APPLED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1977, p. 843-848 Copyright © 1977 American Society for Microbiology
Vol. 34, No. 6 Printed in U.S.A.
Cultural and Physiological Characteristics of Clostridium botulinum Type G and the Susceptibility of Certain Animals to Its Toxin ALBERTO S. CICCARELLI,t DAVID N. WHALEY, LORETTA M. McCROSKEY, DOMINGO F. GIMENEZ,t V. R. DOWELL, JR., AND CHARLES L. HATHEWAY* Bureau of Laboratories, Center for Disease Control, Atlanta, Georgia 30333
Received for publication 13 June 1977
Strain 89 of Clostridium botulinum type G, isolated by Gimenez and Ciccarelli in 1969, was characterized culturally, biochemically, and toxigenically. It was motile, hemolytic, asaccharolytic, weakly proteolytic, lipase and lecithinase negative, and it produced acetic, isobutyric, butyric, and isovaleric acids in peptoneyeast extract-glucose broth. No spores were seen in smears from solid or liquid media. Very low levels of toxin were produced in regular broth cultures, but dialysis cultures yielded 30,000 mouse 50% mean lethal doses (LD50) per ml. Dogs and lambs were resistant to intragastric challenges of up to 75,000 mouse LD50 per kg of body weight. Rhesus monkeys, chickens, and guinea pigs were susceptible to enteral and parenteral challenge with the toxin. These species showed signs of botulism after challenge that were similar to, if not identical with, those observed in other studies with other botulinal toxin types. The lethal doses for monkeys were 33,000 and 120 mouse LD50 per kg, intragastrically and intravenously, respectively; for chickens, 3,125 and 1,200 to 2,600 mouse LD50 per kg, orally and subcutaneously, respectively; and for guinea pigs, 10,000 to 20,000 and 100 mouse LD50 per kg, intragastrically and intraperitoneally, respectively.
Clostridium botulinum type G (strain 89) was isolated by Gimenez and Ciccarelli in 1969 from a cornfield in the Mendoza Province of Argentina (4). The isolate produced a trypsin-activatable, heat-labile, botulinal-like toxin that was not neutralized by antitoxin to any of the known botulinal toxins (types A, B, C, D, E, F). Antitoxin produced against toxoid prepared from the new toxin neutralized only its homologous toxin. The signs appearing in mice and the range of time to death after administering high and low doses of type G toxin were similar to those observed after administering botulinal toxins of types A through F. Strain 89 produced very little toxin, even in special media, and was weakly proteolytic and asaccharolytic. Further studies showed that strain 89 produces a "cryoprotein" that appears to be unrelated to toxic and hemagglutinating factors produced by the organism (1). Our report provides descriptions of additional cultural and physiological characteristics of strain 89 and of the susceptibility of various animal species to type G botulinal toxin. MATERIALS AND METHODS
peptone-yeast extract-glucose (PYG), 5% rabbit blood agar (BA), and modified McClung-Toabe egg yolk agar (EYA) were prepared as described by Dowell and Hawkins (2). The medium used for toxin production in dialysis sac cultures contained 4% proteose peptone (Oxoid, code L46)-1% yeast extract (BBL, 107638)-1% dextrose (Fisher Scientific Co., D16), with pH adjusted to 7.2 with 5 N NaOH before autoclaving. Organism. A subculture of C. botulinum type G (strain 89) was obtained from Lillian V. Holdeman of the Anaerobe Laboratory, Virginia Polytechnic Institute and State University, Blacksburg. Characterization studies. The organism was subcultured in freshly boiled CMG in a loosely capped tube and incubated for 24 h at 370C in a GasPak (BBL) anaerobic jar. It was then streaked on EYA and BA and incubated in a GasPak jar at 37°C for 48 h to obtain isolated colonies. The resulting colonies were examined macroscopically through a hand lens; 40 well-isolated colonies from both media were selected, subcultured individually in CMG, and incubated in an anaerobic glove box (2) containing a mixture of 85% N2-10% H2-5% C02 at 31°C for 5 days. Each of the 40 isolates was characterized according to methods described by Dowell and Hawkins (2). These included microscopic examinations, colonial characteristic observations, a variety of conventional biochemical tests, and metabolic product identification in PYG medium by gas-liquid chromatography. Preliminary toxicity tests. The toxicity of each of the 40 single-colony isolates in CMG medium was
Media. Chopped-meat-glucose medium (CMG), tPresent address: Universidad Nacional de Cuyo, Mendoza, Argentina.
