JOURNAL OF BACTERIOLOGYVol. 88, No. 5, p. 1503-1518 November, 1964Copyright ©1964 American Society for Microbiology

Printed in U.S.A.

EPITHELIAL CELL PENETRATION AS AN ESSENTIAL STEP INTHE PATHOGENESIS OF BACILLARY DYSENTERYEUGENE H. LABREC, HERMAN SCHNEIDER, THOMAS J. MAGNANI,

AND SAMUEL B. FORMALDepartment of Applied Immnnunology, Walter Reed Army Institute of Research, Washington, D.C.

Received for publication 17 July 1964

ABSTRACTLABREc, EUGENE H., HERMAN SCHNEIDER,

THOMAS J. MAGNANI, AND SAMUEL B. FORMAL.Epithelial cell penetration as an essential step inthe pathogenesis of bacillary dysentery. J. Bac-teriol. 88:1503-1518. 1964.-A parent strain ofShigella flexneri 2a and a colonial mutant derivedfrom it were studied in three animal models. Bothstrains were equally virulent for mice when livingcells suspended in hog gastric mucin were injectedby the intraperitoneal route. Feeding the parentstrain to starved guinea pigs, followed by theintraperitoneal injection of opium, resulted in theformation of ulcerative lesions in the intestinaltract and in the death of these animals. When thecolonial variant was fed to similarly preparedanimals, the animals survived and the intestinaltract remained normal. The parent produceddiarrheal symptoms and intestinal lesions afterits oral administration to rhesus monkeys; thevariant caused neither symptoms nor pathology inthis species. Studies were carried out to definethe characteristics present in the parent strain andabsent in the colonial mutant, which would enablethe parent to produce ulcerative lesions of thebowel and death in the guinea pig model or intesti-nal lesions and diarrheal symptoms in the monkey.Neither serological studies nor growth studiesconducted both in vitro and in vivo offered a clueto explain this difference. The virulent parentstrain was shown to penetrate the bowel epi-thelium and enter the lamina propria; the aviru-lent mutant did not do this. Entrance to thelamina propria was by way of the epithelial cellof the mucosa. The avirulent mutant did notpossess the capacity to penetrate this cell. Thisobservation was extended to show that the viru-lent parent possesses the ability to infect andmultiply within HeLa cells; furthermore, theorganisms are able to penetrate epithelial cellsof the guinea pig cornea, causing ulcerativelesions. The avirulent variant possesses neither ofthese capacities. It is suggested that epithelialcell penetration is a major factor in determiningthe pathogenicity of dysentery bacilli.

A major feature in the pathogenesis of bacil-lary dysentery involves the formation of ulcera-tive lesions of the intestinal tract. These lesionshave long been considered to be the result ofsuccessive waves of absorption and excretion oftoxic products of the dysentery bacillus throughthe bowel wall (Flexner and Sweet, 1906; Felsen,1945). Although the parenteral injection of bac-terial endotoxin may cause intestinal lesions insome animal species (Thomas, 1954; Gilbert,1960), this is a situation quite different fromabsorption of this material from the bowel lumen.

Recently, Schneider and Formal (1963) in apreliminary communication described a smoothcolonial variant of a strain of Shigella flexneri 2awhich, when fed to starved guinea pigs, causedneither death nor pathology. Feeding the parentculture resulted in a fatal enteric infection withtypical ulcerative lesions in the intestinal tract(Formal et al., 1958; Schneider and Formal,1963). The present report summarizes experi-ments in which the parent and variant strainswere studied in various in vivo and in vitromodels. From the results of this work. a conceptof the pathogenesis of bacillary dysentery isproposed which differs from that previouslydescribed, and, as a corollary, an insight intosome properties which render a dysentery bacilluspathogenic is presented.

MATERIALS AND METHODSAnimals. White ICR mice, weighing 18 to 25 g,

were used to study virulence of the S. flexneri 2astrains by intraperitoneal challenge. Hartleystrain or Walter Reed strain guinea pigs, weigh-ing 300 to 400 g, were used to study oral infectionswith the test Shigella strains. Monkeys (Macacamulatta) weighing 6 to 8 lb (2.7 to 3.6 kg) werealso used to study oral infections. All monkeyswere held in quarantine for at least 8 weeks priorto delivery.

Cultures. S. flexneri 2a, strain 2457T, which is1503

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virulent for guinea pigs by oral challenge wasused in previous studies (Formal et al., 1958,1963). This strain has a green-gold smooth trans-lucent (T) colonial form when examined underthe microscope by oblique transmitted light(Landy, 1950; Cooper, Keller, and Walters,1957). It segregates a stable orange opaque (0)colonial variant (strain 24570) once in every 104to 105 cell divisions (Schneider and Formal,1963). Reversion from the variant to the paren-tal form has not been observed. The colonialappearance of the parent and mutant is shownin Fig. 1. The parent and the variant strainswere maintained in the lyophilized state, and anew ampoule was opened for each experiment.Before use in any one experiment, the strainswere checked for colonial purity.Media. Meat extract agar was used for routine

cultivation of strains. Brain Heart Infusion(BHI) Broth (Difco) was used to suspend thechallenge organisms. Salmonella-Shigella (SS)Agar (Difco) and MacConkey Agar (Difco) wereused for isolation and enumeration of the testorganisms. BHI broth, or liquid minimal glucosemedium (Falkow, Rownd, and Baron, 1962)supplemented with 10 ,ig/ml of both nicotinicand aspartic acids, was employed for in vitrogrowth studies of the Shigella strains.

