INFECTION AND IMMUNITY, Feb. 1977, p. 539-548Copyright © 1977 American Society for Microbiology

Vol. 15, No. 2Printed in U.S.A.

Use of Fluorescent Antibody in Studies of Immunity toCholera in Infant Mice

M. NEAL GUENTZEL,1 LEANNE H. FIELD, ELIZABETH R. EUBANKS,2 AND L. J. BERRY*Department of Microbiology, The University of Texas, Austin, Texas 78712

Received for publication 27 July 1976

Infant mice 8 days of age were infected orally with virulent, motile, classicalor El Tor strains of Vibrio cholerae and with nonmotile mutants oflow virulencederived from the same strains. At intervals of 8 and 12 h postinfection, frozenthin sections of the ileum were prepared, stained with fluorescein isothiocya-nate-labeled rabbit anti-vibrio antibody, and examined with the fluorescencemicroscope. The motile organisms were present in larger numbers, especially at12 h, and had penetrated the intervillous spaces and crypts of Lieberkuhn morecompletely than nonmotile vibrios. Dilution counts were made on various re-gions of the intestines of infant mice challenged orally 12 h previously witheither motile or nonmotile strains of V. cholerae. Greater numbers of organismswere found, especially in the upper intestinal regions, when motile organismswere used. Low numbers of vibrios, limited mostly to the lumen, were seen inthe ileum of infant mice infected with motile organisms when the infants werethe offspring of mothers that had been immunized with crude flagellar vaccineor a vesicular preparation derived from the vibrio cell surface. The distributionof vibrios in this case was similar to that found in infected infants of unvaccin-ated mothers challenged with nonmotile organisms. Motility appears to enablethe bacteria to better populate the upper regions of the intestinal tract and toavoid the washing effects of secretions and peristalsis. Antibacterial immunitymay function, at least in part, by making it impossible for motile vibrios toaccomplish this widespread distribution within the ileum.

The role of motility in the pathogenesis ofVibrio cholerae has been studied in our labora-tory. Guentzel and Berry (10) examined toxino-genic and prototrophic nonmotile strains thatretained normal surface structures asjudged byphage susceptibility tests. The nonmotilestrains, including both Ogawa and Inaba sero-types of classical and El Tor biotypes, werefound to be less virulent for orally challengedmice than the wild types from which they werederived. Eubanks et al. (3) extended these find-ings by showing that nonmotile vibrios wereless virulent for adult mice infected intraperito-neally with organisms suspended in hog gastricmucin. As we have described most recently (4),crude flagellar vaccines or preparations of ve-sicular material, believed to be composed offlagellar sheaths of material from the vibriocell surface, confer high levels ofpassive immu-nity on infant mice of mothers that had beenvaccinated with either preparation at the timeof mating. By implication, the virulent motile

1 Present address: Division of Allied Health and LifeSciences, The University of Texas at San Antonio, SanAntonio, TX 78285.

2 Present address: Department of Botany and Microbiol-ogy, Arizona State University, Tempe, AZ 85281.

vibrios are immobilized by the antibodies in themilk and thereby permit the baby mice to sur-vive an otherwise lethal challenge.The importance of motility in other animal

models also has been reported. Williams et al.(22) used isolated ileal loops of the rabbit toshow that motile vibrios penetrated into thecrypts of Lieberkuhn in larger numbers than anonmotile mutant. Jones (14) has made similarobservations.On the basis of these findings, it is logical to

assume that an intimate association ofa toxino-genic strain of V. cholerae with the intestinalmucosa is an essential condition for the onset ofthe disease state. Since motility seems to benecessary for this association, the experimentsreported here were undertaken with this inmind. The localization of the bacteria withinthe intestine was determined with the use offluorescein-labeled anti-vibrio antibody. In theabsence of a widespread distribution of vibriosbetween the villi and deep within the crypts ofLieberkuhn, no serious illness was observed.

MATERIALS AND METHODSMice. CFW mice were purchased from Carworth

Division, Charles Rivers Farms, and raised in de-539

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540 GUENTZEL ET AL.

partmental animal facilities. The mice were housedand fed as described previously by Guentzel andBerry (9).

