Vol. 30, No. 1INFECTION AND IMMUNITY, Oct. 1980, p. 224-2300019-9567/80/10-0224/07$02.00/0

Cholera Toxin-Like Toxin Released by Salmonella Species inthe Presence of Mitomycin C

N. CHRISTINE MOLINA AND JOHNNY W. PETERSON*Department ofMicrobiology, University of Texas Medical Branch, Galveston, Texas 77550

Several serotypes of Salmonella were shown to release increased amounts of acholera toxin-like toxin during culture in vitro with mitomycin C (MTC). Filter-sterilized culture supernatants containing the toxin caused elongation of Chinesehamster ovary cells, which could be blocked by heating the supernatants at 1000Cfor 15 min or by adding mixed gangliosides or monospecific cholera antitoxin.When MTC was not added to the Salmonella cultures, little or no toxin wasdetected in crude, unconcentrated culture supernatants. Optimal production oftoxin was observed in the presence of 0.5 tig of MTC per ml in shake flask culturesof Casamino Acids-yeast extract medium, Syncase, or peptone saline at 370C.Meat infusion media (heart infusion and brain heart infusion) plus MTC resultedin poor toxin yield. Culture filtrates frequently could be diluted 1:8 and still resultin elongation of Chinese hamster ovary cells.

The pathogenesis of Salmonella-mediateddiarrheal disease has eluded virtually all inves-tigative attempts designed to probe the patho-genic mechanisms of this notorious group ofenteric pathogens. Unlike Vibrio cholerae andenterotoxigenic species of Escherichia coli, thesalmonella are noted for their invasive capacityof the small intestinal epithelium. Takeuchi (15),using transmission electron microscopy, care-fully examined the penetration of small intes-tinal epithelial cells by Salmonella species. Sub-sequently, Giannella et al. (4) concluded thatepithelial cell invasion was correlated with thecapacity of Salmonella species to cause loss ofwater and electrolytes from the intestine, cul-minating in diarrhea.Attempts to identify an enterotoxin (or exo-

toxin) responsible for the diarrheal response elic-ited by Salmonella species were unsuccessfuluntil the independent reports by Koupal andDeibel in 1975 (9) and Sandefur and Peterson in1976 (12). The enterotoxic principle describedby Koupal and Deibel (9) and more recently bySedlock et al. (14) was associated with the cellwall or outer-membrane fraction and causedfluid loss when administered to infant mice. Thefactor was destroyed at 80°C or above in 30 min,and Sedlock et al. (14) later reported that thefactor produced positive intestinal loops in adultrabbits.

Sandefur and Peterson (12) reported the iso-lation and partial purification of two skin perme-ability factors (PFs) in culture supernatants ofSalmonella typhimurium. The first PF was heatstable and elicited a rapid skin permeabilityresponse, typically producing erythema and flat,

soft edema at the site of intradermal injection.The second factor, referred to as the delayedPF, was heat labile and elicited an edematous,erythematous skin reaction which became max-imal after 18 to 24 h. This Salmonella delayedPF reaction, which was indistinguishable fromthat produced by cholera toxin, could be blockedby both cholera antitoxin and GM, ganglioside(11, 12). The delayed PF was also detectable bythe Chinese hamster ovary (CHO) cell assay ofGuerrant et al. (6). We have previously reportedthe successful use of the CHO cell assay tomeasure the delayed PF from partially purifiedSalmonella preparations (13).The Salmonella delayed PF may be respon-

sible, at least in part, for the loss of fluid andelectrolytes from the small intestine because (i)rabbits immunized with heat-inactivated, puri-fied cholera toxin (procholeragenoid) are pro-tected against fluid loss when their intestinalloops are challenged with live S. typhimurium(11) and (ii) because partially purified delayedPF will result in a fluid accumulation in rabbitintestinal loops that is blocked by monospecificcholera antitoxin (J. W. Peterson, submitted forpublication). Henceforth, we will refer to thedelayed PF as Salmonella toxin.

Previously, it was not possible to detect theheat-labile Salmonella toxin in crude, unproc-essed culture filtrates; however, by using thebasic technique which Isaacson and Moon usedfor E. coli (8), we have found that addition ofmitomycin C (MTC) to Salmonella cultures 2 hafter inoculation caused the release of amountsof toxin from the bacterial cells which weredetectable by the CHO cell assay. We have

224

on August 30, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

SALMONELLA TOXIN 225

examined a variety of Salmonella strains andcultural conditions in an attempt to maximizetoxin production.

