Toxlcon, Vd. 21, No . 6, pp . 783-796, 1983 .

0041-0101/83 53 .00+ .00Printed in Clre~t Brlnln.

® 1983 Pagamon Preis Ltd.

CYTOTONIC ENTEROTOXINS AND CYTOTOXIC FACTORSPRODUCED BY SALMONELLA ENTERITIDIS AND

SALMONELLA TYPHIMURIUM

S. B. BALODA, A. FARIS, K. KROVACEK and T. WADSTRÖM'Department of Bacteriology and Epizootology, College of Veterinary Medicine, Swedish University of

Agricultural Sciences, Biomedicum, Box 583, 5-751 23 Uppsala, Sweden

(Acceptedfor publirntion 26 April 1983)

S . B . BALODA,A. FAats, K. KROVACEK and T . Ww~sTaöM . C~totoaic enterotoxins and cytotoxicfactors produced by Salmonella enteritidts and Salmonella typhLnuriunt . Toxicon 21, 785-796,1983 . - Strains of Sabnondla enteritidisand Sabnondla typhiinwrium isolated from human diarrhealcases produced heat-labile enterotoxin(s) and cytotoxic factors) which elongated, lysed or deformedChinese hamster ovary odla is tissue culture . The tO7Ra(a) caused fhlid accumulation in ligated rabbitgut loops and produced increased skin permeability . Salmonella toxin produced by these strainsdoes not cross-react immunologically with high titer Vibrlo cholerae toxin aatiaera or heat-labileFscherichiacoli enterotoxin antisera used in this study and does sot bind to galactose-Sepharosegel. The activity of the toxin was not inhibited by GM,-ganglioside .

INTRODUCTION

TIC MECI~AxISM by which Salmonella organisms cause diarrhea is poorly understood .Early reports that endotoxin causes loss offluid and electrolytes from the intestinal epitheliain salmonellosis do not seem convincing (PETERSON, 1980). TAYLOR and WILKINS (1961)first observed that strains of Salmonella cause fluid accumulation in rabbit intestinal loops,but they did not test cell-free filtrates in this assay. Later, Caxa~srcl et al. (1974) confirmedthese findings, but found no correlation between the effect of culture filtrates and thatof living cultures in such intestinal loop experiments.

In studies in which newborn mice were fed culture filtrates by the oral route, KOUPALand DEIBEL (1975) identified a all-envelope-associated enterotoxic factor and observedan early onset of fluid secretion similar to that induced by heat-stable enterotoxin ofEscherzchia coli. SANDEFUR and PETERSON (1976), on the other hand, described two skinpermeability factors. They later confirmed that these also induced elongation of Chinesehamster ovary cells, similar to that produced by cholera toxin or heat-labile enterotoxinof E. coli. Their toxin was neutralized by antibodies raised against cholera toxin andE. coliheat-labile enterotoxin and its biological effect on Chinese hamster ovary cells was blockedby GM,-ganglioside (SANDEFUR and PETERSON, 1977 ; PETERSON, 1977 ; PETERSON andSANDEFUR, 1979).

Despite the striking similarities between salmonella toxin, cholera toxin andE. coli heat-labile enterotoxin described in a series of papers (KOUPAL and DEIBEL, 1975 ; $ANDEFURand PETERSON, 1977 ; MOLINA and PETERSON, 1980), Peterson and collaborators found

rI'o whom correspondence should be addressed .

783

78 6

S. B. BALODA, A. FARTS, K. KROVACEK and T. WADSTRÖM

that salmonella toxin was a poor inducer of fluid and electrolyte secretion in rabbit intestinalloop tests .We nowreport on production of cytotonic enterotoxin(s) and cytotoxic factors) among

strains of two Salmonella species commonly isolated from humans with salmonellosis indifferent parts of the world (KUHN et al., 1978). These strains were characterized on thebasis of their enterotoxin production by use of the rabbit intestinal loop test, rabbit skintest and Chinese hamster ovary cells in tissue culture.

MATERIALS AND METHODS

Bacterial strainsSeven strains ofSalmonella enteritidis and two strains of Salmonella typhimurium were obtained from human

patients with acute diarrheal disease in various geographical regions of India (North, Central and South) . Onestrain of Salmonella enteritidis was isolated from a guinea pig (Table 1) . After isolation, the strains were kepteither freeze dried or frozen at -70°C in trypticase soy broth with 15% glycerol .

