INFECTION AND IMMUNITY, May 1977, p. 604-609 Copyright © 1977 American Society for Microbiology
Vol. 16, No. 2 Printed in U.S.A.
Direct Serological Assay for the Heat-Labile Enterotoxin of Escherichia coli, Using Passive Immune Hemolysis DOYLE J. EVANS, JR.,* AND DOLORES G. EVANS Program in Infectious Diseases and Clinical Microbiology, The University of Te-xas Medical School at Houston, Houston, Texas 77030 Received for publication 29 November 1976
Sheep erythrocytes sensitized with the heat-labile enterotoxin (LT) ofEscherichia coli exhibit passive immune hemolysis (PIH) when exposed to specific antitoxin and complement. Thus, PIH serves as the basis for an in vitro serological assay for LT that is sufficiently specific and sensitive to differentiate LT-positive and LT-negative E. coli isolates. The PIH assay for E. coli LT has been performed with the standard Microtiter system and also by a tube method employing the spectrophotometric determination of hemoglobin release. The spectrophotometric method enhances the sensitivity, accuracy, and objectivity of the PIH assay. The increased sensitivity of the spectrophotometric method also facilitates the identification of LT-positive cultures employing polymyxin "miniextracts" of whole overnight (18 h) broth cultures of 2.0% Casamino Acids-0.6% yeast extract-salts medium rather than mini-extracts of cells derived from 3.5-h subcultures. Thus, large numbers of E. coli isolates can be individually tested for LT in less than 24 h after broth inoculation by a rapid in vitro assay which requires anti-LT serum as the only specific reagent. Several varieties of enterotoxigenic Escherichia coli are associated with acute diarrhea in man. These may produce either a heat-labile enterotoxin (LT), a heat-stable enterotoxin, or both (11, 17). Of these enterotoxins, only LT is known to be antigenic (3, 20). Antitoxin prepared against the LT of any one strain neutralizes the biological activity of all other LT-producing strains of E. coli (3, 12-14, 23). Also, E. coli LT and Vibrio cholerae choleragen display considerable immunological cross-identity (3, 12-14, 19, 23). The apparently universal immunological identity of LT is indeed fortuitous in that it has facilitated studies into humoral antitoxin response in relation to enterotoxigenic E. coli-associated diarrhea (17, 21, 22). To further investigations in this important area, we developed an in vitro microtiter assay for detecting humoral antitoxin responses to LT (D. J. Evans, Jr., D. G. Evans, and G. Ruiz-Palacios, Prog. Abstr. Intersci. Conf. Antimicrob. Agents Chemother., 16th, Chicago, Abstr. 368, 1976). The Microtiter assay for anti-LT antibody thus employed by our laboratory utilizes the principle of passive immune hemolysis (PIH), i.e., complement-mediated lysis of LT-sensitized sheep erythrocytes (LT-SRBC) by antitoxin. PIH also provided the basis for an indirect in vitro serological assay for LT which we recently described (5). This lysis inhibition test (LIT) for LT employs preexposure of a standard amount
of rabbit antitoxin to E. coli test preparations before the addition of LT-SRBC and complement, thus allowing soluble LT in a test sample to competitively inhibit the immune hemolysis reaction. Although the LIT assay for LT proved to be highly efficient in detecting LT-producing E. coli isolates and differentiating these from non-enterotoxigenic E. coli, its major drawback is the requirement for purified LT as a reagent. We now describe a direct in vitro assay for LT, also utilizing the principle of PIH, which is simple and rapid in performance, more sensitive than the LIT procedure, and does not require purified LT as a reagent. MATERIALS AND METHODS Bacterial cultures. E. coli strain H-10407, isolated from a case of diarrhea in Dacca, Bangladesh, was used for the large-scale production of LT (6, 7). One thousand E. coli isolates were employed in investigating the specificity and sensitivity of the serological assays for LT. These were isolated from human subjects in Bangladesh, the United States, and Mexico during the course of investigations into the association of enterotoxigenic E. coli with acute diarrhea (2-4, 10). Of these 1,000 isolates, 171 were positive for LT when tested individually by the cultured Y-1 adrenal tumor cell assay of Donta (1), and 829 were negative in the adrenal cell assay. The LT activity of all adrenal cell-positive cultures was neutralized by the hyperimmune rabbit antitoxin prepared against the LT of E. coli H-10407. Stock cul-
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VOL. 16, 1977 tures were maintained on 2.0% peptone-0.5% NaCl2.0% agar slants. Preparations of E. coli LT and antitoxin. The low-molecular-weight LT of E. coli H-10407 was obtained by the polymyxin-release technique (7) and purified by ammonium sulfate fractionation and Affi-Gel 202 (Bio-Rad Laboratories, Richmond, Calif.) chromatography as recently described in detail (8). Antitoxin was obtained by hyperimmunizing adult albino rabbits with increasing doses of LT and bleeding the animals 14 days after the final injection (3). The sera were pooled and stored at -450C. Anti-cholera toxin, a lyophilized horse antiserum, was obtained from Nobuya Ohtomo (Chemosero-therapeutic Research Institute, Kumamoto, Japan). Purified "B" subunit protein of cholera toxin, choleragen, was prepared by W. F. Osborne and S. H. Richardson and was the generous gift of S. H. Richardson. This material was maintained in the lyophilized state until immediately prior to use. Mini-extract preparations. LT was released from E. coli cells by the polymyxin-release technique (7), except that buffer conditions and polymyxin concentration were modified as described below. The term mini-extraction procedure is used in reference to the small-scale preparation of polymyxin "extracts." Two different mini-extraction procedures were employed. The first and basic mini-extraction procedure (ME-I) was as follows. Cultures were prepared by loop inoculation of 10 ml of 2.0% Casamino Acids (Difco)-0.6% yeast extract (Difco)-salts medium (CYE medium; 6) from stock slants. Fifty-milliliter Erlenmeyer flasks were agitated at 140 rpm for 18 h at 37°C in a rotary incubator-shaker. Subcultures were then prepared by inoculating 1.5 ml of the overnight broth culture into 10 ml of modified CYE medium (0.15% instead of 0.6% yeast extract; 7). After a 3.5-h incubation at 37°C as above, the secondary cultures were centrifuged at 12,000 x g for 20 min, and the supernatant fluids were discarded. Each pellet of bacteria was resuspended in 0.8 ml of polymyxin B (Aerosporin, Borroughs Wellcome Co., Research Triangle Park, N. C.) at a concentration of 0.25 mg/ml dissolved in 0.08 M phosphate buffer (pH 6.7) containing 0.9% NaCl and also 0.02% sodium azide as preservative (ME buffer). The cell suspensions were incubated by placing the centrifuge tubes in a 37°C water bath for 15 min, and the cells were then removed by centrifugation at 16,000 x g for 20 min. The resultant supernatants, or ME-I preparations, were recovered by disposable Pasteur pipettes and stored in small vials at 4°C until assayed. In the second mini-extraction procedure (ME-II), cultures were grown as above, except that only 5 ml of CYE medium was used per 50-ml Erlenmeyer flask. At 18 h after inoculation, 5 ml of ME buffer with polymyxin was added to each flask, and shaking was continued at 37°C for an additional 15 min. The supernatants (ME-II preparations) were recovered after centrifugation of the polymyxintreated cultures as above. ME-II preparations were assayed only by the spectrophotometric assay described below. Spectrophotometric method for the assay of E.
