Vol. 58, No. 4

INFECTION AND IMMUNITY, Apr. 1990, p. 930-934 0019-9567/90/040930-05$02.00/0 Copyright © 1990, American Society for Microbiology

Assay for Enterotoxigenic Escherichia coli Heat-Stable Toxin b in Rats and Mice SHANNON C. WHIPP

National Animal Disease Center, Agricultural Research Service, U.S. Department of Agriculture, Ames, Iowa 50010 Received 20 September 1989/Accepted 4 January 1990

The Escherichia coli heat-stable enterotoxin STb is the most prevalent toxin associated with diarrheagenic isolates of porcine origin. This report demonstrates that when endogenous protease activity was blocked with soybean trypsin inhibitor, STb evoked a dose-dependent secretory response in infant mice and jejunal loops of rats. Infant mice were much less sensitive to STb than rats were. The response of rat jejunal loops to STb was linearly related to the log of the dose of STb through 10 twofold dilutions. The anterior 25% of the jejunum was less sensitive than was the remainder of the gut. An incubation period of 2.5 h provided a maximal response. The linear response to STb in rats could be used as a bioassay.

International, Harbor City, Calif.) per liter. After being gently stirred for 1 h at room temperature, the gel was filtered on a Buchner funnel and suspended in 50 ml of distilled water. The gel was then poured into a column (1 by 15 cm). The columns were eluted, using a 250-ml linear gradient elution from 0 to 100% methanol-trifluoroacetic acid (99.8:0.2). The flow rate was 1.5 ml/min. This eluate was evaporated to dryness and suspended in 10% Tris buffer. The eluate was collected in 10-ml fractions. Biological activity, as determined in jejunal loops of pigs, was usually observed in tubes 11 through 15. The relative concentrations of C and STb concentrates (designated C-conc and STbconc, respectively) were expressed as volumes equivalent to that of the original CCF. Thus, a 16x STb-conc indicates that the concentrated eluate was dissolved in a volume equivalent to 1/16 of the original volume of CCF. Animal experiments. Sprague-Dawley rats, weighing 250 to 350 g and obtained from BioLab Corp. (St. Paul, Minn.), were used. Rats were deprived of food and water for 48 h and deprived of water for 24 h before each experiment. Rats were anesthetized with 60 mg of pentobarbitol per kg intraperitoneally, and the intestines were exteriorized through a midline incision. The intestinal lumen was rinsed two times with 3 ml of saline containing 1 mg of TI per ml injected at a site 4 to 6 cm distal to the ligament of Trietz and worked posteriorly with gentle manipulation. Starting 1 to 2 cm distal to the injection site, 5- to 6-cm loops separated by 1- to 2-cm interloops were created with intestinal ligatures. Each loop was injected with 0.5 ml of STb or C diluted as described below. Each preparation injected into loops contained 2 mg of TI per ml. After an appropriate incubation period, the loop was emptied and the fluid volume and loop length were recorded. Results were expressed as milliliters per centimeter plus or minus the standard error (SE). Mixed-breed pigs, 6 to 8 weeks old, were used. Pigs were deprived of food and water overnight before the experiments were performed, and loop studies were conducted as previously described (17), except that the jejunal lumen was rinsed twice with saline (50 ml) containing 200 ,ug of TI per ml before loops were created and each preparation injected in loops contained 200 Vig of TI per ml. Pregnant CF-1 mice were obtained from ARS Sprague-

At least two types of heat-stable enterotoxin are produced by enterotoxic Escherichia coli (ETEC). One of these (STa) evokes a secretory response in infant mice, and this response is the basis for an effective bioassay (2). The second ETEC heat-stable enterotoxin (designated STb [1], STp [11], or ST-I1 [7]) has been primarily associated with ETEC isolated from diarrheic swine, and genes encoding this character have been observed to have the highest prevalence among the toxin genes in porcine ETEC (8, 10). This enterotoxin is also of special interest because it appears to have a unique mechanism of action (3, 14-16). Stimulation of the secretion of fluid and electrolytes by the small intestine is the only biological activity attributable to STb to date, and most attempts to demonstrate an intestinal secretory response to STb in species other than the pig have failed (4, 6, 11). Moreover, the response in the pig intestine has been observed to be inconsistent (12, 18). These characteristics have limited efforts to purify STb or study its mechanism and have made quantitation of STb difficult. It was recently observed that STb is sensitive to trypsin degradation. Moreover, a factor which is variably present in the jejunal lumen of swine intestine was shown to interfere with the response to STb. This factor could be blocked with soybean trypsin inhibitor (TI) (17). When TI was used to block intestinal protease activity, an unequivocal intestinal response to STb was observed in mice, rats, rabbits, and calves (S. C. Whipp, unpublished data). This report explores the potential use of jejunal loops in rats and an infant mouse assay as bioassays for STb. MATERIALS AND METHODS

