INFECTION AND IMMUNITY,0019-9567/99/$04.0010

June 1999, p. 2815–2821 Vol. 67, No. 6

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Evaluation of Salmonella typhimurium Mutants in a Modelof Experimental Gastroenteritis

PAUL EVEREST,1* JULIAN KETLEY,2 SIMON HARDY,3 GILL DOUCE,1† SHAHID KHAN,4

JACQUI SHEA,5 DAVID HOLDEN,5 DUNCAN MASKELL,4 AND GORDON DOUGAN1

Department of Biochemistry, Imperial College of Science, Technology and Medicine,1 and Department of Infectious Diseases,Imperial College School of Medicine, Hammersmith Hospital,5 London; Department of Genetics, University of Leicester,

Leicester2; Department of Pharmacy, University of Brighton, Brighton3; and Department of Clinical VeterinaryMedicine, University of Cambridge Veterinary School, Cambridge,4 United Kingdom

Received 23 October 1998/Returned for modification 21 December 1998/Accepted 5 March 1999

Salmonella typhimurium strains harboring independent, defined mutations in aroA, invA, ssrA, or msbB wereassessed for their ability to induce fluid accumulation, tissue damage, and local inflammation in rabbit ilealloops. Three wild-type strains of S. typhimurium, TML, HWSH, and SL1344, and two mutant strains, S.typhimurium SL1344 ssrA and S. typhimurium SL1344 msbB, consistently induced fluid accumulation in thelumen of loops and inflammation of loop-associated tissues. In contrast, three different S. typhimurium aroAstrains and an invA mutant of SL1344 did not induce significant fluid accumulation in the rabbit ileal loops.However, the S. typhimurium aroA strains did induce an inflammatory infiltrate and some local villus-associated damage, but the invA mutant did not. Histologically, wild-type S. typhimurium, S. typhimuriumSL1344 ssrA, and S. typhimurium SL1344 msbB demonstrated more severe effects on villus architecture than S.typhimurium aroA strains, whereas S. typhimurium invA-infected loops showed no detectable damage. Thissuggests that villus damage most likely contributes to fluid accumulation within the loop.

Salmonella-associated infections can be grouped into twogeneral types according to their clinical features and under-lying pathogenicities (18). Isolates of most Salmonella entericaserovars cause localized enteric infections or gastroenteritisassociated with diarrhea and abdominal pain during which theinfecting salmonella bacteria invade the gut wall but remainpredominantly associated with the gut tissue and local lym-phatics. These Salmonella serotypes can sometimes spreadsystemically in very old, very young, or immunocompromised in-dividuals. Other organisms in the genus Salmonella, includingS. enterica serovar Typhi (S. typhi), cause systemic diseases, in-cluding typhoid, in which bacteria spread to organs of the reti-culoendothelial system, including the liver and spleen (20, 21).

Salmonella is an attractive target organism for scientific in-vestigators interested in the genetic basis of virulence. In par-ticular, the availability of excellent genetic manipulation tech-niques and the murine model of infection has allowed a hugeamount of work to be performed. However, the ready accessi-bility of mice has also resulted in most in vivo studies of sal-monella virulence being carried out in this model. Mouse-viru-lent Salmonella strains cause a systemic disease in mice thatsuperficially resembles human typhoid (5, 6). In this model,mice do not develop diarrhea but do succumb to overwhelmingbacterial growth in deep tissues. With the murine model, manySalmonella genes have been shown to contribute to virulence(7). The identification of attenuating mutations has encour-aged the development of novel live salmonella vaccine strains(2, 5–7, 16, 17, 19, 24, 28, 32–35). Most work toward developingthis vaccine for humans has involved attempts to develop liveoral typhoid vaccines based on genetically defined S. typhi (17).

