TULINUM IN
RIVER
SOIL
589
DISCUSSION
In
the
present
study,
soil
culture
to
demonstrate
nonproteolytic
C. botulinum
was performed with a small size of inoculum. (one gram soil), multiple examinations (five) for each sample, and at lower incubation temperature (25 C). These conditions have been recommended to detect C. botulinumtype E (1). Trypsinization of supernatant fluid from soil culture was needed to detect type B toxin in 71 % of 7 positive sites, while 9% of 53 positive sites was the case for type E toxin. These findings are similar to those in the United Kingdom (17, 18). In this study we surveyed four rivers flowing in the central part of Japan. The habitats of C. botulinumtype E were located from lower regions of the main stream to upper and fine branches. The detection rates, 33 to 82%, were the same level or greatly higher than those of rivers in Hokkaido where Kanzawa et al (11) examined 45 places of 15 rivers and reported that the maximum detection rate was 30%. A major feature of the distribution on the four rivers is concluded to be the prevalence of C. botulinumtype E in the whole river system including upper part or higher location of river. Furthermore, the presence of type E organism was demonstrated in the mountainous district which was the source of the Tedori River and the Nagara River. It that from in
has
its
been
high
surrounding the
type
E
organism
type
For
containing similar pletely
the
inhibited
zole
soil
of
distinct (12). belonged
at
60
The
C for
Clostridium
C.
using
types
CMG
butyricum producing
botulinum
min
type
before E
toxin
which type
nonproteolytic
toxin type
were infant was
bacterium showed B
even
in
proteolytic
enrichment
in
culture in
such
CMG
culture
production
was
was
com-
sulfamethoxa-
selective
agents
for
botulism
(3).
Heating
also as
spreading
A and
culture
toxin
identified
above
and
D-cycloserine,
cultivation were is another
B
toxicity
of
organism
of direct
type
using
the
which feces
for by
The
the
rivers
method
enrichment
containing
patients'
of
down
hypothesis.
B,
drug.
(4 ƒÊg/ml)
from 30
producing
isolates to
the
the
and
washed
mentioned
of
used
isolated
type
without
culture
by been
be
organism
being
findings
origin
although
trimethoprim.
proteolytic
isolates from
soil
could
effective,
culture
and
sample
Our
in
in
(76ƒÊg/ml)
isolation
was
level
terrestrial
nonproteolytic
its
reaches
unsuccessful
organisms of
terrestrial
present
upper
have
a
multiplication
Our in
which
is from
addition,
the
was
E
results
In
support
plates,
the
isolation
D-cycloserine to
to
type
(1).
distributed
EYNA
However,
CMG.
10).
E organisms
on
B (20).
in
(5,
suggested
seem
of type
suspension
botulinum sediments
was widely
district
Isolation
C.
marine
masses
sediments
mountainous
of soil
that in
land
marine
that
of
postulated
incidence
C.
the
inhibitory. botulinum
producing nonproteolytic
type type nature
E
(2),
E toxin and
(2).
Isolation of nonproteolytic type B from food causing botulism or environmental sample has been quite rare (4, 13) and habitat of the bacterium has been unknown although C. botulinumnonproteolytic type B was discovered as the prototype of C. botulinumby van Ermengem about 100 years ago (6). However, markedly high incidence of nonproteolytic type B in aquatic soil is suggested by circumstantial
590
K.YAMAKAWA
AND S.NAKAMURA
evidence in the United Kingdom (17, 18). In the Tedori and the Nagara the distribution of type B organism overlapped with that of type E, and also nonproteolytic type B and type E organisms could be isolated. Therefore, we expect that nonproteolytic type B and type E organisms might share the same niches in aquatic soil. This expectation might be supported by the findings that biochemical properties of the type B and E isolates were nearly identical except toxigenicity. It should be noted that this would be the first report of detection and isolation of type B from soil of Japan. The soil specimen itself seemed to be appropriate to the demonstration of type B toxin and organism, for the method employed for the demonstration of botulinum toxin in this study was ordinary one for type E toxin as mentioned at the beginning of this section, including low incubation temperature (25 C). Finally, detection rates of type C in the rivers examined were similar to those in land soil (20) but lower than those in lake shore soil (16). This work was supported in part by a Grant-in-Aid of Education, Science and Culture of Japan.
