INFECriON AND IMMUNrrY, Mar. 1974, p. 564-570Copyright 0 1974 American Society for Microbiology

Vol. 9, No. 3Printed in U.S.A.

Immunological Study of the Heat-Labile Enterotoxins ofEscherichia coli and Vibrio cholerae

CARLTON L. GYLES

Department of Veterinary Microbiology and Immunology, Ontario Veterinary College, University of Guelph,Guelph, Ontario, Canada

Received for publication 24 October 1973

Immunodiffusion experiments were conducted to associate a precipitin linewith Escherichia coli heat-labile enterotoxin (LT). Wild strains of porcine and ofhuman enteropathogenic E. coli as well as laboratory-derived enterotoxigenicvariants of E. coli K-12 were used for LT antigen preparations. These wereproduced mainly by ultrafiltration and ammonium sulfate precipitation of brothculture supernatants. When antisera with anti-LT activity were reacted withantigen preparations from Ent+ and Ent- variants of E. coli K-12, a line "a" wasgiven by Ent+ but not by Ent- preparations. Line "a" was removed byabsorption of anti-LT serum with antigen preparation from an Ent+ E. coli K-12,but was unaffected when the antigen preparation used to absorb the serum wasfrom an Ent- E. coli K-12. A line identical to "a" was given by antigenpreparations from wild strains of porcine enteropathogenic E. coli reacted withhomologous or heterologous anti-LT sera. One human strain of enteropathogenicE. coli was shown to possess an antigen identical to that which gave rise to line"a." To demonstrate this line it was necessary to use high concentrations ofgammaglobulin and high concentrations of the crude antigen preparations. LTpreparations reacted with anticholera toxin to give a line "c," which showed areaction of partial identity with line "b" produced by reaction of purecholeragenoid and anticholera toxin. Lines "a" and "c" gave reactions ofidentity.

The enterotoxin produced by Vibrio choleraehas been isolated in a pure form (4) and hasbeen well characterized (9, 10). In contrast, theenterotoxins produced by Escherichia coli havebeen neither isolated nor well characterized.Whereas a single enterotoxic substance is pro-duced by V. cholerae, two classes of enterotox-ins, one heat labile and the other heat stable,have been described for E. coli (5, 12, 13).The heat-labile E. coli enterotoxin (LT) bears

an antigenic relationship to the enterotoxin ofV. cholerae (6). Also, LT is determined by aplasmid (11, 12, 14, 15), and it is possible toproduce pairs of E. coli strains that are isogenicexcept that one has an enterotoxin plasmid(Ent+) and the other lacks the plasmid (Ent-).These factors are utilized in the present study,designed to demonstrate a precipitin line for LTand to investigate antigenic relationships be-tween LT and choleragen, between LT fromvarious serotypes of porcine enteropathogenicE. coli, and between LT from human andporcine strains of E. coli. Additionally, electro-phoresis and immunoelectrophoresis are ap-

plied to aid in the characterization of the E. coliheat-labile enterotoxin.

MATERIALS AND METHODSBacterial cultures. E. coli cultures used for the

preparation of antigens and the immunization of pigswere as follows. The Ent- K-12 strains used were 711and CRM. The Ent+ K-12 strains used were711(P307), 711(P155), 711(P130), 711(P25),711(P116), and CRM(P75) (the donor strains arelisted in parentheses). The Ent+ wild strains usedas donors of Ent plasmids were P307, P155, P130,P25, P116, and P75; the Ent+ wild strains of porcineorigin used for the immunization of pigs were P307,P155, P1253, P1108, P3111, and P491; the Ent+ wildstrains used that were of human origin were H10407(antiserum was produced against this strain), H339,and H410. Strain 711 was a nalidixic acid-resistant,lactose-negative, auxotrophic E. coli K-12, and CRMwas a prototrophic colicin-resistant K-12. The Ent+K-12 organisms were produced as previously de-scribed (12, 14). The wild strains of porcine entero-pathogenic E. coli represent nine serological types.All strains with the "P" designation are porcineenteropathogens; those with the "H" are humanenteropathogens.

