Vol. 19, No. 2
INFECTION AND IMMUNrrY, Feb. 1978, P. 752-754 0019-9567/78/0019-0752$02.00/0 Copyright i 1978 American Society for Microbiology
Printed in U.S.A.
Detection of Cholera Enterotoxin Activity in Suckling Hamsters TAE TAKEDA,l* YOSHIFUMI TAKEDA,1 TOSHIO MIWATANI,' AND NOBUYA OHTOMO2 Department of Bacteriology and Serology, Research Institute for Microbial Diseases,' Osaka University, Yamada-kami, Suita, Osaka, and Chemo-Sero-Therapeutic Research Institute,2 Shimizu-cho,
Kumamoto, Japan Received for publication 5 October 1977
For detection of cholera enterotoxin activity, suckling hamster, mouse, and rat models were compared. It was found that not only suckling mice but also suckling rats and hamsters were sensitive to cholera enterotoxin. Among these models, suckling hamsters were the most sensitive and gave positive results with about 1/100 the cholera toxin seen in suckling mice and rats.
The biological activity of cholera enterotoxin Station for Laboratory Animals, Osaka Univerhas been studied in several animal systems, such sity), and Syrian hamsters (Nippon Clea Co.) as infant rabbits (6), dogs (15), ligated rabbit were established in our laboratory. Suckling anileal loops (3), and rabbit skin (2). Methods to imals, 2 to 4 days old, were used throughout measure morphological changes in Chinese ham- this study. Cholera enterotoxin was purified as ster ovary cells (10) and Y-1 adrenal cells (5) previously reported (14). Purified cholera enterhave also been established. Gill (9) developed a otoxin in 0.1 ml of a buffer containing 50 mM method to measure a stimulation of adenylate tris(hydroxymethyl)aminomethane-hydrochlocyclase activity by cholera enterotoxin by using ric acid (pH 7.5), 30 mM NaN3, 1 mM disodium pigeon erythrocytes. Among these models, in- ethylenediaminetetraacetate, and 200 mM NaCl fant rabbits and dogs reflect directly the patho- was administered into the stomach of each anigenesis of cholera enterotoxin, which causes mal by using an appropriate gastric tube. About fluid secretion into intestinal lumen (7). 0.08% Evans blue dye was added to each inocuUjiiye and Kobari (18) reported that suckling lum as a marker. At the desired time interval mice were sensitive to Vibrio cholerae infection, after administration, the suckling animals were and the animals were killed after oral adminis- sacrificed by an inhalation of chloroform. After tration of the organisms. This work was ex- confirming the existence of the dye in intestinal tended by the recent work of Baselski et al. (1), lumen, the entire intestine was removed. The in which it was reported that cholera enterotoxin ratio of (weight of entire intestine)/(total body caused fluid accumulation in intestinal lumen. weight - weight of entire intestine) of each However, Giannella (8) reported that suckling animal was calculated. Ratios more than 0.095 mice were insensitive to the action of cholera were considered to be positive results. enterotoxin. The suckling mouse model has been All suckling animal models used in this study used to detect a heat-stable enterotoxin of Esch- responded to cholera enterotoxin when the anierichia coli and has been considered to be a mals were sacrificed at 17 h or more after the convenient, reproducible, and inexpensive model toxin administration (Table 1). The sensitivity (4, 8, 12). If the suckling mouse model could be of suckling hamsters was about 100 times higher an acceptable assay system for cholera entero- than that of suckling mice and rats. As low as toxin, it would be a more convenient and inex- 0.05 ,ug of the cholera enterotoxin gave positive pensive whole animal system than the others. results in suckling hamsters, whereas 5 ,ug of the The present work was initiated to examine toxin was required for positive results in suckling whether the suckling mouse model can be ap- mice and rats. Sensitivity of suckling mice and plied to detect cholera enterotoxin activity, and rats was almost the same. Moreover, suckling during the course of the study, it was found that hamsters showed positive results with a shorter not only suckling mice but also suckling rats incubation time than that observed in suckling and hamsters could be used for the same pur- mice and rats. pose. Moreover, it was found that suckling hamBaselski et al. (1) reported that a dose as low sters were about 100 times more sensitive than as 0.5 ,Ag of cholera enterotoxin gave positive suckling mice and rats. results in the suckling mouse model. On the Breeding colonies of white mice (Nippon Clea other hand, our results showed that at least 5 Co., Osaka), Sprague-Dawley rats (Breeding ,ug of the toxin was necessary to give positive 752