Bacterial strains. The highly virulent Inabastrain CA401 and El Tor Inaba strain 8233, and aweakly virulent nonmotile strain of each (CA401 M-1 and 82336B3), have been described previously (10).The nonmotile strains were prototrophic and toxino-genic in vivo and in vitro and manifested the samephage susceptibility as the parental strains. All cul-tures were maintained in the lyophilized state andrestored as needed.

Oral challenge. Seven-day-old mice (eight to tenmice per litter) were fasted overnight and then chal-lenged by the oral route as described by Guentzeland Berry (9). In each case, the challenge dose wasapproximately 108 colony-forming units represent-ing approximately 1,000 50% lethal doses for themotile strains. The suckling mice were returned totheir mothers.

Vaccines. The methods of preparation of crudeflagella (CF) from Inaba CA401 and vesicles from anonmotile mutant of this strain have been describedin a separate report (4). HS222, a gift of Paul Actor,Smith Kline and French Laboratories, is an Ogawa-derived subcellular vaccine, the properties of whichhave been described (13). Female CFW mice, 10weeks of age, were administered a single subcuta-neous dose of the respective test vaccines just priorto mating, as described previously (9). Vaccineswere diluted in sterile nonpyrogenic saline (Trav-enol, Inc.) immediately before use. Control animalswere injected with saline alone.

Anti-vibrio antiserum production. Adult rabbitswere bled prior to immunization to obtain normalrabbit serum. Antiserum was produced against liveV. cholerae strain CA401. The antigen was washedfrom an 18-h heart infusion agar culture with salineand adjusted to 100 Klett units. The rabbits wereimmunized according to the following protocol: day1, 0.2 ml subcutaneously; day 5, 0.5 ml subcutane-ously; days 9 and 14, 0.5 ml intravenously; days 14,19, 27, and 35, 1.0 ml intravenously. The rabbitswere bled seven days after the last injection and thetiter was determined by agglutination tests. Thetiter against live CA401 was 1:2,560.

Preparation of anti-mouse IgA. The immuno-globulin A (IgA) secreting MOPC 315 tumor linewas obtained from M. Potter, National Cancer Insti-tute, NIH. Ascites fluid was harvested from BALB/cmice (Jackson Laboratories, Health Research, Inc.)bearing MOPC 315 tumors. IgA was purified accord-ing to a modification (W. Mandy of this department)ofthe procedure of Hashimoto et al. (11). The ascitesfluid was centrifuged for clarification and filtered toremove fibrous clots. In the first step ofthe isolationprocedure, IgA was precipitated with sodium sulfateat a final concentration of 18%. The fraction wasdissolved in saline and reprecipitated with sodiumsulfate at a final concentration of 16%. After over-night dialysis against 0.9% saline, the proteins wereprecipitated again with sodium sulfate at 12.5%.The IgA contained in the supernatent fluid wasprecipitated by adjusting the solution to 16% sodiumsulfate. The precipitated IgA was collected, dis-

INFECT. IMMUN.

solved, and dialyzed against 0.01 M sodium phos-phate buffer, pH 7.5. The IgA was then subjected todiethylaminoethyl-cellulose chromatography, usinga stepwise elution technique in which the molarityof the eluting sodium phosphate buffers (pH 7.5)were increased from 0.01 to 0.3 M. The IgA, whicheluted with 0.3 M buffer, was dialyzed against dis-tilled water and preserved by lyophilization.

Rabbit anti-mouse IgA was prepared according tothe following protocol: day 1, 0.5 mg in each footpad;day 14, 0.5 mg in Freund complete adjuvant givensubscapularly; days 28, 42, and 56, 0.5 mg subcuta-neously. Since no immunoelectrophoretic precipitinbands appeared when purified IgM or IgG was re-acted against the anti-IgA, it is assumed that thecommon light chains in the different classes of im-munoglobulins were not antigenically active underthese conditions. A single precipitin band did formwhen purified IgA or normal mouse serum wastested against the anti-IgA.

Preparation of FA conjugates. Fluorescent anti-body (FA) conjugates were prepared from the nor-mal rabbit serum and the antiserum as outlined byHerbert et al. (12). The sera were precipitated threetimes with 35% saturated ammonium sulfate at250C. The globulin fraction from each was dialyzedat 40C against frequent changes of 0.85% NaCl solu-tion (pH 8.0) until sulfate was no longer detected inthe dialysate. The protein concentration of eachglobulin fraction (and the final conjugates) was de-termined using the method of Lowry et al. (16). Theprotein concentration was adjusted to 20 mg/mlprior to labeling.

