Vol. 16, No. 2 Printed in U.S.A.
INFECTION AND IMMUNITY, May 1977, p. 617-622 Copyright ©) 1977 American Society for Microbiology
Assay of Escherichia coli Heat-Labile Enterotoxin with Vero Cells J. I. SPEIRS,* S. STAVRIC, AND J. KONOWALCHUK Bureau of Microbial Hazards, Food Directorate, Health Protection Branch, Health and Welfare Canada, Ottawa KiA OL2, Canada
Received for publication 16 November 1976
The continuous cell line of African green monkey kidney, Vero, showed characteristic morphological changes in response to culture filtrates from toxigenic strains of Escherichia coli. The response compared favorably with that of Y-1 (mouse adrenal) and CHO (Chinese hamster ovary) cells. Vero cells were the simplest and most economical to maintain in the laboratory. Escherichia coli heat-labile enterotoxin (LT) Earle salts (Connaught Laboratories Ltd.); all meacts on certain cell cultures to induce measura- dia were supplemented with 10% fetal calf serum ble biochemical changes, e.g., increased aden- (Grand Island Biological Co.); the mixtures are reto as growth media. (FlO medium suppleylate cyclase activity (6, 10, 13, 22), increased ferred mented with 15% horse serum and 2.5% fetal calf intracellular levels of cyclic adenosine 3',5'- serum been used previously for the culture of Ymonophosphate (9, 22), and induced synthesis 1 cells has [2]. In our laboratory, cell growth was better of A4,3-keto-steroids (1, 2, 4). With Y-1 (mouse in the presence of fetal calf serum than in horse adrenal) and CHO (Chinese hamster ovary) serum; 10% was as satisfactory as 15 or 20%.) The cells, these changes are accompanied by altera- requirements for maintenance were as follows: Y-1, tions in cell morphology and thus provide a weekly passage and an additional weekly change of growth medium; CHO, twice-weekly passages but simple diagnostic tool (3, 9, 11, 15, 18, 19). The continuous cell line Vero (African green no additional change of medium; and Vero, passages to 3 weeks or every 3 days, as required, monkey kidney) is also morphologically affected extending but no additional change of medium. by LT and shows advantages over other assay Enterotoxin activity was assayed in plastic dishes methods. The study reported here compares the with 24 16-mm-diameter wells (Falcon 3008), using responses of Y-1 (2), CHO (9), and Vero cells to 0.5 ml of cell culture in each well. The normal procethe culture filtrates of various strains of E. coli. dure was to use confluent monolayers of Y-1 and MATERIALS AND METHODS
Enterotoxin production. E. coli enterotoxigenic human or porcine strains H10407, B2C, and B7A were obtained from H. L. DuPont (U.S.A.), and P155, P307, 339, and 711 (P307) were from C. Gyles (Canada). Nontoxigenic strains 711 and K-12 were obtained from C. Gyles and V. N. Iyer (Canada), respectively. Erlenmeyer flasks, 150 ml, containing 20 ml of Trypticase soy broth (TSB) or Evans medium (8) were inoculated and mechanically shaken at 37°C. After 16 to 20 h, the cultures were centrifuged at 17,000 x g for 30 min. The supernatants were filtered through 0.45-,um membrane filters (Millipore Corp.) and stored at 4°C until assay that same day. Filtrate dilutions were made in phosphate-buffered saline (PBS), pH 7.0. Cell culture assay. Stocks of the continuous cell lines Y-1, CHO, and Vero, purchased from the American Type Culture Collection, Rockville, Md., were passaged by trypsinization and grown as monolayers at 36°C in a 5% CO2 atmosphere. Y-1 cells were grown in Ham nutrient mixture F10 (Connaught Laboratories Ltd.), CHO cells were grown in Ham nutrient mixture F12 (Grand Island Biological Co.), and Vero cells were grown in medium 199 with
Vero cells but freshly seeded CHO cells. Y-1 monolayers were obtained by seeding 105 cells per well 4 days before use; growth medium was changed 24 h before and at the time of assay. For Vero monolayers, 105 cells were seeded 1 to 2 days before use; growth medium was replaced with PBS at the time of assay. CHO cells, 104 in medium without serum, were seeded at the time of assay. To 0.5 ml of cell culture, 0.05 ml of bacterial filtrate was added. For controls, cultures received PBS or the appropriate bacterial culture medium. Cultures were incubated at 36°C for 18 to 24 h. Morphological effects were recorded as 1, 2, 3, or 4, corresponding to roughly .25, 50, 75, or >:90 cells affected, respectively. In comparative studies, variations in the above procedure were as follows: at the time of assay, Vero monolayers received growth medium instead of PBS; 3 x 105 cells of Vero in growth medium or PBS were seeded at the time of assay; 3 x 103, 5 x 104, or 3 x 105 cells of CHO in medium without serum or 104 cells in medium containing 0.5, 1, 5, or 10% serum were seeded at the time of assay; Y-1 monolayers received F10 medium supplemented with 0, 5, 10, 15, or 20% serum at the time of assay (monolayers were first rinsed twice with the appropriate medium). Rabbit ileal-loop assay. Ileal-loop assay was performed in duplicate or triplicate in 9-week-old rab617
618
INFECT. IMMUN.