MATERIALS AND METHODSStrains of Salmonella. Several serotypes of Sal-

monella enteritidis were isolated from fecal specimensby the Houston Health Department, Houston, Texas,and sent to our laboratory. The cultures were main-tained without further passage on Trypticase soy agar(BBL Microbiology Systems) slants under sterile min-eral oil. The serotype identity of each numbered iso-late was as follows: S. enteritidis 10016, serotype ja-viana; S. enteritidis 10234, serotype newport; S. enter-itidis 11271, serotype typhimurium; S. enteritidis 9630,serotype newport; S. enteritidis 9633, serotype new-port; and S. enteritidis 9186, serotype newport. S.enteritidis 2000 serotype typhimurium was obtainedfrom the pediatric bacteriology laboratory of the Uni-versity of Texas Medical Branch Children's Hospitaland previously reported to produce the heat-labiletoxin (11). S. enteritidis SR11 serotype typhimuriumwas kindly provided by L. J. Berry, University ofTexas, Austin.

Bacterial cultivation. Casamino Acids-yeast ex-tract broth (pH 7.0) was composed of the following (ingrams per liter of distilled water): Difco CasaminoAcids, 36; Difco yeast extract, 6; and NaCl, 6. Syncasewas prepared with glucose (5 g/liter) instead of su-crosed as described by Finkelstein (2). Peptone salinewas composed of 10 g of Difco peptone per liter and8.5 g of NaCl per liter. Asparagine-glucose mediumwas prepared with and without added lysine (5 g/liter)(1). Trypticase soy broth (BBL Microbiology Systems)was prepared with and without 1% yeast extract. Heartinfusion broth (Difco Laboratories) and brain heartinfusion broth (Difco Laboratories) were used accord-ing to manufacturers directions.MTC (Sigma Chemical Co.; lot no. 96C-0361) was

hydrated with sterile saline and added to Salmonellacultures 2 h after inoculation. Except in the dose-response study, MTC was used at a concentration of0.5 tg/ml. Culture flasks were wrapped in aluminumfoil to protect the MTC from light. Except whereotherwise specified, 20- or 50-ml cultures were grownin 125- or 250-ml screw-top Erlenmeyer flasks, respec-tively, at 37°C in a gyratory shaker incubator (NewBrunswick Scientific Co.) set at 90 to 100 rpm for 18to 20 h. Cells were removed by centrifugation (27,000x g), and the supernatants were filter sterilized with0.2-jim filter units (Nalgene Labware Div., Nalge/Sy-bron Corp.).CHO cell assay. Stock cultures of CHO cells (13)

were grown at 37°C with 4% CO2 in F-12 medium(GIBCO Laboratories) supplemented with 10% fetalcalf serum and 100 U of penicillin G per ml and 100,ug of streptomycin per ml. For the CHO cell elongationtests (6), cells were suspended in F-12 medium con-taining 1% fetal calf serum and 100 U of penicillin Gper ml and 100 jg of streptomycin per ml. A 0.1-mlamount of the suspension containing approximately100 cells was delivered to each well of the microcultureplates. After 3 to 4 h of incubation at 37°C with 4%.C02, 10-tl volumes of toxin dilutions were added to

the appropriate wells; incubation was then continuedunder the same conditions for 18 to 20 h longer. Atthis time, the culture medium was decanted, and thecells were fixed with methanol for 3 to 5 min. Afterthe plates had air dried, Giemsa stain was added toeach well for 15 min. After washing thoroughly withtap water, the plates were air dried. At least 100 cellswere counted in each well, and the percentage ofelongated cells was determined.

Characterization of CHO cell activity. Salmo-nella toxin contained in Trypticase soy broth-MTCculture filtrates as well as in a solution of purifiedcholera toxin (10 ng/ml) (7, 10) was characterized byheat inactivation, exposure to gangliosides, and mix-ture with rabbit antiserum to purified cholera toxin(7, 10). Heat inactivation experiments were performedby boiling 0.5-ml samples of Salmonella culture fil-trates for 15 min. The filtrates and cholera toxinsolution were also combined with an aqueous solutionof mixed gangliosides (10 mg/ml; Sigma Chemical Co.)in a ratio of 1:9 (ganglioside to toxin) and incubated at37°C for 30 min. The toxin solutions were also mixedin a 1:1 ratio with serum or serum dilutions fromimmune or preimmune rabbits and incubated at 37°Cfor 30 min.