Microbiology. Three strains of E. coli of porcine origin were used: strain 123 (serotype 043:K-:H28), which is nontoxigenic; strain 1790 (serotype O9:K+), which produces only STb; and strain 431 (serotype 0101:K30,K99), which produces only STa. These were cultured in brain-heart infusion broth. Sterile crude culture filtrates (CCF) were prepared as previously described (9) and designated control (C), STb, and STa, respectively. For the preparation of concentrates, 2 liters of CCF of C or STb was mixed with 4 g of Bondesil resin (Analytichem 930

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RAT BIOASSAY FOR E. COLI HEAT-STABLE TOXIN b

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TABLE 1. Effect of incubation time on intestinal responses of infant mice to STb Intestinal responses of mice after indicated times (h)

2

Inoculum

Gut/body wt ratio'

16x CC 2x TCC 4x TC 8x TC 16x TC

Slope y Intercept

Correlation coefficient

0.073 0.071 0.088 ± 0.005 0.103 ± 0.006 0.120 ± 0.006

3

4

No. of mice

Gut/body wt ratio'

No. of mice

Gut/body wt ratioa

No. of mice

4

0.067 ± 0.001 0.068b 0.082 ± 0.005 0.106 ± 0.010 0.135 ± 0.019

12

0.062 0.068b 0.083 ± 0.005 0.098 ± 0.011 0.107 ± 0.001

5

4

11 12 7

-0.0160 0.1197 0.9993

4

12 12 8

-0.0265 0.1342 0.9985

5

13 12 8

-0.0120 0.1080 0.9897

a Mean ± SE b Single determination on gut and body pools. c TC, STb-conc.

Dawley (Madison, Wis.). Infant mice obtained from these dams were inoculated by gavage with 0.1 ml of control or toxin-containing preparations when they were 2 to 4 days old. In each assay, the guts and bodies of 3 to 4 mice were pooled to determine a gut/body ratio. In the first experiment, mice were inoculated with 16x C-conc containing 2 mg of TI per ml (n = 7), 16x STb-conc containing 2 mg of TI per ml (n = 6), C (n = 4), or serial 1:2 dilutions of STa from 1:2 (n = 3 to 4 at each point). A 4-h incubation period was used. In the second experiment, mice were treated with 2, 4, 8, and 16x STb-conc containing 2 mg of TI per ml, and incubation periods of 2, 3, and 4 h were used. The numbers of mice at each point are given in Table 1. In the third experiment, 4 and 16x STb-conc, 8x C-conc, and three different concentrations of TI (2, 4, and 8 mg/ml) were used with a 3-h incubation period. There were 3 to 4 mice at each point. Means were compared by using the least significant difference (13).

negligible (0.004 + 0.01 ml/cm) at all sites (Fig. 2). The response to STb was least in the anterior 20% of the jejunum and increased steadily in the next 30% of the jejunum. This response was fairly consistent in the mid portion and tended to decrease in the last 20% of the small intestine. On this basis, responses from loops positioned in the anterior 25% of the small intestine were discarded in subsequent experiments. Dose-response relationships in rat loops. Volumes (0.5 ml each) of twofold dilutions of STb to 1:128 were injected into 5-cm loops in each of 20 rats. STb was diluted with C to minimize changes in osmolarity. Doses were randomly assigned to loops. Eight loops in each of three rats were also injected with C. Rats were killed 2.5 h after the loops were injected. No fluid was recovered from any C loop. There was a linear relationship between the final volume of fluid in the loops and the negative log of the reciprocal of the STb dilution (Fig. 3) from an STb dilution of 1:2 to 1:128. The slope of this relationship was -0.030 ml/cm per twofold dilution, and the correlation coefficient was 0.9964. The