Identification of candidate attenuating mutations for use inexperimental typhoid vaccines has been, in large part, depen-dent on studies of Salmonella typhimurium in the mouse, as S.typhi isolates exhibit low virulence rates in nonhumans. Fewattenuated mutants identified in the mouse have been exam-ined for their role in the diarrhea associated with Salmonellainfections, as the mouse model is inappropriate for this pur-pose. Many of the virulence-associated genes identified byusing the murine model fall into distinct classes. Some encodeenzymes for critical biosynthetic pathways essential to sustainin vivo growth, such as the aro genes, which encode enzymes ofthe chorismate pathway (16, 17). Some encode type III secre-tion systems and are known as pathogenicity islands. Salmo-nella spp. have at least two such loci. One, named Salmonellapathogenicity island 1 (SPI-1), contributes to eucaryotic cellinvasion (8, 22, 25, 26). A second, SPI-2, contributes to survivalin deep tissue (29). Another group of genes are involved in thebiosynthesis of lipopolysaccharide. For example, msbB is in-volved in the biosynthesis of the highly toxic lipid A componentof lipopolysaccharide, and S. typhimurium msbB mutants havean altered pathogenicity in the mouse and a reduced ability toinduce production of tumor necrosis factor alpha, interleukin1, and nitric oxide (23).

In contrast to studies of systemic disease, investigations ofthe contributions of different Salmonella genes to gastroenter-itis and diarrhea are more difficult to undertake because of thelack of readily available experimental systems. The rabbit loopmodel has been utilized as a method for identifying Salmonellastrains that are able to induce fluid accumulation and poten-tially diarrhea (3, 12, 13, 15, 30, 36, 37, 40). For example,S. typhimurium TML has been shown to be a strong inducer offluid accumulation and associated pathology in this model (12,37). We employed the rabbit ileal loop assay to investigate thecontributions of different classes of virulence-associated S. ty-phimurium genes, previously characterized in the murine mod-el, to the pathogenesis of gastroenteritis and diarrhea.

* Corresponding author. Mailing address: Department of InfectiousDiseases, Imperial College School of Medicine, Hammersmith Hospi-tal, London, United Kingdom. Phone: 441813832067. Fax: 441813832074.E-mail: p.everest@ic.ac.uk.

† Present address: Vaccine Research Unit, Medeva Development,Imperial College, London, United Kingdom.

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MATERIALS AND METHODS

Bacterial strains and growth conditions. S. typhimurium TML was isolatedfrom a clinical case of human gastroenteritis, causes diarrhea in monkeys, andhas been shown in previous studies to invade rabbit ileal loop mucosa and causean intense inflammatory response and fluid accumulation within 18 h (10–14, 30,31, 36, 37). S. typhimurium TML aroA was provided by G. Douce (ImperialCollege, London, United Kingdom). S. typhimurium HWSH was initially isolatedfrom a calf dying of systemic salmonellosis (27). S. typhimurium HWSH aroAharbors a deletion mutant in the aroA gene and has been described previously(27). S. typhimurium SL1344 and SL1344 aroA (strain 3261) have been describedpreviously (15). S. typhimurium SL1344 msbB was constructed by inserting aDNA sequence encoding kanamycin resistance into a cloned S. typhimuriummsbB gene and replacing the wild-type msbB gene with the inactivated gene byallelic exchange (23). S. typhimurium SL1344 ssrA is defective in expression of thetype III secretion system encoded within SPI-2. S. typhimurium SL1344 invA wasprovided by Tahir Ali (Imperial College). S. typhimurium SL1344 invA is defec-tive in the expression of SPI-1 (9) (Table 1).

Bacteria were grown statically and aerobically in Luria broth overnight at 37°C.For intestinal infection, bacteria were harvested by centrifugation and resus-pended at a concentration of 108 bacteria/ml in phosphate-buffered saline (PBS)for injection into rabbit ileal loops.