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determined for mice as follows. After each CMG culwas centrifuged at 12,000 x g for 15 min, 0.5 ml of the supernatant fluid was injected intraperitoneally (i.p.)' into each of six 15- to 20-g Swiss white mice (ICR), and the time until death was recorded. Some supernatants killed no mice, but no attempt was made to determine whether these were atoxigenic mutants of the strain. Some killed some of the six mice within 4 days. Others killed all six mice in a mean time of less than 7 h. The culture supernatant that killed the mice in the shortest time was considered to contain the most toxin. Toxin production. A previously described procedure (4) was used to produce type G botulinal toxin with isolate 13-52, the most potent toxin producer. A section of dialysis tubing 13.2 cm wide when flat and 38 cm long) containing a few glass beads was mounted in a plasma collection bottle to which 450 ml of toxin production medium was added, and the system was autoclaved at 121°C for 30 min. Isolate 13-52 was grown in CMG for 20 h at 37°C. After the autoclaved system cooled to approximately 30°C, 50 ml of a 1:250, 0.85% saline dilution of this culture was seeded through the glass tube into the dialysis tubing, and the bottles were incubated at 30 to 31°C for 7 days in Brewer jars evacuated and filled with a mixture of 85% N2-10% H2-5% CO2. A very dense bacterial suspension developed in the dialysis tubing. The contents of the dialysis sacs were pooled in a sterile beaker. From 1 to 3 ml of sterile 0.85% saline was added to the dialysis tubing from each bottle to help remove the contents. The pooled suspensions were centrifuged at 12,000 x g for 15 min, and the recovered supernatant fluid was titrated for toxicity in mice. Serial twofold dilutions were made in gelatin diluent (2). One-half
ture
milliliter of each dilution was inoculated i.p. into each of six 20-g mice. The mice were observed for 4 days, and the deaths were recorded. The toxin content (mouse 50% lethal dose [LD5o] per ml) of the culture fluid was calculated by the Reed and Muench method
(8). activation of toxin. One volume of 1% (Difco, 1:250) solution was mixed with 9 volumes of toxin, and the mixtures were incubated at 37°C for 30 min. Serial twofold dilutions were made with samples of treated and untreated toxin, and 0.5 ml of each dilution was injected into mice. The 50% end-point dilution was calculated in each case by the Reed and Muench method (8). Immunization of rabbits. Toxoid was prepared by adding Formalin to a final concentration of 0.5% in the toxic culture fluid and incubating the mixture at 37°C until it was nontoxic for mice (0.5 ml, i.p., into each of four mice). The toxoid was stored at 4°C. Six New Zealand white rabbits (2.5 to 3.0 kg) were immunized by intravenous (i.v.) injections of toxoid and toxic culture fluid by a schedule (Table 1) that was based on results of preliminary trials. Sera from the final bleeding were pooled after an initial titration showed that their antitoxin levels were similar. Antitoxin titrations. Individual serum and the pool were titrated by making serial dilutions (10-fold for individual serum and twofold for the pool) in gelatin diluent, mixing 1 volume of the diluted serum with 5 volumes of toxin containing 20 LD5o/0.5 ml, Trypsin
aqueous trypsin
TABLE 1. Schedule for i.v. immunization of rabbits with type G botulinal toxoid and toxic culture fluid Day of injection
Quantity of toxoid or toxic culture fluid injected (ml)
Toxoid, 0.50 1 (prebleeding, 20 ml of blood) Toxoid, 1.00 4 Toxoid, 2.00 8 11 Toxoid, 3.00 Toxoid, 4.00 15 Toxoid, 5.00 19 25 (test bleeding, 20 ml of blood) Toxin, 0.25a 29 36 Toxin, 0.50 Toxin, 1.00 42 Toxin, 2.00 52 Toxin, 3.00 59 69 Toxin, 3.00 Toxin, 3.00 79 89 (animals bled and serum harvested) a Toxin, Toxic culture fluid containing 30,000 mouse
LD5o/ml. incubating at 37°C for 30 min, and injecting 0.6 ml of each mixture into each of four mice. The injected mice were observed for 4 days, and the end-point dilution of the serum (at which 50% of the mice survived) was calculated by the Reed and Muench method (8). The pooled type G antitoxin was used in cross-neutralization tests. Cross-neutralization tests. One volume of undiluted type G antitoxin was mixed with 5 volumes of botulinal toxin types A, B, C, D, E, F, and G containing 10 to 20 mouse LD5o/0.5 ml. The mixtures were incubated at 37°C for 30 min, after which 0.6 ml of each was injected into each member of a separate mouse pair. Toxin types A through F were standard toxins in 50% glycerol, which are used to determine antitoxin levels in various sera. Type A, B, C, D, E, F, and G antitoxins were tested for their ability to neutralize 10 to 20 LD5o of type G toxin. Types A through F were those antitoxins distributed by the Center for Disease Control for diagnostic use. Types A, B, C, and E contain 10 IU of toxin per ml, and types D and F contain 8 IU of toxin per ml. The 0.6 ml of toxin-antitoxin mixture injected into mice contained 0.5 ml of toxin (10 to 20 LDso) and 0.1 ml (0.8 or 1.0 IU) of antitoxin. In the case of the type G antitoxin, however, no unitage had been established. Susceptibility of monkeys, chickens, sheep, dogs, and guinea pigs. Various amounts of the crude, nonactivated type G toxin were administered to animals i.v., subcutaneously (s.c.), orally, or intragastrically (i.g.), depending on animal species and body weight. In each experiment the toxin was concurrently titrated in mice to ascertain the exact amount of toxin administered. Inoculation i.g. was performed (for monkeys, sheep, dogs, and guinea pigs) by passing a plastic catheter into the stomach, after which the toxin was
Vo.. 34, 1977
CLOSTRIDIUM BOTULINUTM TYPE G
injected by syringe through the catheter and the catheter was then flushed with 1 ml of gelatin diluent. Chickens were given the toxin orally drop by drop, followed by 1 ml of gelatin diluent in the same manner. Animals given toxin i.g. or orally were fasted for 24 h before it was administered. After toxin treatment, animals were monitored and their signs and symptoms were recorded. Some of the animals that died were necropsied, and some tissue samples were examined histologically.
type and yield of toxin in cultures. Toxin. A 4-day CMG subculture of isolate 13 (from the high toxicity group) contained 43 mouse LD5o/ml, which increased to 512 LD50/ml after trypsin treatment. Dialysis cultures of isolate 13 contained 30,000 mouse LD5o/ml after 7 days of growth. Toxin from these latter cultures was used without trypsin treatment to challenge animals. After 19 months of refrigeration, this crude toxin preparation contained 16,000 LD5o/ml, which increased to 50,800 LD6o/ml after trypsin treatment. Activation of this toxin preparation by trypsin was not determined at the time of preparation. Antitoxin. The pooled rabbit type G antitoxin had a 50% end-point dilution of 1:154 when 0.1 ml of dilutions was tested against 17 LD50 of type G toxin (nonactivated). Therefore, 1 ml of undiluted serum would neutralize 26,200 mouse LD5o of type G toxin. One-tenth milliliter of undiluted type G antitoxin, although having the capacity to neutralize approximately 2,600 mouse LD50 of type G toxin, failed to neutralize 10 to 20 mouse LD50 of type A, B, C, D, E, or F toxin. Specificity of type G toxin. One-tenth milliliter each of antitoxins A through F (0.8 or 1.0 IU) did not neutralize 17 mouse LD50 of type G toxin (nonactivated).