SEROLOGICAL ASSAY FOR E. COLI LT
605
coli LT employing PIH. The following reagents were employed to assay E. coli ME-I and ME-II preparations for LT. Fresh SRBC, stored at 4°C in Alsever solution, were washed twice with 8 volumes of 0.08 M phosphate buffer (pH 6.7) containing 0.9% NaCl and finally resuspended at a concentration of 10% in 0.02 M phosphate buffer (pH 6.7) plus 0.9% NaCl. These cells remained sufficiently stable at 4°C for 48 to 72 h after washing. Lyophilized guinea pig complement (Grand Island Biological Co., Grand Island, N.Y.) was restored as per instructions and then stored at -20°C. Rabbit anti-LT antiserum, prepared as above, was diluted 1:100 in phosphatebuffered saline (PBS, 0.1 M, pH 7.2) and maintained at4°C. The assay for LT in ME-I preparations was performed as follows. Duplicate disposable tubes (1.0 by 5.0 cm) received 40 ,l of 0.02 M phosphate buffer plus 0.9% NaCl (pH 6.7), 10 ,ul of the ME-I preparation, and finally 50 ,ul of a 1.0% suspension of SRBC freshly prepared from the 10% stock suspension by dilution in the 0.02 M phosphate buffer (pH 6.7) plus 0.9% NaCl. These reaction mixtures were incubated with either intermittent or continuous shaking for 30 min at 37°C in order to allow LT, if present, to adsorb to the SRBC. Next, 50 ,lI of antitoxin (1:100 in PBS) was added, and the tubes were incubated for another 30 min at 37°C to allow antibody to react with the LT-SRBC complexes. Next, 50 ,lp of guinea pig complement (diluted 1:10 in PBS) was added, and the tubes were returned to 370C for 60 min. Hemolysis was quantitated as follows. The reaction mixtures (0.2 ml) were diluted 1:10 by the addition of 1.8 ml of PBS, and the tubes were centrifuged at 1,200 rpm for 10 min to sediment the remaining SRBC. Hemoglobin concentration in the supernatant fluids was determined spectrophotometrically at 420 nm employing a Beckman model 26 doublebeam spectrophotometer with PBS in the reference cell. All experiments included controls to correct for nonimmune lysis. The spectrophotometric assay of ME-II mini-extract preparations was the same as for ME-I preparations, except that 25-z1l portions were assayed, adding 25 Al of the ME buffer (minus polymyxin) to compensate for volume and to provide the additional buffering capacity required for ME-Il preparations. The same basic procedure was also employed to define hemoglobin release as a function of antibody (antitoxin) and antigen (purified LT or B subunit of choleragen) concentration. Microtiter method for the assay of E. coli LT by PIH. The PIH assay for E. coli LT in ME-I preparations was performed as follows, employing the Microtiter titration system (Cooke Laboratory Products, Alexandria, Va.). Duplicate twofold dilutions of 0.025-ml portions of ME-I preparations were performed in U-well plates (Linbro Scientific Co., Hamden, Conn.), employing equal volumes of 0.02 M phosphate buffer (pH 6.7) plus 0.9% NaCl. Next, each well received 0.025 ml of washed SRBC (1% suspension in the same 0.02 M phosphate buffer plus saline), and the plate was sealed and incubated at 37°C for 30 min. Each well then received 0.025 ml of the 1:100 dilution of rabbit antitoxin (in PBS plus
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0.02% bovine serum albumin). After another incubation at 37°C for 30 min, each well received 0.025 ml of guinea pig complement (1:10 in PBS). The final incubation was for 60 min at 37°C; however, the optimal time for recording hemolysis was 20 min after the plates were returned to room temperature. The titer recorded was the highest dilution of ME-I z preparation eliciting greater than 50% hemolysis of 0 CMJ the SRBC.
RESULTS Basis for the direct assay of E. coli LT in polymyxin-release preparations by PIH. Previously we described a polymyxin-release, or mini-extraction, procedure which consistently yielded sufficient quantities of LT from E. coli isolates for detection by an in vitro serological assay, the LIT. An indirect method was necessitated because the buffer conditions employed for the release of LT from the E. coli cells were compatible with the antigen-antibody reaction but not with LT sensitization of SRBC. Subsequently, a polymyxin-release buffer (ME buffer) was designed to accommodate LT sensitization of SRBC (see Materials and Methods). Polymyxin mini-extract preparations could then be assayed for LT by a direct method employing PIH. The assay, described in detail in Materials and Methods, includes a sequence of three incubation periods providing for (i) sensitization of SRBC by LT, (ii) interaction of antitoxin with LT-SRBC, and (iii) complementmediated lysis of the SRBC immune complexes, respectively. Characterization of the PIH assay for LT. Early in this work it became apparent that the spectrophotometric determination of hemoglobin released from SRBC via PIH could facilitate characterization of this reaction with regard to specificity and sensitivity. Therefore, hemoglobin release as a function of antigen (LT) and antibody (anti-LT) concentration was investigated. In addition, we also employed the anticholeragen antiserum and the purified preparation of choleragen B subunit protein available to us, since the serological cross-identity of LT and choleragen is well known (3, 12), and it appears that this homology resides in the B subunit portion of the cholera toxin molecule (W. F. Osborne and S. H. Richardson, Abstr. Annu. Meet. Am. Soc. Microbiol. 1976, B73, p. 23). The results shown in Fig. 1 demonstrate that both the specific anti-LT rabbit serum and the horse anti-choleragen serum mediated the PIH of LT-sensitized SRBC to a very similar extent, indicating that the degree of hemolysis is a function of antigen (LT) concentration. Less than 100 ng of LT was detectable with either antiserum.
o
51
>03 z
w 0 0.2 _1
~0.1 500 125 31 NANOGRAMS PROTEIN (LT) FIG. 1. PIH of LT-SRBC as a function of antigen concentration. Rabbit anti-LT serum (circles) and horse anti-cholera toxin serum (triangles) were employed at a 1:200 dilution. Optical density at 420 nm represents hemoglobin release from hemolyzed cells.