RESULTSFigure 1A shows the typical shape of normal

CHO cells as they appeared after staining withthe Giemsa stain. When CHO cells were exposedto Salmonella toxin, they appeared elongated(Fig. 1B). The elongation effect caused by theheat-labile Salmonella toxin from unconcen-trated MTC filtrates was virtually identical inappearance to that caused by cholera toxin (6,13).Figure 2 shows the striking effect of increasing

amounts of MTC on two clinical isolates of S.enteritidis (serotypes newport and typhimu-rium). The dose-response curves indicated thata concentration of 0.5 [Lg/ml in the culture me-dium was optimal for maximum release of theSalmonella toxin after 18 h at 37°C in shakeflask cultures of Trypticase soy broth. The back-ground rate of elongation observed with unin-oculated Trypticase soy broth was approxi-mately 20% with or without addition of MTC;therefore, the effect of the MTC was not uponthe CHO cells but upon the level of toxin pro-duction, toxin release, or both by bacteria. Sim-ilarly, the addition of MTC to crude filtrates hadno effect on the activity of preformed toxin (datanot shown).

Figure 3 shows composite data from a surveyof clinical isolates of Salmonella for toxin pro-duction. Uninoculated Trypticase soy broth,both with and without 0.5 jig of MTC per ml,caused a background elongation level of approx-imately 20%. In contrast, filtrates from five ofseven isolates grown with MTC could be dilutedtwo- to eightfold before the elongation values

VOL. 30, 1980

on August 30, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

226 MOLINA AND PETERSON

A I~~~

1h~

ip

_

9

B

#~4

I9494 wD

Nfh.. %FIG. 1. (A) Normal CHO cells stained with Giemsa stain. (B) Stained CHO cells after treatment with heat-

labile Salmonella toxin in an concentrated MTC filtrate.

returned to base line. Isolates grown withoutMTC yielded no detectable toxin or toxin de-tectable only in undiluted filtrates. Two of theSalmonella strains did not appear to releaseincreased levels of toxin under these conditions.To characterize the factor released from Sal-

monella cells by MTC induction and confirmthat it was the same as that described previouslyby our laboratory, we performed the followingstudy. Figure 4 shows that crude culture filtratesfrom four Salmonella isolates grown at 370C for18 h exhibited MTC-released toxin which washeat labile, inactivated by ganglioside, and neu-tralized by cholera antitoxic serum. The openbars show the CHO cell elongation caused byeach of the four Salmonella filtrates, whereasthe adjacent bars show the significant loss ofactivity after boiling for 15 min and addingmixed gangliosides or antitoxic serum. Incuba-tion with undiluted, preimmune serum resultedin partial inhibition of the toxin activity, but a1:10 dilution of the serum eliminated this inhi-bition (data not shown). Incubation with undi-luted immune serum, on the other hand, causedcomplete inhibition of the Salmonella toxin ac-tivity, and this serum could be diluted as muchas 1:100 without loss of the ability to inhibittoxin activity. Also shown in Fig. 4 is the back-

ground elongation caused by the uninoculatedTrypticase soy culture medium treated in thesame manner. For comparison purposes, choleratoxin behaved in a manner similar to the Sal-monella toxin. Based on these characteristics,the MTC-induced Salmonella toxin was vir-tually identical to that previously studied in thislaboratory (11) and is remarkably similar tocholera toxin.To increase the yield of toxin further, a survey

was performed to determine the best culturemedium for optimal toxin production in the pres-ence of MTC at 370C in 18 h. Figure 5 showsthe extent to which the filtrates from two Sal-monella isolates grown in this medium could bediluted. The poorest toxin yield by both isolateswas observed with brain heart infusion broth,whereas one of the best yields was observed withCasamino Acids-yeast extract medium, which iscomposed of Casamino Acids, yeast extract, andNaCl. If one selects the filtrate dilution whichcauses 50% maximal elongation and equates thisdilution reciprocal with units of toxin, the rela-tive toxin contents of several culture media canbe compared. Shaken cultures of CasaminoAcids-yeast extract medium, Syncase, and pep-tone saline yielded the highest amounts of toxinin 18 h at 370C (Table 1). Lesser amounts of

INFECT. IMMUN.