FIG. 1. Colonial forms of Shigella flexneri 2a.The translucent parent (T) is seen as a less-dense,smooth colony. The opaque variant (0) is seen as a

smooth, more-dense colony. Photographed on an

MEA agar plate with the use of oblique transmittedlight. X7.5.

Oral infection of guinea pigs. The procedure asdescribed by Formal et al. (1958) was employed,except that calcium carbonate, previously usedto neutralize gastric acidity, was not adminis-tered. Animals were starved for 4 days; thechallenge dose containing approximately 5 X 107organims suspended in 10 ml of BHI broth wasfed by stomach tube; 1 ml of tincture of opiumwas injected intraperitoneally after challenge.

Oral infection of monkeys. Upon arrival fromthe quarantine section, the monkeys were placedin individual cages or in restraining chairs. Theywere observed daily for at least 1 week for theoccurrence of diarrhea, and stool specimens werecultured for enteric pathogens on at least threeoccasions. Approximately 18% of the animalswhich were found to carry S. flexneri 4 were notused in the study.

After the period of observation, the monkeyswere challenged by stomach tube with approxi-mately 5 X 1010 of either the parent-T or thevariant-0 organisms suspended in 20 ml of BHIbroth. The animals were observed daily for 7days for diarrheal symptoms. The feces werecultured daily on SS agar to determine the pres-ence of the challenge organisms.Mouse virulence tests. Groups of 20 animals

containing equal numbers of males and femaleswere injected intraperitoneally with 0.5 ml ofserial tenfold dilutions of the test culture sus-pended in 5% hog gastric mucin (pH 7.2). Deathswere recorded for 72 hr after challenge, and theLD5,) and standard error (SE) were estimated bythe method of Miller and Tainter (1944).

Histological procedures. The method for freez-ing and sectioning specimens of intestine forfluorescent antibody studies, and procedures todetermine the specificity of the reaction betweenthe Shigella organisms and the homologousfluorescein-labeled rabbit antibody, were pre-viously described (LaBrec and Formal, 1961).However, several modifications of the methodfor preparing the fluorescein-labeled antiserumwere used. (i) Rabbit antisera were fractionatedwith dry sodium sulfate by the method ofSchur and Becker (1963). The y-globulin ob-tained after final precipitation with 12% (w/v)Na2SO4 usually gave a strong y-globulin bandwhen tested against sheep antirabbit serum byimmunoelectrophoresis. (ii) A 1% globulin solu-tion in saline adjusted to pH 9.0 with 0.5 Msodium carbonate-bicarbonate buffer was con-

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jugated with crystalline fluorescein isothiocyanate(BBL; lot # 302699) at a dye-protein ratio of1:50. The dye, dissolved in acetone, was addedto the chilled globulin solution with constantstirring over a period of 2 hr; stirring was con-tinued overnight. The labeled globulin was thenpassed through a column of Sephadex G-25(Pharmacia Fine Chemicals, Inc., Rochester,Minn.) to remove the unconjugated dye. Thefluorescein-labeled globulin was then dialyzedagainst 0.01 M phosphate-buffered saline to re-store the proper pH (7.2) for the antigen-labeledantibody reactions. Before use, suitably dilutedlabeled globulin was adsorbed one time with 50mg of guinea pig or rabbit liver powder. Rho-damine-labeled bovine serum albumin (Nutri-tional Biochemicals Corp., Cleveland, Ohio),used as a counterstain, was added to the adsorbedconjugate before sterilization by filtration.

Tissues for routine histological examinationwere fixed in 10% neutral formalin with 1%sodium acetate, embedded in paraffin, and sec-tions were cut and stained with hematoxylinand eosin.

Cell-culture methods. HeLa S-3 cell monolayers,obtained from Microbiological Associates, Inc.,Bethesda, Md., were used to study the infectivitycharacteristics of the parent-T and variant-Ostrains in an in vitro biological system. The cellcultures were maintained in prescription bottlesas monolayers grown in Minimal Essential Me-dium (Eagle) supplemented with 10% fetal calfserum, 20 ,umoles/ml of glutamine, 50 units perml of penicillin G, and 50 ,ug/ml of streptomycin(complete medium; CM). The cultures weremaintained in a humidified incubator in an at-mosphere of 5% CO2 and 95% air. For passage,the medium (CM) was removed and the cellswere washed once with 2 ml of 0.25% trypsin(Difco, 1:300) in saline D-1 (trypsin D-1), (Hamand Puck, 1962). The wash was removed andwas replaced with 2 ml of trypsin D-1. Themonolayers were incubated at 37 C with occa-sional shaking until the monolayers loosenedfrom the surface. After further dispersion bvgentle agitation, the cells were dispensed intonew prescription bottles.The cover-slip culture technique of DeMars