Figure 2 shows PIH as a function of antitoxin concentration with SRBC sensitized with 125 ng of LT protein. As expected, these results show that PIH is also a function of antibody concentration. The results presented in Fig. 3 show that purified B subunit of choleragen also sensitized SRBC to PIH. When either anti-LT or anti-choleragen sera were employed at a 1:100 dilution to titrate the B protein, a large prozone effect was evident at concentrations of antigen greater than 10 ng. This prozone effect was less when the sera were employed at a 1:50 dilution. Therefore, excess (free) B protein effectively mediates lysis inhibition, as does the LT of E. coli. Also, the homologous as well as the heterologous antiserum detected as little as 1.0 ng of the B protein of choleragen. Identification of enterotoxigenic E. coli by PIH. Subsequent work has dealt with the problem of differentiating enterotoxigenic (LT-positive) and non-enterotoxigenic isolates of E. coli. In the first series of experiments, approximately 300 isolates of E. coli were employed to prepare polymyxin mini-extracts (ME-I preparations; see Materials and Methods) for the assay of LT by the Microtiter technique employing PIH. It was found that ME-I preparations
VOL. 16, 1977
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positive for LT by the adrenal cell assay were also positive by PIH, with titers ranging from 1:4 to 1:64. However, since microtiter results are difficult to represent other than qualitatively, we sought a technique for recording data in a more objective and quantitative fashion. The spectrophotometric assay, described in Materials and Methods, satisfied this need. Figure 4 shows the results obtained by the spectrophotometric technique with ME-I preparations derived from 168 adrenal cell-positive and 140 adrenal cell-negative E. coli isolates. All but six non-enterotoxigenic isolates (96%) caused the release of less than 30 gg of hemoglobin, whereas all but three enterotoxigenic isolates (98%) caused the release of more than 50 ,ug of hemoglobin from SRBC when 10-,l portions of ME-I preparations were assayed. Thus, Fig. 4 shows that hemoglobin released as a conse160 40 10 2.5 0.6 quence of immune lysis can readily be differenNANOGRAMS PROTEIN (CT--B SUBUNIT) tiated, by spectrophotometric quantitation, FIG. 3. PIH of SRBC sensitized with the B subfrom that which is not complement mediated. unit protein of cholera toxin as a function of antigen Simplification of the polymyxin-release concentration. Rabbit anti-LT serum (circles) and technique. The next series of experiments dealt horse anti-cholera toxin serum (triangles) were emwith the possibility that mini-extract preparaat 1:50 (solid symbols) and at 1:100 (open tions obtained from the overnight (18 h) CYE ployed symbols) dilutions. Optical density at 420 nm reprebroth cultures might contain detectable quanti- sents hemoglobin release from hemolyzed cells. ties of LT. It was first determined that the supernatant fluids from such cultures did not leased LT by the PIH assay. This introduced interfere with the detection of polymyxin-rethe possibility of releasing LT with polymyxin in the presence of the spent culture medium, 0.5 i.e., performing the mini-extraction process with whole cultures rather than with cells previously recovered by centrifugation. z Based upon these preliminary results, a seco 04 ond mini-extraction procedure (ME-Il) was designed and employed in conjunction with the spectrophotometric PIH assay for LT. Figure 5 shows the results obtained with the first 1,000 >0.3 assays performed with ME-II preparations. All of the ME-II preparations derived from adrenal z cell-positive E. coli isolates contained quantiuJ ties of LT that were sufficient for detection by PIH when 25 ul of ME-II preparation was assayed. According to the data shown in Fig. 5, only 0.7% (7 out of 1,000 isolates assayed) might ~0.1 possibly be misidentified if one assumes a 10%o error in the assay. This, according to our experience with the adrenal cell assay for LT, is within the margin of error of any other assay available at the present time. 160 640 2560 10 40 DILUTION of SERUM DISCUSSION FIG. 2. PIH of LT-SRBC as a function of antibody Fresh washed SRBC are readily sensitized concentration. Rabbit anti-LT serum (circles) and horse anti-cholera toxin serum (triangles) were ti- with the low-molecular-weight LT which is retrated with sheep cells sensitized with 125 ng of LT leased from enterotoxigenic E. coli by polyprotein. Optical density at 420 nm represents hemo- myxin. LT-SRBC is rapidly lysed by anti-LT globin release from hemolyzed cells. antibody in a complement-mediated reaction,
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PIH. Having purified the LT of E. coli (8), we obtained specific anti-LT antiserum as a reagent for the detection of LT in polymyxin mini-extract preparations. For technical reasons, the first method to be developed was based on inhibition of lysis in a standardized anti-LT/LT-SRBC reaction system; this assay has been described as the LIT assay. The LIT assay requires purified LT as a reagent and therefore might find limited usefulness. However, the direct PIH method described here requires anti-LT antiserum as the sole specific reagent needed to perform the assay. Parenthetically, it should be noted that the LIT assay procedure might be useful for detecting enterotoxins serologically cross-reactive with LT but which do not react, as LT does, with SRBC. Recent reports of enterotoxin production by other enteric bacteria (11, 15, 16; A. Ljung, S. Blomberg, B. Wretlind, and T. Wadstrom, Abstr. Annu. Meet. Am. Soc. Microbiol. 1976, B74, p. 23) suggest that such enterotoxins may exist. A major advantage of the direct method of detecting LT by PIH is its ready adaptability to either the Microtiter technique or to a tube method employing the spectrophotometric deternination of hemoglobin release. Basically the same results can be obtained employing the Microtiter technique in conjunction with ME-I 70-
[AC IADRENAL CELL
+
60-
U) I- 50 w 0 40 0 m
'U
30H
z 20-
10I
60 100 140 180 220 0 20 MICROGRAMS HEMOGLOBIN RELEASED FIG. 4. Assay for LT in ME-I polymyxin miniextract preparations by the PIH assay with hemoglobin release recorded at 420 nm. Isolates were also assayed by the adrenal cell method; 168 were positive for LT, and 140 were negative for LT by both methods. Hemoglobin release via immune hemolysis was calculated on the basis of LT-negative controls assayed in duplicate in every assay.