for Scientific Research from the Ministry
REFERENCES
1)
2)
3) 4) 5) 6) 7)
8) 9)
10) 11)
12)
Bott, T., Johnson, J. Jr., Foster, E.M., and Sugiyama, H. 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. Cato, E.P., George, W.L., and Finegold, S.M. 1986. Genus Clostridium,p. 1141-1200. In Sneath, P.H.A. (ed), Bergey's manual of systematic bacteriology, Vol. 2, Williams & Wilkins Co., Baltimore. Dezfulian, M., McCroskey, L.M., Hatheway, C.L., and Dowell, V.R., Jr. 1981. Selective medium for isolation of Clostridium botulinum from human feces. J. Clin. Microbiol. 13: 526-531. Dolman, C.E., Tomsich, M., Campbell, C.C.R., and Laing, W.B. 1960. Fish eggs as a cause of human botulism. J. Infect. Dis. 106: 5-19. Dolman, C.E., and Iida, H. 1963. Type E botulism: its epidemiology, prevention, and specific treatment. Can. J. Public Health 54: 293-308. van Ermengem, E. 1897. Ueber einen neuen anaeroben Bacillus und seine Beziehungen zum Botulismus. Ztschr. Hyg. Infektionskr. 26 : 1-56. Hayashi, K., Tokuchi, M., Sakakibara, S., Yamaguchi, A., Kinoshita, Y., and Wakabayashi, T. 1974. Distribution of Clostridium botulinum in Shiga Prefecture. Rep, Shiga Pref. Inst. Pub. Hlth. Environ. Sci. 10 9-17 (in Japanese). Holdeman, L.V., Cato, E.P., and Moore, W.E.C. 1977. Anaerobe Laboratory manual, 4th ed, Virginia Polytechnic Institute and State University, Blacksburg, Virginia. Iida, H. 1968. Epidemiological observation of botulism outbreaks in Japan, p. 357-359. In Herzberg, M. (ed), Proceedings of the first U.S.-Japan conference on toxic micro-organisms, U.S. Government Printing Office, Washington, D.C. Johannsen, A. 1963. Clostridium botulinumin Sweden and the adjacent waters. J. Appl. Bacteriol. 26 : 43-47. Kanzawa, K., Ono, T., Karashimada, T., and Iida, H. 1968. Distribution of Clostridium botulinum type E in Hokkaido, Japan, p. 299-303. In Herzberg, M. (ed), Proceedings of the first U.S.-Japan conference on toxic micro-organisms, U.S. Government Printing Office, Washington, D.C. McCroskey, L.M., Hatheway, C.L., Fenicia, L., Pasolini, B., and Aureli, P. 1986. Characterization
of an
organism
that
produces
type
E botulinal
toxin
but
which
resembles
Clostridium
C. BOTULINUM
13) 14)
15) 16)
17) 18) 19) 20)
IN RIVER
SOIL
591
butyricumfrom the feces of an infant with type E botulism. J. Clin. Microbiol. 23: 201-202. Meyer, K.F., and Gunnison, J.B. 1929. European strains of C. botulinum. J. Infect. Dis. 45: 96-105. Nakamura, S., Shimamura, T., Hayase, M., and Nishida, S. 1973. Numerical taxonomy of saccharolytic clostridia, particularly Clostridium perfringens-like strains: descriptions of Clostridium absonum sp. n. and Clostridium paraperfringens. Int. J. Syst. Bacteriol. 23: 419-429. Nakamura, Y., Iida, H., and Saeki, K. 1952. Report on a botulism encountered in Iwanai. Spec. Report, Hokkaido Inst. Publ. Health 2: 1-5 (in Japanese). Serikawa, T., Nakamura, S., and Nishida, S. 1977. Distribution of Clostridium botulinumtype C in Ishikawa Prefecture, and applicability of agglutination to identification of nontoxigenic isolates of C. botulinum type C. Microbiol. Immunol. 