0 .AOIV

on August 30, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

SALMONELLA TOXIN 227

40 F

30 F

CZ

Co

L-

0

I

0

A.

fI 0-* Salmonella enteritidis Serotype NeI e9633

I v---4 Salmonella enteritidis Serotype tEYI e1 1271l

ewport

phimurium

10 F

0 01 05 1.0

Concentration of Mitomycin C (mcg/ml)

FIG. 2. Dose-response curves of two Salmonellaspecies in response toMTC showing enhanced releaseof the heat-labile toxin as detected by CHO cell elon-gation.

toxin were observed in asparagine-glucose me-dium with or without lysine, Trypticase soybroth with or without yeast extract, and themeat infusion broths, such as heart infusion andbrain heart infusion broths.

DISCUSSIONThe successful use ofMTC for the purpose of

enhancement of toxin release was first reportedby Isaacson and Moon (8) for E. coli heat-labiletoxin. The initial hypothesis regarding the mech-anism of MTC-induced toxin release by E. coliwas that it caused derepression of plasmid geneexpression (8). More recently, Gemski et al. (3)reported that the drug activates a temperatephage in E. coli which causes lysis and releaseof cellular contents. Although it remains to bedetermined if MTC acts through phage lysis or

plasmid gene derepression in Salmonella spe-cies, the drug provides a reliable method forenhancing release of the heat-labile, choleratoxin-like toxin into crude filtrates of most Sal-monella isolates. Since the basis of toxin releasemediated by MTC in Salmonella species re-mains unclear, caution should be taken before

concluding that strains failing to release toxin inresponse to MTC are truly nontoxigenic. Suchstrains might possess the appropriate geneticcapacity for toxin synthesis, but might lack thephage possibly needed for cell lysis and toxinrelease.

Before the use of the MTC technique, relia-bility of toxin production was poor, and detec-tion of toxin in crude, unprocessed filtrates wasnot possible (12, 13). Despite the release of sig-nificant amounts of toxin by MTC, the actualconcentration in the crude filtrates was very lowand usually could be diluted out completely bymaking a 1:10 dilution of the filtrate. If oneassumes that the Salmonella toxin has the samespecific activity as cholera toxin (not yet deter-mined), most of the MTC-induced Salmonellafiltrates examined thus far would contain ap-proximately 0.01 ng of Salmonella toxin per mlas determined by the CHO cell assay. Comparedwith the larger amounts of the closely relatedtoxins produced by V. cholerae and E. coli,Salmonella toxin was produced in exceedinglylow amounts with the current in vitro experi-mental conditions. This probably accounts forour inability to obtain fluid accumulation re-sponses in rabbit intestinal loops after injectionof the unconcentrated, MTC-induced Salmo-nella filtrates (unpublished data). Likewise, wehave been unable to detect Salmonella toxin infiltrates by using more convenient serologicaltests which have been used for cholera toxin,such as the Ouchterlony analysis, radioimmu-noassay, and passive hemagglutination inhibi-tion.With an MTC concentration of 0.5 Ag/ml, a

limited survey of clinical isolates of Salmonellarevealed that five out of seven strains examinedproduced the toxin. Although the amount oftoxin produced was small, it occurred withrather high frequency. The high frequency oftoxin production is logical since Salmonella spe-cies are virtually always associated with diar-rheal disease when present in the gastrointes-tinal tract. In addition, one of the negative iso-lates, strain 2000, had been producing heat-labilePF several months previously, but appeared togradually lose this ability. The reason for thisinstability remains to be determined, and we arenot certain whether this involves loss of someaspect of toxin release or some genetic factor inthe form of a plasmid, chromosomal gene, ortemperate phage.We have examined a number of cultural con-

ditions (in the presence of MTC) which areconducive to toxin production. Since ganglio-sides inactivate the Salmonella toxin, the gan-

