INFECTION AND IMMUNITY, May 1983, p. 701-7070019-9567/83/050701-07$02.00/0Copyright © 1983, American Society for Microbiology

Vol. 40, No. 2

Purification and Characterization of Heat-Stable Enterotoxinfrom Bovine Enterotoxigenic Escherichia coli

ABDUL M. K. SAEED,' N. SRIRANGANATHAN,1* W. COSAND,2 AND D. BURGER1Department of Veterinary Microbiology and Pathology' and Program in Biochemistry and Biophysics,2

Washington State University, Pullman, Washington 99164

Received 28 December 1982/Accepted 27 February 1983

Heat-stable enterotoxin (ST) from Escherichia coli pathogenic for cattle was

mass produced in a chemically defined medium. The toxin was concentrated andpurified by sequentially applying batch adsorption chromatography on AmberliteXAD-2 resin, acetone fractionation, and preparative isoelectric focusing in a

flatbed granulated gel. Reverse-phase high-performance liquid chromatographywas used to purify the toxin further and to eliminate contarhinating ampholytes.The toxin was purified more than 2,000-fold and had a minimal effective dose ofless than 0.5 ng. It was biologically active after heating to 100°C for 30 min andwas not hydrolyzed by trypsin, pronase, and subtilisin, but it was inactivated bytreatment with 0.1 M 2-mercaptoethanol or 4 x i0- M dithiothreitol, suggestingthat disulfide bonds are essential for retaining its biological activity. The aminoacid analysis revealed 18 amino acid residues per molecule, which is in agreementwith the composition of ST from a human strain of enterotoxigenic E. coli. Theamino acid composition of our ST matched the published coding sequence of thelast 18 codons of TnJ618, a transposon isolated from the bovine enterotoxigenicE. coli strain B41 and shown to be present also in some strains of porcineenterotoxigenic E. coli. These findings further support the existence of a form ofST common to bovine, porcine, and human strains of enterotoxigenic E. coli.

Secretory diarrhea of human and other mam-malian neonates is often induced by enterotox-ins from enterotoxigenic strains of Escherichiacoli (ETEC) (9, 12, 17, 19, 20, 25, 26). Most ofthe ETEC isolated from cases of diarrhea inhumans and swine produce either or both of twodistinct classes of enterotoxins, a heat-labile(LT) enterotoxin of high molecular weight whichis readily destroyed at 60°C, and a heat-stable(ST) enterotoxin of low molecular weight (21,26, 27). Whereas some virulence attributes ofETEC, such as the colonization factors, arehighly host species specific, all of the LT toxinsof ETEC examined appear to be immunological-ly related and share antigenic determinants withthe enterotoxin of Vibrio cholerae (5, 8, 11, 30).Much less is known about the ST enterotoxins ofETEC which are poorly antigenic and thought tobe peptides of low molecular weight. Fromswine-pathogenic ETEC strains, at least twospecies of ST have been obtained which aredistinguished by their solubility in water andmethanol and their activity in the guts of swineand suckling mice (3, 34). Because conflictingdata have been published on the molecularweights and amino acid composition of ST pro-duced by ETEC from humans, swine, and cattle(2, 14, 15, 31, 33), we decided to purify ST from

a bovine ETEC strain and compare its charac-teristics with those reported for ST from humanand porcine strains. We describe here proce-dures suitable for large-scale production andpurification of ST from bovine ETEC and thecharacterization and amino acid composition ofthe purified ST peptide.

MATERIALS AND METHODS

E. coli. The ETEC strain B44 was originally isolatedfrom a case of calf scours and was kindly furnished byC. L. Gyles, Ontario Veterinary College, Universityof Guelph, Ontario, Canada.

Nutrient media and growth conditions. Three growthmedia were compared for their efficacy of enterotoxinproduction: the four-amino-acid-Tricine medium ofAlderete and Robertson (1), the asparagine-salt medi-um of Staples et al. (31), and the Casamino Acid-yeastextract medium of Evans et al. (6). The media wereprepared as described by these authors and dispensedin 10-liter volumes into 15-liter carboys. Each carboywas inoculated with 200 ml of a culture of E. coli B44which had been grown for 24 h in the respectivemedium. The carboys were incubated at 39°C andcontinuously purged with filter-sterilized air and oxy-gen at rates of 3,000 and 500 ml/min, respectively. Toprevent foaming, 0.1 ml of Antifoam B Emulsion(Sigma Chemical Co.) was added to each 10 liters ofmedium.