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Preparation of broth supernatant antigens. TwoEnt and six Ent+ E. coli K-12 organisms (see above),six Ent+ porcine enteropathogens (P307, P155, P1253,P1108, P3111, and P491), and the three humanenteropathogens were used for preparation of brothsupernatant antigens. The medium in which theseorganisms were grown was syncase broth (3), modifiedby replacing 0.5% sucrose with 1.0% glucose, 1%Casamino Acids with 2%, and by addition of 0.01%tryptophane. The growth conditions were as de-scribed previously (8) except that the duration ofincubation was 20 h. The cultures were usually grownin 5- or 10-liter batches, and at least two batches wereproduced for each organism. Six batches were pre-pared for E. coli strains 711 and 711(P307).

Cell-free broth culture supernatant was obtainedby centrifugation followed by filtration through a0.45-tsm cellulose acetate membrane filter. Thisbroth culture supernatant was then subjected torepetitive concentration (by passage through a cellu-lose acetate filter with a molecular weight cut-off of30,000) and dilution in 0.1 M tris(hydroxymethyl)-aminomethane (Tris)-chloride buffer, pH 8.0. Theultrafiltration was terminated when approximately99.9% of the completely membrane-permeable con-stituents had been eliminated. The retentate waslyophilized and used as broth supernatant antigen.These preparations were reconstituted in distilledwater at concentrations ranging from 200 to 400mg/ml for use in immunodiffusion, immunoelectro-phoresis, and electrophoresis. Preparations from en-terotoxigenic organisms are referred to as tox+, andpreparations from non-enterotoxigenic organisms arereferred to as tox-.

Four batches of 711(P307) and 711(P155) brothsupernatant, as well as one batch of supernatant fromeach of the strains P491, P307, and H10407, weresubjected to precipitation with ammonium sulfate at80% saturation. The precipitate was dissolved in 0.1M Tris-chloride buffer, pH 8, and subjected to discon-tinuous ultrafiltration and subsequent freeze-dryingas described above.WCL antigen. Whole cell lysate (WCL) was pre-

pared from E. coli 711(P307) as previously reported(6). WCL solution at 2.5 mg per ml of 0.1 M Tris-chloride buffer (pH 8) was treated with solid ammo-nium sulfate to 60% saturation. The precipitate wasdissolved in 0.01 M Tris-chloride buffer, pH 8, anddialyzed against running tap water for 30 h. Aftercentrifugation at 10,000 x g for 10 min, the superna-tant was freeze-dried and used as WCL antigen.

Choleragenoid. Pure choleragenoid (4) was kindlysupplied in the freeze-dried state by R. A. Finkelstein.It was reconstituted in distilled water to a concentra-tion of 100 ,g/ml for use as an antigen in immunodif-fusion experiments.

Antisera. Antisera were prepared in pigs againstthe porcine enteropathogenic E. coli strains P307,P155, P1253, P1108, P3111, and P491, as well asagainst the human enteropathogen, H10407. A singleantiserum was used for each of these strains. Detailsof the vaccination procedure have been described (C.L. Gyles, J. Infect. Dis., in press). Essentially, themethod consisted of multiple intravenous injections of

live cultures into 8- to 12-week-old pigs. Anti-LTtiters of the sera were determined by their ability toneutralize LT preparations in ligated segments ofrabbit intestine (Gyles, in press). The antiserum withthe highest titer was that prepared against E. coliP491.The gammaglobulin was precipitated from each

serum by using 33% ammonium sulfate (1). Thegammaglobulin was then freeze-dried. It was reconsti-tuted in distilled water containing 0.1% sodium azideand stored at 5 C during use. The amount of wateradded effected a five-times concentration, comparedwith serum, for use in immunodiffusion, and a three-times concentration for use in immunoelectrophore-sis.

Anti-choleragenoid serum prepared in a horse (2)and anti-choleragenoid serum absorbed with chole-ragenoid were obtained from R. A. Finkelstein. Pu-rifed horse cholera antitoxin was obtained from theSwiss Serum and Vaccine Institute (SSVI), Bern,Switzerland.