VOL. 30, 1980

on August 30, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

228 MOLINA AND PETERSON

50 r

401-

Sol 11271Sol 10016

*MTC

-MTC

-MTC

- MTC

I

S.i 9630 S.l 9633 Sol 2000

.MTC*MTC

-*MTC

-MTC #~~~~MT

MTC

Undil. 12 14 Undil. 1:2 1 4 Undil 1:2 1:4 Undil. 1:2 1 4

Dilution of Filtrate

FIG. 3. Survey of several clinical isolates of Salmonella for toxin production. Curves showing greater CHOcell elongation in response to MTC are marked +MTC; those cultures incubated without MTC are marked

-MTC.

glioside content of some media (e.g., brain heartinfusion) may account for their poor perform-ance. Since the amount of toxin released in vitrois so small, there still may be nutritional defi-ciencies in the artificial culture media. On theother hand, the efficiency of toxin delivery tothe intestinal epithelium by the Salmonella cellsin vivo may not require release of large quanti-ties of toxin. This is particularly pertinent sincewe do not yet know the relationship betweentoxin release and invasion of the intestinal epi-thelium by Salmonella species. Similarly, we donot understand what triggers release of the toxinin vivo and whether this occurs before or afterepithelial cell invasion. Gianella et al. have re-

ported finding increased levels of cyclic adeno-sine monophosphate in the intestinal epitheliuminfected by diarrhea-inducing strains of Salmo-nella (5). This increase in cyclic adenosinemonophosphate was correlated with increasedadenylate cyclase activity in the infected epithe-lium (5). Since the Salmonella toxin acts on

CHO cells in the same manner as cholera toxin,and since cholera toxin increases cyclic adeno-sine monophosphate levels by stimulation ofadenylate cyclase activity, it seems reasonableto believe that the Salmonella toxin may cause

the elevated adenylate cyclase activity and in-creased cyclic adenosine monophosphate levelsfound in the Salmonella-infected intestinal epi-thelium. In preliminary experiments in our lab-oratory, unconcentrated MTC filtrates whichwere positive in the CHO cell assay did notinduce fluid accumulation in ligated intestinalloops of adult rabbits. Similarly, 30x concen-

trates of MTC filtrates did not result in positiveintestinal loops. The basis for this lack of enter-otoxic response remains to be determined, but ispossibly due to the low concentration of thetoxin in filtrates or some requirement for inva-sion of epithelial cells by live Salmonella cells.Therefore, the precise role of Salmonella toxinin the pathogenesis of salmonellosis remains tobe determined. The reliability of assaying toxin

30

20

10c

4)

C

0c01CD0w

us

0IU

30

20

101

I--

INFECT. IMMUN.

Madly Con1r01 Sal 10234

Sol 9 se

on August 30, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

SALMONELLA TOXIN 229

50

-

c)61)

0.noAnCL0

coCY0wuJ

0C-)

40

30

20

10

Sal Sal Sal Sal Uninoculated Cholera toxin9630 9633 10016 10234 Control 10 ng ml

FIG. 4. Characterization of the toxin in filtratesreleased by several isolates of Salmonella grown inthe presence of MTC. OL MTC-treated filtrate; U,MTC-treated filtrate at 1000C for 15 min; a, MTC-treated filtrate + mixed gangliosides (Sigma); XMTC-treated filtrate + cholera antitoxic serum.

TABLE 1. Effect of culture media on release of toxinby two clinical isolates of Salmonella

CHO cell factor (U/10 IAWi

Culture medium' S. enteriti-dis 10016 se- S. typhimu-rotype ja- rium SR11

viana

CYE 32 16Syncase 12 36Peptone saline 21 24AGL 11 2.5TSB 8 6TSB+1%YE 3 5AG 4 2HI 1.75 2BHI 1.50 1

a One unit is the reciprocal of the culture filtratedilution (10 ph) that will result in 50% maximal elon-gation of CHO cells.

b CYE, Casamino Acids-yeast extract; AGL, aspar-agine glucose with lysine; TSB, Trypticase soy broth;YE, yeast extract; AG, asparagine glucose (withoutlysine); HI, heart infusion; BHI, brain heart infusion.

production in unconcentrated filtrates by usingthe techniques presented in this paper will aidin future studies probing the significance of thetoxin in the pathogenesis of salmonellosis.