Bacterial growth was monitored at hourly intervals

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by quantitation of viable cells, using serial 10-folddilutions of the hourly samples plated on Trypticasesoy agar (BBL Microbiology Systems). At the sametime, parallel samples of growth medium were centri-fuged and filtered through a 0.22-,um Millipore filter.The protein concentration of the filtrate was deter-mined by the Lowry method (18). A calibration curvecalibration curve prepared with known quantities ofbovine serum albumin was employed.ST assay. Serial dilutions of ST-containing medium

were tested by the suckling mouse assay (SMA) ofGiannella (7); a mouse unit (MU) of ST was defined asthe minimum amount of toxin which after intragastricinoculation into 2- to 4-day-old mice induced within 3to 4 h secretory diarrhea as determined by a gut-to-remaining body weight ratio of 0.083 or higher. Thisassay was used to monitor yield and recovery of STthroughout purification steps.

Adsorption chromatography. The adsorption chro-matography procedure is described by Staples et al.(31). Briefly, 10 liters of clarified and filter-sterilizedmedium was percolated through a glass column (8 by30 cm) containing 300 g of Amberlite XAD-2 (Mal-linckrodt Co.). The resin was washed with 2 liters ofdistilled water followed by 1 liter of 1% acetic acid in20% methanol. The toxin was then eluted stepwisefrom the resin, first with 0.5 liter of 1% acetic acid in99% methanol and then with 1% acetic acid in 80%methanol. The toxin was concentrated by evaporatingmost of the solvent under reduced pressure and thenwas freeze-dried.

Acetone fractionation. The lyophilized ST prepara-tion was dissolved in 20 ml of 25% acetic acid. Then 5volumes of acetone was added gradually under stir-ring. The mixture was held for 1 h at room temperatureand centrifuged at 15,000 x g for 30 min. The pelletwas discarded, and the supernatant was concentratedby evaporation and lyophilization.

Preparative isoelectric focusing in granulated gel. Thefreeze-dried samples were dissolved in 20 ml of 1%glycine and dialyzed at 4°C in a Spectrapore 6 dialysistubing with a molecular cutoff of 1,000 daltons (Spec-trum Medical Industries Inc., Los Angeles, Calif.)against 4 liters of 1% glycine. After clarification bycentrifugation, the sample was dissolved in a solutionconsisting of 3.5 ml of Ampholine (LKB Produktor,Bromma, Sweden) (pH range 2.5 to 4.0) and 1.5 ml ofAmpholine (pH range 3.5 to 5.0). The solution wasdiluted to 100 ml with distilled water to which wasslowly added 5 g of prewashed and dried Sephadex G-75 superfine (Pharmacia Fine Chemicals, Inc., Pis-cataway, N.J.). A flatbed was formed by pouring themixture onto a glass plate (10 by 25 cm). Under a lightstream of air, water was allowed to evaporate from thegel at room temperature until a weight loss of about30% of the initial weight of the slurry was obtained.Anodic electrofocusing strips were soaked in 1 Mphosphoric acid, and cathodic strips were soaked in 1M sodium hydroxide and applied to the electrofocus-ing strips at both ends of the gel tray. Electrofocusingwas performed in a LKB 2117 multiphore chamber(LKB Produktor, Bromma, Sweden) with a constantpower setting of 8W and a maximum voltage setting of800 V for 4 h and 1,400 V for 11 h. During electrofocus-ing, water at 4°C was circulated through the coolingplate at a rate of 4 liters/min. At the end of the run, thegradient of the gel was determined by measuring the

pH at 4°C with a microelectrode (Microelectrodes,Inc., Londonderry, N.H.). The gel was divided into 30sections with a fractionation grid, and the focusedmaterial from each section was eluted with 5 ml ofdistilled water, using small plastic columns. The absor-bance of the eluates was measured at 280 nm, and thebiological activity of the eluates was determined bySMA. Toxin-containing fractions were lyophilized andstored.High-performance liquid chromatography (HPLC).