RESULTS Studies on strain 89 and its toxin. Characterization of isolates. The microscopic appearance of the bacterial cells of the 40 pure culture isolates and their colonial characteristics on blood agar were essentially as previously described (4). The bacilli were motile, and gram positive, and no spores were seen in cells from either solid or liquid media (Fig. 1). The composite biochemical reactions and end products produced in PYG medium are shown in Table 2. The results of the biochemical tests agree with those reported by Gimenez and Ciccarelli (4) and by Holdeman and Moore (7). Both small, usually smooth colonies and large, rough colonies were apparent on blood agar (Fig. 2), but no differences were detected in the biochemical characteristics of subcultures from the two colony types. There was no relation between colony
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a
FIG. 1. Photomicrograph of Gram-stained smear of a 48-h BA culture of C. botulinum type G. Note absence of spores.
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TABLE 2. Composite biochemical reactions and end products produced by 40 single-colony i'solates of C. botulinum type G (strain 89)" lResults
TIest
Motility Oxygen tolerance Hemolysis Lecit.hinase Lipase Chopped-meat mediumn Action in milk
H2S production Urease production Indole production Nitrate reduction Gelatin liquefaction Esculin hydrolysis Starch hydrolysis Fermentation of: Glucose Mannitol Lactose Sucrose
Obligate anaerobe + (rabbit blood)
Blackened and slightly digested (7 days) Coagulated, no digestion ++
+ (48 h)
Maltose
Salicin Glycerol Mannose Xylose Rhamnose
DISCUSSION
Arabinose
l'rehalose Volatile fatty acids end products in PYG medium
secondary bacterial infection. Chickens. The results of oral and s.c. administration of type G botulinal toxin to chickens are shown in Table 4. The most noticeable sign of type G botulism in these animals was marked muscular weakness, particularly in the legs and neck. As the illness progressed, the chickens evidenced paralysis and were unable to rise; some developed limberneck. Sheep. Two lambs were challenged i.g. with type G toxin. A 7.5-kg lamb was given 1,000 mouse LD50 of the toxin per kg of body weight, and an 8.8-kg lamb was given 75,000 mouse LD5o/kg of body weight. No signs of botulism were noted in either animal. Dogs. Two dogs (4.08 kg each), which were given 1,000 mouse LD50 and 75,000 mouse LD50 of toxin i.g. per kg, respectively, showed no signs of botulism. Guinea pigs. The susceptibility of guinea pigs to type G toxin administered i.g. and i.p. is shown in Table 5. The signs of type G botulism in these animals were very similar to those observed in mice. These included ruffled hair, difficulty in breathing, generalized weakness, and, ultimately, respiratory paralysis and death.
Acet ic Isobutyric Butyric Isovaleric
Susceptibility of animals to type G botulinal toxin. Monkeys. The results of administering type G botulinal toxin to rhesus monkeys i.g. and i.v. are summarized in Table 3. The signs of type G botulism in monkeys included marked ptosis, muscular weakness (particularly in the neck muscles), respiratory distress (shallow breathing and gasping), oral and nasal discharges, and an inability to eat and drink normally. Although the exact cause of death was difficult to determine, respiratory failure was presumed to be the major factor. The only gross abnormality noted at autopsy was the pale-white appearance of distal portions of the lower lung lobes, from which a frothy, chalky-white fluid could be expressed. The Pathology Department, Center for Disease Control, examined tissue samples from the monkeys microscopically and confirmed that pneumonia was the only significant finding. A chronic interstitial fibrosis of the lungs was attributed to the presence of lung mites. An acute bronchopneumonia was attributed to aspiration of water and food and to
The experiments described here confirm an earlier report (4) that the serological specificity of the toxin of C. botulinum type G (strain 89) is distinct from that of types A through F. The inability to ferment glucose, the negative lipase reaction, and the volatile fatty acid by-products produced in PYG broth cultures distinguish strain 89 from the other types of C. botulinum. Without the iridescent sheen or "pearly layer" caused by lipase on EYA, isolation of this organism from mixed cultures is most difficult. Although this strain is nonproteolytic (i.e., it does not digest milk), it produces isobutyric and isovaleric acids in PYG cultures which the nonproteolytic strains of types B, C, D, E, and F do not produce. It does not produce propionic acid, which is produced by proteolytic strains of types A, B, and F as well as by type C and D strains. Acetic and butyric acids are produced by all seven types of C. botulinum whether proteolytic or not (7). Signs and symptoms occurring in animals after administering type G toxin are similar to those caused by botulinal toxins, types A through F. The toxin from strain 89 is obviously a neurotoxin that affects the same nerves as are affected by previously recognized types of botulinal toxin. Studies with type G toxin have not established that its mode of action is by interfering with acetylcholine release at the myoneural junction. Although it is assumed that
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CLOSTRIDIUM BOTUJLINUJM T'Yl'E G
847
in__~FIG. 2. Colonies of C. botulinum type G on BA after 48-h anaerobic incubation at 35°C. TABLE 3. Results obtained with monkeys challenged with type G botulinal toxin i.g. and i.v. Wt (kg) (kg)outeChallenge Mo Wt dose Monkey of chal- (mouse no.
2 1 3 4 5
2.83 3.07 2.85 1.94 1.69
lne lenge
LD50/kg)
i.g. ig. i.g. ig. i.g.