21: 127-136. Smith, G.R., and Moryson, C.J. 1975. Clostridium botulinum in the lakes and waterways of London. J. Hyg. 75: 371-379. Smith, G.R., Milligan, R.A., and Moryson, C.J. 1978. Clostridium botulinumin aquatic environments in Great Britain and Ireland. J. Hyg. 80: 431-436. Smith, L. DS., and Sugiyama, H. 1988. Botulism, 2nd ed, Charles C Thomas Publisher, Springfield, Illinois. Yamakawa, K., Kamiya, S., Nishida, S., Yoshimura, K., Yu, H., Lu, D., and Nakamura, S. 1988. Distribution of Clostridiumbotulinumin Japan and in Shinkiang district of China. Microbiol. Immunol. 32: 579-587. (Received for Publication, December 25, 1991)
Immunol.
Vol. 36 (6), 583-591, 1992
Prevalence of Clostridium botulinum Type E and Coexistence of C. botulinum Nonproteolytic Type B
in the River Soil of Japan Kiyotaka YAMAKAWA, and Shinichi NAKAMURA* Department of Bacteriology,Schoolof Medicine, Kanazawa University, Kanazawa,
Ishikawa 920, Japan
(Accepted for publication, March 9, 1992)
Abstract Soil samples from 98 sites in the whole systems of four rivers in Japan were examined for the presence of Clostridium botulinum. Type E organism was prevalently shown throughout the whole river systems including upper part ; detection rates of type E toxin in soil culture ranged from 33 to 82% . This type was also detected in soil of adjacent mountainous district . Type B and C toxins were detected at 7% and 9% of the sites examined , respectively. C, botulinum type E and nonproteolytic type B strains were isolated from enrichment cultures of soil samples. These results suggest that the terrestrial origin of type E organism would be considered as one of the reasons for the high incidence of this organism in the sea areas, and prove that C. botulinumnonproteolytic type B exists in the soil of Japan .
Clostridiumbotulinumtype E is an important type in human botulism as well as types A and B. C. botulinumtype E is disseminated in aquatic land of the northern high-latitude areas (19). In Japan, since type E outbreak caused by a foodstuff "izushi" was recognized in Hokkaido, the northern island of the country , in 1951 (15), many investigations have been performed to know occurrence of the bacterium in soil. In these investigations soil of sea shore , lakes and rivers were extensively surveyed and a wide distribution of type E throughout Japan was demonstrated (7, 9, 11,20). In rivers, however, the surveys have been mainly restricted to lower reaches closely related to human behaviors . From a standpoint of the hypothesis that the marine areas are catchment basins in which are concentrated the type E organisms that have multiplied on land and are carried in by the drainage water (4, 10), it is expected that type E organism is present in the upper regions of rivers. In the present study, we performed a survey of C. botulinumtype E in soil of the whole systems of four rivers in Japan to evaluate a distributional state of upper regions. Furthermore, a preliminary survey on mountainous district was conducted . We also report the presence of C. botulinumnonproteolytic type B in the river soil. MATERIALS
AND METHODS
Soil samples. Soil samples were collected from 98 sites of four rivers in Japan 583
584
K.