The toxin was purified further by reverse-phase chro-matography on an Aquapore RP-300 C-18 column (4.6by 25 cm) with a Beckman series 344 system equippedwith model 112 pumps and a model 160 detector(Beckman Instruments, Inc., Fullerton, Calif.). TheST was eluted at 26°C by applying a gradient of 0 to100% methanol containing a constant counterion con-centration of 15 mM phosphoric acid. The effluentswere monitored at 214 nm, and 2-ml fractions werecollected. Fractions which contained peaks of UV-absorbing material were assayed by the SMA, and theST-containing fractions were lyophilized.Amino acid analysis. Samples purified to homogene-

ity by HPLC containing 10 to 20 jig of ST peptideswere lyophilized in 10-ml glass ampoules to whichwere added 2 ml of 6 M HCl in 0.05% mercaptoeth-anol. After the ampoules were repeatedly evacuatedand flushed with nitrogen, they were sealed and heatedin an oven at 110°C for 24 h. The hydrolysates wereanalyzed on a Beckman 121 MB amino acid analyzerat the Bioanalytical Laboratory of Washington StateUniversity. The cysteine content was determined ascystine in the absence of mercaptoethanol.

Carboxyl terminus determination. The amino acid atthe carboxyl terminus was determined by amino acidanalysis after treatment of the ST peptide with car-boxypeptidase A by the method of Narita et al. (23).

Stability. (i) Heat. Volumes of ST (1 ml each)containing 20 mouse units per ml were heated at 65,80, or 100°C for 30 min, plunged into an ice bath, andtested for biological activity by mouse inoculation.The pHs of 1.0-ml samples containing 50 mU of STwere adjusted to pH values of 1, 4, 9, and 12 with 1 MNaOH or 1 M HCl and kept at 37°C for 4 h. Then thesamples were adjusted to pH 7, and aliquots contain-ing approximately 2 mU of ST were given intragastri-cally to suckling mice.

(ii) Enzymes. Enzymatic treatment of ST with tryp-sin, pronase, and subtilisin was performed as de-scribed previously (2, 13).

(iii) Reducing agents. A final concentration of 0.1 M2-mercaptoethanol or 4 x 10-5 M dithiothreitol (Cal-biochem-Behring Corp., La Jolla, Calif.) was added to105 mU of ST. The mixtures were incubated at 370Cfor 1 h and assayed for biological activity by mouseinoculation.

RESULTSToxin production. In a pilot study, the ST

production of ETEC strain B44 in three nutrientmedia was compared. The data (not shown)indicated that ST yield in the aerated fermentorswas higher than in agitated flasks. Similar resultswere obtained when other strains of bovineETEC were used. Of the three nutrient mediacompared, the four-amino-acid-Tricine medium

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B

Om% E

a 0m80

8~.0

0

co 06.,

1400 a

1000 00

0

600 2

200 CO

0 10 20 30 0 10 20 30

Hours HoursFIG. 1. Time course of bacterial growth and ST production by E. coli B44. (A) Asparagine-salts medium. (B)

Four-amino-acid-Tricine medium. The inoculum consisted of 200 ml of 24-h culture of E. coli in the respective

medium. Dots indicate viable counts; bars represent ST concentration. Arrows refer to samples which did not

contain detectable biological activity of ST.

and the asparagine-salts medium yielded moreST with less protein contamination than did theCasamino Acid-yeast extract medium. The as-

paragine-salts medium, however, had the high-est specific activity of ST; hence, it was selectedfor routine production of ST.The results of a time course study of ST

production of strain B44 in the two chemicallydefined media are shown in Fig. 1 and demon-strated that ST production occurred as early as 6h post-inoculation in the four-amino-acid-Tri-cine medium, whereas ST was not detectedbefore 16 h in the asparagine-salts medium. Toestimate the level of impurities in the two ST-preparations, the optical density of the clarifiedsupernatant was measured at 280 nm (OD280),and the ST content was determined by the SMA.After 24 h of incubation, the relative ST activitywas 3,000 MU per OD280 unit in the asparagine-salts medium and 1,575 MU per OD280 unit in

the four-amino-acid-Tricine medium. At 28 h,the relative ST activity was only 2,400 MU per

OD280 unit in the asparagine-salts medium and1,150 MU per OD280 unit in the four-amino-acid-Tricine medium. Therefore, asparagine-salts medium was selected for routine produc-tion of ST, and the cultures were harvested after24 h.