U- 700 0
50
40 Z
30 2010-
5 10 15 20 30 50 70 90 110 130 MICROGRAMS HEMOGLOBIN RELEASED FIG. 5. Assay for enterotoxin (LT) in ME-II polymyxin mini-extract preparations by the PIH assay with hemoglobin release recorded at 420 nm. Isolates were also assayed by the adrenal cell method; 171 were positive for LT, and 829 were negative for LT by both methods. Hemoglobin release via immune hemolysis was calculated on the basis of two LT-negative controls assayed in duplicate in every assay.
preparations (supernatants from polymyxintreated cells) or the spectrophotometric method in conjunction with ME-II preparations (supernatants from polymyxin-treated whole cultures). However, the spectrophotometric method performed with ME-II preparations is not only more sensitive by virtue of the amplification rendered by the high absorbancy of hemoglobin at 420 nm, but it also facilitates an objective rather than a subjective method of recording data. Thus, the increased sensitivity of the spectrophotometric PIH assay aided in the simplification of the polymyxin-release (mini-extraction) procedure, i.e., the use of whole, overnight (18 h) CYE cultures instead of cells derived from young secondary subcultures, without decreasing the sensitivity of the assay. This is an important feature in that LT determinations can be accomplished within 24 h after culture inoculation and in a more economical fashion. By routinely assaying E. coli cultures for LT by both the spectrophotometric PIH assay and the adrenal cell technique, we have found the in vitro serological method to be at least as sensitive as the tissue culture method for detecting LT production. Of course, the possibility remains that some few isolates of potentially
SEROLOGICAL ASSAY FOR E. COLI LT
VOL. 16, 1977
LT-positive E. coli may not produce LT in vitro and therefore may remain undetected by either assay, but this possibility is considered highly unlikely. It should be noted that in 1973, we described a Casamino Acids-based medium supplemented with 0.6% yeast extract (Difco; pH 8.5) which facilitated the production of maximum yields of LT in vitro (6). We highly recommend the use of this culture medium, now referred to as CYE medium, in conjunction with the in vitro PIH assay for LT. This suggestion is based not only on our accumulated experience with this medium but also on the basis of reports by other investigators who have confirmed the effectiveness of this formulation for producing maximum yields of enterotoxin by E. coli (9, 18). In summary, the sensitivity and specificity of the PIH assay for LT has proven sufficient for the identification of enterotoxigenic (LT-positive) E. coli. Also, the spectrophotometric PIH assay can be employed for either qualitative or quantitative determinations of LT according to the needs of the investigator. The extreme sensitivity of the assay demonstrated with the antigenically related B subunit protein of choleragen also suggests other possible uses for this technique. We are hopeful that this serological method will satisfy the needs ofthose investigators who find it impractical or impossible to employ biological assays for LT that depend on the availability of large animal care and/or tissue culture facilities. ACKNOWLEDGMENTS This investigation was supported by contract no. 200-750536 from the Center for Disease Control, Atlanta, Ga., and by Public Health Service grant no. AI-13385 from the National Institute of Allergy and Infectious Diseases. We acknowledge the technical assistance of D'Lynn Satterwhite and Steve Hamilton. K. Boyle typed this manuscript. LITERATURE CITED 1. 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. 2. DuPont, H. L., J. Olarte, D. G. Evans, L. K. Pickering, E. Galindo, and D. J. Evans, Jr. 1975. Comparative susceptibility of Latin American and United States students to enteric pathogens. N. Engl. J. Med.
295:1520-1521.
3. Evans, D. G., D. J. Evans, Jr., and S. L. Gorbach. 1973. Identification of enterotoxigenic Escherichia coli and serum antitoxin activity by the vascular permeability factor assay. Infect. Immun. 8:731-735. 4. Evans, D. J., Jr., and D. G. Evans. 1973. Three characteristics associated with enterotoxigenic Escherichia coli isolated from man. Infect. Immun. 8:322-328. 5. Evans, D. J., Jr., and D. G. Evans. 1977. Inhibition of immune hemolysis: a serological assay for the heat-
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labile enterotoxin of Escherichia coli. J. Clin. Microbiol. 5:100-105. 6. Evans, D. J., Jr., D. G. Evans, and S. L. Gorbach. 1973. Production of vascular permeability factor by enterotoxigenic Escherichia coli isolated from man. Infect. Immun. 8:725-730. 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. J., Jr., D. G. Evans, S. H. Richardson, and S. L. Gorbach. 1976. Purification of the polymyxin-released, heat-labile enterotoxin of Escherichia coli. J. Infect. Dis. 133:S97-S102. 9. Giannella, R. A. 1976. Suckling mouse model for detection of heat-stable Escherichia coli enterotoxin: characteristics of the model. Infect. Immun. 14:95-99. 10. Gorbach, S. L., B. H. Kean, D. G. Evans, D. J. Evans, Jr., and D. Bessudo. 1975. Travelers' diarrhea and toxigenic Escherichia coli. N. Engl. J. Med. 292:933936. 11. Guerrant, R. L., R. A. Moore, P. M. Kirchenfeld, and M. A. Sande. 1975. Role of toxigenic and invasive bacteria in acute diarrhea of childhood. N. Engl. J. Med. 293:567-573. 12. Gyles, C. L. 1974. Immunological study of the heatlabile enterotoxins of Escherichia coli and Vibrio cholerae. Infect. Immun. 9:564-570. 13. Gyles, C. L. 1974. Relationships between heat-labile enterotoxins of Escherichia coli and Vibrio cholerae. J. Infect. Dis. 129:277-383. 14. Holmgren, J., 0. Soderlind, and T. Wadatrom. 1973. Cross-reactivity between heat labile enterotoxins of Vibrio cholerae and Escherichia coli in neutralization tests in rabbit ileum and skin. Acta Pathol. Microbiol. Scand. 81:757-762. 15. Klipstein, F. A., L. V. Holdeman, J. J. Corcino, and W. E. C. Moore. 1973. Enterotoxigenic intestinal bacteria in tropical sprue. Ann. Int. Med. 79:632-641. 16. Klipstein, F. A., H. B. Short, R. F. Engert, L. Jean, and G. A. Weaver. 1976. Contamination of the small intestine by enterotoxigenic coliform bacteria among the rural population of Haiti. Gastroenterology 70:1035-1041. 17. Merson, M. H., G. K. Morris, D. A. Sack, J. G. Wells, J. C. Feeley, R. B. Sack, W. B. Creech, A. Z. Kapikian, and E. J. Gangarosa. 1976. Travelers' diarrhea in Mexico. A prospective study of physicians and family members attending a congress. N. Engl. J. Med. 294:1299-1305. 18. Mundell, D. H., C. R. Anselmo, and R. M. Wishnow. 1976. Factors influencing heat-labile Escherichia coli enterotoxin activity. Infect. Immun. 14:383-388. 19. Nalin, D. R., A. Al-Mahmud, G. Curlin, A. Ahmed, and J. Peterson. 1974. Cholera toxoid boosts serumEscherichia coli antitoxin in humans. Infect. Immun. 10:747-749. 20. Sack, R. B. 1975. Human diarrheal disease caused by enterotoxigenic Escherichia coli. Annu. Rev. Microbiol. 29:333-353. 21. Sack, R. B., N. Hirschhorn, 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. 22. Sack, R. B., B. Jacobs, and R. Mitra. 1974. Antitoxin responses to infections with enterotoxigenic Escherichia coli. J. Infect. Dis. 129:330-335. 23. Smith, N. W., and R. B. Sack. 1973. Immunologic crossreactions of enterotoxins from Escherichia coli and Vibrio cholerae. J. Infect. Dis. 127:164-170.
