INFECTION AND IMMUNITY, Apr. 1973, p. 666-672Copyright © 1973 Amiierican Society for Microbiology

Vol. 7, No. 4P'rinted in U.S.A.

Microbial Colonization of the Intestinal Epitheliumin Suckling Mice

C. P. 1)AVIS, J. S. McALLISTERV,,' AND D. C. SAVAGE

Dh'partmcntt of Microbiology, Untiversity of T'exas, A ustint, T'exas 78712

Coloinizationi by indigenious microorganisms of the mucosal epithelia of the largebowels of suckling mice was followed by microbial culture techniques and by light,fluorescenice, and electron microscopy. Certain microbes colonize in distinctivepatterns the cecal and colonic epithelia in these mice. Coliforms and enterococeicolonize the large bowel 7 to 9 days after birth and reach high population levelsdurinig the second week. During that period, these facultative anaerobes can bedetected by immunofluoreseence techniques in microcolonies in the mucin OIn theepithelium. During the third week, however, after their populations decline to thelow levels characteristic of adult mice, coliforms and enteroccoci can be observedonly infrequently in the mucous layer. Anaerobic fusiform-shaped bacteria appear

in the mucous layers along with the microcolonies of enterococci and coliformsduring the second week after birth. These anaerobes increase in numbers in themucin until they form thick layers oIn the mucosal epithelium by the end of thethird week. They remain in the mucous layer throughout the life of the normalmouse. Anaerobic spiral-shaped microbes also colonize the mucous layer on thececal and colonic epithelium. But these organisms can be detected by immuno-fluoreseenee in 1-week-old mice, well in advance of the time the fusiform-shapedbacteria can be found. In the second week, the latter microbes co-inhabit the mu-

cous layer with the spiral-shaped organisms. The fusiform- and spiral-shapedmicrobes remnain associated in the mucin on the cecal and colonic mucosal epitheliaiIito the adult life of mice.

The gastrointestinial cainals of suckliing micefrom certaiii specific pathogen-free colonies arecolonized in a particular sequence by varioustypes of indigenous bacteria. Lactobacilli andanaerobic streptococci colonize first in 1 or 2 davsafter birth (6, 9). These microbes are founidthroughout the tract, but particularly in thestomach, where they form a layer oIn the keratin-ized stratified squamous epithelium of the non-secreting tissue (6). The lactic acid bacteria arefollowed by coliforms and enterococci a few dayslater (6, 9). The latter bacterial types are followedby oxygen-tolerant and oxygein-intolerant an-aerobic bacteria during the second week afterbirth (3, 6). The oxygen-tolerant bacteria arel)redominantly bacteroides; the oxygen-intolerantforms are fusiform-shaped bacteria of severalgenera. One or more types of these fusiform-shaped bacteria colonize the mucin closely asso-ciated with the epithelium of the mucosa of thececa anid colons of adult mice (6, 7).

Anaerobic spiral-shaped microorganisms alsocan be detected in the ceca and colons of adultmice (3, 4, 7). Both oxygen-tolerant and oxygen-

' Present address: 1749 Louise, St. Paul, Minn. 55106.

intoleranit forms are present (7). In adult aiiimals,at least one type of spiral-shaped microbe colo-nizes the mucous layer intermixed with the fusi-form-shaped bacteria (7). In this paper, we reportthat in suckling mice one type of spiral-shapedmicroorganism colonizes the epithelial mucusduring the first week after birth, and the fusiform-shaped bacteria coloinize at least a week later. Inaddition, we report that coliforms and enterococeialso can be found OIi the epithelial mucin of thececa and colons of baby mice when the populationlevels of these facultative anaerobes are at highlevels during the second week after birth.