RESULTS

STb response with time. In each of 30 rats, three loops were injected with 0.25 ml of STb diluted to 0.5 ml with saline, and 2 loops (the most anterior and the most posterior loops) were injected with 0.25 ml of C diluted to 0.5 ml with saline. Six rats were killed at each of the following times: 1, 1.5, 2, 2.5, and 3 h. Mean rat weight, gut length, and loop length plus or minus the SE were 259 ± 6.0 g, 81.0 ± 1.6 cm, and 5.25 ± 0.09 cm, respectively. After 1.5 h, the amount of fluid recovered from control loops was negligible. The response to STb increased steadily from 1 to 2.5 h, but the response at 3 h was not different from that at 2.5 h (Fig. 1). The response of the most proximal STb loop was significantly less (P < 0.01) than that of the most distal STb loop. Effect of loop position on STb response. Eleven loops were created in each of 11 rats, and 0.25 ml of C or STb diluted to 0.5 ml with saline was injected into alternate loops such that there were 6 STb loops and 5 C loops. The rats were killed 2.5 h after the loops were injected. The mean rat weight, gut length, and loop length plus or minus the SE were 252 + 5.0 g, 83.7 ± 2.2 cm, and 4.84 ± 0.01 cm, respectively. The most proximal loop was 2.5 + 0.5 cm distal to the ligament of Trietz, and the most distal loop was 15.0 + 3.0 cm proximal to the ileocecal valve. The fluid volume in C loops was

0.30 w

+1

E E w E 0.

0.20

0.10

0 0

W0

0.00

1.0

1.5

2.0

2.5

3.0

Time (hours) FIG. 1. The effect of duration of exposure on the responses (means plus or minus the SE) of jejunal loops of rats to the E. coli heat-stable enterotoxin STb. n = 6 at each time period. Symbols: 0, STb; 0, control.

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INFECT. IMMUN.

WHIPP 0.4

04 -

0-H

I

0-

E 2 E

II

cm

0.3

0.2

0 0 0

0.1

.5 la 0.0

0l1 0

20

10

30

40

50

50

70

80

90

100

0

1

% of Distance From Ligament of Trletz to Ileo-Cecal Valve

2

3

4

5

6

7

8

9

10

Number of Two-Fold Dilutions

FIG. 2. The effect of loop position on the responses (means plus or minus the SE) of intestinal loops in rats to the E. coli heat-stable enterotoxin STb. n = 11.

FIG. 4. A dose-response relationship describing maximal intestinal fluid secretory responses (means plus or minus the SE) induced in rat jejunal loops by the E. coli heat-stable enterotoxin STb during a 2.5-h incubation. n = 6.

response to a 1:256 dilution was significantly different from 0 (P < 0.01). Maximal response in rat loops. To demonstrate a maximal response, STb was mixed with lOOx STb-conc (2:1, vol/vol), and serial dilutions of this material were tested in jejunal loops in each of 12 rats. The mean rat weight, gut length, and loop length plus or minus the SE were 263 ± 5.2 g, 90.7 ± 1.3 cm, and 5.0 ± 0.5 cm, respectively. Maximal responses of 0.340 and 0.349 ml/cm were observed with the two highest concentrations of this material (Fig. 4). The slope of the dose-response relationship between 1:2 and 1:1,028 dilutions was -0.030 ml per twofold dilution, and the correlation coefficient was 0.9860. Dose-response relationships in pig loops. For a comparative assessment, 5.0 ml of STb and twofold dilutions of STb were injected into 5-cm loops in each of four pigs. STb was diluted with C. Doses were randomly assigned to loops. Each STb loop was accompanied by an adjacent C loop. The mean final fluid volume in C loops was 0.5 ± 0.05 ml/cm per 2 h. There was a linear relationship between the final fluid volume and

the negative log of the reciprocal of the STb dilution from undiluted STb to the 1:128 dilution (Fig. 5). The slope of this relationship was -0.101 ml per twofold dilution of STb, and the correlation coefficient was 0.9913. The values associated with the response to a 1:64 dilution were significantly different from control values (P < 0.01). Response in infant mice. Gut/body weight ratios of infant mice inoculated with 16x C-conc (0.068 + 0.002) were not different from those of mice inoculated with C (0.067) and were similar to control values observed previously (19). The gut/body ratio of infant mice was linearly related to the negative log of the reciprocal of the STa dilution between 1:2 and 1:32. The maximal and minimal ratios observed with STa were 0.185 and 0.088, with a slope of -0.0232 and a correlation coefficient of 0.9402. Gut/body ratios of mice injected with 16x STb-conc were 0.159 + 0.003. The response was linearly related to the negative log to the base 2 of the dose (Table 1) at incubation periods of 2, 3, and 4 h with a high correlation coefficient (0.9893, 0.9985, and 1.6

OA -

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I.s8 0.