Ileal loop procedure. Specific-pathogen-free New Zealand White rabbitsweighing less than 2 kg were anesthetized. The peritoneal cavity was opened bya sterile surgical technique, and the bowel was carefully washed with prewarmedPBS and clamped at least 20 cm distal to the ligament of Trietz. The requirednumber of loops (loop length, 5 cm; interloop length, 5 cm) was measured, andthe bowel was clamped proximal to the ileo-cecal junction. The length of bowelmaking the loops was cut at both ends, and all cut surfaces were clamped. Theremaining intestine was reanastomosed and placed back into the peritonealcavity. The resected ends of the isolated intestine were closed with sutures, and,to avoid compromising the integrity of blood supply to the tissue, the requirednumber of loops was constructed by tying with ligatures. While closing the looptie, we injected the bacterial suspension containing approximately 108 viablebacteria in a 0.5-ml volume of PBS into the proximal end of the loop. Positive-control loops were inoculated with 1 mg of cholera toxin (CT) (a gift from M.-G.Pizza, Instituto Richerche Immunologique Siena, Siena, Italy) in 0.5 ml of PBS;negative-control loops received 0.5 ml of PBS alone. The resected intestine wasreplaced into the peritoneal cavity, the cavity was closed, and the animal wasallowed to recover. After 18 h, the animal was anesthetized, and the loops wereremoved and weighed, and loop fluids were placed in sterile containers.

Analysis of fluids from loops. Fluid from infected loops was measured by vol-ume, and the consistency, in terms of color and viscosity, was noted. Bacterial andcellular contents (erythrocytes and leukocytes) of the fluid were determined by wetpreparation and Gram and Giemsa staining. The predominant cell types in the fluidexudate were determined by differential counts after Giemsa staining. Loop fluidswere centrifuged to remove cellular components and assayed for bicarbonate,pH, and hemoglobin levels with a laboratory biochemical analyzer (Radiometer,Copenhagen, Denmark) and for total protein by the method of Bradford (1).

Histopathology of intestinal tissue. After postmortem removal, the loop tissuewas weighed and cut longitudinally to release any fluid contained within. Anygross changes were noted, and the mucosal side was inspected for macroscopictissue damage. Loop tissue was washed in PBS, and small samples of loop tissuewere placed into 10% formaldehyde in PBS at pH 7.2. Tissue was wax embedded,and thin-cut sections were stained with hematoxylin and eosin and examinedunder bright-field illumination with a Nikon Axiophot microscope.

Quantitation of bacteria in loops and fluids. Loop fluid was assessed fornumber of viable bacteria by dilution and surface-viable counting by using amodification of the technique of Miles et al. (25). Loop tissue samples taken postmortem were cultured for S. typhimurium to demonstrate the presence of bac-teria on or within loop tissue. Tissue samples (0.5 g [wet weight]) were washedwith PBS before homogenization. Tissue was homogenized in 5 ml of Luria brothin a stomacher, and the resulting suspension was serially diluted for surface vi-able counting. All samples were plated on Luria agar.

RESULTS

Fluid secretion in rabbit ileal loops induced by differentwild-type S. typhimurium strains and their mutant derivatives.Different S. typhimurium mutant derivatives and the appropri-ate S. typhimurium wild-type controls were inoculated intoligated rabbit ileal loops, and the level of fluid accumulationover the next several hours was noted. Wild-type S. typhimu-rium TML, HWSH, and SL1344 consistently caused significantlevels of fluid to accumulate within the gut lumen comparedto uninfected control loops. The loop-derived fluid was bloodstained and, when examined microscopically, found to containmany erythrocytes and polymorphonuclear leukocytes (PMNs).Fluid taken from loops infected with S. typhimurium HWSHcontained blood clots which were not detectable in similarloops infected with other Salmonella strains. The volumes offluids varied between 3 and 6 ml for the inoculations of differ-ent wild-type strains (Fig. 1). Loops infected with SL1344 ssrAor SL1344 msbB were macroscopically indistinguishable fromloops infected with wild-type S. typhimurium controls. In con-trast, the S. typhimurium aroA mutants and S. typhimuriumSL1344 invA failed to induce any significant fluid accumula-tion. However, on the apical, lumen-exposed surface of loopsinfected with S. typhimurium aroA derivatives, a thin layer ofpus was consistently associated with the mucosal surface,which, upon microscopic examination, was shown to be com-posed mainly of sheets of PMNs. Interestingly, this thin layerof pus was not observed when the invA mutant was tested.The increase in tissue weights for the wild-type S. typhimu-rium-infected loops reflected the fluid contained within them(Fig. 2).