Fluorescein isothiocyanate (FITC) (InternationalBiological Supplies, Inc., Melbourne, Fla.) used inthe labeling was certified as 99.9% pure by the Bio-logical Stain Commission. The globulin fractionswere labeled by the dialysis method. The labelingreaction was carried out for 18 h at 250C, pH 9.5, in0.05 M Na2HPO4. Unreacted fluorescent materialwas removed by extensive dialysis against 0.01 Mphosphate-buffered saline, pH 9.0, at 40C. Merthiol-ate at a final concentration of 1:10,000 was added tothe conjugates, and they were stored at 40C.FITC was determined as protein-bound FITC by

absorbance at 490 nm in 0.1 N NaOH and related toa pure fluorescein diacetate reference standard (17).The fluorescein-to-protein ratios of the conjugateswere calculated from the FITC and protein measure-ments and expressed as micrograms of protein-bound FITC per milligram of protein. The fluores-cein-to-protein ratios of the normal rabbit serumconjugate and the anti-CA401 conjugate were 12.8and 11.8, respectively. The conjugates were diluted1:16 in 0.01 M phosphate-buffered saline, pH 9.0, foruse in FA staining of intestinal sections.

Preparation and staining of sections. The intes-tines were removed from infant mice and immedi-ately placed in a 0.9% NaCl solution. A 2-cm ilealsegment (2 cm proximal to the cecum) was ligatedand excised from the intestine. The excised ilealsegment was quick-frozen in O.C.T. compound (Lab.Tech. Products) in a dry-ice and acetone bath. Ap-proximately 30 sections (10 to 12 gm thick) were cutfrom each ileal segment using an International

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FLUORESCENT STUDIES OF CHOLERA IN MICE

cryostat. Sections were allowed to air dry on glassslides and then were fixed for 90 s in absolute meth-anol.

Several sections in each group were randomlychosen as controls and stained with the normal rab-bit serum conjugate. All sections were stained for 30min at 250C in a moist chamber. Slides were rinsedin phosphate-buffered saline (pH 9.0) and allowedto air dry and were mounted in buffered glycerolsaline, pH 9.0.

Slides were read with a Zeiss Universal fluores-cence microscope equipped with an HBO 200 mer-cury arc lamp. Filters used were a BG 38 (red-absorbing filter), a BG 12 (primary filter), and Zeiss43/56 (secondary filters). Kodak Tri X black-and-white film was used for photomicrography.

RESULTSDistribution of vibrios in the ileum of mice

8 h postinfection. Infant mice, infected withthe highly virulent, motile Inaba strain CA401,had a number of fluorescing bacteria in theileal lumen 8 h postinfection. Some had pene-trated into intervillous spaces, as can be seen inFig. la. In Fig. lb, a comparatively small num-ber of vibrios located primarily in the lumenare visible at 8 h. This picture is typical ofthoseseen with mice infected with a nonmotile mu-tant of strain CA401. This difference in thenumber and distribution of vibrios in intestinesofmice infected with either motile or nonmotileorganisms was typical of numerous sections.

Distribution of vibrios in the ileum of mice12 h postinfection. When infected infant micewere sacrificed 12 h after oral challenge withthe motile CA401 strain of V. cholerae, obser-vations similar to those in Fig. 2a and b wereobtained. Numerous bacteria were found in thelumen, as the close-up shown in Fig. 2a makesevident. Many had penetrated into intervillousspaces and also deep into the crypts. The lattercan be seen especially in Fig. 2b. Some distor-tion in the structure of the mucosa is visible inboth plates, and this is believed to be due to thediarrhea that was present at the time. Figure2c is a photograph of an ileal section from aninfant mouse 12 h after infection with a nonmo-tile mutant of strain CA401. Bacteria were visi-ble in the lumen and a few had penetrated intoa crypt, but it was also true that many sectionshad no visible fluorescing organisms. Presum-ably the vibrios were unable to make closecontact with the mucosal structures and wereswept out of the intestine more rapidly andcompletely than the motile parent strain.