1A

03

E

1.2

sL

1.0-

o

.8

0.2

0.1

.-I

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0.6 0

1

2 7 8 3 4 6 5 Number of Two-Fold Dilutions FIG. 3. A dose-response relationship describing intestinal fluid secretions (means plus or minus the SE) induced in jejunal loops in rats by the E. coli heat-stable enterotoxin STb during a 2.5-h incubation. n = 21.

0

1

7 2 3 4 5 6 8 Number of Two-Fold Dilutions FIG. 5. A dose-response relationship describing intestinal fluid secretions (means plus or minus the SE) induced in jejunal loops in pigs by the E. coli heat-stable enterotoxin STb during a 20-h incubation. n = 4.

RAT BIOASSAY FOR E. COLI HEAT-STABLE TOXIN b

VOL. 58, 1990

0.9897, respectively). The dose-response relationship with the highest correlation coefficient and the steepest slope was observed after a 3-h incubation period. Increasing the concentration of TI did not alter the response. Gut/body weight ratios in mice inoculated with 16x STb-conc were 0.111, 0.111, 0.115, and 0.103 with 1, 2, 4, and 8 mg of TI per ml, respectively. Similarly, the gut/body weight ratios induced by 4x STb-conc were 0.077, 0.074, 0.087, and 0.079 with 1, 2, 4, and 8 mg of TI per ml, respectively. DISCUSSION

These data demonstrate that the intestinal response to STb is dose related in rats. In jejunal loops in rats, the response increased with time until 2.5 h, after which there was no further increase. This is not necessarily a characteristic of the response of the intestinal mucosa to STb, since it may reflect protease activity overcoming the TI block. The dose-response relationship was linear over a fairly broad range of dilutions (0 to 1:1,028); in fact, it was necessary to add STb-conc to the CCF (Stb) to demonstrate an unequivocal maximal response in the rat. The rat intestine was less responsive to STb than was the pig intestine in the sense that the slope of the dose-response relationship was lower in the rat (0.030 versus 0.097 ml/cm of intestine per twofold dilution of STb). On the other hand, intestinal loops in rats require a smaller volume and can therefore detect smaller quantities of STb than can intestinal loops in pigs. An enzyme-linked immunosorbent assay for STb has been described previously (5); however, bioassays for STb will continue to be necessary to detect biological activity, and the only biological activity attributable to STb at this time is the intestinal secretory response. In preliminary experiments, we could not detect a response to CCF in infant mice by using the same preparations that evoked the responses shown in rats (Fig. 3) and pigs (Fig. 5). Since detectable responses were observed with 1:64 and 1:256 dilutions in pigs and rats, respectively, it was apparent that more STb was required to induce a detectable response in an infant mouse than in an intestinal loop in a pig or rat. With a more concentrated preparation (STb-conc), we observed a response that was dose related, with high correlation coefficients after 2-, 3-, and 4-h incubation periods (Table 1). The response to 4x STb-conc was significantly greater than that to C. In a separate titer determination in pig loops, 4x STb-conc was estimated to contain twice as much STb as the CCF contained (data not shown). Thus, relative to the rat loop, several times as much STb was required to induce a detectable response in the infant mouse. This insensitivity to STb did not appear to be a function of protease activity, since varying the concentration of TI in the inoculum from 1 to 8 mg/ml had no apparent effect on the response evoked by STb. The intestinal response of infant mice to STb was linearly related to the log of the dose through the highest dose tested, so the effective range of this dose-response relationship was presumably not defined. An incubation period of 3 h gave a greater response and a dose-response relationship with a steeper slope than did an incubation period of either 2 or 4 h. These characteristics suggest that the infant mouse model could serve as a bioassay for STb, although the relatively large concentrations of STb required to induce a detectable response make this a less useful model than intestinal loops in rats or pigs. We conclude that STb elicits an intestinal secretory response in species other than the pig when endogenous

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intestinal protease activity is blocked with soybean TI, that the sensitivity of the intestinal response to STb varies between species, and that jejunal loops of rats can be used as a bioassay to determine relative quantities of STb. The data also indicate that, although STb induces an intestinal secretory response in infant mice, the response is less sensitive than that in rats or pigs. ACKNOWLEDGMENTS The author is indebted to Robert W. Morgan for excellent technical assistance and to Annette L. Bates for assistance in preparation of the manuscript. LITERATURE CITED 1. Burgess, M. N., R. J. Bywater, C. M. Cowley, N. A. Mullan, and P. M. Newsome. 1978. Biological evaluation of a methanol soluble, heat-stable Escherichia coli enterotoxin in infant mice,