Control loops injected with CT contained an average of 20ml of non-blood-stained fluid, and leukocytes were absent fromthese fluids. Loops injected with PBS contained no free fluid.

Histopathology of rabbit ileal loops infected with differentS. typhimurium wild-type strains, S. typhimurium SL1344 msbB,and S. typhimurium SL1344 ssrA. Tissue was taken from allloops and studied for histological changes. Normal tissue takenfrom a loop inoculated with PBS is shown in Fig. 3A. Contrast-ing tissue taken from a loop infected with wild-type HWSH isshown in Fig. 3B. The changes shown are typical of the histo-logical changes observed in all loops infected with the wild-type strains. In all cases, villi were shortened, and in some casesvilli were completely absent in tissue samples taken from loopsinfected with any of the S. typhimurium wild-type strains (Fig.3C and E). There were large infiltrates of PMNs present inthe damaged villus structure of the lamina propria and on theluminal surface of the mucosa. The lamina propria and sub-mucosal blood vessels also contained large numbers of infil-trating PMNs. The villus enterocyte layer was severely dam-aged. There was bleeding into the submucosa and free blood inthe lumen. Tissue edema was evident, unlike in PBS-injectedcontrols (Fig. 3A). Loops infected with wild-type S. typhimu-rium exhibited dilatation of the crypts, suggesting an active se-cretory response to infection. Tissue taken from loops infectedwith either SL1344 ssrA or SL1344 msbB (Fig. 3D) showedmorphological changes identical to those observed in loopsinfected with the appropriate wild-type control.

Histopathology of rabbit ileal loops infected with S. typhi-murium invA and the different S. typhimurium aroA mutants.Intestinal tissue infected with S. typhimurium aroA strains ex-hibited some histopathological similarities to intestinal tissueinfected with wild-type S. typhimurium (Fig. 3E and F). How-ever, there was a discernible difference in the degree of tissuedamage to the villi. The villus structures were shortened butretained reasonable tissue architecture, unlike the flattened

TABLE 1. Strains used in this study

S. typhimurium Mutation

TML .................................S. typhimurium isolated from clinical case ofgastroenteritis (10)

TML aroA........................aroA mutant of TMLHWSH..............................Wild-type S. typhimuriumHWSH aroA ....................aroA mutant of HWSHSL1344 .............................Wild-type S. typhimuriumSL1344 aroA ....................aroA mutant of SL1344SL1344 msbB...................msbB mutant of SL1344 (lipid A mutant)SL1344 ssrA .....................ssrA mutant of SL1344 (SPI-2 mutant)SL1344 invA ....................invA mutant of SL1344 (SPI-1 mutant)

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mucosa exhibited after wild-type S. typhimurium infection.Large numbers of PMNs were present within the lamina pro-pria of S. typhimurium aroA-infected tissue, demonstrating anongoing inflammatory response. Loops infected with S. typhi-murium aroA strains showed significantly less crypt dilatationthan wild-type-infected tissue. No loops infected with S. typhi-murium invA showed significant and reproducible differencesfrom uninfected control loops (Fig. 3G).