Absorption of antisera. Samples of the anti-LTgammaglobulin from E. coli P491 were absorbed withbroth supernatant antigen preparations from theEnt- E. coli 711 and the Ent+ E. coli 711(P307).Freeze-dried gammaglobulin (50 mg) was dissolved in10 ml of distilled water, and 50 mg of the antigenpreparation was added. After incubation at 37 C for 2h, the material was centrifuged at 40,000 x g for 20min, and the processes of adding antigen, incubation,and centrifugation were repeated. The absorbed gam-maglobulin was then passed through a 0.45-Am cellu-lose acetate membrane filter and concentrated bydialysis against Ficoll (Pharmacia, Uppsala, Swe-den). Five-times concentrated gammaglobulin prepa-rations were used for immunodiffusion and three-times concentrated material was used for immuno-electrophoresis.

The SSVI chlora antitoxin was absorbed with E.coli 711 antigen preparation as follows. The 711antigen (30 mg) was added to 1 ml of the antitoxin.Two series of incubation and centrifugation werecarried out as described above, and the material wasfiltered through a 0.45-,um cellulose acetate mem-brane filter.

Immundiffusion. Immunodiffusion tests were con-ducted in 0.65% agarose in 0.05 M Gelman high-reso-lution buffer (Tris-barbital-sodium barbital, pH 8.8)as well as in 2% special Noble agar (Difco) at a pH of7.0 to 7.2 (Hyland Immuno-Plates). A 0.1% concentra-tion of sodium azide was used as a preservative inboth cases. With the agarose slides, wells contained 10ILliters and were 5 mm apart. With the Immuno-Plates, wells contained 8 Mliters and were 5 mm apart,center to center.

Immunodiffusion reactions were allowed to developat room temperature, and the plates were observedover a 7-day period. Initially, tests were conducted inthe agarose medium only, but once the Immuno-Plates proved satisfactory, tests were repeated inthese plates so that all tests were carried out in bothmedia.

Immunoelectrophoresis. Immunoelectrophoresiswas performed at room temperature on microscope

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slides overlaid with 0.65% agarose in 0.05 M high-reso-lution buffer, pH 8.8, containing 0.1% sodium azide.The electrode vessels contained 0.1 M high/reso-lution buffer, pH 8.8. E. coli antigen preparation(10 Aliters) was placed in the wells, and electro-phoresis was conducted for 60 min at 9 V/cm. Concen-trated anti-LT gammaglobulin or cholera antitoxin(60 iliters) was placed in the troughs, and the slideswere incubated at room temperature and observed for7 days.On two occasions, after electrophoresis of a sample

of enterotoxic E. coli 711(P307) antigen, the agarosewas divided into seven 1-cm sections, so that thesample well was the center of one section. Thesesections of agarose were extracted with high-resolu-tion buffer and tested in ligated segments of rabbitintestine.

Electrophoresis. Electrophoresis was carried outon cellulose acetate strips (Sepraphore III, Gelman,Ann Arbor, Mich.) in 0.05 M high-resolution buffer,pH 8.8, by using 300 V for 60 min. After electrophore-sis, the strips were stained with Ponceau S. In twocases, an enterotoxic antigen preparation was appliedas two adjacent spots. After electrophoresis, the stripwas divided longitudinally and one-half was stained.The unstained half was cut into seven fractionscorresponding to stained bands, and each fraction waseluted in high-resolution buffer.

Ligated intestine tests. Tests for enterotoxicitywere conducted in ligated segments of rabbit intestine(7). The antigen preparations as well as materialeluted from agarose and from cellulose acetate stripswere tested for enterotoxicity.

Experiments. Immunodiffusion tests were de-signed to: (i) compare the precipitin lines given byEnt+ and Ent- E. coli antigen preparations reactedwith antisera against Ent+ E. coli; (ii) determine theeffect of absorption of an anti-enterotoxic serum withantigen preparation from Ent+ and Ent- E. coli onthe lines produced in reactions with an Ent+ E. coliantigen preparation; (iii) compare the precipitin linesgiven by Ent+ and Ent- E. coli antigen preparationsreacted with cholera antitoxin; (iv) compare thereactions of antigens from Ent+ porcine and humanstrains of E. coli reacted with cholera antitoxin; (v)compare the reactions of enterotoxin antigen prepara-tions in homologous and heterologous anti-LT sera;and (vi) associate enterotoxic activity with a fractionof antigen preparation subjected to electrophoresis.