SPEIRS, STAVRIC, AND KONOWALCHUK
bits by the method of Kasai and Burrows (14). Two milliliters of filtrate was used for each 10-cm loop; after 18 h, the fluid accumulation was measured and expressed as milliliters of fluid per centimeter of gut. A ratio of 1.0 or more was considered positive. Fractionation of bacterial filtrates. A pool of 175 ml of bacterial filtrate of strain H10407, grown in Evans medium, was fractionated by filtration, in series, through 76-mm Diaflo membranes XM300, XM100A, PM30, and UM10 (Amicon Corp., Lexington, Mass.). These membranes are designed to restrict macromolecules in excess of molecular weight 300,000, 100,000, 30,000, and 10,000, respectively. Ten milliliters of filtrate from each membrane was retained for assay; the remaining fluid was filtered through the next grade of membrane. Effect of endotoxin on cell cultures. Commercial lyophilized lipopolysaccharide B (endotoxin) from E. coli 026:B6 (Difco) was dissolved in distilled water to a concentration of 20 mg/ml. A 0.05-ml volume of this solution or of three successive fourfold dilutions of it were added in duplicate to 0.5 ml of freshly seeded cells of Y-1 or Vero in growth medium or of CHO cells in growth medium or medium without serum. Cultures were examined at 24, 48, and 72 h for visual changes.
RESULTS In preliminary experiments, Vero cells showed a morphological response to filtrates of E. coli H10407. Response occurred in the presence of growth medium or PBS with either established monolayers or freshly seeded cells. The most-defined effect was observed when the growth medium of confluent monolayers was replaced with PBS just prior to the addition of bacterial filtrates; the affected cells appeared enlarged, thick-walled, and refractile, with several filamentous tendrils (Fig. 1). This method of assay was used for further studies. When cells, freshly seeded in PBS, were treated with toxigenic filtrates, the morphological changes resembled those of CHO cells (9) (see Fig. 3); i.e., control cells were round whereas treated cells were flattened and elongated (Fig. 2). Vero, Y-1, and CHO cells responded to filtrates of various known LT-producing E. coli strains but not to nontoxigenic strains (Table 1). Some variation from experiment to experiment occurred in the optimum response with all three cell types (Table 2). Undiluted filtrates of strain H10407, grown in two different media, elicited response in 50 to 100% of the Y1, CHO, or Vero cells within 18 h; the mostfrequent response in Y-1 cells was 100% and that in CHO or Vero cells was 75%. No increase in the degree of effect was discernible after 18 h. The first evidence of response to H10407 filtrates was noted after 2 h with Y-1 and Vero cells and after 4 h with CHO cells. Near-maxi-
~
b~~~~~
~
T
.~~~~~~~~/* I
m
o
woa F ~~~~~~~~~~~.