200,000 100,000 100,000 33,000 11,000
Result
Died (9 days) Died (6 days) Died (5 days) Died (4 days) Surviveda
i.v. 120 Died (7 days) 8 2.86 7 3.23 i.v. 40 Surviveda 6 2.57 i.v. 13 Surviveda a Minor signs of botulism (ptosis, muscle weakness) were observed.
this is the mode of action for all of the types of botulinal toxin (9), the experimental evidence to support this was obtained primarily with type A toxin. In our experiments animals were challenged with nonactivated type G toxin. It is quite possible that toxin administered orally or i.g. was at least partially activated by enzymes in the gastrointestinal tract. If parenterally administered toxin had had the benefit of activation, the ratios between oral or i.g. and parenteral lethal doses might have been greater. Nonactivated type G toxin was also used in cross-neu-
TABLE 4. Susceptibility of White Leghorn roosters to type G botulinal toxin given orally and s.c. Dose of toxin
Chicken
(mouse
Route of
2.11 1.92 1.79 1.43 1.66 1.46 1.72 1.37 1.48
100,000 50,000 25,000 12,500 6,250 3,125 3,125 1,562 1,562
Oral Oral Oral Oral Oral Oral Oral Oral Oral
Died, 18 h Died, 24 h Died, 48 h Died, 72 h Died, 96 h Died, 168 h Died, 144 h Survived Survived
1.55 1.55
12,000 5,600 2,600 1,200 1,200
S.C.
120
S.C. S.C. S.C. S.C. S.C.
Died, 24 h Died, 96 h Died, 168 h Died, 48 h Survived Survived
120
s.c.
Survived
no.
Wt (kg)
3 2 1 9 10 11 15 12 16 6 8 7 5 13 4 14
1.37 1.56 1.85 1.66 1.67
LD5o/kg inoculation of body wt)
Result
tralization tests. Thus, a greater amount of type G toxin (perhaps proportionate to the activation factor) was in the mixtures with antitoxins when neutralization was attempted. On the other hand, the type G antitoxin neutralization power would probably have been greater (in terms of
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TABLE 5. Susceptibility ofguinea pigs to type G botulinal toxin by i.g. and i.p. challenge Dose of toxin
(mouseLD5o/ Route of kg of body
challenge
No. of animals
Mean wt (g)
Deathsl
80,000 40,000 20,000 10,000 5,000
i.g. i.g. i.g. i.g. i.g.
1 3 3 2 2
700 710 787 765 685
1/1 3/3 3/3 1/2 0/2
1,000 100 40 20 10
i.p. i.p.
1 3 3 3 3
690 680 740 717 780
1/1 3/3 0/3 0/3 0/3
i.p. i.p.
i.p.
LD5o neutralized
jected
per ml) had it been tested against activated toxin. The i.g. and i.v. lethal doses of type G toxin (33,000 and 120 mouse LDso/kg, respectively) for rhesus monkeys are quite similar to the lethal doses of type A toxin (30,000 and 40 mouse LD5o/kg, i.g. and i.v., respectively) reported by Herrero et al. (6) for this species. Chickens were quite susceptible to type G toxin (Table 4), with a ratio of oral to parenteral lethal dose of less than 3. Gross and Smith (5) found that chickens were susceptible to type A, B, C, and E toxins but were resistant to type D and F toxins. They found low ratios of oral to i.v. lethal doses of Calpha, and especially C-beta toxins for four species of gallinaceous birds. There was no indication that activated toxins were used in those studies. It has been shown that the toxin of Cbeta is activated by trypsin (3). Activation in vivo after oral challenge may be a factor in the low oral-to-parenteral lethal dose ratios for both type G and C-beta toxins. Nevertheless, if the activation factor for the type G toxin in our studies would have been as high as 30, its oral toxicity for chickens would be similar to that of the C and E toxins to which chickens are the most susceptible (5). In our limited experiments dogs and lambs appeared to be rather resistant
to ingested type G toxin. Guinea pigs are susceptible to the toxin administered i.g. as well as i.p. The relatively high susceptibilities of monkeys and chickens to orally or i.g. administered type G toxin indicate that C. botulinum type G is a potential hazard when it is present. It has been isolated a second time in Argentina (D. F. Gimenez, unpublished data). Primate susceptibility raises the question of human susceptibility. That type G botulism has not been found to occur naturally can perhaps be attributed to the low toxigenicity of the type G strains studied to date. Type G cultures produce 40 LD50/ml in media in which type A strains can produce 10,000 to 1,000,000 LD50/ml. However, we have found that under dialysis culture conditions, a type G strain can produce up to 90,000 LD50/ml, indicating that should such suitable conditions for toxin production occur naturally or in a food containing C. botulinum type G, this type of botulism could result. LITERATURE CITED 1. Ciccarelli, A. S., and D. F. Gimenez. 1972. Cryoprotein produced by Clostridium botulinum type G. Infect. Immun. 5:985-986. 2. Dowell, V. R., Jr., and T. M. Hawkins. 1973. Laboratory methods in anaerobic bacteriology, CDC laboratory manual, Publication no. (CDC) 77-8272. Department of Health, Education and Welfare, Center for Disease Control, Atlanta. 3. Eklund, M. W., and F. T. Poysky. 1972. Activation of a toxic component of Clostridium botulinum types C and D by trypsin. Appl. Microbiol. 24:108-113. 4. Gimenez, D. F., and A. S. Ciccarelli. 1970. Another type of Clostridium botulinum. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. 215:221-224. 5. Gross, W. B., and L. DS. Smith. 1971. Experimental botulism in gallinaccus birds. Avian Dis. 15:716-722. 6. Herrero, B. A., A. E. Ecklund, C. S. Streett, D. F. Ford, and J. K. King. 1967. Experimental botulism in monkeys a clinical pathological study. Exp. Mol. Pathol. 6:84-95. 7. Holdeman, L V., and W. E. C. Moore. 1972. Anaerobe laboratory manual. VPI Anaerobe Laboratory, Virginia Polytechnic Institute and State University, Blacksburg. 8. Reed, L J., and H. Muench. 1938. A simple method for estimating 50 percent endpoints. Am. J. Hyg. 27:493-497. 9. Smith, L. DS. 1977. Botulism: the organism, its toxins, the disease. Charles C Thomas, Publisher, Springfield, m.
Vol. 34, No. 6 Printed in U.S.A.