MATERIALS AND METHODSMice. ARS Ha/ICR (A. R. Gibco, Madison,

Wis.) specific pathogen-free (SPF) females werebred in our own colony. Pregnant SPF CF-1(Carworth-Bioquest, New City, N.Y.) and CD-1(Charles River, Wilmington, Mass.) females werepurchased directly from the suppliers. Animalsfrom the three colonies were kept separated fromeach other at all times in our laboratory. Theywere housed in plastic boxes with Isocaps (Iso-cage, Carworth) containing Ad-Sorb Dri (AlliedMills, Chicago, Ill.) in separate flexible film iso-lators (Standard Safety Equipment Co., Pala-

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COLONIZATION OF INTESTINAL EPITHELIUM6

tine, Ill.) previously sterilized with 2% peraceticacid. All entering and exiting air was filteredthrough glass wool. Lab Blox (Allied Mills) andacidified water (7) were given ad libitum. The dateof birth of the infants was recorded so that experi-ments could be conducted with animals of preciselyknown age.

Preparations of bowel specimens. Animalswere killed with either chloroform vapors or CO2and autopsied. Ceca and colons were removed andtreated in one of three ways. For bacteriologicalculturinig, the ceca, with contents intact, were

weighed and then homogenized with Teflon grind-ers in 5 ml of sterile charcoal water (8). For im-munofluorescence testing, the ceca and colonswith contents intact were frozen at -22 C in 2%methyl cellulose in saline (6). For electron mi-croscopy, pieces of ceca and colon from individualmice were homogenized together in 2 ml of coldphosphate-buffered 2% glutaraldehyde.

Bacteriological culture techniques. Thehomogenates described above were diluted incharcoal water in 10-fold steps. Calibrated loopfulsof each dilution were then spread onto Tergitol-7-triphenyl tetrazolium medium (T7T) selective forcoliform bacteria and methylene blue medium se-

lective for enterococci (9). Inoculated plates ofboth media were incubated aerobically at 37 C for24 h. Estimates of the bacterial populations con-

tained in each gram of cecal tissue were madefrom counts of colonies appearing on the media (9).

Histological methods. Segments of colons or

whole ceca were frozen with contents intact in 2%methyl cellulose in saline. The tissues were sec-

tioned at 4,um on a microtome cryostat and fixedonto slides in absolute methanol. These slideswere stored overnight at 4 C. Slides were thenprocessed for either immunofluorescence testingor tissue Gram staining.

Reagents for immunofluorescence. Rabbitantiserum containing antibodies to a spiral-shapedbacterium isolated from Ha/ICR mice (7) was

used in an indirect immunofluorescence test forspiral-shaped microbes. The preparation of thisantibody and its absorption with fusiform-shapedbacteria and mouse liver powder have been de-scribed (7). Normal rabbit serum and a prepara-

tion of goat anti-rabbit IgG labeled with fluores-cein isothiocyanate (FITC; FITC-labeled goatanti-rabbit IgG, kindly supplied by W. J. Mandy)were used in the indirect test. The labeled anti-serum was also absorbed as described above.

FITC-labeled immunoglobulin preparationsprepared from the sera of rabbits immunized withcoliforms or enterococci were used for direct im-munofluorescence testing for these bacterial types.The coliform and enterococcal vaccines were pre-

pared from an Escherichia coli strain and a Strepto-coccus species isolated from Ha/ICR mice. Thebacterial cells of both types were grown in quan-

tity in penicillin assay broth (Difco), centrifuged,and washed three times with phosphate-bufferedsaline (PBS). The coliform cells in PBS were heat-killed at 60 C for 30 min and then adjusted to ap-

proximately 2 X 1010 cells/ml. The enterococcalcells were killed with 1.2%o formaldehyde in PBS,washed three times with PBS, and stored in 0.1%formaldehyde in PBS. This enterococcal vaccinewas adjusted to approximately 4 X 10' cells/ml.

Sera containing antibodies to the E. coli strainwere obtained from 2.5-kg rabbits given threeintravenous injections of 1 X 1010 cells of thecoliform vaccine. The rabbits were rested for 4days between each injection and bled 5 days afterthe last injection. Sera from individual rabbitswere pooled. Pooled sera used to prepare the im-munofluorescence reagent agglutinated the coli-form in suspension to a titer of 1:512.

Sera containing antibodies to the enterococcalStreptococcus strain were obtained from 2.5-kgrabbits given 12 intravenous injections rangingfrom 1 X 10' to 4 X 10' cells of the enterococcalvaccine. The rabbits were injected on each of threesuccessive days and then rested for 9 days. Thissequence was repeated four times for each animal;the rabbits were bled 6 days after the last injec-tion. Individual sera were pooled. Pooled seraused to prepare the immunofluorescence reagentagglutinated the enterococcal cells to a titer of1:2,048.