an TSB
AOnrl B iued11 nPS 13 ' Ab
o3 as ih eo el. No CHO cels an£p FIG. 1. Vero monolayers incubated for 24 h in a filtrate of E. coli Hi 0407 grown in TSB. Filtrate (A) treated and TSBwihedtxnitafnlcocnrto (control) (B) diluted 1:10 in PBS. x133. 4
of 0.002 to 0.1 mg/ml.1 mum response was noted at 4 h with Y-1 and Vero cells, and cellular changes persisted at least 5 days with Y-1 cells, up to 2 days with CHO cells, and up to 3 days with Vero cells. No cellular changes were seen in any of these cells treated with endotoxin at a final concentration of 0. 002 to 0. 1 mg/ml. Two media were compared for production of LT by assaying serial dilutions of several H10407 filtrates (Table 3). TSB was inferior to Evans medium. Compared with that of Y-1 cells, the titer for CHO and Vero cells was greater but varied with the filtrate preparation. Heating these filtrates at 980C for 15 mi negated activity in each cell type; heating at 650C for 15 min reduced activity by 75%. Filtrates of three LT-producing E. coli strains, grown in Evans medium, were titrated by the methods of cell cultures and rabbit ileal loops. The end points in Vero cells were 16 times greater than those in Y-1 cells but four times less than those in CHO cells (Table 4). In rabbit ileal loops, undiluted filtrates from all strains invoked fluid accumulation, but the response to filtrates diluted 1:4 was inconsistent.
ASSAY OF E. COLI HEAT-LABILE ENTEROTOXIN
VOL. 16, 1977
To determine whether the high titer in CHO cells was due to the comparatively low concentration of cells used in the test (104/well), cell numbers were adjusted to 3 x 103, 1 x 104, 5 x
619
TABLE 2. Morphological response of cell cultures to different filtrates of E. coli H10407 grown in two different mediaa Medium
TSB TSB TSB TSB TSB Evans Evans Evans
Y-1
CHO
Vero
4b 4 4 4 2 4 4 3
3 2 3 3 3 3 3 2
2 3 2 4 3 4 3 3
a 2, ca. 50%; 3, ca. 75%; and 4, .90% cells affected. Readings were recorded 18 h postincubation. bDuplicate or triplicate cultures yielded similar results.
TABLE 3. Titration in cell culture of filtrates of E. coli H10407 grown in two media Medium
as
O
.0
t
.s e
0
&
V0.
4
_za 6
-W
0
*.D v
*"
0 0
TSB TSB Evans 16 1,024 256 Evans 16 1,024 256 Evans 16 64 64 Evans 16 256 64 a Duplicate or triplicate cultures yielded similar results. Readings were recorded 24 h postincubation. TABLE 4. Titration in cell culture of filtrates of toxigenic E. coli strains grown in Evans mediuma Strain
FIG. 2. Freshly seeded Vero cells, in PBS, incubated for 24 h in a filtrate of E. coli H10407 grown in TSB (A) or, for a control, TSB (B). Each was diluted 1:10 in PBS. x133. TABLE 1. Morphological response of three cell lines to filtrates of toxigenic and nontoxigenic E. coli strains grown in TSBa Strain
Toxigen-
Y-1
CHO
Vero
H10407 339 P155 P307 711 (P307) B7A B2C
+ + + + + + +
4b 4 4 4 3 3 4
2 2 2 3 2 1 1
3 3 3 3 3 2 3
711
-
-
-
-
K-12
-
-
-
-
icity
a -, No response; 1, 300,000, our separation by membrane filtration indicated that the active components were smaller. Mo-
ASSAY OF E. COLI HEAT-LABILE ENTEROTOXIN
VOL. 16, 1977
621
lecular weights of 20,000 (7), 100,000 (5, 17), 200,000 (20), and >300,000 (12, 16) have been reported for LT. .f .I a,
VIz~~~~~~I
ACKNOWLEDGMENT
.
S1,,
j
i
%
We are grateful to Deborah Jeffrey for competent technical assistance.
\
LITERATURE CITED
XCb ,,
to, 4
AP
_11
._
X. '- 1.4
16 a,,
FIG. 4. Freshly seeded CHO cells, in medium F12 with 10% serum, incubated for 4 days in a filtrate of E. coli H10407 grown in TSB (A) or, for a control, TSB (B). Each was diluted 1:10 in PBS. x133.
OJ CONTROL (NO ULTRAFILTRATION) n11 XM300 XM IOOA PM30 1/64 z
0
1/16 z 0
t31/4
Y-I CHO VERO RL FIG. 5. Titration of E. coli H10407 ultrafiltrates in cell lines and rabbit ileal loops. Readings were recorded 24 h postinoculation for the former and 18 h for the latter.