YAMAKAWA
AND S. NAKAMURA
during 1986 and 1990: 38 sites 1 to 80 km from the mouth of a river (0 to 1,200 m above sea level) in the Tedori River (August 1986 and August 1987), 18 sites 66 to 160 km (50 to 750 m) in the Nagara River (November 1989), 22 sites 1 to 245 km (0 to 800 m) in the Tenryu River (July 1990) and 20 sites 123 to 245 km (200 to 1,200 m) in the Chikuma River (July 1990). These rivers are located in the central mountainous part of Honshu, the major island of the country (Fig. 1). Soil specimens were collected at some distance of 1 to 20 km. At each site, a pair of soil samples was taken; one from waterside and the other from several meters apart from the waterside. Soil samples were also collected from 15 sites on a mountainous district of Mt. Hakusan in August 1988. The height of these sites was between 1,000 and 2,700 m (near the peak) above sea level. Specimens were collected at some distance of 0.5 to 3 km.
Fig. 1. Location of four rivers examined in Japan. 1, The Tedori River; 2, The Nagara River; 3, The Tenryu River; 4, The Chikuma River; 5, Mt. Hakusan.
C. BOTULINUM
IN
RIVER
SOIL
585
About 100 g specimen was sampled from soil at a depth of 10 to 15 cm and stored at 4 C until use. Detectionof botulinumtoxin. Presence of C. botulinumin soil was demonstrated according to the method of Yamakawa et al (20) except cultivation condition. Five 1-g portions of each soil sample (ten 1-g portions per each site) were inoculated into tubes containing chopped meat-glucose (CMG) medium (8) and incubated anaerobically under CO2 gas at 25 C for 7 days. The culture was frozen, thawed, and then each supernatant fluid from the culture was examined for botulinum toxins with and without trypsinization. When at least one culture of a site showed botulinum toxicity, the site was expressed as positive for the corresponding type of C. botulinum. Isolation of C. botulinum typeE and nonproteolytictypeB. Isolation of C. botulinum from soil was performed using the samples in which botulinum toxin was detected in the original experiment. Culture of additive 1-g portions of the soil sample in CMG or CMG containing D-cycloserine (Sigma Chemical Co., St. Louis, Mo., U.S.A.) (250 pg/ml) was done for isolation of type E or nonproteolytic type B, respectively, under the same conditions as mentioned above. A toxic culture was then diluted with pre-reduced diluent (8), and 0.1 ml of 10-5- or 10-6-fold diluted culture was spread on 5% egg yolk-containing nutrient agar (EYNA) stored anaerobically overnight. The plates were placed in anaerobe jars with some amount of silica gel and cold catalyst pellets, and incubated at 25 C for 4 to 6 days under an atmosphere of 75% H2 and 25% CO2 gas. Colonies of nonproteolytic C. botulinumon EYNA plate were 1 to 5 mm in size, circular to irregular and white or milky, and surrounded with a wide (about 1 cm or more) opaque zone and pearly layer of similar size. Suspected colonies with these characters were fished into liver broth and incubated at 25 C for 1 to 2 days. Cultures showing marked gas production in liver broth were checked on purity by re-cultivation on EYNA plates and subjected to a test of toxigenicity in CMG medium. Biochemicalproperties of the isolates. Tests for biochemical properties were performed according to the description of Nakamura et al (14). Gas-chromatographic analysis of fermentation products in PYG medium was performed according to the method of Holdeman et al (8). RESULTS
Surveyof Four Rivers and the Related MountainousDistrict A wide distribution of C. botulinumtype E was demonstrated in the soil of the Tedori and the Nagara (Fig. 2). Type E toxin was detected in the lower to upper reaches of these rivers, and even in mountainous branches. Detection rates of type E toxin were 39% (15 sites) of examined sites in the Tedori and 33% (6 sites) in the Nagara. The most upper site yielding the toxic culture was 75 km from the mouth of a river (1,000 m above sea level) in the Tedori, and 160 km (750 m) in the Nagara. Type B toxin was detected at 3 (8%) and 4 (22%) sites in the Tedori and the Nagara, respectively, mainly in the upper reaches. Samples from two of
586
K. YAMAKAWA
Fig.2.