Analysis of fluid from rabbit ileal loops. The fluids collectedfrom loops infected with different S. typhimurium derivativeswere subjected to biochemical analysis (Table 2). The totalprotein content of the fluid was high, reflecting the increasedcellular content and, presumably, leakage of plasma proteins

from damaged capillaries, in contrast to the secretory nature ofthe fluid from CT-treated loops. There was also a high hemo-globin content in the fluid, reflecting the visible presence ofblood. Bicarbonate concentrations in the fluids from S. typhi-murium-infected tissue were within the normal range for rabbitblood (16.2 to 31.8 mM). Fluid pH ranged from 4.5 to 5.2, incontrast to the alkaline pH of 8 in CT-induced fluid, reflectingthe high bicarbonate levels in the CT-induced fluids.

Microbiological analysis of infected rabbit ileal loops. Therewere large numbers of wild-type and mutant S. typhimuriumstrains within all loops where fluid accumulation occurred (Fig.4). Further, viable S. typhimurium strains could be recoveredfrom homogenized intestinal mucosa from all infected loops

FIG. 1. Volumes of fluid recovered from infected rabbit ileal loops 18 h after infection. (a) Fluid recovery in control loops inoculated with either CT as a positivecontrol or PBS as a negative control. (b) Fluid accumulation in loops immunized with wild type S. typhimurium strain TML, HWSH, or SL1344 or the equivalent aroAmutant. (c) Fluid accumulation in loops inoculated with S. typhimurium in which deletions have been engineered in the msbB, invA, or ssrA gene. These mutants wereall created in wild-type S. typhimurium SL1344, which is included for comparison. Error bars represent standard errors of the means. WT, wild type.

FIG. 2. Weight of infected loops recovered from animals 18 h after infection. (a) Weight of control loops inoculated with either CT as a positive control or PBSas a negative control. (b) Weight of loops immunized with wild-type S. typhimurium strain TML, HWSH, or SL1344 or the equivalent aroA mutant. (c) Weight of loopsinoculated with S. typhimurium msbB, invA, or ssrA. These mutants were all created in wild-type S. typhimurium SL1344, which is included for comparison. Error barsrepresent standard deviations. WT, wild type.

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(Fig. 5). The numbers of S. typhimurium mutants and wild-typecontrols present in the tissues were similar in all cases, with theexception of the S. typhimurium invA mutant. Counts of thesebacteria from infected loops were significantly below those ofthe corresponding wild-type S. typhimurium control.

DISCUSSIONHere we demonstrate that genes associated with S. typhi-

murium virulence in the murine model of typhoid fever show

significant differences in their influences on S. typhimuriumvirulence in the rabbit ileal loop model of gastroenteritis. Wild-type strains of S. typhimurium SL1344, TML, and HWSH andmutants SL1344 ssrA and SL1344 msbB exhibited similar pat-terns of pathogenicity in the rabbit ileum with respect to levelsof fluid secretion, histological damage, inflammation intensity,and PMN infiltration. These data suggest that SPI-2 and a fullytoxic lipid A component of endotoxin are not required for theinduction of fluid accumulation in this model. Further, these

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data indicate that these genes are not essential for the induc-tion of diarrhea in gastroenteritis. In contrast, S. typhimuriumTML aroA, HWSH aroA, and SL1344 aroA each induced sig-nificant tissue inflammation but no significant fluid accumula-tion. In addition, S. typhimurium SL1344 invA was found to beessentially avirulent in this assay, inducing no significant fluidaccumulation or tissue damage, which is in agreement withpublished studies showing that SPI-1 function is required forfluid secretion and tissue inflammation in calf ileal loops (38, 39).