1. Donta, S. T. 1974. Differentiation between the steroidogenic effects of cholera enterotoxin and adrenocorticotropin through use of a mutant adrenal cell line. J. Infect. Dis. 129:728-731. 2. 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. 3. Donta, S. T., D. A. Sack, R. B. Wallace, H. L. DuPont, and R. B. Sack. 1974. Tissue-culture assay of antibodies to heat-labile Escherichia coli enterotoxins. N. Engl. J. Med. 291:117-121. 4. Donta, S. T., and D. M. Smith. 1974. Stimulation of steroidogenesis in tissue culture by enterotoxigenic Escherichia coli and its neutralization by specific antiserum. Infect. Immun. 9:500-505. 5. Dorner, F. 1975. Escherichia coli enterotoxin purification and partial characterization. J. Biol. Chem. 250:8712-8719. 6. Dorner, F., and P. Mayer. 1975. Escherichia coli enterotoxin: stimulation of adenylate cyclase in broken-cell preparations. Infect. Immun. 11:429-435. 7. Evans, D. J., Jr., D. G. Evans, and S. L. Gorbach. 1974. Polymyxin B-induced release of low-molecularweight, heat-labile enterotoxin from Escherichia coli. Infect. Immun. 10:1010-1017. 8. Evans, D. G., D. J. Evans, Jr., and N. F. Pierce. 1973. Differences in the response of rabbit small intestine to heat-labile and heat-stable enterotoxins of Escherichia coli. Infect. Immun. 7:873-880. 9. 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. 10. Hewlett, E. L., R. L. Guerrant, D. J. Evans, Jr., and W. B. Greenough, III. 1974. Toxins of Vibrio cholerae and Escherichia coli stimulate adenyl cyclase in rat fat cells. Nature (London) 249:371-373. 11. Isaacson, R. E., and H. W. Moon 1975. Induction of heat-labile enterotoxin synthesis in enterotoxigenic Escherichia coli by mitomycin C. Infect. Immun. 12:1271-1275. 12. Jacks, T. M., B. J. Wu, A. C. Braemer, and D. E. Bidlack. 1973. Properties of the enterotoxic component in Escherichia coli enteropathogenic for swine. Infect. Immun. 7:178-189. 13. Kantor, H. S., P. Tao, and C. Wisdom. 1974. Action of Escherichia coli enterotoxin: adenylate cyclase behavior of intestinal epithelial cells in culture. Infect. Immun. 9:1003-1010. 14. Kasai, G. J., and W. Burrows. 1966. The titration of cholera toxin and antitoxin in the rabbit ileal loop. J. Infect. Dis. 116:606-614. 15. Kwan, C. N., and R. M. Wishnow. 1974. Escherichia coli enterotoxin-induced steroidogenesis in cultured adrenal tumor cells. Infect. Immnun. 10:146-151. 16. Lariviere, S., C. L. Gyles, and D. A. Barnum. 1973. Preliminary characterization ofthe heat-labile enterotoxin of Escherichia coli F11(P155). J. Infect. Dis. 128:312-320.
622
SPEIRS, STAVRIC, AND KONOWALCHUK
17. Mollby, R., S. G. Hjalmarsson, and T. Wadstrom. 1975. Separation of E. coli heat-labile enterotoxin by preparative isotachophoresis. FEBS Lett. 56:30-33. 18. Sack, D. A., and R. B. Sack. 1975. Test for enterotoxigenic Escherichia coli using Y1 adrenal cells in miniculture. Infect. Immun. 11:334-336. 19. Sack, R. B., N. Hirschborn, W. E. Woodward, D. A. Sack, and R. A. Cash. 1975. Antibodies to heat-labile Escherichia coli enterotoxin in Apaches in Whiteriver, Arizona. Infect. Immun. 12:1475-1477. 20. Schenkein, I., R. F. Green, D. S. Santos, and W. K.
INFECT. IMMUN.
Maas. 1976. Partial purification and characterization of a heat-labile enterotoxin ofEscherichia coli. Infect. Immun. 13:1710-1720. 21. Willingham, M. C., and I. Pastan. 1975. Cyclic AMP and cell morphology in cultured fibroblasts. J. Cell Biol. 67:146-159. 22. Zenser, T. V., and J. F. Metzger. 1974. Comparison of the action of Escherichia coli enterotoxin on the thymocyte adenylate cyclase-cyclic adenosine monophosphate system to that of cholera toxin and prostaglandin E. Infect. Immun. 10:503-509.