Distribution
positive
sites;○,type
positive
site;C2,
of C.
botulinum E
C2
in
AND S. NAKAMURA
the
rivers
toxin-negative
toxin-positive
of
sites;B, site;*,type
Tedori
type B
B
isolation
and
Nagara.●,type
toxin-positive sites;★,type
E
sites;C1, E
C isolation
toxinI
toxinsites.
the 7 type B-positive sites, one in the Tedori and the other in the Nagara, contained both types B and E. In these 7 sites, a total of 10 cultures were type B toxin-positive. Trysinization was effective on detecting type B toxin in soil cultures. In 8 cultures the toxicity was not detected before trypsinization and increased to 4 to 40 MLD/ml after trypsinization. In other two cultures the toxicity was 4 MLD/ml before the treatment, and 80 and 400 MLD/ml after the treatment. A mountainous district, a common source of these rivers, was surveyed. The type E toxin was detected at 3 (20%) sites. These sites were not directly related to a river stream and their heights were 1,000, 1,000 and 1,700 m above sea level, respectively. In comparison with the results of the Tedori and the Nagara, the other two rivers showed extremely high incidence of type E. Detection rates of type E toxin
C. BOTULINUM
IN RIVER
SOIL
587
were 82% (18 sites) in the Tenryu and 80% (16 sites) in the Chikuma (Fig. 3). The toxin-yielding sites were distributed throughout the river systems. The most upper site yielding toxic culture was 245 km from the mouth of a river (800 m above sea level) in the Tenryu, and 330 km (1,200 m) in the Chikuma. Unlike the Tedori and the Nagara, no type B toxin was found in these rivers. Detection rates of type C (Ci or C2 toxin) were 3% (1 site) in the Tedori, 6% (1 site) in the Nagara, 14% (3 sites) in the Tenryu and 20% (4 sites) in the Chikuma. Isolationof C. botulinum TypesB and E from River Soil Type E organisms could be isolated from soil by the method of enrichment culture using CMG at 4 sites (refer to Fig. 2) in the Tedori in 1987. Five to 90% of the suspected colonies on EYNA plates were demonstrated to be type E in each
Fig.3.
Distribution
positive
sites;○,
posltlve
slte.
of C.botulinum type
E
in
toxin-negative
the
rivers
of
sites;C1,
Tenryu
and
Chikuma.●,type
C I toxin-positive
sites;C2,
E
toxin.
C2
toxin-
588
K. Table
YAMAKAWA 1.
Properties
AND S. NAKAMURA of C. botulinum
isolates
site.
Cultural and biochemical properties of these isolates are given in Table 1. Isolation of type B organisms from soil taken at a site (refer to Fig. 2) in the Tedori in 1987 was unsuccessful by the method for type E. Type B organisms could. be isolated by the modified method of enrichment culture using D-cycloserinecontaining CMG from soil taken in 1989 at a site of the Nagara (refer to Fig. 2), culture of which exhibited 40 MLD/ml of type B toxicity. A total of 128 isolates from
the culture,
which
showed
colonial
characters
of nonproteolytic
C. botulinum,
were examined for the toxigenicity and 4 isolates produced type B toxin. In July 1990 soil was taken at the same site of the Tedori stated above and isolation was attempted again by the modified method. Type B toxicity was detected at 400 MLD/ml in this soil culture and 5 toxigenic isolates were obtained out of 36 suspected isolates tested. Cultural and biochemical properties of the toxigenic isolates showed nonproteolytic nature and extremely high similarity to those of the type E isolates (Table 1).