Bacterial culturesEach Salmonella strain was grown in a casaminoacid yeast extract medium (MOLINA and PerEtsoN, 1980) .

Overnight cultures on a shaking table (20 rev/min, 18 hr, 37°C) were centrifuged (18,000 s, 30 min) and cell-free culture supernatants (crude Salmonella enterotoxin, 3.0 mg/ml protein) were sterile filtered (Millipore® ,0.22 lan). These crude Salmonella enterotoxin samples were concentrated (£mal protein cone. 5.8 mg/ml) by dialysisagainst polyethylene glywl (15,000-20,000 mol.wt) and were either used immediately or stored at -20°C forless than seven days before assays wereperformed . The terms `Salmorrclta enterotoxin' and `concentrated Salmonellaenterotoxin' throughout the text refer tothe `crude' preparations of cell-free culture supernatants and concentratedcell-free culture supernatants, respectively .

Rabbit intestinal loop and skin testsThese tests were performed in young rabbits (1 .6-2 .0 kg) as previously described (Twn.ox et al., 1958 ; Cxw~a,

1965). Each experiment was carried out with concentrated Salmonella enterotoxin . Aheat-labile enterotoxin froman E. coli strain 1628-15 was used as a reference toxin .

Tissue culture assaysSalmonella enterotoxin was assayed after serial twofold or tenfold dilutions inthe Chinese hamster ovary cells

test (GueeRwxr et al ., 1974) and in Yl adrenal cell cultures (Dorrrw et al., 1974) with cholera toxin andE. coliheat-labile enterotoxin as positive controls .

Neutralization experimentsThese were performed on the rabbit skin and Yl adrenal cell cultures . Crude Salmonella enterotoxin, cholera

toxin and E. coli heat-labile enterotoxin were preincubated (37°C, 3 hr) with standard amounts of antibodiesagainst cholera toxin andE. coli heat-labile enterotoxin, containing 60 and 64 mg protein/ml, respectively. Theantibody preparations used for neutralization could detect as low as 1 ng of cholera toxin and 5 ng of E. coliheat-labile enterotoxin in the coagglutination test when Staphylococcus aureus strain Cowan 1 was coated withhomologous antisera (RÖxNSexc and WwnsrttöM, 1983).

ImmunoassaysImmunodiffusion in agarose gels with standard cholera toxin, E. coli heat-labile enterotoxin and Salmonella

emerotoxin were performed as previously described (Ouct~rrexwnv, 1968). In brief, 1 N~ agarose was layeredover the glass slides (4 x 4 cm) and the desired number of wells were cut in the gel after it solidified . Ten micro&tensof one of the respective antigens was put in each of the outer wells while the central well received the desiredantisera against cholera toxin or E. rnll heat-labile enterotoxin. The immunodiffusion plates were incubated ina humid chamber for three days and were observed each day for immunoprecipitation lines .

Coagglutination tests to detect enterotoxin in culture fluids and all lysates were performed using ooagglutinationreagents prepared according to the method described by RörnvsERC and Wwns~raöM (1983). One milliliter of al0ab heat killed and formalin treated suspension of Staphylocnocvs aunes strain Cowan 1 was washed in phosphatebuffered saline (0 .01 Msodium phosphate buffer, pH 7.4, in 0.14 MNaCI). The suspension was sensitized with0.1 ml of an optimal dilution of antisera against E. coli heat-labile enterotoxin or antisera against cholera toxinin phosphate buffered saline . The sensitization mixtures were incubated for 1 hr at room temperature and thenthe bacteria were washed in phosphate buffered saline . A4~ solution of methylene blue in phosphate bufferedsaline was added to this mixture and it was further incubated for 1 hr at room temperature . Finally, after washingwith phosphate buffered saline, the reagents were diluted to a working suspension of gala in phosphatebufferedsaline and stored at 4°C.

Salmonella Cytotonic Enterotoxins

787

For coagglutination tests, Salmonella strains cultured on blood agar or Salmondla enterotoxin were used . Aloopful of bacteria from a blood agar plate was suspended in 100 Pl of saline containing 2 mg/ml of polymyxinB and incubated in a water bath at 37°C for 1 hr. Later, a non-ionic detergent (Triton X-100, final concentration0.1 qs in 100 Pl of the above sample) was added and the mixture was further incubated for 10 min to obtain celllysates . The lysates were then centrifuged (3000 g, 20 min) and equal volumes of all lysates (or Salmonellaenterotoxin) andcoagglutination reagent (25 pl ofeach) were mixed on a glass slide or Phadebact cellulose papercards (Pharmacia Diagnostia, Uppsala, Sweden). The results wen read within 2 min, and a positive result wasindicated by coagglutination due to aggregation of coated staphylococci .