In infant mice infected orally with an El TorInaba strain, 8233, and a nonmotile mutantderived from it, observations similar to thosejust described were made. This can be seen inFig. 3a and b. The motile form was present in

very large numbers throughout the cross-sec-tional area 12 h after infection (Fig. 3a), andswollen and distorted villi were evident. Nononmotile organisms were visible at this timeanywhere in the ileum, except for a few in thelumen (Fig. 3b). The difference in the appear-ance of the villi in this section compared to thatin Fig. 3a is quite dramatic. Swollen and edem-atous structures were evident in the intestinecontaining the motile vibrios.

Effect of passive immunity on the numberand distribution of motile vibrios in theileum. Offspring nursed by mothers that hadbeen vaccinated at the time ofmating with 1 jigof CF or with 1 pug of vesicles were challengedorally with the virulent CA401 strain. Thislevel of either vaccine was just sufficient toprovide essentially complete protection againsthomologous challenge (4). The number of flu-orescing vibrios seen in sections of ileum ofinfants sacrificed 12 h later was small and lim-ited to the lumen, or in some cases no fluoresc-ing vibrios were observed. The characteristicappearance in such animals can be seen in Fig.4a and especially in the close-up photograph inFig. 4b. The appearance in these sections issimilar to that of Fig. 2c and 3b taken frommice challenged with nonmotile mutants.An even higher level of protection was appar-

ent in the intestines of infants suckled by moth-ers that had been vaccinated with 10 ,ug offlagella or vesicles. A typical section of ileum isshown in Fig. 4c, where no fluorescing orga-nisms can be seen. In offspring of mice given alevel of immunogen sufficient to prevent mor-tality in 50% of the animals, the appearance ofvibrios in the gut ranged from that seen withmotile organisms in nonimmune animals tothat seen with nonmotile organisms in nonim-mune animals. The presence of antibody in themilk is believed to immobilize the vibrios eitherthrough direct action on the flagella or else byclumping the organisms. The photographs, es-pecially Fig. 4a, show the presence of clumps,but these were seen also in some sections frommice that were not passively immunizedthrough vaccination of the mothers. Whateverthe basis for the reduction in number of fluor-escing bacteria and the prevention of theirpenetration into intervillous spaces and crypts,the antibody was clearly effective.

Reversal of passive immunity by priortreatment with anti-mouse IgA. One-tenthmilliliter of rabbit anti-mouse IgA was givenorally 30 min prior to challenge with 108 colony-forming units of Inaba CA401 to infant micesuckled by immunized mothers. A comparisonwas made between the percentage of survivorsat 36 h in infants given anti-IgA serum and

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542 GUENTZEL ET AL.

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FIG. 1. Distribution of V. cholerae in the ileum of infant mice at an early point (8 h) in the infection. (a)Motile classical strain. x390. (b) Nonmotile classical strain. x390. The organisms (Fig. 1-5) appear asbright spots, which may be located in the lumen, between the villi, or at the base of villi in the crypts ofLieberkuhn.

those given control serum. The results are pre-sented in Table 1. When mothers were immu-nized with 10 tug of CF, anti-IgA reduced survi-vors from 100 to 85%, a change that is notstatistically significant. Vaccination of themothers with 1 ug of CF protected 97% of theoffspring, but this was reduced to 19% survivor-

ship in those given anti-IgA. It seems obvious,therefore, that the former group of mice wasprotected by an excess of IgA in the intestine,even in the presence of the rabbit antiserum.This was not the case when mothers were vacci-nated with the 1-rig dose of CF. Similar resultswere obtained when the HS222 vaccine was

FIG. 2. Distribution of V. cholerae in the ileum of infant mice at a later point (12 h) in the infection. (a)Close-up of lumen, motile classical strain. x390. (b) Photomicrograph showing extensive distribution of themotile strain. x248. (c) Nonmotile classical strain. x390.

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FIG. 3. Comparison of the distribution of El Tor vibrios in the ileum of infant mice at 12 h. (a) Motilestrain. x248. (b) Nonmotile. x248.

administered at a level of 1 gg to the mothers atthe time of mating. The use of anti-IgAdecreased survivorship from 52 to 0%. On thebasis of these data, it seems clear that thepassive immunity conferred by immunizedmothers on their offspring can be reversed atleast in part by anti-mouse IgA. The contribu-

tion of other immunoglobulins in this situationis not known.Figure 5 shows that the reversal ofprotection

by anti-IgA in passively immunized infant miceis accompanied by a distribution of vibrios inthe ileum similar to that seen in nonimmuneanimals. The photomicrograph shows the pres-

FIG. 4. Effect ofpassive immunization on the distribution of motile classical V. cholerae at 12 h postinfec-tion. Ileal sections of offspring of mothers immunized with: (a) 1 ig of CF, x248; (b) 1 Mg ofCF, x390; (c)10 ug of vesicles, x248.