Vol. 16, No. 2 Printed in U.S.A.
Direct Serological Assay for the Heat-Labile Enterotoxin of Escherichia coli, Using Passive Immune Hemolysis DOYLE J. EVANS, JR.,* AND DOLORES G. EVANS Program in Infectious Diseases and Clinical Microbiology, The University of Te-xas Medical School at Houston, Houston, Texas 77030 Received for publication 29 November 1976
Sheep erythrocytes sensitized with the heat-labile enterotoxin (LT) ofEscherichia coli exhibit passive immune hemolysis (PIH) when exposed to specific antitoxin and complement. Thus, PIH serves as the basis for an in vitro serological assay for LT that is sufficiently specific and sensitive to differentiate LT-positive and LT-negative E. coli isolates. The PIH assay for E. coli LT has been performed with the standard Microtiter system and also by a tube method employing the spectrophotometric determination of hemoglobin release. The spectrophotometric method enhances the sensitivity, accuracy, and objectivity of the PIH assay. The increased sensitivity of the spectrophotometric method also facilitates the identification of LT-positive cultures employing polymyxin "miniextracts" of whole overnight (18 h) broth cultures of 2.0% Casamino Acids-0.6% yeast extract-salts medium rather than mini-extracts of cells derived from 3.5-h subcultures. Thus, large numbers of E. coli isolates can be individually tested for LT in less than 24 h after broth inoculation by a rapid in vitro assay which requires anti-LT serum as the only specific reagent. Several varieties of enterotoxigenic Escherichia coli are associated with acute diarrhea in man. These may produce either a heat-labile enterotoxin (LT), a heat-stable enterotoxin, or both (11, 17). Of these enterotoxins, only LT is known to be antigenic (3, 20). Antitoxin prepared against the LT of any one strain neutralizes the biological activity of all other LT-producing strains of E. coli (3, 12-14, 23). Also, E. coli LT and Vibrio cholerae choleragen display considerable immunological cross-identity (3, 12-14, 19, 23). The apparently universal immunological identity of LT is indeed fortuitous in that it has facilitated studies into humoral antitoxin response in relation to enterotoxigenic E. coli-associated diarrhea (17, 21, 22). To further investigations in this important area, we developed an in vitro microtiter assay for detecting humoral antitoxin responses to LT (D. J. Evans, Jr., D. G. Evans, and G. Ruiz-Palacios, Prog. Abstr. Intersci. Conf. Antimicrob. Agents Chemother., 16th, Chicago, Abstr. 368, 1976). The Microtiter assay for anti-LT antibody thus employed by our laboratory utilizes the principle of passive immune hemolysis (PIH), i.e., complement-mediated lysis of LT-sensitized sheep erythrocytes (LT-SRBC) by antitoxin. PIH also provided the basis for an indirect in vitro serological assay for LT which we recently described (5). This lysis inhibition test (LIT) for LT employs preexposure of a standard amount
of rabbit antitoxin to E. coli test preparations before the addition of LT-SRBC and complement, thus allowing soluble LT in a test sample to competitively inhibit the immune hemolysis reaction. Although the LIT assay for LT proved to be highly efficient in detecting LT-producing E. coli isolates and differentiating these from non-enterotoxigenic E. coli, its major drawback is the requirement for purified LT as a reagent. We now describe a direct in vitro assay for LT, also utilizing the principle of PIH, which is simple and rapid in performance, more sensitive than the LIT procedure, and does not require purified LT as a reagent. MATERIALS AND METHODS Bacterial cultures. E. coli strain H-10407, isolated from a case of diarrhea in Dacca, Bangladesh, was used for the large-scale production of LT (6, 7). One thousand E. coli isolates were employed in investigating the specificity and sensitivity of the serological assays for LT. These were isolated from human subjects in Bangladesh, the United States, and Mexico during the course of investigations into the association of enterotoxigenic E. coli with acute diarrhea (2-4, 10). Of these 1,000 isolates, 171 were positive for LT when tested individually by the cultured Y-1 adrenal tumor cell assay of Donta (1), and 829 were negative in the adrenal cell assay. The LT activity of all adrenal cell-positive cultures was neutralized by the hyperimmune rabbit antitoxin prepared against the LT of E. coli H-10407. Stock cul-
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VOL. 16, 1977 tures were maintained on 2.0% peptone-0.5% NaCl2.0% agar slants. Preparations of E. coli LT and antitoxin. The low-molecular-weight LT of E. coli H-10407 was obtained by the polymyxin-release technique (7) and purified by ammonium sulfate fractionation and Affi-Gel 202 (Bio-Rad Laboratories, Richmond, Calif.) chromatography as recently described in detail (8). Antitoxin was obtained by hyperimmunizing adult albino rabbits with increasing doses of LT and bleeding the animals 14 days after the final injection (3). The sera were pooled and stored at -450C. Anti-cholera toxin, a lyophilized horse antiserum, was obtained from Nobuya Ohtomo (Chemosero-therapeutic Research Institute, Kumamoto, Japan). Purified "B" subunit protein of cholera toxin, choleragen, was prepared by W. F. Osborne and S. H. Richardson and was the generous gift of S. H. Richardson. This material was maintained in the lyophilized state until immediately prior to use. Mini-extract preparations. LT was released from E. coli cells by the polymyxin-release technique (7), except that buffer conditions and polymyxin concentration were modified as described below. The term mini-extraction procedure is used in reference to the small-scale preparation of polymyxin "extracts." Two different mini-extraction procedures were employed. The first and basic mini-extraction procedure (ME-I) was as follows. Cultures were prepared by loop inoculation of 10 ml of 2.0% Casamino Acids (Difco)-0.6% yeast extract (Difco)-salts medium (CYE medium; 6) from stock slants. Fifty-milliliter Erlenmeyer flasks were agitated at 140 rpm for 18 h at 37°C in a rotary incubator-shaker. Subcultures were then prepared by inoculating 1.5 ml of the overnight broth culture into 10 ml of modified CYE medium (0.15% instead of 0.6% yeast extract; 7). After a 3.5-h incubation at 37°C as above, the secondary cultures were centrifuged at 12,000 x g for 20 min, and the supernatant fluids were discarded. Each pellet of bacteria was resuspended in 0.8 ml of polymyxin B (Aerosporin, Borroughs Wellcome Co., Research Triangle Park, N. C.) at a concentration of 0.25 mg/ml dissolved in 0.08 M phosphate buffer (pH 6.7) containing 0.9% NaCl and also 0.02% sodium azide as preservative (ME buffer). The cell suspensions were incubated by placing the centrifuge tubes in a 37°C water bath for 15 min, and the cells were then removed by centrifugation at 16,000 x g for 20 min. The resultant supernatants, or ME-I preparations, were recovered by disposable Pasteur pipettes and stored in small vials at 4°C until assayed. In the second mini-extraction procedure (ME-II), cultures were grown as above, except that only 5 ml of CYE medium was used per 50-ml Erlenmeyer flask. At 18 h after inoculation, 5 ml of ME buffer with polymyxin was added to each flask, and shaking was continued at 37°C for an additional 15 min. The supernatants (ME-II preparations) were recovered after centrifugation of the polymyxintreated cultures as above. ME-II preparations were assayed only by the spectrophotometric assay described below. Spectrophotometric method for the assay of E.