Cultural and Physiological Characteristics of Clostridium botulinum Type G and the Susceptibility of Certain Animals to Its Toxin ALBERTO S. CICCARELLI,t DAVID N. WHALEY, LORETTA M. McCROSKEY, DOMINGO F. GIMENEZ,t V. R. DOWELL, JR., AND CHARLES L. HATHEWAY* Bureau of Laboratories, Center for Disease Control, Atlanta, Georgia 30333
Received for publication 13 June 1977
Strain 89 of Clostridium botulinum type G, isolated by Gimenez and Ciccarelli in 1969, was characterized culturally, biochemically, and toxigenically. It was motile, hemolytic, asaccharolytic, weakly proteolytic, lipase and lecithinase negative, and it produced acetic, isobutyric, butyric, and isovaleric acids in peptoneyeast extract-glucose broth. No spores were seen in smears from solid or liquid media. Very low levels of toxin were produced in regular broth cultures, but dialysis cultures yielded 30,000 mouse 50% mean lethal doses (LD50) per ml. Dogs and lambs were resistant to intragastric challenges of up to 75,000 mouse LD50 per kg of body weight. Rhesus monkeys, chickens, and guinea pigs were susceptible to enteral and parenteral challenge with the toxin. These species showed signs of botulism after challenge that were similar to, if not identical with, those observed in other studies with other botulinal toxin types. The lethal doses for monkeys were 33,000 and 120 mouse LD50 per kg, intragastrically and intravenously, respectively; for chickens, 3,125 and 1,200 to 2,600 mouse LD50 per kg, orally and subcutaneously, respectively; and for guinea pigs, 10,000 to 20,000 and 100 mouse LD50 per kg, intragastrically and intraperitoneally, respectively.
Clostridium botulinum type G (strain 89) was isolated by Gimenez and Ciccarelli in 1969 from a cornfield in the Mendoza Province of Argentina (4). The isolate produced a trypsin-activatable, heat-labile, botulinal-like toxin that was not neutralized by antitoxin to any of the known botulinal toxins (types A, B, C, D, E, F). Antitoxin produced against toxoid prepared from the new toxin neutralized only its homologous toxin. The signs appearing in mice and the range of time to death after administering high and low doses of type G toxin were similar to those observed after administering botulinal toxins of types A through F. Strain 89 produced very little toxin, even in special media, and was weakly proteolytic and asaccharolytic. Further studies showed that strain 89 produces a "cryoprotein" that appears to be unrelated to toxic and hemagglutinating factors produced by the organism (1). Our report provides descriptions of additional cultural and physiological characteristics of strain 89 and of the susceptibility of various animal species to type G botulinal toxin. MATERIALS AND METHODS
peptone-yeast extract-glucose (PYG), 5% rabbit blood agar (BA), and modified McClung-Toabe egg yolk agar (EYA) were prepared as described by Dowell and Hawkins (2). The medium used for toxin production in dialysis sac cultures contained 4% proteose peptone (Oxoid, code L46)-1% yeast extract (BBL, 107638)-1% dextrose (Fisher Scientific Co., D16), with pH adjusted to 7.2 with 5 N NaOH before autoclaving. Organism. A subculture of C. botulinum type G (strain 89) was obtained from Lillian V. Holdeman of the Anaerobe Laboratory, Virginia Polytechnic Institute and State University, Blacksburg. Characterization studies. The organism was subcultured in freshly boiled CMG in a loosely capped tube and incubated for 24 h at 370C in a GasPak (BBL) anaerobic jar. It was then streaked on EYA and BA and incubated in a GasPak jar at 37°C for 48 h to obtain isolated colonies. The resulting colonies were examined macroscopically through a hand lens; 40 well-isolated colonies from both media were selected, subcultured individually in CMG, and incubated in an anaerobic glove box (2) containing a mixture of 85% N2-10% H2-5% C02 at 31°C for 5 days. Each of the 40 isolates was characterized according to methods described by Dowell and Hawkins (2). These included microscopic examinations, colonial characteristic observations, a variety of conventional biochemical tests, and metabolic product identification in PYG medium by gas-liquid chromatography. Preliminary toxicity tests. The toxicity of each of the 40 single-colony isolates in CMG medium was
Media. Chopped-meat-glucose medium (CMG), tPresent address: Universidad Nacional de Cuyo, Mendoza, Argentina.