F,fJect of heating, pH andenzymesThe Salmonella enterotoxins wen heated at 65°C and 100°C for 30 min and the samples were rapidly cooled

and assayed for enterotoxic activity on Chinese hamster ovary cells and rabbit skin . Concentrated Salmonellaenterotoxins were incubated for 4 hr after adjusting the pH of separate samples to 3, 4, S, 9, 10 or 11 . Afterincubation, the samples wen neutralized and the protein content was adjusted to the cornsponding protein contentof the original non-concentrated Salmonella enterotoxin (3 mg/ml) by adding the necessary amount of sterilephysiological saline . Sensitivity of Salmonella enterotoxin to enzymes was tested by incubating 1 .0 ml ofconcentrated toxin sample with 1.0 ml each oflysozyme (1 .0 mg/ml in distilled water) and protease (1 .0 mg/mlin 0.001 M HCl) overnight at 37°C . Later these toxins were assayed on Chinese hamster ovary cells .

Binding to galactose gelsThe galactose agarose gel was prepared according to the method described by Poxw~ and Eassort (1973) .

Small teflon columns (10.0 mm diameter, 9.0 cm height) were packed with 2.0 ml ofthe coupled gel . The columnwas equilibrated with TEAN buffer (G~.en~rrrs and Ftru~tsrenv, 1978) and 1.0 ml each of Salmonella andE. tollheat-labile enterotoxin were eluted through the column using TEAN buffer containing 0.2 M n(+)galactose .Fractions of 2.0 ml were collected and tested on Chinese hamster ovary cells.

Binding to gangliosidesCrude gangliosides type II and III were used (3 .0 pg/ml) . One milliliter each ofthe enterotoxin and the respective

ganglioside were incubated at 37°C for 30 min and then assayed on the Chinese hamster ovary cells for activity .

Protein determinationBovine serum albumin was used as the standard for protein estimation by the method of LowxY et al . (1951) .

ChemicalsCasaminoacids and yeast extract were purchased from Difoo, Detroit, MI, USA. Protease (Streptomyces griseus),

lysozyme, bovine brain gangliosides type II and III, polymyxin B, Triton X-100 and bovine serum albumin wereobtained from Sigma Chemical Co ., St. Louis, MO, U.S.A . Polyethylene glycol 20,000 technical grade was fromKEBO, Stockholm, Sweden . Tissue culture media were from Flow laboratories, Irvine, Scotland . Sepharose®6B from Pharmacia Fme Chemicals, Uppsala, Sweden, and galactose from F'luka, Buchs, Switzerland, were used .Cholera toxin was kindly donated by the Swiss Serum Institute, Basel, Switzerland, and E toll heat-labileenterotoxin and the corresponding antiserum by B. Rönnberg .

RESULTS

The culture supernatants of Salmonella enteritidis and Salmonella typhimurium werescreened for enterotoxin production bythe Chinese hamster ovary cells test in tissue culture(Table 1) . Eight of the ten strains caused cell elongation very similar to that induced bycholera toxin orE. coli heat-labile enterotoxin (Fig . 1), while two strains lysed or deformedthe cells . The effects of sterile all-free culture supernatants were studied on rabbit intestinalloops. Since the original non~oncentrated culture supernatants did not show positive rabbitdeal loop ratios, the supernatants were concentrated and tested on deal loops. All the ninestrains tested showed positive deal loop reactions to varying degrees (Tables 2 and 3) . Fiveof these strains showing a positive deal loop ratio were selected for studies on rabbit skin .All these strains caused induration and blueing and/or blanching around the inoculumsite (Fig . 2, Table 4) . The central necrosis was rarely observed with E coli heat-labileenterotoxin.

Immunodiffusion in agarose gel showed no spur or full immunoprecipitate with antiseraagainst cholera toxin or E. coli heat-labile enterotoxin, even when extremely high titers

78 8 S. B. BALODA, A. FARIS, K. KROVACEK and T. WADSTRÖM

TABLE 1 . DETECCIONOF ELONGATION, LYTIC AND DEFORMATIVE FACTORS PRODUCED BY SELECTED STRAINS

OF SQImone!la IN CHINESE HAMSTER OVARY CELL ASSAY

'Morphological changes of Chinese hamster ovary cells in tissue culture observed 18 hr after additionof the respective toxin samples . EF =elongation factor (+ + + = 754b of the cells are elongated;++ = 25-75~o cells are elongated; + = up to 25% cells are elongated); LF = lytic factor,representing total lysis of the cells ; DF = defonaative factor, indicating the deformationofthe normalcell shape.