Purification by adsorption chromatography on

Amberlite XAD-2 resin. Most of the ST bound tothe resin, and recovery of approximately 90% ofthe ST was obtained by stepwise elution with 1%acetic acid in 99 and 80% methanol (Table 1).

Acetone fractionation. The addition of acetoneto partially purified ST dissolved in acetic acidresulted in further purification: 71% of the STwas dissolved in the acetone phase, and 85% ofthe total protein content was precipitated (Table1).

Preparative isoelectric focusing. At completion

TABLE 1. Purification of ST from bovine ETEC strain B44

a ST to~tal Sp act Minimal Purifi- ST re-Procedure Protein' mu (MU per effective cation covery(mg) X106 mg of

ds n) (odprotein) ds n) (od %

Filtrate of culture super- 10,000 18 1,800 555.5 1 100natant (10 liters)

Amberlite XAD-2 chro- 850 16.5 19,000 52.0 11 91matography

Acetone fractionation 122 12.8 105,000 9.5 58 71Preparative isoelectric 4.04 5 1,200,000 0.8 685 28

focusingbReverse-phase HPLC 0.75c 3.8 5,067,000 0.2 2,775 21

a Measured by the Lowry method (18).b Only fraction 20 from isoelectric focusing was further processed by HPLC.c Measured by both the Lowry and the ninhydrin methods (10, 22).

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Ec0Go

cm.0

0co

0.00

.0

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2.0 00

1.0 2

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A* Fraction number *CFIG. 2. Preparative isoelectric focusing of ST in a flatbed gel. The gel was Sephadex G-75 superfine poured

onto a glass plate (10 by 25 cm). The pH gradient was 2.4 to 4.5. The load of 122 mg of acetone-fractionated STwas mixed with the gel slurry. Focusing was at 800 V for 4 h followed by 1,400 V for 11 h. A, Anode; C, cathode.The pH gradient (A) was measured by inserting a microelectrode into the gel. The absorbance (A) was measuredat 280 nm. The hatched bars represent the ST concentrations determined by SMA.

of the run, six to eight bands were identified inthe gel under UV illumination. Biological assayof the 30 fractions indicated that the toxin hadfocused into one major zone having a pl of 4.3and one minor zone with a pl of 4.0 (Fig. 2). Ofthe ST loaded 39% was recovered from themajor zone. The minimal effective dose was 0.81ng per MU (Table 1). Small quantities of STwere also detected in fractions which focusedbetween pH 2.2 and 4.0. No ST was found infractions beyond pH 4.3. Similar electrofocusingpatterns were observed with four batches oftoxin and over a wide range of ST loads. In theseruns, the ST-containing fractions always fo-cused at pH 4.3 and could be identified bymeasuring the pH of the gel.HPLC. Fraction 20 from isoelectric focusing

which contained the major portion of ST waschromatographed on the reverse-phase column.Two peaks of biologically active material wereobtained (fourth and sixth in Fig. 3). Peak 4contained the majority of the ST load in a stateof high purity (Table 1) and proved to be homo-geneous on rechromatography.Amino acid analysis. Hydrolysis of the ST

peptide into its amino acid constituents pro-duced values close to integers for all residuesexcept for cysteine (Table 2). The analysisshowed that the ST had 18 amino acid residuesand that 6 of these residues were cysteine. Nobasic amino acids were present. Digestion withcarboxypeptidase released only 1 mol of tyro-sine per mol of toxin from the carboxy terminusof the peptide.

Stability. Purified ST remained biologically

active after heating at 65 and 100°C for 30 minand at pH values of 1, 4, 9, and 12. It resistedenzymatic hydrolysis by trypsin, pronase, andsubtilisin. However, it was completely inactivat-

EC 2.0I-C4.4-.

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80

60 *

40 s0

20 'D

20 40 60

Time in minutesFIG. 3. Purification of ST by reverse-phase chro-

matography, using a methanol gradient ranging from 0to 60%. The load was approximately 1 mg of peptide(fraction 20 from isoelectric focusing); the column wasan Aquapore RP-300 C-18; the mobile phase consistedof solvent a, 15 mM phosphoric acid in water, andsolvent b, 15 mM phosphoric acid in methanol; theflow rate was 1 ml/min for 80 min. Symbols:absorbance at 214 nm;----, methanol concentra-tion. Peaks 4 and 6 contained ST, as determined bySMA.