(personal communication) was used to preparemonolayers for infection experiments. Five cleansterile no. 1 cover slips, 18 mm in diameter, were

immobilized onto the bottom surface of a plastic

tissue culture dish (60 mm; Falcon Plastics, LosAngeles, Calif.) by drawing a flap of plastic overthe edge of the cover slip with a hot spatula.Each dish was then seeded with approximately105 cells, and the CM was changed at 18 hr andevery 48 hr thereafter; 24 hr prior to infection,the CM was removed and replaced with CMlacking antibiotics. A second change of CM with-out antibiotics was made just prior to infection.Infection of the HeLa cell monolayers was ac-complished by simple addition of washed bacteriato yield a final concentration of 3 X 107 bacteriaper ml, and the monolayers were incubated.After 3 hr, the CM lacking antibiotics was re-moved, the monolayers were washed three timeswith Hank's Balanced Salt Solution (BSS), andfresh CM without antibiotics was added. Thewashing procedure was repeated every 2 hr tominimize extracellular multiplication of the in-fecting Shigella strains. Usually, cover-slip sam-ples were taken at 3, 5, and 7 hr after inoculationof the Shigella strains. The cover-slip preparationswere washed in three changes of saline, fixed in amixture of 3:1 methanol and acetic acid, andthen stained with Giemsa stain at pH 6.5 (0.01M phosphate buffer).

Agglutinin and agglutinin-adsorption tests.Antisera against the parent-T and the variant-Ostrains were prepared in rabbits by intravenousinoculation of suspensions of living cells. To de-termine agglutinin titers, 0.5 ml of saline suspen-sion of living cells was added to 0.5 ml of serialtwofold dilutions of antiserum. The tubes wereincubated at 52 C for 18 hr, and the agglutinintiters were recorded.For reciprocal agglutinin-adsorption tests, 4 ml

of a 1:20 dilution of each antiserum was adsorbedtwo times with 1011 of the appropriate living cellsfor periods of 1 hr at room temperature. Afterfinal adsorption, the serum was separated fromthe bacteria by centrifugation, and was testedfor agglutinins as described above.Immunodiffusion studies. Antisera prepared as

above were tested by the Ouchterlony method todetermine the qualitative antigenic structure ofthe two strains. Heavy suspensions of living cellsor cell juice from sonically disrupted bacteriawere used as antigens. The precipitin bands wereallowed to develop for 1 week at room tempera-ture in a humid atmosphere.In vitro growth studies. A total of 105 cells of

either the parent or the mutant strains were

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FIG. 2. Ileum of a guinea pig starved 4 days, fedthe virulent parent strain of Shigella fiexneri 2a, andsacrificed 24 hr after challenge. Thinning of theepitheliuin can be seen, with some loss of epithelialcells. There is fusion of the villi. The lamina propriacontains large numbers of inflammatory cells. Thevillus-crypt ratio is altered, indicating a high turn-over of epithelial cells. Goblet cells are empty. X95.

FIG. 3. Ileuni of a guiinea pig starved 4 days, fedthe acirulent variant strain of Shigella flexneri 2a,and sacrificed 24 hr postchallenge. T'he normalmucosal architecture is maintained. The villus-crypt ratio is normal. The epithelial cells are normlalin polarity and height, with a well-defined brushbor der. Goblet cells are futll. X95.

inoculated into 15 ml of BHI broth or liquidminimal glucose medium. At various intervals,samples were removed and viable cell countswere determined.

RESULTS

Oral infection of guinea pigs with the parentand variant strains. Formal et al. (1958) reportedthat starved guinea pigs are susceptible to oral in-fection with S. flexneri 2a, strain 2457T, providinga drug such as opium is injected after challenge.In this exlerimental model, oral challenge with107 dysentery bacilli usually results in the deathof approximately 80 to 90%c of the animals, andulcerative lesions of the intestines similar tothose seen in human infections are observed. Onthe other hand, similarly treated guinea pigssurvive oral challenge with Escherichia coli, andthe intestinal mucosa of these animals remainsnormal.The virulence of the lparent-r strain was com-

pared with that of the variant-O strain by feed-ing the cultures to starved guinea pigs. In aseries of experiments, 54 of 60 animals fed theparent-T strain succumbed, whereas only 1 of44 of those fed the variant-O died. It is readilyapparent that this mutation, expressed pheno-typically by colonial variation from translucentto opaque, is also accoml)anied by a loss ofvirulence for the starv-ed guinea pig.The ability of these strains to l)roduce his-

tological changes in the guinea pig intestine wascompared in similar feeding experiments. Ani-mals fed either of the strains were sacrificed 24hr after challenge. Figure 2 shows the typicalulcerative lesion of the intestine seen in animalsfed the virulent parent culture. This lesion ischaracterized by a heavy infiltration of inflam-matory cells, congestion and extravasation oferythrocytes, and a general loss of villus archi-tecture. The histological picture in animals in-fected with the avirulent variant is strikinglydifferent, as can be seen in Fig. 3 where the in-testinal tract is essentially normal. The 0-variant,therefore, not only has lost its ability to killstarved guinea pigs but also is incapable of caus-ing significant histological alteration of the in-testinal mucosa.