VOiL. 19, 1978
NOTES
753
TABLE 1. Effect of cholera enterotoxin on fluid accumulation in intestines of suckling hamsters, mice, and rats Amt of cholera enterotoxin inoculated per animal (jg)
Suckling hamsters Uninoculateda Buffere 0.025 0.05 0.1
Suckling mice Uninoculated Buffer 3 5 10
Time of incubation (h) 3
7
17
24
0.048 ± 0.004b 0.082 ± 0.009 0.086 ± 0.007 0.079 ± 0.005 0.085 ± 0.008
0.047 ± 0.005 0.076 ± 0.008 0.087 ± 0.001 0.088 ± 0.003 0.134 ± 0.013
0.039 ± 0.004 0.058 ± 0.003 0.060 ± 0.005 0.087 ± 0.015 0.102 ± 0.020
0.044 ± 0.001 0.044 ± 0.001 0.081 ± 0.015 0.102 ± 0.009 0.106 ± 0.011
0.051 ± 0.002 0.055 ± 0.003 0.052 ± 0.004 0.063 ± 0.005 0.065 ± 0.004
0.051 ± 0.005 0.052 ± 0.001 0.060 ± 0.009 0.071 ± 0.006 0.081 ± 0.012
0.048 ± 0.002 0.055 ± 0.006 0.084 ± 0.023 0.078 ± 0.011 0.138 ± 0.025
0.050 ± 0.007 0.051 ± 0.001 0.085 ± 0.017 0.118 ± 0.029 0.120 ± 0.009
Suckling rats
Uninoculated
0.047 ± 0.004 0.042 ± 0.002 0.045 ± 0.004 0.048 ± 0.002 0.043 ± 0.003 0.043 ± 0.002 0.045 ± 0.001 0.046 ± 0.003 0.062 ± 0.005 0.087 ± 0.014 3 0.056 ± 0.006 0.065 ± 0.009 0.127 ± 0.012 0.070 ± 0.012 0.056 ± 0.008 0.065 ± 0.014 5 0.137 ± 0.040 0.099 ± 0.014 0.055 ±0.006 0.065 ± 0.012 10 a Animals were separated from mothers and administred nothing. b Numbers are expressed as the mean of (weight of entire intestine)/(total body weight - weight of entire intestine) ± standard deviation of five animals. Numbers in boldface represent positive results. c A buffer solution containing 50 mM tris(hydroxymethyl)aminomethane-hydrochloric acid (pH 7.5), 30 mM NaN3, 1 mM disodium ethylenediaminetetraacetate, and 200 mM NaCl was administered.
Buffer
results. This discrepancy may either be due to four, was quite reproducible as reported by Basdifferences in toxin preparation or differences elski et al. (1), and statistically reliable results in stability of the toxin in solution. Differences were obtained with five animals. Recently, Lepot and Banweli (13) reported of calculation of the ratio may also reflect the effective dose. The fluid accumulation ratio as that infant hamsters were useful for detecting reported by Baselski et al. (1) is rather low. an activity of cholera enterotoxin. They used They considered a ratio of less than 0.065 as the animals weighing about 90 to 110 g and negative and more than 0.075 as positive. On inoculated more than 75 ,ug of the cholera enterthe other hand, we considered a ratio of more otoxin in 1.5 ml of a buffer solution to get posithan 0.095 as positive, since in some cases, es- tive results. Suckling hamsters gave positive repecially in suckling hamsters, a high ratio of sults with about 1/2,500 the toxin required by around 0.085 was seen at an early incubation infant hamsters (Table 1). The suckling hamster model reported in this time. This is probably due to differences in weighing the guts. Baselski et al. (1) weighed paper is sensitive, reproducible, and convenient both the stomach and entire intestine, whereas for detecting cholera enterotoxin activity, and thus may be applicable for studying the pathowe weighed only the entire intestine, as has been applied for E. coli heat-stable enterotoxin (4). genesis of cholera enterotoxin and V. cholerae Also, we noticed that in suckling hamsters, ab- infection. Also, an application of this model to sorption of the administered solution was slower other enterotoxins, such as E. coli heat-labile than that in suckling mice and rats, which was enterotoxin, V. parahaemolyticus toxin (11), shown when a control solution (a buffer) was Salnonella typhinurium toxin (16), and Shigella dysenteriae toxin (17) may give interesting administered (Table 1). results. in was administration oral applied Although this study, direct administration of the toxin LITERATURE CiTED into the stomach of the animals by use of a 1. Baselski, V., R. Briggs, and C. Parker. 1977. Intestinal needle and syringe was also applicable. Oral fluid accumulation induced by oral challenge with administration, however, was more precise, since Vibrio cholerae or cholera toxin in infant mice. Infect. in some cases direct injection of the toxin soluImmun. 15:704-712. tion would cause leakage. Weighing animals in2. Craig, J. P. 1965. A permeability factor (toxin) found in cholera stools and culture filtrates and its neutralization dividually, rather than in groups of three or
754
3. 4.