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TABLE 2. Amino acid analysis of ST purified frombovine ETEC strain B44

Experimental AssumedAmino acid residues per Asition

molecule composition

Alanine 1.% 2Asparaginea 1.99 2Cysteine 5.10 6bGlutamic acid 1.01 1Glycine 1.03 1Leucine 1.02 1Phenylalanine 0.98 1Proline 0.98 1Threonine 0.99 1Tyrosine 1.97 2

a Determined as aspartic acid from the peptidehydrolysate. However, we believe that it representsasparagine in the peptide, which is consistent with theamino acid composition of ST reported by Staples etal. (31) and that encoded by the transposon Tn1681described by So and McCarthy (29).

b Cysteine was determined as cystine in the absenceof mercaptoethanol. The experimental result of 5.10cysteine residues per molecule does not seem incon-sistent with an assumed composition of six cysteineper molecule of ST.

ed by treatment with 0.1 M 2-mercaptoethanolor with 4 x 10-5 M dithiothreitol.

DISCUSSIONSynthetic media have been effectively em-

ployed for the production of ST from human andporcine strains of ETEC (1). However, the yieldof toxin was shown to vary from medium tomedium (32), and it was uncertain which of therecommended media would produce the highestyields of ST from bovine ETEC. In our experi-ence, the asparagine-salts medium of Staples etal. (31) was optimal because it produced highyields of ST with the lowest protein contamina-tion. Confirming the observations of others (31),we found that oxygen is essential for obtaininghigh yields of ST.

Batch adsorption onto Amberlite XAD-2, anunderivatized polystyrene resin, was advanta-geous for concentration of ST from a humanstrain ofETEC (31), and, as shown in this study,it was equally effective for concentrating STfrom bovine ETEC strains. Contrary to reportsof others (2, 10, 31, 33), we were not able topurify ST by conventional column chromatogra-phy, as it bound irreversibly to chromatographymatrices of DEAE-cellulose and polyacrylamidegels. Alternatively, we found that using prepara-tive isoelectric focusing of ST in a flatbed gel(24) allowed the purification of large quantities(up to 200 mg of protein) of ST in a single step.Purification by preparative isoelectric focusingwas convenient, fast, and of high resolution.

Most of the ST was concentrated in one bandwhich focused at a pH of 4.3. It was readilyrecovered from the gel by elution with distilledwater. The purity of the electrofocused prepara-tion was comparable to or greater than thatreported for human or porcine ST obtained byconventional chromatography.

Application of HPLC provided a powerfultool for further purification of the toxin as itseparated the residual ampholytes and otherimpurities from the sample. Although the major-ity of ST focused at a pH of 4.3 in a single band,reverse-phase chromatography indicated that itcontained two chromatographically differentspecies which eluted from the column as a majorand a minor peak with 40 and 42% methanol,respectively. Only the ST peptide from the ma-jor peak was available in sufficient quantity forfurther characterization. The homogeneity ofpeak no. 4 was evidenced by peak symmetry,homogeneity on rechromatography on the re-versed phase column with various gradients (notshown), by the integer values in the amino acidanalysis, and by the high specific activity of theST. We estimated that the minimum molecularweight of ST peptide from strain B44 was 1,900daltons and contained 18 amino acid residues.Its molecular weight and amino acid composi-tion resembled closely those described by Sta-ples et al. (31) from a human strain of ETEC, butit was much smaller and of different compositionthan the 4,000- to 5,000-dalton toxin reported forporcine and human ST by several laboratories(2, 33). So and McCarthy (29) published thenucleotide sequence of the transposon TnJ681,which determined ST production in bovineETEC strain B41. The presence of a similar oridentical transposon in ST-producing ETEC ofporcine origins was identified by restriction en-zyme analysis (16, 28). Moreover, Staples et al.(31) determined the amino acid composition of18-amino acid ST peptide produced by an ETECpathogenic for humans and showed that it wasconsistent with the 18 continuous codons pre-ceding the termination codon of the transposonTn1681. In addition, the N-terminal amino acidof this ST peptide was identical to that predictedfrom the coding sequence of the transposonTn1681, but when the ST peptide was se-quenced, it was found that its amino acid se-quence differed from the transposon nucleotidesequence in two positions: the amino acid of thecarboxy terminal was asparagine instead of tyro-sine, and the 11th amino acid was found to betyrosine instead of an asparagine (4). Each ofthese differences could be the result of a pointmutation involving a base of one codon each.The ST from the bovine ETEC that we havecharacterized in this paper has an amino acidcomposition consistent with both the human ST