Peritoneal infection of mnice with the parent andvariant strains. Others (Cooper et al., 1954;Watkins, 1960) reported that colonial variantsof Shigella strains differed in their ability to kill

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TABLE 1. Virulence for mice of the parent and colonial variant strains of Shigella flexneri 2a*

Challenge dosetStrain LD50 SE

104 103 102

Parent ................ 33/40t 26/40 12/40 2.4 X 102 4. 2 X 102

Variant .... .. 35/40 25/40 18/40 3. 5 X 102 5.5 X 102

* Data were pooled from two experiments.t Challenge was suspended in 0.5% hog gastric mucin, and was injected intraperitoneally in 0.5-ml

volumes.t Death/total.

mice when injected by the intraperitoneal route.Similar mouse virulence assays were performedin our laboratory The parent-T and variant-Ostrains suspended in hog gastric mucin were in-oculated into separate groups of mice to seewhether the differences in virulence noted fororally challenged guinea pigs could be extendedto this laboratory model. The results (Table 1)show that the T and 0 strains are equally viru-lent for mice challenged in this manner. Thetoxicity of the parent and variant cultures wasalso compared. Weighed amounts of acetone-killed and dried cells suspended in saline wereinjected intraperitoneally, and deaths whichoccurred within 3 days were recorded. The LD50for cells of both the parent and the variant strainswas 8 mg.

Oral infection of monkeys with the parent andvariant cultures. The parent culture of S. flexneri2a and its variant were fed to Rhesus monkeysto determine whether either or both would causesymptoms associated with bacillary dysentery.A suspension of approximately 5 X 1010 parentorganisms was fed by stomach tube to each often monkeys. Observations for signs of illnessand stool cultures were made daily. Within 48hr of challenge, three of the animals had frankdysentery with blood and mucus and a fourthexhibited simple diarrhea. The translucent formof S. flexneri 2a was isolated from the diarrhealstools. Of the six monkeys which appeared nor-mal over the period of the experiment, three shedthe parent-T organisms on only the day afteroral challenge. The stools of the other threemonkeys were negative throughout the courseof the experiment.The variant, on the other hand, produced no

apparent symptoms when five doses of 5 X 1010cells were fed to 25 monkeys at intervals of 3

days. The stools remained normal, and variantcells were recovered from the feces of all animalsat one time or another over the course of theexperiment, but never on more than 2 consecu-tive days.

Histological examination of tissues from ani-mals infected with the parent strain, and exhibit-ing symptoms of classical bacillary dysenterv,showed typical lesions of the colon Histologicalalterations of the ileum were observed, but ulcera-tive lesions were rare (Fig. 4 and 5). In contrast,the intestinal tracts of monkeys fed 18 consecu-tive doses of the variant culture were normal(Fig. 6 and 7).Thus, the parent-T strain fatally infects mice

when injected intraperitoneally, causes deathand bowel pathology when fed to starved guineapigs, and causes clinical symptoms of dysenteryand intestinal lesions when administered orallyto monkeys. The 0-variant, though equallyvirulent for mice when inoculated intraperi-toneally, causes neither death nor pathology inthe guinea pig model, and is ineffective in pro-ducing either symptoms or pathology in monkeys.A series of studies were then carried out in an

attempt to define the characteristics possessed bythe parent form, and absent or masked in thevariant culture, which are responsible for thepathogenicity of the parent for starved guineapigs and monkeys.

Serological studies. Experiments were conductedto determine whether mutation from the trans-lucent to the opaque colonial form in S. flexneri2a, strain 2457T, was accompanied by any dis-cernible change in the antigenic mosaic of thetwo strains. First, antisera prepared in rabbitsagainst the living T and 0 cells were tested inreciprocal adsorption tests. Qualitative differ-ences in the surface antigens were not apparent

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FIG. 4. Colon of a monkey killed 3 days after oral challenge with the virulent parent strain of Shigellaflexneri 2a. Mucosal ulcerations and focal crypt abscesses containing large numbers of inflammatory cellsare numerous. The tubular glands are elongated. Goblet cells are empty. X110.

FIG. 5. Terminal ileum of a monkey killed 3 days after oral challenge with the virulent parent strain ofShigella flexneri 2a. The villus tips are swollen and edematous. There is thinning of the epithelium, and thebrush border is indistinct. Goblet cells are diminished in number. High epithelial cell turnover is indicatedby the altered villus-crypt ratio. Large numbers of inflammatory cells can be seen in the lamina propria. X180.

(Table 2). Preliminary experiments with use ofthe Ouchterlony immunodiffusion technique werealso carried out with both live and sonicallydisrupted cells of the T and 0 strains as antigens

and sera from rabbits immunized with live cellsof either strain. Each antigen preparation wastested against antisera from four rabbits (twoantisera for each strain). Analysis by this method

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TABLE 2. Reciprocal agglutinin adsorption tests withantisera against Shigella fiexneri 2a strain 2457

and its colonial variant

Antiserum prepared against

Parent Variant

Test antigen Adsorbed Adsorbedby by

Unadsorbed U_nad-_=c|sorbed 9Cd~~~~~~.

Parent. 10,240* 40 40 5,120 40 40Variant... 10,240 40 40 10,240 40 40

* Values equal reciprocal of highest dilution ir,which agglutination occurred.

revealed no qualitative antigenic differencesbetween the T and 0 strains.

Growth studies. Earlier studies (Formal et al.,1963) indicated that multiplication in the smallintestine is an important factor in initiating thedisease in starved guinea pigs. To determinewhether the loss of pathogenicity of the 0-vari-ant is a function of generation time, in vitroand in vivo growth studies were conducted. Thein vitro growth rate of the two strains was com-pared in a complex medium, BHI broth, and ina synthetic medium, minimal glucose broth. Theresults (Fig. 8) demonstrate that the rates ofgrowth of the parent and variant strains do notdiffer in these media.The ability of the two strains to grow in vivo

was assessed in the experimental guinea pigmodel. In the first experiment, starved guineapigs were orally infected with 5 X 107 cells ofeither strain, and groups were killed 8 and 24 hrpostchallenge. The entire small intestine wasremoved and ground in a mortar with sterilesand and saline. The total volume was noted, ten-

FIG. 6. Colon of a monkey fed 18 consecutivedaily doses of the avirulent variant strain of Shigellaflexneri 2a and sacrificed 24 hr after the last dose.The mucosal architecture is normal. X180.