LHMC,New Delhi-obtained from the Department ofMicrobiology, Lady Hardinge MedicalCollege, New Delhi, India .

TABLE 2. RABBIT ILFwi LOOP RI:ACI70NS PRODUCED BY CONCENTRATED CELL-FREE

CULTURE SUPERNATAN75 OF SELECTED Salmonella sTRAnvs

'In two earlier experiments when concentrated cell-free culture supernatants fromall these strains were injected into rabbit deal loops, the rabbits died. However, whenthe experiments were repeated using the concentrated culture supernatants from twostrains only, the rabbits did not die (Table 3) .t The deal loop ratio represents the average of three different experimental

observations .

of antibodies (sufficient to detect 1- S ng of cholera toxin andE coli heat-labile enterotoxinin the coagglutination test) were used against crude Salmonella enterotoxin preparations(see Materials and Methods) . No neutralization was observed on rabbit skin (Table S) orin the Y1 adrenal cell system . The enterotoxin did not show a positive coagglutination withStaphylococcus aureus strain Cowan 1 coated with high titer antisera against cholera toxinor E. coli heat-labile enterotoxin.

Strain Organism Ileal loop ratio" f(fluid volume length of loop)

15879 S. enteritidis 1 .31S. typhimurium 1 .18

287/80 S. typhimurlum 1 .4327657 S. enteritidis 1 .0327036 S. enteritidis 1 .0224952 S. enteritidis 0.8625279 S. enteritidis not tested30612 S. enteritidis 1 "~74/80 S. enteritidis `1 .4427655 S. enteritidis 1.031628-15 E. coü (positive control) 1.37- Physiological saline 0.00

Strain Species Source/origin Region of India B1010giCaleffect"

27036 S. enteritidis 8 month human femalechild Tamil Nadu EF (+ + +)

27657 S. enterttidis 8 month human femalechild Tamil Nadu EF (+ +)

15879 S. enteritidis Adult human Karnataka EF (++ +)24952 S. enteritidis 1 month homes female

child Maharashtra EF (+)25279 S. enteritidis 4 month human male child Tamil Nadu EF (+)287/80 S. typhimurium Adult human LHMC, New Delhi f EF (+++)30612 S. enteritidis Guinea pig Himachal Pradesh LF/DF74/80 S. enteritidis Adult human LHMC, New Delhi LF- S. typhimurium Adult human LHMC, New Delhi EF (+ +)

27655 S. enteritldis Human male child Tamil Nadu EF (+)

789

Fia . l a. ELONOATION oF CrmvesE HAMSrER ovARY CEi.is BY Salmonella enterltldLs sTRAnv 27036.b . LY3L9 OF CHINFSE HAINSIER OVARY CELLS BY CELLFRHE CULTURE 3UPEANATANI'S OF SQImoneUa

enterltidis sTRAnv 74/80 .IVIagnificadon x 1000

Fia . 1 continued . c . ÜNTREATED No~w~ CHINESE HAMSTER OVARY CELLS .M8~1I1C8ti0II X IOOO.

FYa. 2 . RAHErr sxnv sHOwnva aLUEwa (erres 2- 4, 7 - 9, 11- i5 and 16 - 22) erm rmcaos~s (erres1, S, 6 and 10) cwusen av Salmonella enterotoxlns.

E. rnll enterotoxin on site 25 shows blueing but no nxroais. SaJmonelbenteritidisendotoxin (site26) and physiological saline (site 28) gave negative akin reactions.

Salmonella G~totonic 13nterotoxins

7g3

TABLE 3. FLUID ACCUMULATION INDUCE BY DIFFERENTCONCENTRATIONSOF CEI,IrFREECULTURE SUPERNATANTSOFTWO Salmonela STRAIIiSIN THE LIOATEDRABBIT E~lfFSrINAL

LOOP

"The cell-free ailture supernateats was ooncenhated by dialysis against polyethyleneglycol (15,000-20,000 mol.wt)

1' Loop ratio = volume of fluid in the loop/length of the loop . The results shownare the average of three experiments .