pigs, rabbits, and calves. 2. Dean, A. G., Y.-C. Ching, R. G. 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. 3. Guerrant, R. L., and C. S. Weikel. 1988. Pharmacologic pathways leading to intestinal secretion: stimulation by enterotoxins and by C-kinase activators, p. 181-188. In S. Kuwahara and N. F. Pierce (ed.), Advances in research on cholera and related diarrheas, vol. 4. KTK Scientific Publishers, Tokyo. 4. Gyles, C. L. 1979. Limitations of the infant mouse test for Escherichia coli heat-stable enterotoxin (ST). Can. J. Comp. Med. 43:371-379. 5. Handl, C., B. Ronnberg, B. Nilsson, E. Olsson, H. Jonsson, and J.-I. Flock. 1988. Enzyme-linked immunosorbent assay for Escherichia coli heat-stable enterotoxin type II. J. Clin. Microbiol. 26:1555-1560. 6. Kennedy, D. J., R. N. Greenberg, J. A. Dunn, R. Abernathy, J. A. Ryerse, and R. L. Guerrant. 1984. Effects of Escherichia coli heat-stable enterotoxin STb on intestines of mice, rats, rabbits, and piglets. Infect. Immun. 46:639-643. 7. Lee, C. H., S. L. Moseley, H. W. Moon, S. C. Whipp, C. L. Gyles, and M. So. 1983. Characterization of the gene encoding heat-stable toxin II and preliminary molecular epidemiological studies of enterotoxigenic Escherichia coli heat-stable toxin II producers. Infect. Immun. 42:264-268. 8. Monckton, R. P., and D. Hasse. 1988. Detection of enterotoxigenic Escherichia coli in piggeries in Victoria by DNA hybridization using K88, LT, ST1, and ST2 probes. Vet. Microbiol. 16:273-281. 9. Moon, H. W., P. Y. Fung, R. E. Isaacson, and G. D. Booth. 1979. Effects of age, ambient temperature, and heat-stable Escherichia coli enterotoxin on intestinal transit in infant mice. Infect. Immun. 25:127-132. 10. Moon, H. W., R. A. Schneider, and S. L. Moseley. 1986. Comparative prevalence of four enterotoxin genes among Escherichia coli isolated from swine. Am. J. Vet. Res. 47:210-212. 11. Olsson, E., and 0. Soderlind. 1980. Comparison of different assays for definition of heat-stable enterotoxigenicity of Escherichia coli porcine strains. J. Clin. Microbiol. 11:6-15. 12. Rose, R., S. C. Whipp, and H. W. Moon. 1987. Effects of Escherichia coli heat-stable enterotoxin b in small intestinal villi in pigs, rabbits, and lambs. Vet. Pathol. 24:71-79. 13. Steel, R. G., and J. H. Torrie. 1960. Principles and procedures of statistics. McGraw-Hill Book Co., New York. 14. Weikel, C. S., and R. L. Guerrant. 1985. STb enterotoxin of Escherichia coli: cyclic nucleotide-independent secretion. Microbial toxins and diarrhoeal disease. CIBA Found. Symp.

112:94-115. 15. Weikel, C. S., S. A. Long-Krug, and R. L. Guerrant. 1986. E. coli STb: a new mechanism for secretory diarrhea? p. 333341. In S. Kuwahara and N. F. Pierce (ed.), Advances in research on cholera and related diarrheas, vol. 3. KTK Scien-

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tific Publishers, Tokyo. 16. Weikel, C. S., H. N. Nellans, and R. L. Guerrant. 1986. In vivo and in vitro effects of a novel enterotoxin, STb, produced by Escherichia coli. J. Infect. Dis. 153:893-900. 17. Whipp, S. C. 1987. Protease degradation of Escherichia coli heat-stable, mouse-negative, pig-positive enterotoxin. Infect. Immun. 55:2057-2060.

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18. Whipp, S. C., E. Kokue, R. W. Morgan, and H. W. Moon. 1987. Functional significance of histologic alterations induced by Escherichia coli pig-specific, mouse-negative, heat-stable enterotoxin (STb). Vet. Res. Commun. 11:41-55. 19. Whipp, S. C., H. W. Moon, and N. C. Lyon. 1975. Heat-stable Escherichia coli enterotoxin production in vivo. Infect. Immun. 12:240-244.

Assay for enterotoxigenic Escherichia coli heat-stable toxin b in rats and mice.

The Escherichia coli heat-stable enterotoxin STb is the most prevalent toxin associated with diarrheagenic isolates of porcine origin. This report dem...
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