Immunofluorescence reagents were prepared bythe same procedure from the anticoliform, anti-streptococcal, and normal rabbit sera. Globulinswere precipitated from the sera with ammoniumsulfate, conjugated with FITC, and dialyzed (1).The conjugated globulins were diluted 1:1 withPBS for use in the direct fluorescent-antibodystain. Both reagents specifically stained theirhomologous antigen. They did not stain other in-digenous microorganisms from Ha/ICR mice in-cluding Lactobacillus species, slow lactose-fer-menting E. coli and Bacteroides species. Both re-agents stained slightly the animal tissue in his-tological sections of the bowels. This small amountof nonspecific fluorescence did not interfere in theprocess of localizing the bacteria on the mucosalepithelium.

Immunofluorescence testing. Ceca or colonswere frozen and sectioned (6). For indirect testingfor spiral-shaped microbes, the sections wereflooded with either normal rabbit'serum or serumcontaining antibodies to the spiral-shaped bac-terium, and then incubated for 30 min at 37 C inhigh humidity (1). The sections were then washedwell with PBS and flooded with FITC goat anti-rabbit IgG. The slides were again incubated for 30min at 37 C and again washed well with PBS. Fordirect testing for coliforms or enterococci, thesections were flooded with one of the immuno-fluoresence reagents prepared as described above,incubated at 37 C, and then washed well withPBS. Cover slips were mounted onto all the slideswith PBS glycerine. All sections were examinedwith a Leitz Ortholux fluorescence microscopefitted with a mercury vapor light source, 3-mmBC 12 excitor filter and a K460 barrier filter (E.Leitz, New York, N.Y.).

Electron microscopy. A drop of homogenized

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DAVIS, McALLISTER, AND SAVAGE

cecum and colon in phosphate-buffered 2% glutar-aldehyde was placed onto Formvar-coated grids.After a few minutes, the excess solution was gentlyremoved with filter paper, and a drop of 0.1 to 1%phosphotungstic acid was placed on the grid. Thephosphotungstic acid was removed within 30 s

with filter paper. The grids were viewed in a Hi-tachi HU-8 electroii microscope.

RESULTS

Colonization of the cecal epithelium bycoliforms and enterococci. Enterococci andcoliforms can be cultured from ceca or colons ofbaby Ha/ICR mice beginining about 9 days afterbirth (Table 1). The populations of both bacterialtypes usually increase to levels of 106 to 107 bac-teria per g of whole bowel by the 11th or 12thday after birth. and remain at those levels untilthe 19th to the 21st day. Thereafter, they dropto levels of 105 to < 103 organisms per g, charac-teristic of adult mice (6).

Immunofluorescence detection of the coliformsanld eIiterococci in the mucus on the cecal or

colonic epithelium of the baby mice parallels theculture results (Table 1). On about the 10th dayafter birth, a few of these bacteria can be seen inthe mucous layer and also in the lumen. There-after, for 7 or 8 days, microcolonies of each typecould be seen with ease in the lumen and in themucus adjacent to the epithelial cells of themucosa. Microcolonies coinsist of several closelyassociated fluorescinig cells. When the bacterialpopulation levels dropped to adult levels in mice19 to 21 days old, the enterococci and coliformscould be observed only infrequently in the lumenand in the mucus onl the epithelium. At this time,and in 30-day-old animals, they could be seen

occasionally as single cells mixed in with massiveiiumbers of fusiform- and spiral-shaped bacteria.