- CYE Salmonella 10016 Filtrate

0-- CYE Salmonella SR 11 Filtrate

*-* BHI Salmonella 1001 6 Filtrate3-4 BHI Salmonella SR 11 Filtrate

* BHI

l

10

Undiluted

* CYE

2 4 8 16 32 UninoculatedMTC-mediumDilution Reciprocal Control

FIG. 5. Dilution curves of Casamino Acids-yeastextract (CYE) and brain heart infusion (BHI) mediaafter culture with two clinical isolates ofSalmonella.

ACKNOWLEDGMENTSThis study was supported by contract DAMD17-77-C-7054

from the U.S. Army Medical Research and DevelopmentCommand. N.C.M. is a recipient of a predoctoral fellowshipfrom the James W. McLaughlin Fund.

LITERATURE CITED

1. Callahan, L. T., Iml, and S. H. Richardson. 1973. Bio-chemistry of Vibrio cholerae virulence. III. Nutritionalrequirements for toxin production and the effects of pHon toxin elaboration in chemically defined media. Infect.Immun. 7:567-572.

2. Finkelstein, R. A., P. Atthasampunna, M. Chulasa-maya, and P. Charunmethee. 1966. Pathogenesis ofexperimental cholera: biologic activities of purified pro-choleragen. J. Immunol. 96:440-449.

3. Gemski, P., A. D. O'Brien, and J. A. Wolhieter. 1978.Cellular release of heat-labile enterotoxin of Esche-richia coli by bacteriophage induction. Infect. Immun.19:1076-1082.

4. Giannella, R. A., S. B. Formal, G. J. Dammin, and H.Collins. 1973. Pathogenesis of salmonellosis. Studies offluid secretion, mucosal invasion, and morphologic re-actions in rabbit ileum. J. Clin. Invest. 52:441-453.

5. Giannella, R. A., R. E. Gots, A. N. Charney, W. B.Greenough III, and S. B. Formal. 1975. Pathogenesisof Salmonella-mediated intestinal fluid secretion. Ac-tivation of adenylate cyclase and inhibition by indo-methacin. Gastroenterology 69:1238-1245.

6. Guerrant, R. L., L. L. Brunton, T. C. Schnaitman, L.I. Rebhun, and A. G. Gilman. 1974. Cyclic adenosinemonophosphate and alteration of Chinese hamsterovary cell morphology: a rapid, sensitive in vitro assayfor the enterotoxins of Vibrio cholerae and Escherichiacoli. Infect. Immun. 10:320-327.

50

40C

cJ6)u6)C

0° 300CY)c00)

a 20IC-)

VOL. 30, 1980

on August 30, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

230 MOLINA AND PETERSON

7. Hejtmancik, K. E., J. W. Peterson, D. E. Markel, andA. Kurosky. 1977. Radioimmunoassay for the anti-genic determinants of cholera toxin and its components.Infect. Immun. 17:621-628.

8. Isaacson, R. E., and H. W. Moon. 1975. Induction ofheat-labile enterotoxin synthesis in enterotoxigenicEscherichia coli by mitomycin C. Infect. Immun. 12:1271-1275.

9. Koupal, L. R., and R. H. Deibel. 1975. Assay, charac-terization, and localization of an enterotoxin producedby Salmonella. Infect. Immun. 11:14-22.

10. Kurosky, A., D. E. Markel, and J. W. Peterson. 1977.Covalent structure of the chain of cholera enterotoxin.J. Biol. Chem. 252:7257-7264.

11. Peterson, J. W., and P. D. Sandefur. 1979. Evidence of

INFECT. IMMUN.

a role for permeability factors in the pathogenesis ofsalmonellosis. Am. J. Clin. Nutr. 32:197-209.

12. Sandefur, P. D., and J. W. Peterson. 1976. Isolation ofskin permeability factors from culture filtrates of Sal-monella typhimurium. Infect. Immun. 14:671-679.

13. Sandefur, P. D., and J. W. Peterson. 1977. Neutrali-zation of Salmonella toxin-induced elongation ofChinese hamster ovary cells by cholera antitoxin. Infect.Immun. 15:988-992.

14. Sedlock, D. M., L. R. Koupal, and R. H. Deibel. 1978.Production and partial purification of Salmonella en-terotoxin. Infect. Immun. 20:375-380.

15. Takeuchi, A. 1971. Penetration of the intestinal epithe-lium by various microorganisms. Curr. Top. Pathol. 54:1-27.

on August 30, 2020 by guest

http://iai.asm.org/

Dow

nloaded from