FIG. 7. Ileum of a monkey fed 18 daily doses ofthe avirulent variant strain of Shigella flexneri2a and sacrificed 24 hr after the last dose. Themucosal architecture is essentially normal, with onlya slight hypercellularity of the lamina propria.Normal polarity of the epithelial cells is seen, andthe brush border is well defined. The normal villus-crypt ratio is preserved and goblet cells are filled.X180.

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-BHI were accomplished by use of aseptic technique,

0O T-BHI with no significant interference with the intestinal,o'oA blood supply. Immediately thereafter, 106 cells

of the proper culture were injected intraduo-denally, and the incision was closed. At variousintervals after the completion of the operation,

, / the animals were killed, and the total numbers ofdysentery bacilli in the small intestine were

8os *xII determined. The growth curves constructed from10sOi/ these data (Fig. 10) demonstrate that, under the

z / conditions of this experiment, the avirulent0 I~~~~~~~~~~x0 --MIN4 GLU NA

Or | / / zw1 0--MIN GLUNA mutant multiplied as well as did the virulent0

o/ X?IT-MINGLUNA parent.C X// , Fluorescent antibody studies. This series of

17 8x <'

10 L

I 0

0~ ~~~1'3/0/ / or

E

o0 00

6 I0 20~~~~~~~~~~~~~~~~~~~~~~10 2 3 1/2 4 1/2 5 1/2. Hours

HOURS FIG. 9. In vivo growth of the virulent parent andFIG. 8. In vitro growth rate of the virulent parent the avirulent variant strains of Shigella flexneri 2a

and the avirulent variant strains of Shigella flexneri in the small intestine of guinea pigs starved 4 days2a in a complex medium (BHI) and a synthetic and then fed 5 X 10' cells of either the parent ormedium (Min Glu). The points on the curves repre- variant strains. T'he points on the curves representsent the number of viable cells per milliliter. the geometric mean of the number of organisms

recovered from the entire small intestine of four ani-

fold serial dilutions were made, and the appropri- mals.ate dilutions were spread on the surface of SS 1010agar plates. S. flexneri colonies, confirmed by sero-logical typing, were then enumerated (Fig. 9). 9 _At 8 hr postchallenge, the avirulent 0-variant

'O

appeared to maintain itself in the small bowelas well as the virulent parent. However, at 24 W

hr after challenge, the 0-variant was present in E /much smaller numbers than was the virulent 107-parent. XBecause the avirulent mutant was unable to z/

maintain itself in the small bowel as well as theparent culture, it was considered possible that it 6' ! _, _, _,_ _is more sensitive than the parent strain to some 0 4 8 12 24factors present in the small intestine. This con- Hours

cept was evaluated by comparing the growth of FIG. 10. In vivo growth of the virulent parent andthe avirulent variant of Shigella flexneri 2a in thethe two cultures in the small intestines of starved l

guinea pigs subjected to ligation of the terminal ligated, and the organisms were inoculated into theileum. This, in effect, produced a closed tube for duodenum. The points on the curves represent thebacterial growth in the presence of any possible geometric mean of the organisms recovered from twoinhibitory substance. Laparotomy and ligation animals.

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growth experiments indicates that the avirulentculture multiplies as well as the parent understatic conditions both in vitro and in vivo wherethe terminal ileum is ligated. Under conditionsof the experimental infection where the organismswere fed and intestinal motility was inhibitedby opium, the 0-variant appeared to do as wellas the parent for only 8 hr. By 24 hr, when theeffects of the opium had worn off, the virulentparent was present in numbers 100-fold largerthan the avirulent mutant. It is likely that thevariant strain was cleared from the small intes-tine by virtue of that organ's motility (Dixon,1960; Formal et al., 1963). On the other hand,the recovery of the virulent parent in relativelyundiminished numbers from the small intestinecan be explained by the fact that it possessesthe ability to penetrate the mucosal epitheliumearly in the infection and to multiply in thelamina propria, thus rendering it resistant to thecleansing effects of bowel motility. This phenom-enon, mucosal penetration, might represent astep in the formation of intestinal ulcers, thecharacteristic lesion of classical bacillary dysen-tery (LaBrec and Formal, 1961). In view of theinability of the variant strain to cause intestinalpathology in either guinea pigs or monkeys, astudy with the use of fluorescent antibody meth-ods was undertaken to determine whether theavirulence associated with the 0-variant is aresult of a loss in ability to penetrate the epi-thelial barrier of the intestinal mucosa.