INFECTION AND IMMUNITY, May 1977, p. 617-622 Copyright ©) 1977 American Society for Microbiology
Assay of Escherichia coli Heat-Labile Enterotoxin with Vero Cells J. I. SPEIRS,* S. STAVRIC, AND J. KONOWALCHUK Bureau of Microbial Hazards, Food Directorate, Health Protection Branch, Health and Welfare Canada, Ottawa KiA OL2, Canada
Received for publication 16 November 1976
The continuous cell line of African green monkey kidney, Vero, showed characteristic morphological changes in response to culture filtrates from toxigenic strains of Escherichia coli. The response compared favorably with that of Y-1 (mouse adrenal) and CHO (Chinese hamster ovary) cells. Vero cells were the simplest and most economical to maintain in the laboratory. Escherichia coli heat-labile enterotoxin (LT) Earle salts (Connaught Laboratories Ltd.); all meacts on certain cell cultures to induce measura- dia were supplemented with 10% fetal calf serum ble biochemical changes, e.g., increased aden- (Grand Island Biological Co.); the mixtures are reto as growth media. (FlO medium suppleylate cyclase activity (6, 10, 13, 22), increased ferred mented with 15% horse serum and 2.5% fetal calf intracellular levels of cyclic adenosine 3',5'- serum been used previously for the culture of Ymonophosphate (9, 22), and induced synthesis 1 cells has [2]. In our laboratory, cell growth was better of A4,3-keto-steroids (1, 2, 4). With Y-1 (mouse in the presence of fetal calf serum than in horse adrenal) and CHO (Chinese hamster ovary) serum; 10% was as satisfactory as 15 or 20%.) The cells, these changes are accompanied by altera- requirements for maintenance were as follows: Y-1, tions in cell morphology and thus provide a weekly passage and an additional weekly change of growth medium; CHO, twice-weekly passages but simple diagnostic tool (3, 9, 11, 15, 18, 19). The continuous cell line Vero (African green no additional change of medium; and Vero, passages to 3 weeks or every 3 days, as required, monkey kidney) is also morphologically affected extending but no additional change of medium. by LT and shows advantages over other assay Enterotoxin activity was assayed in plastic dishes methods. The study reported here compares the with 24 16-mm-diameter wells (Falcon 3008), using responses of Y-1 (2), CHO (9), and Vero cells to 0.5 ml of cell culture in each well. The normal procethe culture filtrates of various strains of E. coli. dure was to use confluent monolayers of Y-1 and MATERIALS AND METHODS
Enterotoxin production. E. coli enterotoxigenic human or porcine strains H10407, B2C, and B7A were obtained from H. L. DuPont (U.S.A.), and P155, P307, 339, and 711 (P307) were from C. Gyles (Canada). Nontoxigenic strains 711 and K-12 were obtained from C. Gyles and V. N. Iyer (Canada), respectively. Erlenmeyer flasks, 150 ml, containing 20 ml of Trypticase soy broth (TSB) or Evans medium (8) were inoculated and mechanically shaken at 37°C. After 16 to 20 h, the cultures were centrifuged at 17,000 x g for 30 min. The supernatants were filtered through 0.45-,um membrane filters (Millipore Corp.) and stored at 4°C until assay that same day. Filtrate dilutions were made in phosphate-buffered saline (PBS), pH 7.0. Cell culture assay. Stocks of the continuous cell lines Y-1, CHO, and Vero, purchased from the American Type Culture Collection, Rockville, Md., were passaged by trypsinization and grown as monolayers at 36°C in a 5% CO2 atmosphere. Y-1 cells were grown in Ham nutrient mixture F10 (Connaught Laboratories Ltd.), CHO cells were grown in Ham nutrient mixture F12 (Grand Island Biological Co.), and Vero cells were grown in medium 199 with
Vero cells but freshly seeded CHO cells. Y-1 monolayers were obtained by seeding 105 cells per well 4 days before use; growth medium was changed 24 h before and at the time of assay. For Vero monolayers, 105 cells were seeded 1 to 2 days before use; growth medium was replaced with PBS at the time of assay. CHO cells, 104 in medium without serum, were seeded at the time of assay. To 0.5 ml of cell culture, 0.05 ml of bacterial filtrate was added. For controls, cultures received PBS or the appropriate bacterial culture medium. Cultures were incubated at 36°C for 18 to 24 h. Morphological effects were recorded as 1, 2, 3, or 4, corresponding to roughly .25, 50, 75, or >:90 cells affected, respectively. In comparative studies, variations in the above procedure were as follows: at the time of assay, Vero monolayers received growth medium instead of PBS; 3 x 105 cells of Vero in growth medium or PBS were seeded at the time of assay; 3 x 103, 5 x 104, or 3 x 105 cells of CHO in medium without serum or 104 cells in medium containing 0.5, 1, 5, or 10% serum were seeded at the time of assay; Y-1 monolayers received F10 medium supplemented with 0, 5, 10, 15, or 20% serum at the time of assay (monolayers were first rinsed twice with the appropriate medium). Rabbit ileal-loop assay. Ileal-loop assay was performed in duplicate or triplicate in 9-week-old rab617
618
INFECT. IMMUN.