Giannella et al. and Gots et al. (11–15) observed a closecorrelation between fluid secretion in rabbit ileal loops and thevirulence for humans of gastroenteritis-causing S. typhimurium.Stephen et al. (4, 30, 31, 36, 37) showed that strains of S.typhimurium in the rabbit ileal loop model could be separatedinto three different groups: invasive and diarrheagenic, inva-sive and nondiarrheagenic, and noninvasive and nondiarrhea-genic. Strains SL1027 and LT-7 were invasive but nondiarrhea-genic and failed to cause a net secretion of chloride in Ussingchambers. This is in contrast to strains isolated from cases ofhuman gastroenteritis, including S. typhimurium TML (30),which were found to be invasive in the rabbit ileal loop, tocause fluid secretion, and to induce chloride secretion and

depressed sodium absorption in Ussing chambers. S. typhi-murium TML was also shown to cause cell damage at villus tipsand to induce a large influx of PMNs into the mucosa. Struc-tural damage to villus tips led to shortened villi, which weresuspected to contribute to diarrhea by altering absorption/secretion ratios (30, 31). Fluid secretion in rabbit loops in-fected with wild-type S. typhimurium was not observed in theabsence of leukocytes, but this leukocyte influx, by itself, didnot induce fluid secretion (36, 37).

Fluid accumulation induced by wild-type S. typhimurium wasassociated with damaged and shortened villi. Although S. ty-phimurium infection had reduced the villi to almost flattenedmucosa by 18 h, most villi retained their structural integrityduring S. typhimurium aroA infection, although some short-ened villi were detectable. Both S. typhimurium wild type andaroA derivatives induced large influxes of PMNs into the sub-mucosal tissue and lamina propria and onto the surface of themucosa. Thus, S. typhimurium aroA derivatives behave like theinvasive, non-fluid-accumulating strains described by Stephenet al. (30). Loops infected with S. typhimurium invA, which ispoorly invasive in tissue culture cells, had normal-looking villiand no inflammatory infiltrate, whereas S. typhimurium SL1344ssrA, required for virulence beyond the mucosa in mice, wasnot impaired in its ability to induce infiltration and damage.

FIG. 3. Histological sections showing cross sections (magnification, 340) ofloop-associated tissues stained with hematoxylin and eosin. (A) Uninfected loopshowing normal villus height. (B) Loop infected with wild-type S. typhimuriumHWSH. Large numbers of inflammatory cells are highlighted in the laminapropria (small arrow) and on top of damaged villi in the lumen (small arrow).Dilation of the crypts suggests an active secretory response (large arrow). Iden-tical histology was also observed with wild-type strains TML and SL1344. (C)Loop infected with S. typhimurium SL1344 showing large numbers of inflamma-tory cells (arrow), predominantly neutrophils (architecture as described above).(D) Loop infected with S. typhimurium SL1344 msbB. Histological changes wereconsistent with those described above for tissues infected with wild-type strains.Similar effects were observed with S. typhimurium SL1344 ssrA. (E) Loop in-fected with S. typhimurium TML. There is extensive villus damage and a largenumber of inflammatory cells in the lamina propria (arrow). (F) Loop infectedwith S. typhimurium TML aroA. Villus shortening and architectural damage areless severe than in panel E, and significantly less crypt dilation is apparent. Tissuetaken from loops infected with either SL1344 aroA or HWSH aroA displayed iden-tical histology. (G) Submucosal loop infected with S. typhimurium SL1344 invA.

TABLE 2. Analysis of fluid production in Salmonella-infected rabbit ileal loops

Strain or agentinoculated

Positive loops(n 5 4)a Mean fluid volb Mean bicarbonate

concnc pHdAvg protein concne

Total Hemoglobinf

CT 4 20 (15–25) 90.1 (70–118) 8 0.3 0.1TML (WT)g 4 2.5 (1.5–3.5) 30.6 (12–46) 5 1.8 0.7HWSH (WT) 4 1.75 (0.5–3) 26.3 (18–39) 5 3.0 0.5SL1344 (WT) 4 3.75 (3–4.5) 30 (18–42) 5.2 2.4 0.35TML aroA 0 No fluid to testHWSH aroA 0 No fluid to testSL1344 aroA 0 No fluid to testSL1344 msbB 4 1 (0.5–1.5) 21 (7.4–35) 4.5 3.5 0.8SL1344 ssrA 4 4.5 (3–6) 25 (11–39) 5 3.5 0.9SL1344 invA 0 No fluid to testPBS control 0 NDh