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546 GUENTZEL ET AL.

ence of a large number of fluorescing bacteriaextensively distributed throughout the edema-tous ileum.

Effect of motility on the number and distri-bution of vibrios in the intestinal tract. Infantmice, 8 days of age, were infected orally with 5x 107 colony-forming units of the motile El TorInaba strain 8233 and with the nonmotile mu-tant derived from it. Twelve hours later, themice were sacrificed and the intestines wereremoved and divided into four sections. Thesewere made up of: (i) the duodenum, jejunum,

TABLE 1. Effect of anti-IgA on susceptibility ofpassively immunized mice to V. cholerae

Dose % Survi- Survivors/Immunogen ( ng)tiIgAa vors at 36 total(pg) ~~h

CF 10 - 100 34/3410 + 85 11/131 - 97 32/331 + 19 5/27

HS222 1 - 52 23/441 + 0 0/10

Saline - 0 0/20+ 0 0/10

Unchal- + 100 10/10lengedcontrola Rabbit anti-mouse IgA (0.1 ml) was given orally

30 min prior to oral challenge.

INFECT. IMMUN.

and proximal ileum; (ii) the 2-cm section ofileum used for the FA experiments; (iii) theremainder of the ileum plus the cecum; and (iv)the large intestine. Each segment was homoge-nized in 5 ml of sterile saline, and appropriatedilutions were plated on Verwey's dextrin heartinfusion medium (19). The results obtainedwith seven mice in each group are shown inFig. 6 and 7. Greater numbers of motile thannonmotile organisms were present in all seg-ments of the intestine, but the smallest differ-ence was seen in the large intestine. In thesegment of ileum used for the photographs,there were nearly 100 times as many motilevibrios as nonmotile. This is the order of magni-tude one might expect on the basis of what thephotomicrographs indicate by rough estima-tion. The difference is greater than 100 in the'duodenum, jejunum, and proximal ileum andless in the ileum and cecum. Less than a 10-folddifference was found in the large intestine.

DISCUSSIONThere is no definitive explanation of why mo-

tility seems to be essential for the virulence ofV. cholera. Data from our laboratory based ondifferent types of evidence indicate, however,that it is. Virulence in motile strains seems tocorrelate with a deeper penetration of the bac-teria into the crypts of the intestine and with alarger population of organisms present. Thereis the question, then, of whether the organismsreach the deeper recesses of the ileum because

FIG. 5. Effect of anti-IgA on the distribution of motile classical V. cholerae in passively immunized mice.Ileal section of offspring of mother immunized with 1 pg of CF.

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FLUORESCENT STUDIES OF CHOLERA IN MICE 547

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of their swimming power, their ability to re-

spond positively to chemotactic stimuli, or be-cause of an attachment site between mucosalcells and flagellar structures. From the scan-ning electron photomicrographs of Nelson et al.(18), the major site of attachment of vibrios tomucosal surfaces appears to be along the sidesof the organism rather than at the end wherethe single polar flagellum emerges. Moreover,the lack of ability of isolated flagella given inadvance of oral challenge to reduce mortality,as shown in the preceding paper (4), makes itunlikely that flagella serve as the major attach-ment site. There is always some uncertainty,when mutants of a given type are isolated,whether characters for which there has been noselection have also changed. It was with this in

mind that Guentzel and Berry (10) subjectedthe nonmotile strains to a variety of tests de-signed to identify additional mutations, butnone was detected. Moreover, the one common

characteristic that resulted in loss of virulencein a number of mutants was the loss of theirability to swim. Were some other mutation re-sponsible, it would not likely show up in all ofthe nonmotile strains, unless it were closelylinked genetically.The difference in the numer of colony-form-

ing units obtained by culturing homogenizedintestines of infant mice infected with motilevibrios versus the number obtained when chal-lenge was with nonmotile strains is as great as