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and the last 18 codons of the ST coded bytransposon TnJ681. However, the bovine STdescribed here has a tyrosine as its carboxyterminal, which is consistent with the amino acidsequence coded for by the transposon describedpreviously (29) rather than the human ST se-quence (4). In agreement with other reports onST, we found that the bovine ST peptide ofETEC strain B44 was rich in cysteine. It shouldbe pointed out that the high cysteine content ofST is consistent with what is believed to becharacteristic of many peptide toxins, exhibitingunusual resistance to heat and to enzymatichydrolysis. Disulfide bonds provide rigidity to apeptide which might otherwise exist in solutionas an almost random coil. It is hoped that theavailability of highly purified ST from bovineETEC will make it possible to relate the molecu-lar structures of STs from ETEC of variousspecies. Moreover, such purified ST will enableus to develop a probe for the investigation of thepathogenesis of secretory diarrhea caused byST.

Recently, Moseley et al. (22a) reported thenucleotide sequence of a plasmid coding for anST enterotoxin from a human strain of entero-toxigenic E. coli and found partial homologywith the nucleotide sequence of the transposonTn1681 described by So and McCarthy (29).These findings further support the existence ofan ST peptide (ST la) common to ETEC strainspathogenic for humans and animals. In addition,hybridization studies by the same authors withDNA probes prepared from the ST la and ST lbcoding sequences point to the existence of asubstantial degree of heterogeneity among STplasmids from field strains of human ETECstrains.

ACKNOWLEDGMENTSThis work was supported in part by U.S. Department of

Agriculture Formula Funding 1OA-3073-0465, and in part bythe Departmental Research Funds of the Department ofVeterinary Microbiology and Pathology, Washington StateUniversity.We thank F. R. Abinanti for his valuable suggestions.

LITERATURE CITED1. Alderete, J. F., and D. C. Robertson. 1977. Nutrition and

enterotoxin synthesis by enterotoxigenic strains of Esche-richia coli: defined medium for production of heat-stableenterotoxin. Infect. Immun. 15:781-788.

2. Alderete, J. F., and D. C. Robertson. 1978. Purificationand chemical characterization of the heat-stable entero-toxin produced by porcine strains of enterotoxigenicEscherichia coli. Infect. Immun. 19:1021-1030.

3. Burgess, M. N., N. A. Mullan, and P. M. Newsome. 1980.Heat-stable enterotoxins from Escherichia coli P16. In-fect. Immun. 28:1038-1040.

4. Chan, S. K., and R. Giannella. 1981. Amino acid sequenceof heat-stable enterotoxin produced by Escherichia colipathogenic for man. J. Biol. Chem. 256:7744-7746.

5. Dallas, W. S., and S. Falkow. 1980. Amino acid sequencehomology between cholera toxin and Escherichia coliheat-labile toxin. Nature (London) 288:499-501.

6. Evans, D. G., D. J. Evans, Jr., and N. F. Pierce. 1973.Differences in the response of rabbit small intestine toheat-labile and heat-stable enterotoxins of Escherichiacoli. Infect. Immun. 7:873-880.

7. Giannella, R. A. 1976. Suckling mouse model for detec-tion of heat-stable Escherichia coli enterotoxin: character-istics of the model. Infect. Immun. 14:95-99.

8. Gill, D. M., J. D. Clements, D. C. Robertson, and R. A.Finkelstein. 1981. Subunit number and arrangement inEscherichia coli heat-labile enterotoxin. Infect. Immun.33:677-682.

9. Gyles, C. L. 1971. Heat-labile and heat-stable forms of theenterotoxins from E. coli strains enteropathogenic forpigs. Ann. N.Y. Acad. Sci. 176:314-322.

10. Hirs, C. H. W. 1967. Detection of peptides by chemicalmethods. Methods Enzymol. 11:325-329.

11. Holmgren, J. 1973. Comparison of the tissue receptors forVibrio cholerae and Escherichia coli enterotoxins bymeans of gangliosides and natural cholera toxoid. Infect.Immun. 8:851-859.