a Loops showing fluid present in the lumen when opened postmortem were considered positive.b Total volume in milliliters collected from each loop divided by the number of positive loops (numbers in parentheses are ranges).c Total bicarbonate content (in millimolar units) of the fluids present in the loops divided by the number of positive loops (numbers in parentheses are ranges).d pH of fluids in the loop. These were found to be consistent for each strain tested.e Total protein content in the fluids (in milligrams per milliliter) divided by the number of positive loops.f Total hemoglobin content in the fluids (in milligrams per milliliter) divided by the number of positive loops.g WT, wild type.h ND, not done.

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Thus, these data indicate that the main determinant of fluidaccumulation within the rabbit ileal loop could be invasion-induced damage to the villus structure. Wallis et al. (37), afterperforming ileal loop experiments in cattle, proposed that vil-lus height reduction resulted in the loss of the upper absorptiveregion of the villus, which in turn led to physiological secretion.They also proposed that the regeneration of damaged villicould give rise to hypersecretion. This is not observed in theS. typhimurium aroA-infected loops, as villus damage is appar-ently not severe enough to reduce the absorptive capability ofthe ileum or elicit hypersecretion due to villus regeneration.The other main histological difference observed in our studywas that in ileal tissue infected with wild-type S. typhimurium,

the crypts were markedly dilated compared to the crypts inS. typhimurium aroA-infected tissue, where no crypt dilationwas observed. In view of the fact that fluid accumulation wasobserved only in wild-type S. typhimurium-infected loops, cryptsecretion may be another contributor to fluid accumulation inthis model. The fact that S. typhimurium aroA derivatives failedto induce fluid accumulation may be due to the large PMNinfiltrate within the tissues controlling infection before moreextensive villus damage can occur.

One aim of this study was to determine which mutationsmight be incorporated into candidate live oral S. typhimuriumvaccine strains suitable for use in humans. It is essential thatany candidate live vaccine be incapable of causing diarrhea. By

FIG. 4. S. typhimurium viable counts in fluid recovered from ileal loops. (a) PBS control. (b) Viable count of bacteria in the fluid recovered from loops infectedwith one of three wild-type S. typhimurium strains: TML, HWSH, or SL1344. (c) Viable count of bacteria in the fluid recovered from loops infected with S. typhimuriummsbB, invA, or ssrA. These mutants were all created in wild-type S. typhimurium SL1344, which is included for comparison. Error bars represent standard deviations.WT, wild type.

FIG. 5. S. typhimurium viable counts from infected ileal loop tissue homogenates. (a) PBS control loop was not cross-infected by S. typhimurium during the courseof the experiment. (b) Viable count of bacteria in loops infected with wild-type S. typhimurium strain TML, HWSH, or SL1344 or the equivalent aroA mutant. (c) Viablecount of bacteria in loops infected with S. typhimurium msbB, invA, or ssrA. These mutants were all created in wild-type S. typhimurium SL1344, which is included forcomparison. Error bars represent standard deviations. WT, wild type.

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using the rabbit ileal loop, we have demonstrated that the aroAand invA (SPI-1) mutations are appropriate candidate genesfor this purpose, whereas in this assay ssrA (SPI-2) and msbBgave no evidence of producing an attenuating effect. The in-ability of S. typhimurium invA mutants to induce local inflam-mation may suggest that they are poorly immunogenic, but thiscan only be evaluated properly in volunteer studies.

ACKNOWLEDGMENTS

We acknowledge the technical support and advice of the BiomedicalServices staff at Leicester University.

G.D. and P.E. were supported by a program grant from the Well-come Trust. J.K. was a Royal Society University Research Fellow atthe University of Leicester. S.H. acknowledges support from the Well-come Trust (047407/Z/96).

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