5.
6. 7.
8. 9. 10.
11.
INFECT. IMMUN.
NOTES
by convalescent cholera sera. Nature (London) 207:614-616. De, S. N. 1959. Enterotoxicity of bacteria-free culture filtrate of Vibrio chokrae. Nature (London) 183:1533-1534. Dean, A. G., Y.-C. Ching, R. Q. Williams, and L. B. Harden. 1972. Test for Escherichia coli enterotoxin using infant mice: application in a study of diarrhea in children in Honolulu. J. Infect. Dis. 125:407-411. Donta, S. T., H. W. Moon, and S. C. Whipp. 1974. Detection of heat-labile Escherichia coli enterotoxin with the use of adrenal cells in tissue culture. Science 183:334-336. Dutta, N. K., M. V. Panse, and D. R. Ku}ikarn. 1959. Role of cholera toxin in experimental cholera. J. Bacteriol. 78:594-595. Finkelstein, R. A. 1973. Cholera. CRC Crit. Rev. Microbiol. 2:553-623. Giannella, R. A. 1976. Suckling mouse model for detection of heat-stable Escherichia coli enterotoxin: characteristics of the model. Infect. Immun. 14:95-99. Gill, M. 1975. The involvement of nicotinamide adenine dinucleotide in the action of cholera toxin in vitro. Proc. Natl. Acad. Sci. U.S.A. 72:2064-2068. Guerrant, R. L, L. L Brunton, T. C. Schnaitman, L I. Rebhun, and A. G. Gilman. 1974. Cyclic adenosine monophosphate and alteration of Chinese hamster ovary cell morphology: a rapid, sensitive in vitro assay for the enterotoxins of Vibrio cholerae and Escherichia coli. Infect. Immun. 10:320-327. Honda, T., IL Shimizu, Y. Takeda, and T. Miwatani. 1976. Isolation of a factor causing morphological
12. 13.
14.
15. 16. 17.
18.
changes of Chinese hamster ovary cells from the culture filtrate of Vibrio parahaemolyticus. Infect. Immun. 14:1028-1033. Jacks, T. M., and B. J. Wu. 1974. Biochemical properties of Escherichia coli low-molecular-weight, heat-stable enterotoxin. Infect. Immun. 9:342-347. Lepot, A., and J. G. Banweil. 1976. The Syrian hamster: a reproducible model for studying changes in intestinal fluid secretion in response to enterotoxin challenge. Infect. Immun. 14:1167-1171. Ohtomo, N., T. Muraoka, H. Inoue, HL Sasaoka, and H. Takahashi. 1974. Preparation of cholera toxin and immunization studies with cholera toxoid, p. 132-142. In Proceedings of the 9th Joint Conference of U.S.Japan Cooperation in Medical Science Progress, Cholera Panel. National Institutes of Health, Bethesda, Md. Sack, R. B., and C. C. J. Carpenter. 1969. Experimental canine cholera. II. Production by cell free culture filtrates of Vibrio chokrae. J. Infect. Dis. 119:150-157. Sandefur, P. D., and J. W. Peterson. 1976. Isolation of skin permeability factors from culture filtrates of SabnoneUa typhimurium. Infect. Immun. 14:671-679. Takeda, Y., K. Okamoto, and T. Miwatani. 1977. Toxin from the culture filtrate of ShigeUa dysenteriae that causes morphological changes in Chinese hamster ovary cells and is distinct from the neurotoxin. Infect. Immun. 18:546-548. Ujiiye, A., and K. Kobari. 1970. Protective effect on infections with Vibrio cholerae in suckling mice caused by the passive immunization with milk of immune mothers. J. Infect. Dis. 121:s50-s55.