RESULTSAntigen preparations. The yield of solid

material from the broth supernatants rangedfrom 100 to 200 mg of broth per liter. Wildstrains of E. coli, particularly those which weremucoid, usually gave larger quantities of mate-rial than did the E. coli K-12 organisms. Prepa-rations of the Ent- E. coli K-12 were tested at2-mg doses in ligated segments of rabbit intes-tine and were always negative. Preparations ofthe Ent+ organisms were always positive at a1-mg dose and were tested at lower doses until a

level was found at which the ligated rabbit loopresponse was less than 1 ml of fluid per cm ofintestine. The most active preparation was abroth supernatant preparation of E. coli711(P307) that gave reactions in excess of 1ml/cm at a dose of 0.125 mg. There was onlyslight improvement in the specific activity ofthe enterotoxic broth supernatant antigen prep-arations as a result of the ammonium sulfateprecipitation. The ammonium sulfate-precipi-tated WCL compared favorably with the brothsupernatant preparations in enterotoxicity.Immunodiffusion tests. P491 antiserum was

reacted with broth supernatant antigen prepa-rations from the Ent- E. coli K-12 strains andfrom the Ent+ variants of E. coli K-12. Aprecipitin line, "a," was observed to be given byall the preparations from the Ent+ K-12 strainsand from all the strains of porcine enteropatho-gens and not by any of the preparations from theEnt- K-12 strains. This line is demonstrated inFig. 1. By reacting antigen preparations fromstrains 711 and 711(P307) with the six antiseraagainst strains of porcine enteropathogenic E.coli, it was shown that a line identical to line"a" could be given by each antiserum. Line"a" was best developed, however, by use of theP491 antiserum.P491 antisera in the unabsorbed state, ab-

FIG. 1. Reaction of one non-enterotoxic (tox) andthree porcine enterotoxic (tox+) antigen preparationswith antiserum (AS) against a porcine enterotoxigenicE. coli (P491). The antigen wells (1 to 4) containedbroth supernatant antigens prepared from E. colistrains 711, 711(P307), 711(P155), and 711(P116),respectively. Line "a" is given by the enterotoxicantigens but not by the non-enterotoxic antigen.

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sorbed with an Ent- 711 antigen preparation,and absorbed with an Ent' 711(P307) antigenpreparation were reacted with an antigen prepa-ration from E. coli strain 711(P307). The resultsof these tests are shown in Fig. 2. A lineconsistent with line "a" is given by the unab-sorbed P491 antiserum and by P491 antiserumabsorbed with the Ent- antigen preparation butnot by P491 antiserum absorbed with the Ent+antigen preparation.When antigen preparations from the Ent- E.

coli strain 711 and from the Ent+ strain711(P307) and choleragenoid were reacted withthe SSVI anticholera toxin, the lines shown inFig. 3 were obtained. Line "b" represents thereaction of pure choleragenoid with its anti-body. Two lines were obtained with the en-terotoxigenic antigen preparation and one linewas obtained with the non-enterotoxigenicpreparation. The line common to the tox+ andtox- antigen preparations was unrelated to thecholera toxin line. The line "c," which was notgiven by the preparation from the Ent- organ-ism, gave a reaction of partial identity with thecholera toxin line, "b." The pattern illustratedin Fig. 3 was obtained for all preparations ofEnt+ and Ent- E. coli K-12, in reactions withSSVI anticholera toxin.When anti-choleragenoid serum was used

instead of purified horse cholera antitoxin(SSVI) in the pattem illustrated in Fig. 3, the

FIG. 2. Reaction of an enterotoxic 711(P307) anti-gen preparation (center well) with unabsorbed anti-enterotoxic P491 serum (well 2), with P491 absorbedwith 711(P307) antigen (well 1), and with P491absorbed with 711 antigen (well 3). The line, whichappears to be line "a" (Fig. 1), is not given by theP491 serum absorbed with 711(P307) antigen.