G-uinea pigs experimentally infected witheither the parent or the mutant strain were killedat various intervals after challenge, and frozensections of intestinal specimens treated withspecific fluorescein-labeled antibody to S. flexneri2a were examined. In confirmation of our pre-vious work, fluorescing dysentery bacilli wereobserved in the lamina propria of the intestinewithin 8 hr after the animals were fed the viru-lent parent strain (Fig. 11). By 24 hr postchal-lenge, the full range of intestinal involvementdescribed above was observed (Fig. 12). Incontrast, penetration of the intestinal mucosawas not observed in specimens from guinea pigsfed the avirulent mutant and sacrificed 6 and 8hr postchallenge, although moderate to largenumbers of specifically fluorescing bacilli were

seen in the lumen (Fig. 13). Bacteria were notobserved in the intestinal mucosa, very fewfluorescing cells were seen in the intestinal lumen,

and the villus architecture was normal 24 hrafter challenge with the avirulent strain.From the above observations, it was considered

that transmigration of dysentery bacilli acrossthe epithelial barrier may be the primary mech-anism involved in the pathogenesis of the disease.It was of major importance, then, to determinewhether this phenomenon occurred by passagethrough individual epithelial cells or betweenthem. Figure 14 clearly demonstrates the pres-ence of dysentery bacilli in an intestinal epithelialcell of a guinea pig fed the virulent parent strain.This was not an isolated observation, but was aconsistent feature of the experimental infection.It is likely that penetration into the epithelialcell and passage into the lamina propria areactive rather than passive processes. Althoughthe avirulent variant is present in relativelylarge numbers in the intestinal lumen, and is inintimate contact with the epithelial surface, itwas never observed within epithelial cells or inthe lamina propria (Fig. 13). Thus, one mighttentatively consider the chain of events in theinfection to consist of penetration of the mucosalepithelial cell (Fig. 14), entrance into the laminapropria by passage through this cell (Fig. 11),multiplication within the mucosa, and subse-quent ulcer formation (Fig. 12). The avirulentvariant has lost the ability to initiate this chainof events.A similar study was carried out in monkeys

with the use of tissues from animals killed afterinfection with either the parent or mutant strain.Animals fed the virulent parent culture and ex-hibiting symptoms of clinical bacillary dysenterywere killed. Frozen sections of intestinal speci-mens were examined by the fluorescent antibodytechnique. Specifically fluorescing dysenterybacilli were seen in the lamina propria of thececum and colon in the presence of an apparentlyintact mucosal epithelium. Most of the specimensexhibited severe ulcerations of the mucosa andfocal crypt abscesses containing large numbersof fluorescing Shigella (Fig. 15). Although thesechanges obscured the mucosal architecture, thephenomenon of epithelial cell penetration byvirulent Shigella was seen sufficiently often to bealso a consistent feature of the disease process inthe monkey (Fig. 16). Again, we failed to observefluorescing dysentery bacilli either in the laminapropria or in the luminal contents of monkeysfed 18 consecutive daily doses of the 0-variant

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FIG. 14. Frozen sections of ileum from four guinea pigs killed 8 hr after feeding the virulent parent strainof Shigella flexneri 2a. All sections were treated with fluorescein-labeled rabbit anti-S. flexneri 2a globulin.Several examples of penetration of the mucosal epithelium by virulent dysentery bacilli can be seen (arrows).Fluorescing Shigella can be seen in the epithelial cells (a, b, d), although the brush border appears intact.Original magnification, X240 (a and b); original magnification, X540 (c and d).

FIG. 11. Frozen section of ileum from a starved guinea pig fed the virulent parent strain of Shigella flex-neri 2a and killed 8 hr after challenge. The section was treated with fluorescein-labeled rabbit anti-S. flexneri2a globulin. Note the specifically fluorescing bacilli in the lamina propria of a single villus (A; see whitearrows). B denotes the primary fluorescence of inclusions in macrophages normally present in the intestine.The black arrows point to other shigellae between two villi. Original magnification, X540.

FIG. 12. Frozen section of ileum, from a starved guinea pig fed the virulent parent Shigella flexneri 2aand killed 24 hr after challenge. The section was treated with fluorescein-labeled rabbit anti-S. flexneri 2aglobulin. A typical ulcerative lesion with complete loss of the normal architecture is seen. The lesion con-tains fluorescing Shigella, inflammatory cells, and necrotic debris. L denotes the intestinal lumen. Originalmagnification, X240.

FIG. 13. Frozen section of ileum from a starved guinea pig fed the avirulent variant strain of Shigellaflexneri 2a and killed 8 hr after challenge. The section was treated with fluorescein-labeled rabbit and anti-S.flexneri 2a globulin. Specifically fluorescent variant-0 Shigella are seen in the intestinal lumen (L), betweenthe villi, and in the infoldings of the villus epithelium (arrows). Original magnification, X240.

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FIG. 15. Frozen section of the colon from a monkey

fed the virulent parent strain and killed 3 days later.The section was treated with fluorescein-labeled

and killed 24 hr after the last dose was admin-istered.

Studies utith the corneal epithelium. For a num-ber of y-ears, workers have differentiated strainsof Shigella on the basis of their ability to cause akeratoconjunctivitis after inoculation of the con-junctival sac (Zoeller and Manoussaki, 1924;Serenev, 1955; Kerekes, 1962). An essentialfeature of this infection is the invasion of theepithelial cell of the cornea (Pieehaud, Szturm-Rubinsten, and 1'iechaud, 1958), which resultsin an ulcerative lesion. When this test was carriedout in guinea pigs, it was found that a positivereaction was obtained with the parent strainwhen approximately 5 X 107 cells were used asan infecting dose.The virulent lparent invaded the epithelial

cell of the cornea (Fig. 17), and an ulcerativelesion resulted (Fig. 18). On the other hand, thecornea of eyes inoculated with as many as 5 X 109cells of the avirulent mutant strain remainednormal, and organisms were never observedwithin the epithelial cells.