TABLE 4. THE EFFECT OF CELLFREE CULTURE SUPERNATANTS OF Salmonella sPEC>FS oN RASHIT sxnv:AFSPONSE OF SKIN IN THE FORM OF INDURATION, HLUEING, NECROSIS OA BLANCE~iO

"The cell-free culture supernatants were oonoeatrated to 10, 20, 30 and 30% of the original vohlmeof the supernatant using dialysis against polyethylene glycol (15,000-20,000 mol.wt).

tPresena of haemorrhagic necrosed tissue around the injxtion site is denoted by the (+) sign ;the ( -)sign indicates no a8sociated necrosis . Similarly, bleaching ofakin (whitish skin patch aroundthe injection site) is shown by the (+) sign; the (-) sign indicates no bleaching. These results arebased upon three studies .

The toxin is heat-labile and loses all activity on heating for 30 min at 65°C and 100°C.The toxin is unaffected by lysozyme but is sensitive to protease and to low and high pH(Table ~. There is a complete loss of activity, as detectod on rabbit slân and Chinese hamsterovary cells in tissue culture, after incubation at pH 3, 4, 10 or 11, but only a,slight decreasein activity at pH S and 9.Thetoxin is not inhibited by gangliosides or does not bind to the galactose -Sepharose

gel, as tested on adrenal cells and Chinese hamster ovary cells. Most of the toxin (80%)is eluted in the second and third fraction from the galactose -Sepharose gel.

Sample designation(concentration)"

Indurationrange (mm)

Blueingrange (mm)

Necrosis ~ Blanchingf

27036 (10-30%) 16-26 16.5-23.5 + +27036 (supernatant) 24 .0 24 .0 - +27657 (10-50%) 20-28 13.3-20.0 + +27657 (supernatant) 28 .5 18 .5 + -15879 (10-50%) 23-29 16-24 + +15879 (supernatant) 23 .0 17 .0 + -

287/80 (supernatant) 22 .0 13 .0 + -S. typhimurium (10-30%) 9-18 5.5-14.0 - -S. typhimurlum(supernatant) 10 .5 7.0 + -S. enteritidis (cndotoxin,0.01 mg/ml) 0.00 0.00 - -Physiological saline 0.00 0.00 - -1628-15 E. rnli 25 .0 21 .0 - -

Organism Strain % of original supernatant volume" Y.oopratio

S. typhimurium - 10 1.1730 -30 1 .70

Originel supernatant 0.00S. enteritldis 27036 10 1 .30

30 1 .50SO 1.49

Original supernatant 0.00E coli . 1628-13 Original supernatant 1 .37- - Physiological saline 0.00

794 S. B. BALODA, A. FARIS, K. KROVACEK and T. WADSTRÖM

TwBLE 5 . NEUTRALIZATION TESTS ON SQhrtortella ENrEROTOXIN AS DETECTED IN THERABBIT SKIN PF3RAII3AHILITY TFST

"The results shown are the average of three experiments .t 0 = no antitoxin .

TwBLe 6. SeNSrmmr oP Salmondla F

ROTOXIN TO HEAT, pH wNn P.NZrnos

"The degra of Chinese hamster ovary all dongadon is q»antitated as follows : + + + = 755 of the cellsare elongated ; + + = 25-75~ of the cells elongated; + = up to 25% of the cells show elongation; - _no eongadon observed in Chinese hamster ovary cells (negative result) . The results in this experiment are theaverage of thra experiments .

DISCUSSION

All nine Salmonella strains of the two species, which were isolated from different casesof salmonellosis, produced heat-labile enterotoxin, as shownby the various bioassays usodin this study. The following findings for the heat-labile enterotoxin of Salmonella suggestthat it should be classed as a cytotonic enterotoxin: (1) positive 18 hr intestinal loop assay;(2) positive rabbit skin test, with blueing and blanching (Fig. 2) ; (3) morphological changesin the two tissue culture systems similar to those produced by highly purified cholera toxin,as well as E. coli heat-labile enterotoxin (Fig . 1) . This view is also supported by the rapidinactivation of Salmonella enterotoxin at 6S°C and its sensitivity to lowpH and treatmentwith protease.