FIG. 3. Reaction of SSVI anticholera toxin (centerwell) with pure choleragenoid (well 3), a porcineenterotoxic antigen (711(P307), well 2), and a non-enterotoxic antigen (711, well 3). Line "c" is given bythe enterotoxic but not the non-enterotoxic antigenand shows a reaction of partial identity with line "b,"the cholera toxin precipitin line.

following results were obtained. The precipitinlines were the same as those shown in Fig. 3except that the line common to the tox+ andtox- antigen preparations was absent, and thetwo lines that appeared (corresponding to lines"b" and "c") were weaker. Immunodiffusiontests were carried out to demonstrate that thesetwo lines obtained with anti-choleragenoidserum were identical to the corresponding linesproduced with the SSVI cholera antitoxin.Of the antigen preparations from the human

enteropathogenic E. coli, only those from strainH10407 were highly reactive in rabbit gut loops.Those from strain H339 required a 0.5-mg doseto give reactions of the order of 1 ml/cm, andthose from strain H410 were much less active.When preparations from these three strainswere reacted with P491 antiserum, a line identi-cal to line "a" was given by the H10407 prepara-tions but not by preparations from strains H410and H339.When 711(P307), H10407, and choleragenoid

antigens were tested against SSVI anticholeratoxin (Fig. 4), a line identical to line "c"9 wasgiven by the H10407 preparation. Antigen prep-arations from H10407 reacted with P491 an-tiserum to give a line identical to line "a." Aline related to line "a" was not observed whenthe H10407 preparations were reacted withantiserum against E. coli H10407.

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FIG. 4. Reaction of SSVI anticholera toxin (centerwell) with choleragenoid (well 1), a porcine en-terotoxic antigen preparation (well 2), and a humanenterotoxic antigen preparation (well 3). There is areaction of identity between line "c" and the outer-most line given by the human enterotoxic antigenpreparation.

When E. coli antigen preparations weretested against homologous antiserum, as manyas six lines usually appeared. With certaincombinations, however, only a few lines werevisible and were often accompanied by a massof precipitated material. Figure 5 illustratessuch a homologous reaction involving antigenand antisera from E. coli P491. This homolo-gous reaction gave a line identical to line "a,"given by 711(P116) antigen and P491 an-tiserum. Line "a" is identical to line "c," givenby 711(P116) antigen and SSVI anticholeratoxin.When choleragenoid was reacted with anti-

LT sera, a weak precipitin line was obtainedwith two (P491 and P3111) of seven antisera.This precipitin line was always very close to theantiserum well and gave a reation of partialidentity with the cholera toxin line. Because ofthe weakness of the line and its closeness to theantiserum well, it was not possible to determineits relationship to line "a."Immunoelectrophoresis. Reactions of broth

supernatant antigens from E. coli 711 and811(P307) and of WCL antigen from 711(P307)are illustrated in Fig. 6 and 7. When P491antiserum absorbed with 711 antigen wasused (Fig. 6), a single line close to the pointof sample application was given by antigenpreparations from the Ent+ but not by anti-gen preparations from the Ent- organisms.

FIG. 5. Reactions of porcine anti-enterotoxicserum (P491) and SSVI anticholera toxin (anti-chol)with homologous and heterologous antigen prepara-tions. The cholera toxoid line, "b," has a reaction ofpartial identity with line "c," given by the enterotoxicantigen 711(P116) (well 4) and anticholera toxin. Line"c" is identical to line "a," given by 711(P116) andP491 antiserum. Line "c" is also identical to a linegiven by antigen P491 (well 3) and antiserum P491.

FIG. 6. Precipitin line given by electrophoresedenterotoxic antigen preparations reacted with P491antiserum absorbed with E. coli 711 antigen prepara-tion. Well 1 contained 10 .liters of E. coli 711 brothsupernatant antigen preparation; well 2 contained 10uliters of 711(P307) broth supernatant antigen, andwell 3 contained 10 utliters of 711(P307) lysate antigen.The antiserum troughs contained 60 uliters of three-times concentrated P491 antiserum absorbed with E.coli 711 antigen preparation.