Colonization of cecal and colonic epitheliaby fusiform-shaped bacteria. Fusiform-shapedbacteria are known to colonize the mucous layeron the epithelium of the murine large bowel inthe second week after birth. These microbes packthe mucous layer in massive numbers; their popu-lations quickly reach high levels in the bowel andremain at those levels throughout the life of theanimal (5, 6). Using tissue Gram stains in thisstudy, we first detected these bacteria, mixed inwith coliforms and enterococci in the mucouslayer, at the end of the first or beginning of thesecond week after birth (Table 2). By the 11thday, these fusiform-shaped microbes are easilydetectable in most of the animals (Fig. 1).Colonization of cecal and colonic epithelia

by spiral-shaped microorganisms. Spiral-shaped organisms can be observed by immuno-fluorescenice as early as 3 days after birth inCD-1 and CF-1 mice (Table 3). They appear asearly as 5 days after birth in Ha/ICR mice. In3- to 7-day-old animals, they can be seen occa-sionally as single cells or in small microcolonies inthe mucus on the cecal or colonic epithelium.During the next week, however, they can befound with ease in the mucous layer. At no timecan these microbes be seen with ease in the lumenof the cecum or colon.

Until the 10th or 11th day after birth, thesespiral-shaped organisms were virtually alone inthe mucous layer. By the 11th day, however,some coliforms, enterococci, and fusiform-shapedbacteria occasionally could be seen along with thespiral-shaped microbes in the mucin in somemice. By about 2 weeks after birth, almost every

animal examined showed fusiform- and spiral-shaped organisms in the mucous layer adjacentto the mucosal epithelial cells. This pattern was

TABLE 1. Detection of coliforms anid entterococci colonizintg the large bowels of baby Ha/ICR micea

Days after birthBacterial type Detection method

3 5 7 9 11 13 15 17 19 21

Coliforms Immunofluorescence 0/3b 0/6 1/9 0/9 3/9 4/9 5/9 6/9 6/6 2/3Culture ND 0/3 0/6 1/6 3/6 4/6 3/5 5/6 3/3 2/3

Enterococci Immunofluorescence ND 0/3 0/6 0/6 2/6 1/6 4/6 3/6 3/3 2/3Culture ND 0/3 0/6 1/6 2/6 5/6 4/5 1/6 3/3 1/3

a Coliforms and enterococci were detected by two methods. (i) Frozen tissue sections of ceca or colonswere treated with either FITC-anticoliform antibody or FITC-antienterococcal antibody. (ii) Homoge-nized pieces of colon or cecum and colon together were diluted in charcoal water (8) and spread ontoplates of T7T or methylene blue medium; population levels above 103 bacteria per g of whole bowel aredetected by this method.

bRatio represents the number of mice in which the bacterial type was detected/number of animalsexamined. ND, Not done.

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observed as well in weanlings and adults of allthree strains of mice examined.

Immunofluorescence was used to locate these

TABLE 2. Detectiotn of fusiform-shaped bacteriacolonizinig the ceca and colons of Ha/ICR, CD-I,

atnd CF-I micea

Days after birthstan Area of.Mouse strain bowel

3 5 7 9 11 30

Ha/IClI Cecum 0/5b 0/5 1/5 1/5 4/5 NI)Colon 0,'5 0/5 0/5 0/5 4/5 ND

CD-1 Cecum 0/5 0/5 0/5 0/5 3/5 5/5Colon 0/5 0/5 1/5 0/5 1/5 5/5

CF-1 Cecum 0/5 0/5 0/5 0/5 4/5 5/5Colon 0/5 0/5 1/5 1/5 3/5 5/5

a Fusiform-shaped bacteria were detected infrozen histological sections of ceca and colonsstained by a tissue Gram stain (6).

b Ratio represents the number of animals pos-sessing fusiform-shaped bacteria/number of ani-mals examined.

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i 42t small, spiral-shaped organisms because theycannot be seen readily in frozen histologicalsections (7). On rare occasionls, however, theycan be viewed with dark-field optics when themucus in suckling mice is not yet densely l)opu-lated with other microorganisms (Fig. 2).Detection of spiral-shaped microbes in

cecal homogenates from suckling mice byelectron microscopy. Ceca with their contentsintact were removed from suckling mice, ho-mogeniized, anid inegatively stainied with PTA.With an electroin microscope, spiral-shapedmicroorganisms could be detected in these ho-nmogeniates onily 2 to 4 days before fusiform-shaped organiisms could be observed (Table 4), incontrast to findings with the immunofluorescencetechnique. Thus, the technique is not as sensitiveas immunlofluorescence in detecting the microbesin the intestinal tract. The spiral-shaped microbesprobably would be detectable in mice youngerthan 7 days if the total volume of the cecalhomogeinates were negatively stained. Far toomany grids would have to be examined by elec-tron microscopy, however, for this procedure tobe practical.The spiral-shaped microorganiisms from all

three strains of suckling mice are similar in their

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FIG. 1. Fusiform-shaped bacteria, in the mucin between the mucosal epithelium to the right anzd the massof microbes in the lument to the left, in a frozent sectiont of the colont of an 11-day-old CD-I mouse. X 2,200.