Tissue culture studies. If a major differencebetween the parent and the mutant strain isreflected by the ability of the parent to penetrateepithelial cells, it seemed possible that this differ-ence might be shown in a less complex system.Accordingly, a study of the behavior of the p)arentand mutant strains in HeLa cell culture wasundertaken. HeLa cell cultures were infectedwith either the parent or the mutant strain asdescribed above. Both strains multiplied equallywell in the cell culture medium. However, 5 hrafter inoculation of the cell cultures with theparent strain, bacteria were observed within theHeLa cells (Fig. 19), and, by 7 hr, degenerationof the cell culture was evident. On the otherhand, the avirulent mutant was not observed

rabbit anti-Shigella flexneri 2a globulin. The photo-graph shows a mlicroulcer with many fluorescingShigella in the lamina propria, in epithelial cells,and phagocytosed inflammnnatory cells. L denotes theintestinal lumen. Original magnification, X240.

FIG. 16. Frozen section of colon from a monkeyfed the virulent parent strain and sacrificed 48 hrlater. T'he section was treated with fluorescein-labeled rabbit anti-Shigella flexneri 2a globulin. Cdenotes the lumiien of a tubular gland. The arrow atE points to specifically fluorescing Shigella inepithelial cells. 7'he other arrows show individualshigellae in the lamiiina propria. Original magnifica-tion, X>240.

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PATHOGENESIS OF BACILLARY DYSENTERY.: s; l-

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1t .. .._ w,

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FIG. 17. Section of eye from a guinea pig killed 24 hr after inoculation of the conjunctiva with the virulentparent strain of Shigella fiexneri 2a. Numerous bacilli can be seen in the corneal epithelium. X896.

FIG. 18. Section of eye from a guinea pig killed 24 hr after inoculation of the conjunctiva with the virulentparent strain of Shigella fiexneri 2a. Note the intact corneal epithelium at A and an ulcerated portion at B.Edema and leukocytic infiltration of the corneal stroma are prominent (C). Ulcerative lesions of the con-junctiva can also be seen (D). X53.

within HeLa cells, and 7 hr after inoculation themonolayer appeared no different from uninfectedcontrols (Fig. 20).

DISCUSSION

These studies emphasize several factors con-cerned with the pathogenesis of bacillary dys-entery. First, they demonstrate quite clearly thatability of a dysentery strain to infect fatally

mice after intraperitoneal injection does notnecessarily correlate with its capacity to provokesymptoms when administered by the oral route.The parent and the variant used in this studywere equally virulent for mice, but only theparent was pathogenic when orally administeredto monkeys or to starved guinea pigs. Indeed,the latter two species appeared to react no differ-ently to the avirulent variant than had the ani-

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FIG. 19. Cover-slip monolayer of HeLa S-3 cells 5 hr after inoculation with the virulent parent strain ofShigella flexneti 2a. Several HeLa cells containing nunmerous bacilli can be seen. Giemsa stain. X640.

FIG. 20. Cover-slip monolayer of HeLa S-3 cells 7 hr after inoculation with the avirulent variant strain ofShigella flexneri 2a. The morphology of the HeLa cells is preserved, and no bacilli can be seen in the illustra-tion. A lower miagnification is used to show a larger field. Giemsa stain. Original magnification, X240.

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PATHOGENESIS OF BACILLARY DYSENTERY

mals been fed E. coli cultures isolated from nor-mal individuals. Thus, the standard mouse viru-lence test, as used over the years, is of limitedvalue for study of the pathogenesis of bacillarydysentery.On the positive side, our results give further

insight into the mechanism of ulcer formationand, from this, a beginning to an explanationconcerning the characteristics which make adysentery bacillus pathogenic. Earlier workers(Flexner and Sweet, 1906; Felsen, 1945) suggestedthat, in dysentery infections, lesions of the in-testines gradually developed after several wavesof absorption and excretion of stable toxic bac-terial products through the bowel wall. However,we fed monkeys large numbers of acetone-killedand dried dysentery bacilli daily for 30 days,with no detectable unfavorable effects (Formal,unpublished data); on the basis of our presentfindings, it seems that an alternative mechanismfor the formation of ulcerative lesions is justified.LaBrec and Formal (1961), using the fluores-

cent antibody technique, previously observeddysentery bacilli in the lamina propria of theileum, cecum, and colon of experimentallyinfected guinea pigs; the epithelium of the mucosaoverlying these regions appeared to be intact.This finding was confirmed in the present study,and was extended to show that a similar situa-tion also occurs in a natural host, the monkey,when it is fed the virulent parent, i.e., dysenterybacilli in the lamina propria in the presence of anintact mucosal epithelium. It seemed most likelythat the dysentery bacilli passed either betweenthe epithelial cells of the mucosa or through them.Our observations with the fluorescent antibodytechnique, and those of Takeuchi (unpublisheddata) with electron microscopic techniques, dem-onstrate clearly that virulent dysentery bacillihave a capacity to enter the epithelial cell ofthe bowel mucosa. One may now construct atentative chain of events in the evolution of anulcerative lesion. First, the dysentery bacilluspenetrates the epithelial cell (at present we donot know the mechanism). The bacteria reachthe lamina propria via this cell. At this stage,the bacteria are present in the lamina propria,while the epithelial barrier remains intact. Herethe organisms multiplv further, and may elab-orate toxic products, resulting in the death ofthe epithelium and ulcer formation.