SEROLOGICAL ASSAY FOR E. COLI LT
605
coli LT employing PIH. The following reagents were employed to assay E. coli ME-I and ME-II preparations for LT. Fresh SRBC, stored at 4°C in Alsever solution, were washed twice with 8 volumes of 0.08 M phosphate buffer (pH 6.7) containing 0.9% NaCl and finally resuspended at a concentration of 10% in 0.02 M phosphate buffer (pH 6.7) plus 0.9% NaCl. These cells remained sufficiently stable at 4°C for 48 to 72 h after washing. Lyophilized guinea pig complement (Grand Island Biological Co., Grand Island, N.Y.) was restored as per instructions and then stored at -20°C. Rabbit anti-LT antiserum, prepared as above, was diluted 1:100 in phosphatebuffered saline (PBS, 0.1 M, pH 7.2) and maintained at4°C. The assay for LT in ME-I preparations was performed as follows. Duplicate disposable tubes (1.0 by 5.0 cm) received 40 ,l of 0.02 M phosphate buffer plus 0.9% NaCl (pH 6.7), 10 ,ul of the ME-I preparation, and finally 50 ,ul of a 1.0% suspension of SRBC freshly prepared from the 10% stock suspension by dilution in the 0.02 M phosphate buffer (pH 6.7) plus 0.9% NaCl. These reaction mixtures were incubated with either intermittent or continuous shaking for 30 min at 37°C in order to allow LT, if present, to adsorb to the SRBC. Next, 50 ,lI of antitoxin (1:100 in PBS) was added, and the tubes were incubated for another 30 min at 37°C to allow antibody to react with the LT-SRBC complexes. Next, 50 ,lp of guinea pig complement (diluted 1:10 in PBS) was added, and the tubes were returned to 370C for 60 min. Hemolysis was quantitated as follows. The reaction mixtures (0.2 ml) were diluted 1:10 by the addition of 1.8 ml of PBS, and the tubes were centrifuged at 1,200 rpm for 10 min to sediment the remaining SRBC. Hemoglobin concentration in the supernatant fluids was determined spectrophotometrically at 420 nm employing a Beckman model 26 doublebeam spectrophotometer with PBS in the reference cell. All experiments included controls to correct for nonimmune lysis. The spectrophotometric assay of ME-II mini-extract preparations was the same as for ME-I preparations, except that 25-z1l portions were assayed, adding 25 Al of the ME buffer (minus polymyxin) to compensate for volume and to provide the additional buffering capacity required for ME-Il preparations. The same basic procedure was also employed to define hemoglobin release as a function of antibody (antitoxin) and antigen (purified LT or B subunit of choleragen) concentration. Microtiter method for the assay of E. coli LT by PIH. The PIH assay for E. coli LT in ME-I preparations was performed as follows, employing the Microtiter titration system (Cooke Laboratory Products, Alexandria, Va.). Duplicate twofold dilutions of 0.025-ml portions of ME-I preparations were performed in U-well plates (Linbro Scientific Co., Hamden, Conn.), employing equal volumes of 0.02 M phosphate buffer (pH 6.7) plus 0.9% NaCl. Next, each well received 0.025 ml of washed SRBC (1% suspension in the same 0.02 M phosphate buffer plus saline), and the plate was sealed and incubated at 37°C for 30 min. Each well then received 0.025 ml of the 1:100 dilution of rabbit antitoxin (in PBS plus
606
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0.02% bovine serum albumin). After another incubation at 37°C for 30 min, each well received 0.025 ml of guinea pig complement (1:10 in PBS). The final incubation was for 60 min at 37°C; however, the optimal time for recording hemolysis was 20 min after the plates were returned to room temperature. The titer recorded was the highest dilution of ME-I z preparation eliciting greater than 50% hemolysis of 0 CMJ the SRBC.
RESULTS Basis for the direct assay of E. coli LT in polymyxin-release preparations by PIH. Previously we described a polymyxin-release, or mini-extraction, procedure which consistently yielded sufficient quantities of LT from E. coli isolates for detection by an in vitro serological assay, the LIT. An indirect method was necessitated because the buffer conditions employed for the release of LT from the E. coli cells were compatible with the antigen-antibody reaction but not with LT sensitization of SRBC. Subsequently, a polymyxin-release buffer (ME buffer) was designed to accommodate LT sensitization of SRBC (see Materials and Methods). Polymyxin mini-extract preparations could then be assayed for LT by a direct method employing PIH. The assay, described in detail in Materials and Methods, includes a sequence of three incubation periods providing for (i) sensitization of SRBC by LT, (ii) interaction of antitoxin with LT-SRBC, and (iii) complementmediated lysis of the SRBC immune complexes, respectively. Characterization of the PIH assay for LT. Early in this work it became apparent that the spectrophotometric determination of hemoglobin released from SRBC via PIH could facilitate characterization of this reaction with regard to specificity and sensitivity. Therefore, hemoglobin release as a function of antigen (LT) and antibody (anti-LT) concentration was investigated. In addition, we also employed the anticholeragen antiserum and the purified preparation of choleragen B subunit protein available to us, since the serological cross-identity of LT and choleragen is well known (3, 12), and it appears that this homology resides in the B subunit portion of the cholera toxin molecule (W. F. Osborne and S. H. Richardson, Abstr. Annu. Meet. Am. Soc. Microbiol. 1976, B73, p. 23). The results shown in Fig. 1 demonstrate that both the specific anti-LT rabbit serum and the horse anti-choleragen serum mediated the PIH of LT-sensitized SRBC to a very similar extent, indicating that the degree of hemolysis is a function of antigen (LT) concentration. Less than 100 ng of LT was detectable with either antiserum.
o
51
>03 z
w 0 0.2 _1
~0.1 500 125 31 NANOGRAMS PROTEIN (LT) FIG. 1. PIH of LT-SRBC as a function of antigen concentration. Rabbit anti-LT serum (circles) and horse anti-cholera toxin serum (triangles) were employed at a 1:200 dilution. Optical density at 420 nm represents hemoglobin release from hemolyzed cells.