VOL. 7, 1973 669

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DAVIS, McALLISTER, AND SAVAGE

TABLE 3. Immunofluorescent detectiont of spiral-shaped microbes colonizing the ceca or colons of babyand adult mice"

Days after birthMouse strain

3 5 7 9 11 13 15 17 21 30 50

Ha/ICR 0/8b 1/6 5/14 11/14 14/14 9/9 9/9 9/9 3/3 ND 5/5CD-1 3/5 5/5 5/5 5/5 5/5 ND ND ND ND 5/5 NDCF-1 3/5 3/5 5/5 5/5 5/5 NI) ND ND ND 5/5 ND

Spiral-shaped microbes were detected by indirect immunofluorescence in frozen sections of ceca orcolons of individual mice (7). If the organisms were present in any section, the mouse was recorded aspossessing spirals.

b Ratio indicates the number of mice possessing spiral-shaped microbes in their ceca or colons/niumberof ceca and colons examined.

FIG. 2. Fusiform- and spiral-shaped microbes as

visualized by dark-field microscopy near the mucosalepithelium in a frozen section of the colon of a CD-isuckling mouse. X5,100.

morphology (Fig. 3a). They have polar flagellaand usually have two to seven "S" curves alongthe cell. They range from approximately 2.0 to7.0 ,um in length and from 0.15 to 0.30 ,um indiameter. Most of the organisms have one to fivecircular bodies inside the protoplasmic cylinider.A few of these bodies show variable electrondensities, especially in those microbes seen inhomogenates from Ha/ICR mice (Fig. 3b).

DISCUSSIONThe colonization of the intestinal canals by

enterococci, coliforms, and fusiform-shaped bac-teria has been described for mice from severaldifferent colonies (2, 5, 6, 9). The coliforms andenterococci begin to colonize the large bowels ofsuckling mice around the end of the first week of

TABLE 4. Detectiont of spiral and fusiform-shapedmicrobes in ceca of suckling mice by

electron microscopy"

Morphologi- Days after birthMouse strain cal type

of microbe 5 7 9 11

Ha/ICR Spiral 0/5b 2/5 2/5 5/5Fusiform 0/5 0/5 0/5 3/5

CD)-l Spiral 0/5 3/5 5/5 5/5Fusiform 0/5 0/5 1/5 3/5

CF-1 Spiral 0/5 1/5 3/5 5/5Fusiform 0/5 0/5 1/5 5/5

"Spiral- and fusiform-shaped microbes weredetected in negatively stained cecal homogenatesexamined in an electron microscope.

b Ratio represents the number of mice possess-ing the morphological type/number of mice ex-amined.

life, reach high population levels from about the10th to 17th days after birth, and then fall tolow levels during the third week. During thetimes the populations of these facultative an-aerobes are at high levels, microcolonies of gram-negative and gram-positive bacteria can be foundin the mucin on the mucosal epithelium of thececum and colon (6). Our findings from immuno-fluorescence studies confirm an earlier suspicioIl(6) that these microcolonies are composed ofcoliform and enterococcal bacteria.

Fusiform-shaped microorganisms begin to col-onize the ceca and colons during the second weekafter birth. Their populations quickly reach highlevels at which they remain throughout the lifeof the animal (5, 6). These microbes have beenidentified as members of the genera Fusobac-terium, Clostridium, and Eubacterium (3). Somespecies of these anaerobic genera colonize the

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FIG. 3. Spiral-shaped bacteria in negativelystained homogenates of cecal mucosa from Ha/lICRsuckling mice. a, Bar represents 1 pm. X23,000. b,Bar represents 0.1 /um. X56,000.

mucin on the mucosal epithelium of the cecum andcolon. In doing so, apparently they displace thecoliforms and enterococci from the mucin in thesuckling mice.