SPEIRS, STAVRIC, AND KONOWALCHUK
bits by the method of Kasai and Burrows (14). Two milliliters of filtrate was used for each 10-cm loop; after 18 h, the fluid accumulation was measured and expressed as milliliters of fluid per centimeter of gut. A ratio of 1.0 or more was considered positive. Fractionation of bacterial filtrates. A pool of 175 ml of bacterial filtrate of strain H10407, grown in Evans medium, was fractionated by filtration, in series, through 76-mm Diaflo membranes XM300, XM100A, PM30, and UM10 (Amicon Corp., Lexington, Mass.). These membranes are designed to restrict macromolecules in excess of molecular weight 300,000, 100,000, 30,000, and 10,000, respectively. Ten milliliters of filtrate from each membrane was retained for assay; the remaining fluid was filtered through the next grade of membrane. Effect of endotoxin on cell cultures. Commercial lyophilized lipopolysaccharide B (endotoxin) from E. coli 026:B6 (Difco) was dissolved in distilled water to a concentration of 20 mg/ml. A 0.05-ml volume of this solution or of three successive fourfold dilutions of it were added in duplicate to 0.5 ml of freshly seeded cells of Y-1 or Vero in growth medium or of CHO cells in growth medium or medium without serum. Cultures were examined at 24, 48, and 72 h for visual changes.
RESULTS In preliminary experiments, Vero cells showed a morphological response to filtrates of E. coli H10407. Response occurred in the presence of growth medium or PBS with either established monolayers or freshly seeded cells. The most-defined effect was observed when the growth medium of confluent monolayers was replaced with PBS just prior to the addition of bacterial filtrates; the affected cells appeared enlarged, thick-walled, and refractile, with several filamentous tendrils (Fig. 1). This method of assay was used for further studies. When cells, freshly seeded in PBS, were treated with toxigenic filtrates, the morphological changes resembled those of CHO cells (9) (see Fig. 3); i.e., control cells were round whereas treated cells were flattened and elongated (Fig. 2). Vero, Y-1, and CHO cells responded to filtrates of various known LT-producing E. coli strains but not to nontoxigenic strains (Table 1). Some variation from experiment to experiment occurred in the optimum response with all three cell types (Table 2). Undiluted filtrates of strain H10407, grown in two different media, elicited response in 50 to 100% of the Y1, CHO, or Vero cells within 18 h; the mostfrequent response in Y-1 cells was 100% and that in CHO or Vero cells was 75%. No increase in the degree of effect was discernible after 18 h. The first evidence of response to H10407 filtrates was noted after 2 h with Y-1 and Vero cells and after 4 h with CHO cells. Near-maxi-
~
b~~~~~
~
T
.~~~~~~~~/* I
m
o
woa F ~~~~~~~~~~~.