C. BO TULINUM IN
RIVER
SOIL
589
DISCUSSION
In
the
present
study,
soil
culture
to
demonstrate
nonproteolytic
C. botulinum
was performed with a small size of inoculum. (one gram soil), multiple examinations (five) for each sample, and at lower incubation temperature (25 C). These conditions have been recommended to detect C. botulinumtype E (1). Trypsinization of supernatant fluid from soil culture was needed to detect type B toxin in 71 % of 7 positive sites, while 9% of 53 positive sites was the case for type E toxin. These findings are similar to those in the United Kingdom (17, 18). In this study we surveyed four rivers flowing in the central part of Japan. The habitats of C. botulinumtype E were located from lower regions of the main stream to upper and fine branches. The detection rates, 33 to 82%, were the same level or greatly higher than those of rivers in Hokkaido where Kanzawa et al (11) examined 45 places of 15 rivers and reported that the maximum detection rate was 30%. A major feature of the distribution on the four rivers is concluded to be the prevalence of C. botulinumtype E in the whole river system including upper part or higher location of river. Furthermore, the presence of type E organism was demonstrated in the mountainous district which was the source of the Tedori River and the Nagara River. It that from in
has
its
been
high
surrounding the
type
E
organism
type
For
containing similar pletely
the
inhibited
zole
soil
of
distinct (12). belonged
at
60
The
C for
Clostridium
C.
using
types
CMG
butyricum producing
botulinum
min
type
before E
toxin
which type
nonproteolytic
toxin type
were infant was
bacterium showed B
even
in
proteolytic
enrichment
in
culture in
such
CMG
culture
production
was
was
com-
sulfamethoxa-
selective
agents
for
botulism
(3).
Heating
also as
spreading
A and
culture
toxin
identified
above
and
D-cycloserine,
cultivation were is another
B
toxicity
of
organism
of direct
type
using
the
which feces
for by
The
the
rivers
method
enrichment
containing
patients'
of
down
hypothesis.
B,
drug.
(4 ƒÊg/ml)
from 30
producing
isolates to
the
the
and
washed
mentioned
of
used
isolated
type
without
culture
by been
be
organism
being
findings
origin
although
trimethoprim.
proteolytic
isolates from
soil
could
effective,
culture
and
sample
Our
in
in
(76ƒÊg/ml)
isolation
was
level
terrestrial
nonproteolytic
its
reaches
unsuccessful
organisms of
terrestrial
present
upper
have
a
multiplication
Our in
which
is from
addition,
the
was
E
results
In
support
plates,
the
isolation
D-cycloserine to
to
type
(1).
distributed
EYNA
However,
CMG.
10).
E organisms
on
B (20).
in
(5,
suggested
seem
of type
suspension
botulinum sediments
was widely
district
Isolation
C.
marine
masses
sediments
mountainous
of soil
that in
land
marine
that
of
postulated
incidence
C.
the
inhibitory. botulinum
producing nonproteolytic
type type nature
E
(2),
E toxin and
(2).
Isolation of nonproteolytic type B from food causing botulism or environmental sample has been quite rare (4, 13) and habitat of the bacterium has been unknown although C. botulinumnonproteolytic type B was discovered as the prototype of C. botulinumby van Ermengem about 100 years ago (6). However, markedly high incidence of nonproteolytic type B in aquatic soil is suggested by circumstantial
590
K.YAMAKAWA
AND S.NAKAMURA
evidence in the United Kingdom (17, 18). In the Tedori and the Nagara the distribution of type B organism overlapped with that of type E, and also nonproteolytic type B and type E organisms could be isolated. Therefore, we expect that nonproteolytic type B and type E organisms might share the same niches in aquatic soil. This expectation might be supported by the findings that biochemical properties of the type B and E isolates were nearly identical except toxigenicity. It should be noted that this would be the first report of detection and isolation of type B from soil of Japan. The soil specimen itself seemed to be appropriate to the demonstration of type B toxin and organism, for the method employed for the demonstration of botulinum toxin in this study was ordinary one for type E toxin as mentioned at the beginning of this section, including low incubation temperature (25 C). Finally, detection rates of type C in the rivers examined were similar to those in land soil (20) but lower than those in lake shore soil (16). This work was supported in part by a Grant-in-Aid of Education, Science and Culture of Japan.
for Scientific Research from the Ministry
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