INFECTION AND IMMUNrrY, Feb. 1978, P. 752-754 0019-9567/78/0019-0752$02.00/0 Copyright i 1978 American Society for Microbiology
Printed in U.S.A.
Detection of Cholera Enterotoxin Activity in Suckling Hamsters TAE TAKEDA,l* YOSHIFUMI TAKEDA,1 TOSHIO MIWATANI,' AND NOBUYA OHTOMO2 Department of Bacteriology and Serology, Research Institute for Microbial Diseases,' Osaka University, Yamada-kami, Suita, Osaka, and Chemo-Sero-Therapeutic Research Institute,2 Shimizu-cho,
Kumamoto, Japan Received for publication 5 October 1977
For detection of cholera enterotoxin activity, suckling hamster, mouse, and rat models were compared. It was found that not only suckling mice but also suckling rats and hamsters were sensitive to cholera enterotoxin. Among these models, suckling hamsters were the most sensitive and gave positive results with about 1/100 the cholera toxin seen in suckling mice and rats.
The biological activity of cholera enterotoxin Station for Laboratory Animals, Osaka Univerhas been studied in several animal systems, such sity), and Syrian hamsters (Nippon Clea Co.) as infant rabbits (6), dogs (15), ligated rabbit were established in our laboratory. Suckling anileal loops (3), and rabbit skin (2). Methods to imals, 2 to 4 days old, were used throughout measure morphological changes in Chinese ham- this study. Cholera enterotoxin was purified as ster ovary cells (10) and Y-1 adrenal cells (5) previously reported (14). Purified cholera enterhave also been established. Gill (9) developed a otoxin in 0.1 ml of a buffer containing 50 mM method to measure a stimulation of adenylate tris(hydroxymethyl)aminomethane-hydrochlocyclase activity by cholera enterotoxin by using ric acid (pH 7.5), 30 mM NaN3, 1 mM disodium pigeon erythrocytes. Among these models, in- ethylenediaminetetraacetate, and 200 mM NaCl fant rabbits and dogs reflect directly the patho- was administered into the stomach of each anigenesis of cholera enterotoxin, which causes mal by using an appropriate gastric tube. About fluid secretion into intestinal lumen (7). 0.08% Evans blue dye was added to each inocuUjiiye and Kobari (18) reported that suckling lum as a marker. At the desired time interval mice were sensitive to Vibrio cholerae infection, after administration, the suckling animals were and the animals were killed after oral adminis- sacrificed by an inhalation of chloroform. After tration of the organisms. This work was ex- confirming the existence of the dye in intestinal tended by the recent work of Baselski et al. (1), lumen, the entire intestine was removed. The in which it was reported that cholera enterotoxin ratio of (weight of entire intestine)/(total body caused fluid accumulation in intestinal lumen. weight - weight of entire intestine) of each However, Giannella (8) reported that suckling animal was calculated. Ratios more than 0.095 mice were insensitive to the action of cholera were considered to be positive results. enterotoxin. The suckling mouse model has been All suckling animal models used in this study used to detect a heat-stable enterotoxin of Esch- responded to cholera enterotoxin when the anierichia coli and has been considered to be a mals were sacrificed at 17 h or more after the convenient, reproducible, and inexpensive model toxin administration (Table 1). The sensitivity (4, 8, 12). If the suckling mouse model could be of suckling hamsters was about 100 times higher an acceptable assay system for cholera entero- than that of suckling mice and rats. As low as toxin, it would be a more convenient and inex- 0.05 ,ug of the cholera enterotoxin gave positive pensive whole animal system than the others. results in suckling hamsters, whereas 5 ,ug of the The present work was initiated to examine toxin was required for positive results in suckling whether the suckling mouse model can be ap- mice and rats. Sensitivity of suckling mice and plied to detect cholera enterotoxin activity, and rats was almost the same. Moreover, suckling during the course of the study, it was found that hamsters showed positive results with a shorter not only suckling mice but also suckling rats incubation time than that observed in suckling and hamsters could be used for the same pur- mice and rats. pose. Moreover, it was found that suckling hamBaselski et al. (1) reported that a dose as low sters were about 100 times more sensitive than as 0.5 ,Ag of cholera enterotoxin gave positive suckling mice and rats. results in the suckling mouse model. On the Breeding colonies of white mice (Nippon Clea other hand, our results showed that at least 5 Co., Osaka), Sprague-Dawley rats (Breeding ,ug of the toxin was necessary to give positive 752