an TSB
AOnrl B iued11 nPS 13 ' Ab
o3 as ih eo el. No CHO cels an£p FIG. 1. Vero monolayers incubated for 24 h in a filtrate of E. coli Hi 0407 grown in TSB. Filtrate (A) treated and TSBwihedtxnitafnlcocnrto (control) (B) diluted 1:10 in PBS. x133. 4
of 0.002 to 0.1 mg/ml.1 mum response was noted at 4 h with Y-1 and Vero cells, and cellular changes persisted at least 5 days with Y-1 cells, up to 2 days with CHO cells, and up to 3 days with Vero cells. No cellular changes were seen in any of these cells treated with endotoxin at a final concentration of 0. 002 to 0. 1 mg/ml. Two media were compared for production of LT by assaying serial dilutions of several H10407 filtrates (Table 3). TSB was inferior to Evans medium. Compared with that of Y-1 cells, the titer for CHO and Vero cells was greater but varied with the filtrate preparation. Heating these filtrates at 980C for 15 mi negated activity in each cell type; heating at 650C for 15 min reduced activity by 75%. Filtrates of three LT-producing E. coli strains, grown in Evans medium, were titrated by the methods of cell cultures and rabbit ileal loops. The end points in Vero cells were 16 times greater than those in Y-1 cells but four times less than those in CHO cells (Table 4). In rabbit ileal loops, undiluted filtrates from all strains invoked fluid accumulation, but the response to filtrates diluted 1:4 was inconsistent.
ASSAY OF E. COLI HEAT-LABILE ENTEROTOXIN
VOL. 16, 1977
To determine whether the high titer in CHO cells was due to the comparatively low concentration of cells used in the test (104/well), cell numbers were adjusted to 3 x 103, 1 x 104, 5 x
619
TABLE 2. Morphological response of cell cultures to different filtrates of E. coli H10407 grown in two different mediaa Medium
TSB TSB TSB TSB TSB Evans Evans Evans
Y-1
CHO
Vero
4b 4 4 4 2 4 4 3
3 2 3 3 3 3 3 2
2 3 2 4 3 4 3 3
a 2, ca. 50%; 3, ca. 75%; and 4, .90% cells affected. Readings were recorded 18 h postincubation. bDuplicate or triplicate cultures yielded similar results.
TABLE 3. Titration in cell culture of filtrates of E. coli H10407 grown in two media Medium
as
O
.0
t
.s e
0
&
V0.
4
_za 6
-W
0
*.D v
*"
0 0
TSB TSB Evans 16 1,024 256 Evans 16 1,024 256 Evans 16 64 64 Evans 16 256 64 a Duplicate or triplicate cultures yielded similar results. Readings were recorded 24 h postincubation. TABLE 4. Titration in cell culture of filtrates of toxigenic E. coli strains grown in Evans mediuma Strain
FIG. 2. Freshly seeded Vero cells, in PBS, incubated for 24 h in a filtrate of E. coli H10407 grown in TSB (A) or, for a control, TSB (B). Each was diluted 1:10 in PBS. x133. TABLE 1. Morphological response of three cell lines to filtrates of toxigenic and nontoxigenic E. coli strains grown in TSBa Strain
Toxigen-
Y-1
CHO
Vero
H10407 339 P155 P307 711 (P307) B7A B2C
+ + + + + + +
4b 4 4 4 3 3 4
2 2 2 3 2 1 1
3 3 3 3 3 2 3
711
-
-
-
-
K-12
-
-
-
-
icity
a -, No response; 1, 300,000, our separation by membrane filtration indicated that the active components were smaller. Mo-
ASSAY OF E. COLI HEAT-LABILE ENTEROTOXIN
VOL. 16, 1977
621
lecular weights of 20,000 (7), 100,000 (5, 17), 200,000 (20), and >300,000 (12, 16) have been reported for LT. .f .I a,
VIz~~~~~~I
ACKNOWLEDGMENT
.
S1,,
j
i
%
We are grateful to Deborah Jeffrey for competent technical assistance.
\
LITERATURE CITED
XCb ,,
to, 4
AP
_11
._
X. '- 1.4
16 a,,
FIG. 4. Freshly seeded CHO cells, in medium F12 with 10% serum, incubated for 4 days in a filtrate of E. coli H10407 grown in TSB (A) or, for a control, TSB (B). Each was diluted 1:10 in PBS. x133.
OJ CONTROL (NO ULTRAFILTRATION) n11 XM300 XM IOOA PM30 1/64 z
0
1/16 z 0
t31/4
Y-I CHO VERO RL FIG. 5. Titration of E. coli H10407 ultrafiltrates in cell lines and rabbit ileal loops. Readings were recorded 24 h postinoculation for the former and 18 h for the latter.
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