Spiral-shaped microbes have been detectedrepeatedly in the large bowels of mice (3-5, 7).Such organisms have been shown to colonize the

mucous layer in the large bowels (7). In ho-mogenates from suckling CD-1 mice viewed byphase-contrast microscopy, the spiral-shapedbacteria could be observed in the bowels of miceas early as 12 days after birth (5). The specificimmunofluorescence technique is a more sensitivemethod of detection, however, and allows us notonly to discern the locatioin of the spiral-shapedbacteria in the large bowel but also to detect thespirals when they are present in low numbersainid further obscured by mucus (7). Usinig thistechnique, we find that the spiral-shaled organ-ismiis appear durinig the first week after birth aindlocalize in the mucous layer adjacent to themucosal epithelium about 2 to 5 days before theepithelium is colonized by fusiform-shaped or-ganisms. Electron microscopy of negativelystained cecal homogenates showed a similarcolonization pattern, except that the spiral-shaped microbes could not be detected beforethe end of the first week after birth. The microbescould not be detected earlier, in all probabilitybecause they were present at population levelstoo low to be detected by electron microscopy.The negative stains indicated that the mor-

phology was similar in the spiral-shaped organ-isms from the three different types of mice. Theorgaiiisms were approximately 2 to 7 um longwith two to seven spirals, and they usually hadtwo polar flagella. Maniy also had compartmentedcircular bodies within the protoplasmic cylinder.The spiral-shaped microbe used to make the

vaccine employed in this study was anaerobicbut not oxygen-intolerant (7). Although thespiral-shaped microbes have yet to be culturedfrom suckling mice, they also may be anaerobicbut not oxygen-intolerant. Oxygen tolerance maybe a factor in the ability of these spiral-shapedmicrobes to colonize suckling mice early in life.All microbes, including lactobacilli, anaerobicstreptococci, coliforms, enterococci, and thesespiral-shaped bacteria, that colonize the murinegastrointestinal canal during the first week afterbirth, may be involved in developing an environ-ment in the large bowel that favors the later es-tablishment of the oxygen-intolerant fusiform-shaped anaerobes. The microaerophilic, faculta-tively anaerobic, and oxygen-tolerant anaerobicbacteria may contribute to the environment byremoving oxygen, lowering the oxidation-reduc-tion potential, and providing certain nutrientsessential to the oxygen-intolerant anaerobes. Thespiral-shaped microbes may be especially impor-tant in the development of this ecosystem.

LITERATURE CITED1. Goldman, M. 1968. Fluorescent antibody methods.

Academic Pres Inc., New York.

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2. Gordon, J. H., and R. Dubos. 1971. Observations onthe normal gastrointestinal flora of the mouse.Ann. N.Y. Acad. Sci. 176:30-39.

3. Gordon, J. H., and R. Dubos. 1970. The anaerobicbacterial flora of the mouse cecum. J. Exp. Med.132:251-260.

4. Lee, A., J. Gordon, and R. Dubos. 1968. Enumera-tion of the oxygen sensitive bacteria usually pres-ent in the intestines of healthy mice. Nature(London) 220:1137-1139.

5. Lee, A., J. Gordon, C. Lee, anid R. Dubos. 1971. Themouse intestinal nmicroflora with emphasis on thestrict anaerobes. J. Exp. Med. 132:339-352.

6. Savage, D. C., R. Dubos, and R. W. Schaedler. 1968.

r[I,R, AND SAVAGE INFECT. IMMUNITY

The gastrointestinal epithelium and its auto-chthonous bacterial flora. J. Exp. Med. 127:67-75.

7. Savage, D. C., J. S. McAllister, and C. P. Davis.1971. Anaerobic bacteria on the mucosal epithe-lium of the murine large bowel. Infect. Immunity4:492-502.

8. Schaedler, R. W., and R. J. Dubos. 1962. The fecalflora of various strains of mice. Its bearing on theirsusceptibility to endotoxin. J. Exp. Med.115:1149-1160.

9. Schaedler, R. W., R. Dubos, and R. Costello. 1965.The development of the bacterial flora in thegastrointestinal tract of mice. J. Exp. Med.122:59-66.

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