VOiL. 19, 1978
NOTES
753
TABLE 1. Effect of cholera enterotoxin on fluid accumulation in intestines of suckling hamsters, mice, and rats Amt of cholera enterotoxin inoculated per animal (jg)
Suckling hamsters Uninoculateda Buffere 0.025 0.05 0.1
Suckling mice Uninoculated Buffer 3 5 10
Time of incubation (h) 3
7
17
24
0.048 ± 0.004b 0.082 ± 0.009 0.086 ± 0.007 0.079 ± 0.005 0.085 ± 0.008
0.047 ± 0.005 0.076 ± 0.008 0.087 ± 0.001 0.088 ± 0.003 0.134 ± 0.013
0.039 ± 0.004 0.058 ± 0.003 0.060 ± 0.005 0.087 ± 0.015 0.102 ± 0.020
0.044 ± 0.001 0.044 ± 0.001 0.081 ± 0.015 0.102 ± 0.009 0.106 ± 0.011
0.051 ± 0.002 0.055 ± 0.003 0.052 ± 0.004 0.063 ± 0.005 0.065 ± 0.004
0.051 ± 0.005 0.052 ± 0.001 0.060 ± 0.009 0.071 ± 0.006 0.081 ± 0.012
0.048 ± 0.002 0.055 ± 0.006 0.084 ± 0.023 0.078 ± 0.011 0.138 ± 0.025
0.050 ± 0.007 0.051 ± 0.001 0.085 ± 0.017 0.118 ± 0.029 0.120 ± 0.009
Suckling rats
Uninoculated
0.047 ± 0.004 0.042 ± 0.002 0.045 ± 0.004 0.048 ± 0.002 0.043 ± 0.003 0.043 ± 0.002 0.045 ± 0.001 0.046 ± 0.003 0.062 ± 0.005 0.087 ± 0.014 3 0.056 ± 0.006 0.065 ± 0.009 0.127 ± 0.012 0.070 ± 0.012 0.056 ± 0.008 0.065 ± 0.014 5 0.137 ± 0.040 0.099 ± 0.014 0.055 ±0.006 0.065 ± 0.012 10 a Animals were separated from mothers and administred nothing. b Numbers are expressed as the mean of (weight of entire intestine)/(total body weight - weight of entire intestine) ± standard deviation of five animals. Numbers in boldface represent positive results. c A buffer solution containing 50 mM tris(hydroxymethyl)aminomethane-hydrochloric acid (pH 7.5), 30 mM NaN3, 1 mM disodium ethylenediaminetetraacetate, and 200 mM NaCl was administered.
Buffer
results. This discrepancy may either be due to four, was quite reproducible as reported by Basdifferences in toxin preparation or differences elski et al. (1), and statistically reliable results in stability of the toxin in solution. Differences were obtained with five animals. Recently, Lepot and Banweli (13) reported of calculation of the ratio may also reflect the effective dose. The fluid accumulation ratio as that infant hamsters were useful for detecting reported by Baselski et al. (1) is rather low. an activity of cholera enterotoxin. They used They considered a ratio of less than 0.065 as the animals weighing about 90 to 110 g and negative and more than 0.075 as positive. On inoculated more than 75 ,ug of the cholera enterthe other hand, we considered a ratio of more otoxin in 1.5 ml of a buffer solution to get posithan 0.095 as positive, since in some cases, es- tive results. Suckling hamsters gave positive repecially in suckling hamsters, a high ratio of sults with about 1/2,500 the toxin required by around 0.085 was seen at an early incubation infant hamsters (Table 1). The suckling hamster model reported in this time. This is probably due to differences in weighing the guts. Baselski et al. (1) weighed paper is sensitive, reproducible, and convenient both the stomach and entire intestine, whereas for detecting cholera enterotoxin activity, and thus may be applicable for studying the pathowe weighed only the entire intestine, as has been applied for E. coli heat-stable enterotoxin (4). genesis of cholera enterotoxin and V. cholerae Also, we noticed that in suckling hamsters, ab- infection. Also, an application of this model to sorption of the administered solution was slower other enterotoxins, such as E. coli heat-labile than that in suckling mice and rats, which was enterotoxin, V. parahaemolyticus toxin (11), shown when a control solution (a buffer) was Salnonella typhinurium toxin (16), and Shigella dysenteriae toxin (17) may give interesting administered (Table 1). results. in was administration oral applied Although this study, direct administration of the toxin LITERATURE CiTED into the stomach of the animals by use of a 1. Baselski, V., R. Briggs, and C. Parker. 1977. Intestinal needle and syringe was also applicable. Oral fluid accumulation induced by oral challenge with administration, however, was more precise, since Vibrio cholerae or cholera toxin in infant mice. Infect. in some cases direct injection of the toxin soluImmun. 15:704-712. tion would cause leakage. Weighing animals in2. Craig, J. P. 1965. A permeability factor (toxin) found in cholera stools and culture filtrates and its neutralization dividually, rather than in groups of three or
754
3. 4.