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determined for mice as follows. After each CMG culwas centrifuged at 12,000 x g for 15 min, 0.5 ml of the supernatant fluid was injected intraperitoneally (i.p.)' into each of six 15- to 20-g Swiss white mice (ICR), and the time until death was recorded. Some supernatants killed no mice, but no attempt was made to determine whether these were atoxigenic mutants of the strain. Some killed some of the six mice within 4 days. Others killed all six mice in a mean time of less than 7 h. The culture supernatant that killed the mice in the shortest time was considered to contain the most toxin. Toxin production. A previously described procedure (4) was used to produce type G botulinal toxin with isolate 13-52, the most potent toxin producer. A section of dialysis tubing 13.2 cm wide when flat and 38 cm long) containing a few glass beads was mounted in a plasma collection bottle to which 450 ml of toxin production medium was added, and the system was autoclaved at 121°C for 30 min. Isolate 13-52 was grown in CMG for 20 h at 37°C. After the autoclaved system cooled to approximately 30°C, 50 ml of a 1:250, 0.85% saline dilution of this culture was seeded through the glass tube into the dialysis tubing, and the bottles were incubated at 30 to 31°C for 7 days in Brewer jars evacuated and filled with a mixture of 85% N2-10% H2-5% CO2. A very dense bacterial suspension developed in the dialysis tubing. The contents of the dialysis sacs were pooled in a sterile beaker. From 1 to 3 ml of sterile 0.85% saline was added to the dialysis tubing from each bottle to help remove the contents. The pooled suspensions were centrifuged at 12,000 x g for 15 min, and the recovered supernatant fluid was titrated for toxicity in mice. Serial twofold dilutions were made in gelatin diluent (2). One-half
ture
milliliter of each dilution was inoculated i.p. into each of six 20-g mice. The mice were observed for 4 days, and the deaths were recorded. The toxin content (mouse 50% lethal dose [LD5o] per ml) of the culture fluid was calculated by the Reed and Muench method
(8). activation of toxin. One volume of 1% (Difco, 1:250) solution was mixed with 9 volumes of toxin, and the mixtures were incubated at 37°C for 30 min. Serial twofold dilutions were made with samples of treated and untreated toxin, and 0.5 ml of each dilution was injected into mice. The 50% end-point dilution was calculated in each case by the Reed and Muench method (8). Immunization of rabbits. Toxoid was prepared by adding Formalin to a final concentration of 0.5% in the toxic culture fluid and incubating the mixture at 37°C until it was nontoxic for mice (0.5 ml, i.p., into each of four mice). The toxoid was stored at 4°C. Six New Zealand white rabbits (2.5 to 3.0 kg) were immunized by intravenous (i.v.) injections of toxoid and toxic culture fluid by a schedule (Table 1) that was based on results of preliminary trials. Sera from the final bleeding were pooled after an initial titration showed that their antitoxin levels were similar. Antitoxin titrations. Individual serum and the pool were titrated by making serial dilutions (10-fold for individual serum and twofold for the pool) in gelatin diluent, mixing 1 volume of the diluted serum with 5 volumes of toxin containing 20 LD5o/0.5 ml, Trypsin
aqueous trypsin
TABLE 1. Schedule for i.v. immunization of rabbits with type G botulinal toxoid and toxic culture fluid Day of injection
Quantity of toxoid or toxic culture fluid injected (ml)
Toxoid, 0.50 1 (prebleeding, 20 ml of blood) Toxoid, 1.00 4 Toxoid, 2.00 8 11 Toxoid, 3.00 Toxoid, 4.00 15 Toxoid, 5.00 19 25 (test bleeding, 20 ml of blood) Toxin, 0.25a 29 36 Toxin, 0.50 Toxin, 1.00 42 Toxin, 2.00 52 Toxin, 3.00 59 69 Toxin, 3.00 Toxin, 3.00 79 89 (animals bled and serum harvested) a Toxin, Toxic culture fluid containing 30,000 mouse
LD5o/ml. incubating at 37°C for 30 min, and injecting 0.6 ml of each mixture into each of four mice. The injected mice were observed for 4 days, and the end-point dilution of the serum (at which 50% of the mice survived) was calculated by the Reed and Muench method (8). The pooled type G antitoxin was used in cross-neutralization tests. Cross-neutralization tests. One volume of undiluted type G antitoxin was mixed with 5 volumes of botulinal toxin types A, B, C, D, E, F, and G containing 10 to 20 mouse LD5o/0.5 ml. The mixtures were incubated at 37°C for 30 min, after which 0.6 ml of each was injected into each member of a separate mouse pair. Toxin types A through F were standard toxins in 50% glycerol, which are used to determine antitoxin levels in various sera. Type A, B, C, D, E, F, and G antitoxins were tested for their ability to neutralize 10 to 20 LD5o of type G toxin. Types A through F were those antitoxins distributed by the Center for Disease Control for diagnostic use. Types A, B, C, and E contain 10 IU of toxin per ml, and types D and F contain 8 IU of toxin per ml. The 0.6 ml of toxin-antitoxin mixture injected into mice contained 0.5 ml of toxin (10 to 20 LDso) and 0.1 ml (0.8 or 1.0 IU) of antitoxin. In the case of the type G antitoxin, however, no unitage had been established. Susceptibility of monkeys, chickens, sheep, dogs, and guinea pigs. Various amounts of the crude, nonactivated type G toxin were administered to animals i.v., subcutaneously (s.c.), orally, or intragastrically (i.g.), depending on animal species and body weight. In each experiment the toxin was concurrently titrated in mice to ascertain the exact amount of toxin administered. Inoculation i.g. was performed (for monkeys, sheep, dogs, and guinea pigs) by passing a plastic catheter into the stomach, after which the toxin was
Vo.. 34, 1977
CLOSTRIDIUM BOTULINUTM TYPE G
injected by syringe through the catheter and the catheter was then flushed with 1 ml of gelatin diluent. Chickens were given the toxin orally drop by drop, followed by 1 ml of gelatin diluent in the same manner. Animals given toxin i.g. or orally were fasted for 24 h before it was administered. After toxin treatment, animals were monitored and their signs and symptoms were recorded. Some of the animals that died were necropsied, and some tissue samples were examined histologically.
type and yield of toxin in cultures. Toxin. A 4-day CMG subculture of isolate 13 (from the high toxicity group) contained 43 mouse LD5o/ml, which increased to 512 LD50/ml after trypsin treatment. Dialysis cultures of isolate 13 contained 30,000 mouse LD5o/ml after 7 days of growth. Toxin from these latter cultures was used without trypsin treatment to challenge animals. After 19 months of refrigeration, this crude toxin preparation contained 16,000 LD5o/ml, which increased to 50,800 LD6o/ml after trypsin treatment. Activation of this toxin preparation by trypsin was not determined at the time of preparation. Antitoxin. The pooled rabbit type G antitoxin had a 50% end-point dilution of 1:154 when 0.1 ml of dilutions was tested against 17 LD50 of type G toxin (nonactivated). Therefore, 1 ml of undiluted serum would neutralize 26,200 mouse LD5o of type G toxin. One-tenth milliliter of undiluted type G antitoxin, although having the capacity to neutralize approximately 2,600 mouse LD50 of type G toxin, failed to neutralize 10 to 20 mouse LD50 of type A, B, C, D, E, or F toxin. Specificity of type G toxin. One-tenth milliliter each of antitoxins A through F (0.8 or 1.0 IU) did not neutralize 17 mouse LD50 of type G toxin (nonactivated).