Figure 2 shows PIH as a function of antitoxin concentration with SRBC sensitized with 125 ng of LT protein. As expected, these results show that PIH is also a function of antibody concentration. The results presented in Fig. 3 show that purified B subunit of choleragen also sensitized SRBC to PIH. When either anti-LT or anti-choleragen sera were employed at a 1:100 dilution to titrate the B protein, a large prozone effect was evident at concentrations of antigen greater than 10 ng. This prozone effect was less when the sera were employed at a 1:50 dilution. Therefore, excess (free) B protein effectively mediates lysis inhibition, as does the LT of E. coli. Also, the homologous as well as the heterologous antiserum detected as little as 1.0 ng of the B protein of choleragen. Identification of enterotoxigenic E. coli by PIH. Subsequent work has dealt with the problem of differentiating enterotoxigenic (LT-positive) and non-enterotoxigenic isolates of E. coli. In the first series of experiments, approximately 300 isolates of E. coli were employed to prepare polymyxin mini-extracts (ME-I preparations; see Materials and Methods) for the assay of LT by the Microtiter technique employing PIH. It was found that ME-I preparations
VOL. 16, 1977
SEROLOGICAL ASSAY FOR E. COLI LT ENTEROTOXIN
607
positive for LT by the adrenal cell assay were also positive by PIH, with titers ranging from 1:4 to 1:64. However, since microtiter results are difficult to represent other than qualitatively, we sought a technique for recording data in a more objective and quantitative fashion. The spectrophotometric assay, described in Materials and Methods, satisfied this need. Figure 4 shows the results obtained by the spectrophotometric technique with ME-I preparations derived from 168 adrenal cell-positive and 140 adrenal cell-negative E. coli isolates. All but six non-enterotoxigenic isolates (96%) caused the release of less than 30 gg of hemoglobin, whereas all but three enterotoxigenic isolates (98%) caused the release of more than 50 ,ug of hemoglobin from SRBC when 10-,l portions of ME-I preparations were assayed. Thus, Fig. 4 shows that hemoglobin released as a conse160 40 10 2.5 0.6 quence of immune lysis can readily be differenNANOGRAMS PROTEIN (CT--B SUBUNIT) tiated, by spectrophotometric quantitation, FIG. 3. PIH of SRBC sensitized with the B subfrom that which is not complement mediated. unit protein of cholera toxin as a function of antigen Simplification of the polymyxin-release concentration. Rabbit anti-LT serum (circles) and technique. The next series of experiments dealt horse anti-cholera toxin serum (triangles) were emwith the possibility that mini-extract preparaat 1:50 (solid symbols) and at 1:100 (open tions obtained from the overnight (18 h) CYE ployed symbols) dilutions. Optical density at 420 nm reprebroth cultures might contain detectable quanti- sents hemoglobin release from hemolyzed cells. ties of LT. It was first determined that the supernatant fluids from such cultures did not leased LT by the PIH assay. This introduced interfere with the detection of polymyxin-rethe possibility of releasing LT with polymyxin in the presence of the spent culture medium, 0.5 i.e., performing the mini-extraction process with whole cultures rather than with cells previously recovered by centrifugation. z Based upon these preliminary results, a seco 04 ond mini-extraction procedure (ME-Il) was designed and employed in conjunction with the spectrophotometric PIH assay for LT. Figure 5 shows the results obtained with the first 1,000 >0.3 assays performed with ME-II preparations. All of the ME-II preparations derived from adrenal z cell-positive E. coli isolates contained quantiuJ ties of LT that were sufficient for detection by PIH when 25 ul of ME-II preparation was assayed. According to the data shown in Fig. 5, only 0.7% (7 out of 1,000 isolates assayed) might ~0.1 possibly be misidentified if one assumes a 10%o error in the assay. This, according to our experience with the adrenal cell assay for LT, is within the margin of error of any other assay available at the present time. 160 640 2560 10 40 DILUTION of SERUM DISCUSSION FIG. 2. PIH of LT-SRBC as a function of antibody Fresh washed SRBC are readily sensitized concentration. Rabbit anti-LT serum (circles) and horse anti-cholera toxin serum (triangles) were ti- with the low-molecular-weight LT which is retrated with sheep cells sensitized with 125 ng of LT leased from enterotoxigenic E. coli by polyprotein. Optical density at 420 nm represents hemo- myxin. LT-SRBC is rapidly lysed by anti-LT globin release from hemolyzed cells. antibody in a complement-mediated reaction,
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PIH. Having purified the LT of E. coli (8), we obtained specific anti-LT antiserum as a reagent for the detection of LT in polymyxin mini-extract preparations. For technical reasons, the first method to be developed was based on inhibition of lysis in a standardized anti-LT/LT-SRBC reaction system; this assay has been described as the LIT assay. The LIT assay requires purified LT as a reagent and therefore might find limited usefulness. However, the direct PIH method described here requires anti-LT antiserum as the sole specific reagent needed to perform the assay. Parenthetically, it should be noted that the LIT assay procedure might be useful for detecting enterotoxins serologically cross-reactive with LT but which do not react, as LT does, with SRBC. Recent reports of enterotoxin production by other enteric bacteria (11, 15, 16; A. Ljung, S. Blomberg, B. Wretlind, and T. Wadstrom, Abstr. Annu. Meet. Am. Soc. Microbiol. 1976, B74, p. 23) suggest that such enterotoxins may exist. A major advantage of the direct method of detecting LT by PIH is its ready adaptability to either the Microtiter technique or to a tube method employing the spectrophotometric deternination of hemoglobin release. Basically the same results can be obtained employing the Microtiter technique in conjunction with ME-I 70-
[AC IADRENAL CELL
+
60-
U) I- 50 w 0 40 0 m
'U
30H
z 20-
10I
60 100 140 180 220 0 20 MICROGRAMS HEMOGLOBIN RELEASED FIG. 4. Assay for LT in ME-I polymyxin miniextract preparations by the PIH assay with hemoglobin release recorded at 420 nm. Isolates were also assayed by the adrenal cell method; 168 were positive for LT, and 140 were negative for LT by both methods. Hemoglobin release via immune hemolysis was calculated on the basis of LT-negative controls assayed in duplicate in every assay.