Although the ability to penetrate the intestinal

epithelial cell and reach the lamina propria is acharacteristic of our virulent parent strain, it isnot an attribute of the nonpathogenic variant.This difference also was seen in other experi-mental models. The parent strain penetratedand multiplied in cultured HeLa cells; the 0-vari-ant was not observed to enter these cells. Theparent entered the epithelial cells of the corneaof the guinea pig, multiplied, and ulcerativelesions resulted. When exposed to the variantstrain, the cornea remained normal, and bac-teria were not observed in the epithelial cells.The only difference in the parent and the vari-

ant strains which we were able to detect is theability of the parent to invade epithelial cells.It is possible that this characteristic is a majorfactor in endowing the parent culture with thecapacity to cause disease. Once the organismreaches the lamina propria, its effect may berelatively nonspecific if the subsequent changeswhich occur are due largely to the effect of bac-terial endotoxin (Thomas, 1954; Gilbert, 1960),a component of bacteria belonging to a varietyof genera. If this is the case, then it is conceivablethat epithelial cell penetration and at least limitedsurvival in the lamina propria are the necessaryattributes for pathogenicity of dysentery bacilli,and are characterisitics which set them apartfrom nonpathogenic E. coli strains and avirulentShigella. Because, to date, there has been noreasonable means to explain the pathogenicityof dysentery bacilli, these possibilities should beconsidered.

LITERATURE CITED

COOPER, M. L., H. M. KELLER, AND E. W. WAL-TERS. 1957. Microscopic characteristics ofcolonies of Shigella flexneri 2a and 2b and th3irrelation to antigenic composition, mousevirulence and immunogenicity. J. Immunol.78:160-171.

DIXON, J. M. S. 1960. The fate of bacteria in thesmall intestine. J. Pathol. Bacteriol. 79:131-140.

FALKOW, S., R. RoWND, AND L. S. BARON. 1962.Genetic homology between Escherichia coliK-12 and Salmonella. J. Bacteriol. 84:1303-1312.

FELSEN, J. 1945. Bacillary dysentery, colitis andenteritis. W. B. Saunders Co., Philadelphia.

FLEXNER, S., AND J. E. SWEET. 1906. The patho-genesis of experimental colitis and the rela-tion of colitis in animal and man. J. Exptl.Med. 8:514-535.

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FORMAL, S. B., G. D. ABRAMS, H. SCHNEIDER, ANDH. SPRINZ. 1963. Experimental Shigella in-fections. VI. Role of the small intestine in anexperimental infection in guinea pigs. J.Bacteriol. 85:119-125.

FORMAL, S. B., G. J. DAMMIN, E. H. LABREC,AND H. SCHNEIDER. 1958. ExperimentalShigella infections: characteristics of a fatalinfection produced in guinea pigs. J. Bacteriol.75:604-610.

GILBERT, R. P. 1960. Mechanisms of the hemo-dynamic effects of endotoxin. Physiol. Rev.40:245-279.

HAM, R. G., AND T. T. PUCK. 1962. Quantitativecolonial growth of isolated mammalian cells,p. 90-119. In S. P. Colowick and N. 0. Kap-lan [ed.], Methods in enzymology. AcademicPress, Inc., New York.

KEREKES, L. 1962. Colonial variants of Shigellaflexneri. Acta Microbiol. Acad. Sci. Hung.9:123-132.

LABREC, E. H., AND S. B. FORMAL. 1961. Experi-mental Shigella infections. IV. Fluorescentantibody studies of an infection in guineapigs. J. Immunol. 87:562-572.

LANDY, M. 1950.The visual identification of V andW form colonies in Salmonella cultures. PublicHealth Rept. 65:950-951.

MILLER, L. C., AND M. L. TAINTER. 1944. Estima-tion of the E.D.50 and its error by means oflogarithmic-probit graph paper. Proc. Soc.Exptl. Biol. Med. 57:862-876.

PIfCHAUD, M., S. SZTURM-RuBINSTEN, AND D.PIlfCHAUD. 1958. Evolution histologique dela k4rato-conjonctivite A bacillus dysente-riques du cobaye. Ann. Inst. Pasteur 94:298-309.

SCHNEIDER, H., AND S. B. FORMAL. 1963. Spon-taneous loss of guinea pig virulence in a strainof Shigella flexneri 2a. Bacteriol. Proc., p. 66.

SCHUR, P. H., AND E. L. BECKER. 1963. Comple-ment fixing properties of pepsin treated rab-bit and sheep antibodies. Science 141:360-361.

SERENEY, B. 1955. Experimental Shigella con-junctivitis. Acta Microbiol. Acad. Sci. Hung.2:293-296.

THOMAS, L. 1954. The physiologic disturbancesproduced by endotoxin. Ann. Rev. Physiol.16:467-490.

WATKINS, H. M. S. 1960. Some attributes ofvirulence in Shigella. Ann. N.Y. Acad. Sci.88:1167-1186.

ZOELLER, C., AND C. MANOUSSAKI. 1924. Experi-mental Shigella keratoconjunctivitis. Compt.Rend. Soc. Biol. 91:257-258.

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