RESULTS Studies on strain 89 and its toxin. Characterization of isolates. The microscopic appearance of the bacterial cells of the 40 pure culture isolates and their colonial characteristics on blood agar were essentially as previously described (4). The bacilli were motile, and gram positive, and no spores were seen in cells from either solid or liquid media (Fig. 1). The composite biochemical reactions and end products produced in PYG medium are shown in Table 2. The results of the biochemical tests agree with those reported by Gimenez and Ciccarelli (4) and by Holdeman and Moore (7). Both small, usually smooth colonies and large, rough colonies were apparent on blood agar (Fig. 2), but no differences were detected in the biochemical characteristics of subcultures from the two colony types. There was no relation between colony
845
a
FIG. 1. Photomicrograph of Gram-stained smear of a 48-h BA culture of C. botulinum type G. Note absence of spores.
846
Ai,i,i.. ENVIRON. MICROBIOL.
CICCARELLI ET AL.
TABLE 2. Composite biochemical reactions and end products produced by 40 single-colony i'solates of C. botulinum type G (strain 89)" lResults
TIest
Motility Oxygen tolerance Hemolysis Lecit.hinase Lipase Chopped-meat mediumn Action in milk
H2S production Urease production Indole production Nitrate reduction Gelatin liquefaction Esculin hydrolysis Starch hydrolysis Fermentation of: Glucose Mannitol Lactose Sucrose
Obligate anaerobe + (rabbit blood)
Blackened and slightly digested (7 days) Coagulated, no digestion ++
+ (48 h)
Maltose
Salicin Glycerol Mannose Xylose Rhamnose
DISCUSSION
Arabinose
l'rehalose Volatile fatty acids end products in PYG medium
secondary bacterial infection. Chickens. The results of oral and s.c. administration of type G botulinal toxin to chickens are shown in Table 4. The most noticeable sign of type G botulism in these animals was marked muscular weakness, particularly in the legs and neck. As the illness progressed, the chickens evidenced paralysis and were unable to rise; some developed limberneck. Sheep. Two lambs were challenged i.g. with type G toxin. A 7.5-kg lamb was given 1,000 mouse LD50 of the toxin per kg of body weight, and an 8.8-kg lamb was given 75,000 mouse LD5o/kg of body weight. No signs of botulism were noted in either animal. Dogs. Two dogs (4.08 kg each), which were given 1,000 mouse LD50 and 75,000 mouse LD50 of toxin i.g. per kg, respectively, showed no signs of botulism. Guinea pigs. The susceptibility of guinea pigs to type G toxin administered i.g. and i.p. is shown in Table 5. The signs of type G botulism in these animals were very similar to those observed in mice. These included ruffled hair, difficulty in breathing, generalized weakness, and, ultimately, respiratory paralysis and death.
Acet ic Isobutyric Butyric Isovaleric
Susceptibility of animals to type G botulinal toxin. Monkeys. The results of administering type G botulinal toxin to rhesus monkeys i.g. and i.v. are summarized in Table 3. The signs of type G botulism in monkeys included marked ptosis, muscular weakness (particularly in the neck muscles), respiratory distress (shallow breathing and gasping), oral and nasal discharges, and an inability to eat and drink normally. Although the exact cause of death was difficult to determine, respiratory failure was presumed to be the major factor. The only gross abnormality noted at autopsy was the pale-white appearance of distal portions of the lower lung lobes, from which a frothy, chalky-white fluid could be expressed. The Pathology Department, Center for Disease Control, examined tissue samples from the monkeys microscopically and confirmed that pneumonia was the only significant finding. A chronic interstitial fibrosis of the lungs was attributed to the presence of lung mites. An acute bronchopneumonia was attributed to aspiration of water and food and to
The experiments described here confirm an earlier report (4) that the serological specificity of the toxin of C. botulinum type G (strain 89) is distinct from that of types A through F. The inability to ferment glucose, the negative lipase reaction, and the volatile fatty acid by-products produced in PYG broth cultures distinguish strain 89 from the other types of C. botulinum. Without the iridescent sheen or "pearly layer" caused by lipase on EYA, isolation of this organism from mixed cultures is most difficult. Although this strain is nonproteolytic (i.e., it does not digest milk), it produces isobutyric and isovaleric acids in PYG cultures which the nonproteolytic strains of types B, C, D, E, and F do not produce. It does not produce propionic acid, which is produced by proteolytic strains of types A, B, and F as well as by type C and D strains. Acetic and butyric acids are produced by all seven types of C. botulinum whether proteolytic or not (7). Signs and symptoms occurring in animals after administering type G toxin are similar to those caused by botulinal toxins, types A through F. The toxin from strain 89 is obviously a neurotoxin that affects the same nerves as are affected by previously recognized types of botulinal toxin. Studies with type G toxin have not established that its mode of action is by interfering with acetylcholine release at the myoneural junction. Although it is assumed that
VOf,. 34,197-7
CLOSTRIDIUM BOTUJLINUJM T'Yl'E G
847
in__~FIG. 2. Colonies of C. botulinum type G on BA after 48-h anaerobic incubation at 35°C. TABLE 3. Results obtained with monkeys challenged with type G botulinal toxin i.g. and i.v. Wt (kg) (kg)outeChallenge Mo Wt dose Monkey of chal- (mouse no.
2 1 3 4 5
2.83 3.07 2.85 1.94 1.69
lne lenge
LD50/kg)
i.g. ig. i.g. ig. i.g.