Retxnt studies on strains of Salmonella enteritidisin the USA suggest that the enterotoxinsof these strains, which seem to be much more potent in the Chinese hamster ovary cellassay than in rabbit intestinal loop tests (PETERSON, 1980), are neutralized by cholera toxinantiserum and that rabbits immunized with detoxified cholera toxin are resistant toSalmonella toxin. Contrary to these results, none of the strains in this study showed anyimmunological cross-reaction, either in two immunoassays (immlmodiffusion andcoagglutination tests) or in three bioassays (rabbit intestinal loop, rabbit skin test and Y1

Treatment

Rabbit skin27036

Induration Blueing

response by strain287/80

Induration Blueing

Chinese hamster ovary cellelongation" by strain

27036 287/80

Heating at :65°C 0 0 0 0 - -100°C 0 0 0 0 - -

pH 3-4 0 0 0 0 - -S 15 13 21 19 .3 + + + +9 18 .8 18 24 20 + + + +

10-11 0 0 0 0 - -Lysozyme 25 25 32 28 .3 + + + + + +Protase 0 0 0 0 - -Untrated toxin

(control) 26 24.7 34 31 .3 + + + + + +

Strain Enterotoxin Antitoxin Induration Blueing

27036 S. ertterltldls 0 ~ 27 .3 27 .5V, choleroe 45 .0 36 .0E. coli 27 .5 23 .0

- V. cholerae 0 28 .5 18 .0V. choolrae 0.0 0.0

T-63 E. rnli. 0 25 .0 21 .0E. coli 0.0 0.0

Salmonella Cytotonic Enterotoxina

79S

adrenal cell cultures). We cannot explain the discrepancy between the test results of ourstudy and these observations . Moreover, a recent report on purification of heat-labileenterotoxin from one strain of Salmonella wien (CAPRIOLL et al., 1982) also suggests aclose relationship between cholera toxin, E. colt heat-labile enterotoxin and Salmonellaenterotoxin.

Recent investigations with monoclonal antibodies directed against the A and B subunitsof cholera toxin andE. colt heat-labile enterotoxin suggest that both these subunits of thetwo toxins show a much greater immunological heterogeneity than was previously believed(HOLMES and TV1~DY, 1982 ; TSUJI et al., 1982). Moreover, recent studies with monoclonalantibodies andDNA hybridization probes point to the existence of enterotoxigenicE. coltstrains of human and animal origin that are either related or are non-related (R. Holmes,personal communication) . Recent data frôm ourown laboratory have shown that anotherenterotoxigenic entempathogen (Aeromonas hydrophila), which is genetically closely relatedto Vibrio cholerae, produces a cytotonic enterotoxin which is immunologically non-relatedto cholera toxin but induces a response in the cellular synthesis of cAMP-related metabolites,such as steroids (LJLTNGH et al., 1982a, 19826) . These findings should encourage furtherstudies on the mechanism of action of Aeromonas and Salmonella enterotoxins, whichwill become possible when purified preparations of these toxins are available. However,GIANELLA et al. (1975) demonstrated activation of the adenyl cyclase system in the rabbitintestinal mucosa as a result of experimental Salmonella infections, and concluded thata different mechanism from that previously observed for both V. cholerae andenterotoxigenic E. colt induces fluid accumulation in ligated rabbit intestinal loops, sincepretreatement of the rabbit with indomethacin completely inhibited the ability of Salmonellato cause fluid accumulation . Thus, the relative importance of mucosal invasion andenterotoxin production in the pathogenesis of Salmonella diarrheal disease must be exploredfurther . Recent reports on a Shiga-like enterotoxin in Salmonella (K1~TYI et al., 1979) andin the so called enteropathogenic strains of E. colt (SCHEFTEL et al., 1980) add to theindications that the cytotoxic factors found in some of the strains in this study requirefurther investigation. Such cytotoxic factors can disguise biological effects caused bycytotonic enterotoxins in the Y1 adrenal and Chinese hamster ovary cell assays, as alsorecently described in Aeromonas hydrophila (LJUNGH et al ., 1982a, 1982b) .

Aabiowkdgernertts - For the supply ofSalmoneUa strains we are indebted to Dr S . N. Swx®vw, Director, CentralResearch Institute, ICaaauli, H.P., India, and to Mr F. Y . Soaott, Departmwt ofMicrobiology, Lady HardingeMedical College, New Delhi, India. We are grateful to Mr B. Rötvxaeaa for kindly supplying ua withcoagglutinatioa reagents and the -Sepherose gel. The skilful technical assistance of Ms Lwn.w Earassowis gratefully acknowledged. This study was supported by the grants from the Swedish Medical Research Council(16 x 04723) and the Swedish Institute (to S.B.B .).

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