5.
6. 7.
8. 9. 10.
11.
INFECT. IMMUN.
NOTES
by convalescent cholera sera. Nature (London) 207:614-616. De, S. N. 1959. Enterotoxicity of bacteria-free culture filtrate of Vibrio chokrae. Nature (London) 183:1533-1534. Dean, A. G., Y.-C. Ching, R. Q. Williams, and L. B. Harden. 1972. Test for Escherichia coli enterotoxin using infant mice: application in a study of diarrhea in children in Honolulu. J. Infect. Dis. 125:407-411. Donta, S. T., H. W. Moon, and S. C. Whipp. 1974. Detection of heat-labile Escherichia coli enterotoxin with the use of adrenal cells in tissue culture. Science 183:334-336. Dutta, N. K., M. V. Panse, and D. R. Ku}ikarn. 1959. Role of cholera toxin in experimental cholera. J. Bacteriol. 78:594-595. Finkelstein, R. A. 1973. Cholera. CRC Crit. Rev. Microbiol. 2:553-623. Giannella, R. A. 1976. Suckling mouse model for detection of heat-stable Escherichia coli enterotoxin: characteristics of the model. Infect. Immun. 14:95-99. Gill, M. 1975. The involvement of nicotinamide adenine dinucleotide in the action of cholera toxin in vitro. Proc. Natl. Acad. Sci. U.S.A. 72:2064-2068. Guerrant, R. L, L. L Brunton, T. C. Schnaitman, L I. Rebhun, and A. G. Gilman. 1974. Cyclic adenosine monophosphate and alteration of Chinese hamster ovary cell morphology: a rapid, sensitive in vitro assay for the enterotoxins of Vibrio cholerae and Escherichia coli. Infect. Immun. 10:320-327. Honda, T., IL Shimizu, Y. Takeda, and T. Miwatani. 1976. Isolation of a factor causing morphological
12. 13.
14.
15. 16. 17.
18.
changes of Chinese hamster ovary cells from the culture filtrate of Vibrio parahaemolyticus. Infect. Immun. 14:1028-1033. Jacks, T. M., and B. J. Wu. 1974. Biochemical properties of Escherichia coli low-molecular-weight, heat-stable enterotoxin. Infect. Immun. 9:342-347. Lepot, A., and J. G. Banweil. 1976. The Syrian hamster: a reproducible model for studying changes in intestinal fluid secretion in response to enterotoxin challenge. Infect. Immun. 14:1167-1171. Ohtomo, N., T. Muraoka, H. Inoue, HL Sasaoka, and H. Takahashi. 1974. Preparation of cholera toxin and immunization studies with cholera toxoid, p. 132-142. In Proceedings of the 9th Joint Conference of U.S.Japan Cooperation in Medical Science Progress, Cholera Panel. National Institutes of Health, Bethesda, Md. Sack, R. B., and C. C. J. Carpenter. 1969. Experimental canine cholera. II. Production by cell free culture filtrates of Vibrio chokrae. J. Infect. Dis. 119:150-157. Sandefur, P. D., and J. W. Peterson. 1976. Isolation of skin permeability factors from culture filtrates of SabnoneUa typhimurium. Infect. Immun. 14:671-679. Takeda, Y., K. Okamoto, and T. Miwatani. 1977. Toxin from the culture filtrate of ShigeUa dysenteriae that causes morphological changes in Chinese hamster ovary cells and is distinct from the neurotoxin. Infect. Immun. 18:546-548. Ujiiye, A., and K. Kobari. 1970. Protective effect on infections with Vibrio cholerae in suckling mice caused by the passive immunization with milk of immune mothers. J. Infect. Dis. 121:s50-s55.