200,000 100,000 100,000 33,000 11,000
Result
Died (9 days) Died (6 days) Died (5 days) Died (4 days) Surviveda
i.v. 120 Died (7 days) 8 2.86 7 3.23 i.v. 40 Surviveda 6 2.57 i.v. 13 Surviveda a Minor signs of botulism (ptosis, muscle weakness) were observed.
this is the mode of action for all of the types of botulinal toxin (9), the experimental evidence to support this was obtained primarily with type A toxin. In our experiments animals were challenged with nonactivated type G toxin. It is quite possible that toxin administered orally or i.g. was at least partially activated by enzymes in the gastrointestinal tract. If parenterally administered toxin had had the benefit of activation, the ratios between oral or i.g. and parenteral lethal doses might have been greater. Nonactivated type G toxin was also used in cross-neu-
TABLE 4. Susceptibility of White Leghorn roosters to type G botulinal toxin given orally and s.c. Dose of toxin
Chicken
(mouse
Route of
2.11 1.92 1.79 1.43 1.66 1.46 1.72 1.37 1.48
100,000 50,000 25,000 12,500 6,250 3,125 3,125 1,562 1,562
Oral Oral Oral Oral Oral Oral Oral Oral Oral
Died, 18 h Died, 24 h Died, 48 h Died, 72 h Died, 96 h Died, 168 h Died, 144 h Survived Survived
1.55 1.55
12,000 5,600 2,600 1,200 1,200
S.C.
120
S.C. S.C. S.C. S.C. S.C.
Died, 24 h Died, 96 h Died, 168 h Died, 48 h Survived Survived
120
s.c.
Survived
no.
Wt (kg)
3 2 1 9 10 11 15 12 16 6 8 7 5 13 4 14
1.37 1.56 1.85 1.66 1.67
LD5o/kg inoculation of body wt)
Result
tralization tests. Thus, a greater amount of type G toxin (perhaps proportionate to the activation factor) was in the mixtures with antitoxins when neutralization was attempted. On the other hand, the type G antitoxin neutralization power would probably have been greater (in terms of
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APPI. ENVIRON. MICROBIOL.
CICCARELLI ET AL.
TABLE 5. Susceptibility ofguinea pigs to type G botulinal toxin by i.g. and i.p. challenge Dose of toxin
(mouseLD5o/ Route of kg of body
challenge
No. of animals
Mean wt (g)
Deathsl
80,000 40,000 20,000 10,000 5,000
i.g. i.g. i.g. i.g. i.g.
1 3 3 2 2
700 710 787 765 685
1/1 3/3 3/3 1/2 0/2
1,000 100 40 20 10
i.p. i.p.
1 3 3 3 3
690 680 740 717 780
1/1 3/3 0/3 0/3 0/3
i.p. i.p.
i.p.
LD5o neutralized
jected
per ml) had it been tested against activated toxin. The i.g. and i.v. lethal doses of type G toxin (33,000 and 120 mouse LDso/kg, respectively) for rhesus monkeys are quite similar to the lethal doses of type A toxin (30,000 and 40 mouse LD5o/kg, i.g. and i.v., respectively) reported by Herrero et al. (6) for this species. Chickens were quite susceptible to type G toxin (Table 4), with a ratio of oral to parenteral lethal dose of less than 3. Gross and Smith (5) found that chickens were susceptible to type A, B, C, and E toxins but were resistant to type D and F toxins. They found low ratios of oral to i.v. lethal doses of Calpha, and especially C-beta toxins for four species of gallinaceous birds. There was no indication that activated toxins were used in those studies. It has been shown that the toxin of Cbeta is activated by trypsin (3). Activation in vivo after oral challenge may be a factor in the low oral-to-parenteral lethal dose ratios for both type G and C-beta toxins. Nevertheless, if the activation factor for the type G toxin in our studies would have been as high as 30, its oral toxicity for chickens would be similar to that of the C and E toxins to which chickens are the most susceptible (5). In our limited experiments dogs and lambs appeared to be rather resistant
to ingested type G toxin. Guinea pigs are susceptible to the toxin administered i.g. as well as i.p. The relatively high susceptibilities of monkeys and chickens to orally or i.g. administered type G toxin indicate that C. botulinum type G is a potential hazard when it is present. It has been isolated a second time in Argentina (D. F. Gimenez, unpublished data). Primate susceptibility raises the question of human susceptibility. That type G botulism has not been found to occur naturally can perhaps be attributed to the low toxigenicity of the type G strains studied to date. Type G cultures produce 40 LD50/ml in media in which type A strains can produce 10,000 to 1,000,000 LD50/ml. However, we have found that under dialysis culture conditions, a type G strain can produce up to 90,000 LD50/ml, indicating that should such suitable conditions for toxin production occur naturally or in a food containing C. botulinum type G, this type of botulism could result. LITERATURE CITED 1. Ciccarelli, A. S., and D. F. Gimenez. 1972. Cryoprotein produced by Clostridium botulinum type G. Infect. Immun. 5:985-986. 2. Dowell, V. R., Jr., and T. M. Hawkins. 1973. Laboratory methods in anaerobic bacteriology, CDC laboratory manual, Publication no. (CDC) 77-8272. Department of Health, Education and Welfare, Center for Disease Control, Atlanta. 3. Eklund, M. W., and F. T. Poysky. 1972. Activation of a toxic component of Clostridium botulinum types C and D by trypsin. Appl. Microbiol. 24:108-113. 4. Gimenez, D. F., and A. S. Ciccarelli. 1970. Another type of Clostridium botulinum. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. 215:221-224. 5. Gross, W. B., and L. DS. Smith. 1971. Experimental botulism in gallinaccus birds. Avian Dis. 15:716-722. 6. Herrero, B. A., A. E. Ecklund, C. S. Streett, D. F. Ford, and J. K. King. 1967. Experimental botulism in monkeys a clinical pathological study. Exp. Mol. Pathol. 6:84-95. 7. Holdeman, L V., and W. E. C. Moore. 1972. Anaerobe laboratory manual. VPI Anaerobe Laboratory, Virginia Polytechnic Institute and State University, Blacksburg. 8. Reed, L J., and H. Muench. 1938. A simple method for estimating 50 percent endpoints. Am. J. Hyg. 27:493-497. 9. Smith, L. DS. 1977. Botulism: the organism, its toxins, the disease. Charles C Thomas, Publisher, Springfield, m.