FIG. 7. Precipitin line given by electrophoresedenterotoxic antigen preparations reacted with SSVIanticholera toxin absorbed with E. coli 711 antigenpreparation. The wells contained the same antigens asin Fig. 6. The antiserum troughs contained 60 ,ilitersof anticholera toxin absorbed with E. coli 711 antigenpreparation.

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When the antiserum was SSVI anticholeratoxin absorbed with E. coli 711 antigen, a

similar line was observed (Fig. 7). With unab-sorbed P491 antiserum, three lines were givenby the 711(P307) antigens and two by the 711antigens. When the antigens were tested againstanti-choleragenoid serum, a single line at theorigin was given by 711(P307) and no line was

given by 711. No line was given when anti-choleragenoid serum absorbed with cholerage-noid was used.

Electrophoresis. Seven bands were detectedon stained strips of cellulose acetate on which711(P307) antigen preparations were subjectedto electrophoresis. The same number of bandswas recognized when 711 antigen preparationswere examined. Tests of materials eluted fromunstained half-strips showed that the en-

terotoxic activity was present in a fraction thatremained at the point of sample application.Similar results were obtained when fractionswere tested after elution from agarose slidesthat had been subjected to electrophoresis.

DISCUSSIONA precipitin line, "a," was demonstrated to

be due to an antigen present in porcine entero-pathogenic E. coli. It was shown by immunodif-fusion tests of antigens prepared from Ent+ andEnt- strains of E. coli K-12 and by studies withabsorbed sera that this antigen is attributableto the presence of the enterotoxin plasmid.Furthermore, line "a" was identical to line "c,"which was given by reactions of anticholeratoxin with antigens from enterotoxigenic strainsof E. coli, but not by reactions with antigensfrom non-enterotoxigenic strains. Line "c" (andtherefore line "a") has a reaction of partialidentity with the cholera toxin precipitin line"b." These findings strongly suggest that line"a" represents the reaction of E. coli heat-labileenterotoxin with its antibody.The antigen giving rise to line "a" appeared

to be identical in enterotoxic preparations fromvarious serotypes of porcine enteropathogenicE. coli. There also appeared to be identitybetween the antigen in porcine and in humanstrains of enteropathogenic E. coli in reactionswith antisera against porcine LT.The observation of a spur formation between

line "b" (the cholera toxoid line) and line "c" isconsistent with the finding (Gyles, in press)that E. coli LT is neutralized by high dilutionsof anticholera toxin, but cholera toxin is notneutralized to any significant extent by anti-E.coli LT. In that study it was also shown thatabsorption of cholera antitoxin with E. coli LT

preparation removed the anti-E. coli LT butnot the anticholera toxin activity of the serum.The results of both of these investigationsindicate that there are antigens common to E.coli LT and V. cholerae enterotoxin that inducethe formation of antibody that neutralizes theE. coli LT but not the V. cholerae enterotoxin.The immunoelectrophoretic studies, com-

bined with the tests for enterotoxicity of variouselectrophoresed fractions of toxin preparations,suggest that the line seen at the point of sampleapplication is due to E. colC*enterotoxin. Failureto detect a band on cellulose acetate strips thatcould be associated with enterotoxin may bedue to the presence of comparatively smallamounts of toxin in the antigen preparations.Furthermore, a band at the point of applicationis easily obscured. It appears that preparativeelectrophoresis or other methods relying on thecharge of the molecule may be useful in at-tempts to improve the specific activity of brothsupernatant preparations.

Failure to detect a line that could be associ-ated with enterotoxin in antigen preparationsfrom the two human enteropathogens H410 andH339 was likely due to a low content of toxin inthese preparations. When antigen preparationswith higher specific activity are produced, theprobability of demonstrating a precipitin linefor enterotoxin in most strains of human entero-pathogenic E. coli will be enhanced.

ACKNOWLEDGMENTSThe author is grateful to R. A. Finkelstein, S. L. Gorbach,

H. Williams Smith, S. Lariviere, and to the National Insti-tutes of Health for various contributions to this study. MarionWilke provided excellent technical assistance.

The research was supported in part by Canadian MedicalResearch Council grant MA4235.

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