12. Isaacson, R. E., H. M. Moon, and R. Schneider. 1978.Distribution and virulence of Escherichia coli in the smallintestines of calves with and without diarrhea. Am. J. Vet.Res. 39:1750-1755.

13. Jacks, T. M., and B. J. Wu. 1974. Biochemical propertiesof Escherichia coli low-molecular-weight, heat-stable en-terotoxin. Infect. Immun. 9:342-347.

14. Kapltany, R. A., A. Scoot, G. W. Forsyth, S. L. McKen-zie, and R. W. Worthington. 1979. Evidence for two heat-stable enterotoxins produced by enterotoxigenic Esche-richia coli. Infect. Immun. 24:965-966.

15. Klipstein, F. A., and R. F. Engert. 1978. Immunologicalrelationship of different preparations of coliform entero-toxins. Infect. Immun. 21:771-778.

16. Lathe, R., P. Hirth, M. DeWilde, N. Harford, and J. P.Lecocq. 1980. Cell-free synthesis of enterotoxin of E. colifrom a cloned gene. Nature (London) 284:473-474.

17. Levine, M. M., E. S. Caplan, D. Waterman, R. A. Cash,R. B. Hornick, and M. J. Snyder. 1977. Diarrhea causedby Escherichia coli that produce only heat-stable entero-toxin. Infect. Immun. 17:78-82.

18. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folin phenolreagent. J. Biol. Chem. 193:265-275.

19. Martinez, A. F., C. L. Gyles, and D. G. Butler. 1980.Escherichia coli heat-stable enterotoxins in feces andintestines of calves with diarrhea. Am. J. Vet. Res.41:1143-1149.

20. Moon, H. W., B. Nagy, R. E. Isaacson, and I. 0rskov.1977. Occurrence of K99 antigen on Escherichia coliisolated from pigs and colonization of pig ileum by K99+enterotoxigenic E. coli from calves and pigs. Infect.Immun. 15:614-620.

21. Moon, H. W., S. C. Whipp, and S. M. Scantvedt. 1976.Etiologic diagnosis of diarrheal diseases of calves: fre-quency and methods for detecting enterotoxin and K99antigen production by Escherichia coli. Am. J. Vet. Res.37:1025-1029.

22. Moore, S. 1968. Amino acid analysis: aqueous dimethylsulfoxide as solvent for the ninhydrin reaction. J. Biol.Chem. 213:6281-6283.

22a.Moseley, S. L., J. W. Hsrdy, M. I. Huq, P. Echeverria,and S. Falkow. 1983. Isolation and nucleotide sequencedetermination of a gene encoding a heat-stable enterotox-in of Escherichia coli. Infect. Immun. 39:1167-1174.

23. Narita, K., H. Matsuo, and T. Nakajima. 1975. End-groupdetermination, p. 86-91. In S. B. Needleman (ed.), Pro-tein sequence determination, 2nd ed. Springer Verlag,New York.

24. Radola, B. J. 1974. Isoelectric focusing in granulated gels.II: Preparative isoelectricfocusing. Biochim. Biophys.Acta 386:181-195.

25. Sack, R. B. 1975. Human diarrheal diseases caused byenterotoxigenic Escherichia coli. Annu. Rev. Microbiol.29:333-351.

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26. Smith, H. W., and C. L. Gyles. 1970. The relationshipbetween two apparently different enterotoxins producedby enteropathogenic strains of E. coli of porcine origin. J.Med. Microbiol. 3:387-401.

27. Smith, H. W., and M. A. Lingood. 1972. Further observa-tions on Escherichia coli enterotoxins with particularregard to those produced by atypical piglet strains and bycalf and lamb strains: the transmissible nature of theseenterotoxins and of a K antigen possessed by calf andlamb strains. J. Med. Microbiol. 5:243-250.

28. So, M., F. Heffron, and B. J. McCarthy. 1979. The E. coligene encoding heat stable toxin is a bacterial transposonflanked by inverted repeats of IS1. Nature (London)277:453-456.

29. So, M., and B. J. McCarthy. 1980. Nucleotide sequence ofbacterial transposon Tn 1681 encoding a heat-stable (ST)toxin and its identification in enterotoxigenic Escherichiacoli strains. Proc. Natl. Acad. Sci. U.S.A. 77:4011-4015.

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VOL. 40, 1983

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