U- 700 0
50
40 Z
30 2010-
5 10 15 20 30 50 70 90 110 130 MICROGRAMS HEMOGLOBIN RELEASED FIG. 5. Assay for enterotoxin (LT) in ME-II polymyxin mini-extract preparations by the PIH assay with hemoglobin release recorded at 420 nm. Isolates were also assayed by the adrenal cell method; 171 were positive for LT, and 829 were negative for LT by both methods. Hemoglobin release via immune hemolysis was calculated on the basis of two LT-negative controls assayed in duplicate in every assay.
preparations (supernatants from polymyxintreated cells) or the spectrophotometric method in conjunction with ME-II preparations (supernatants from polymyxin-treated whole cultures). However, the spectrophotometric method performed with ME-II preparations is not only more sensitive by virtue of the amplification rendered by the high absorbancy of hemoglobin at 420 nm, but it also facilitates an objective rather than a subjective method of recording data. Thus, the increased sensitivity of the spectrophotometric PIH assay aided in the simplification of the polymyxin-release (mini-extraction) procedure, i.e., the use of whole, overnight (18 h) CYE cultures instead of cells derived from young secondary subcultures, without decreasing the sensitivity of the assay. This is an important feature in that LT determinations can be accomplished within 24 h after culture inoculation and in a more economical fashion. By routinely assaying E. coli cultures for LT by both the spectrophotometric PIH assay and the adrenal cell technique, we have found the in vitro serological method to be at least as sensitive as the tissue culture method for detecting LT production. Of course, the possibility remains that some few isolates of potentially
SEROLOGICAL ASSAY FOR E. COLI LT
VOL. 16, 1977
LT-positive E. coli may not produce LT in vitro and therefore may remain undetected by either assay, but this possibility is considered highly unlikely. It should be noted that in 1973, we described a Casamino Acids-based medium supplemented with 0.6% yeast extract (Difco; pH 8.5) which facilitated the production of maximum yields of LT in vitro (6). We highly recommend the use of this culture medium, now referred to as CYE medium, in conjunction with the in vitro PIH assay for LT. This suggestion is based not only on our accumulated experience with this medium but also on the basis of reports by other investigators who have confirmed the effectiveness of this formulation for producing maximum yields of enterotoxin by E. coli (9, 18). In summary, the sensitivity and specificity of the PIH assay for LT has proven sufficient for the identification of enterotoxigenic (LT-positive) E. coli. Also, the spectrophotometric PIH assay can be employed for either qualitative or quantitative determinations of LT according to the needs of the investigator. The extreme sensitivity of the assay demonstrated with the antigenically related B subunit protein of choleragen also suggests other possible uses for this technique. We are hopeful that this serological method will satisfy the needs ofthose investigators who find it impractical or impossible to employ biological assays for LT that depend on the availability of large animal care and/or tissue culture facilities. ACKNOWLEDGMENTS This investigation was supported by contract no. 200-750536 from the Center for Disease Control, Atlanta, Ga., and by Public Health Service grant no. AI-13385 from the National Institute of Allergy and Infectious Diseases. We acknowledge the technical assistance of D'Lynn Satterwhite and Steve Hamilton. K. Boyle typed this manuscript. LITERATURE CITED 1. 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. 2. DuPont, H. L., J. Olarte, D. G. Evans, L. K. Pickering, E. Galindo, and D. J. Evans, Jr. 1975. Comparative susceptibility of Latin American and United States students to enteric pathogens. N. Engl. J. Med.
295:1520-1521.
3. Evans, D. G., D. J. Evans, Jr., and S. L. Gorbach. 1973. Identification of enterotoxigenic Escherichia coli and serum antitoxin activity by the vascular permeability factor assay. Infect. Immun. 8:731-735. 4. Evans, D. J., Jr., and D. G. Evans. 1973. Three characteristics associated with enterotoxigenic Escherichia coli isolated from man. Infect. Immun. 8:322-328. 5. Evans, D. J., Jr., and D. G. Evans. 1977. Inhibition of immune hemolysis: a serological assay for the heat-
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labile enterotoxin of Escherichia coli. J. Clin. Microbiol. 5:100-105. 6. Evans, D. J., Jr., D. G. Evans, and S. L. Gorbach. 1973. Production of vascular permeability factor by enterotoxigenic Escherichia coli isolated from man. Infect. Immun. 8:725-730. 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. J., Jr., D. G. Evans, S. H. Richardson, and S. L. Gorbach. 1976. Purification of the polymyxin-released, heat-labile enterotoxin of Escherichia coli. J. Infect. Dis. 133:S97-S102. 9. Giannella, R. A. 1976. Suckling mouse model for detection of heat-stable Escherichia coli enterotoxin: characteristics of the model. Infect. Immun. 14:95-99. 10. Gorbach, S. L., B. H. Kean, D. G. Evans, D. J. Evans, Jr., and D. Bessudo. 1975. Travelers' diarrhea and toxigenic Escherichia coli. N. Engl. J. Med. 292:933936. 11. Guerrant, R. L., R. A. Moore, P. M. Kirchenfeld, and M. A. Sande. 1975. Role of toxigenic and invasive bacteria in acute diarrhea of childhood. N. Engl. J. Med. 293:567-573. 12. Gyles, C. L. 1974. Immunological study of the heatlabile enterotoxins of Escherichia coli and Vibrio cholerae. Infect. Immun. 9:564-570. 13. Gyles, C. L. 1974. Relationships between heat-labile enterotoxins of Escherichia coli and Vibrio cholerae. J. Infect. Dis. 129:277-383. 14. Holmgren, J., 0. Soderlind, and T. Wadatrom. 1973. Cross-reactivity between heat labile enterotoxins of Vibrio cholerae and Escherichia coli in neutralization tests in rabbit ileum and skin. Acta Pathol. Microbiol. Scand. 81:757-762. 15. Klipstein, F. A., L. V. Holdeman, J. J. Corcino, and W. E. C. Moore. 1973. Enterotoxigenic intestinal bacteria in tropical sprue. Ann. Int. Med. 79:632-641. 16. Klipstein, F. A., H. B. Short, R. F. Engert, L. Jean, and G. A. Weaver. 1976. Contamination of the small intestine by enterotoxigenic coliform bacteria among the rural population of Haiti. Gastroenterology 70:1035-1041. 17. Merson, M. H., G. K. Morris, D. A. Sack, J. G. Wells, J. C. Feeley, R. B. Sack, W. B. Creech, A. Z. Kapikian, and E. J. Gangarosa. 1976. Travelers' diarrhea in Mexico. A prospective study of physicians and family members attending a congress. N. Engl. J. Med. 294:1299-1305. 18. Mundell, D. H., C. R. Anselmo, and R. M. Wishnow. 1976. Factors influencing heat-labile Escherichia coli enterotoxin activity. Infect. Immun. 14:383-388. 19. Nalin, D. R., A. Al-Mahmud, G. Curlin, A. Ahmed, and J. Peterson. 1974. Cholera toxoid boosts serumEscherichia coli antitoxin in humans. Infect. Immun. 10:747-749. 20. Sack, R. B. 1975. Human diarrheal disease caused by enterotoxigenic Escherichia coli. Annu. Rev. Microbiol. 29:333-353. 21. Sack, R. B., N. Hirschhorn, 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. 22. Sack, R. B., B. Jacobs, and R. Mitra. 1974. Antitoxin responses to infections with enterotoxigenic Escherichia coli. J. Infect. Dis. 129:330-335. 23. Smith, N. W., and R. B. Sack. 1973. Immunologic crossreactions of enterotoxins from Escherichia coli and Vibrio cholerae. J. Infect. Dis. 127:164-170.