anticipated from visualization with FA (seeFig. 6 and 7 versus the photomicrographs).The close association of vibrios seen in sectionsof intestine must have retarded the rate atwhich they were swept along the intestinaltract. A combination of greater retention plustime for multiplication would account for thenumerical differences seen visually and by dilu-tion counts.A marked reduction in the number of motile

vibrios and their presence in intervillous spacesand crypts was observed in baby mice passivelyimmunized at a level sufficient to prevent mor-

tality in these animals (Fig. 4). Antibody mightfunction by directly preventing attachment ofthe organisms to the mucosal surface. Freterand his associates (5-7) observed that specificantibody reduced adherence of V. cholerae invitro and in ligated ileal loops of rabbits.Antibody could also prevent attachment indi-

rectly, as suggested by Verwey (20, 22), by im-mobilizing the organisms and thus reducingtheir penetration of a mucous barrier and sub-sequent migration into the intervillous spaces.This immobilization of vibrios may depend on

cross-linking and physical aggregation of theorganisms, as the results of Verwey and co-workers (20, 22) and Rowley and co-workers (1,21) suggest. We observed more extensiveclumping of vibrios in sections from immunizedbaby mice than in control animals, althoughapparent clumping was sometimes observedeven in the latter case.The reduction in numbers of vibrios and their

restriction to the lumen in ileal sections wassimilar in baby mice born to mothers immu-nized with CF or vesicular (predominately lipo-polysaccharide) preparations. It is significantthat antisomatic antibody can prevent migra-tion of vibrios through soft agar and mucousgels (20, 22). Specific antiflagellar antibody isat least as protective, if not more protective,against oral challenge than antisomatic anti-

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548 GUENTZEL ET AL.

body in passively immunized baby mice (1, 21).Antibody ofeither class may inhibit mobility byimmobilizing the flagellum directly. Benensonet al. (2) observed in 1964 that antisomatic anti-body quickly rendered motile vibrios nonmotilein wet mounts of the stools of cholera patients.These results, coupled with the observationsreported in the present study, suggest that pre-vention of attachment by antibody can occur byprocesses other than by directly blocking anadhesive substance on the vibrio surface.The reversal of the passive immunity in in-

fant mice with anti-mouse IgA is not unex-pected. Fubara and Freter (8) have found thatpurified secretary IgA preparations derivedfrom orally vaccinated animals, and free of de-monstrable IgG or IgM, protected isolated ilealloops of rabbits against fluid accumulationafter challenge with homologous organisms.Our observations do not exclude a contributionof other classes of immunoglobulins to the im-munity we observe.

It is our hypothesis that the ability of motilestrains to penetrate between villi and intocrypts helps the organisms avoid removal byintestinal secretions and the peristaltic actionof the digestive tract. The vibrio mucinase mayassist the organism in the penetration ofmucusbarrier. Adsorption of the bacteria to the mu-

cosal epithelium may be mediated by the slimeenvelope or hemagglutinin described by Lank-ford and Legsomburana (15). It has not beenproven, but it can be suggested that the anti-body that is passively acquired by infant micefrom their mothers immobilizes the vibrios andalso may agglutinate them and in one or bothways convert motile forms into functionallynonmotile ones. This is subject to experimentalverification and will be investigated.

ACKNOWLEDGMENT

This work was supported in part by Public Health Serv-ice grant AI-10466 from the National Institute of Allergyand Infectious Diseases.

LITERATURE CITED

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2. Benenson, A. S., M. R. Islam, and W. B. GreenoughIII. 1964. Rapid identification of Vibrio cholerae bydark-field microscopy. Bull. W.H.O. 30:827-831.

3. Eubanks, E. R., M. N. Guentzel, and L. J. Berry. 1976.Virulence factors involved in the intraperitoneal in-fection of adult mice with Vibrio cholerae. Infect.Immun. 13:457-463.

4. Eubanks, E. R., M. N. Guentzel, and L. J. Berry. 1977.Evaluation of surface components of Vibrio cholera

as protective immunogens. Infect. Immun. 15:533-538.

5. Freter, R. 1969. Studies of the mechanism of action ofintestinal antibody in experimental cholera. Tex.Rep. Biol. Med. 27(Suppl. 1):299-316.

6. Freter, R. 1970. Mechanism of action of intestinal anti-body in experimental cholera. II. Antibody-mediatedantibacterial reaction at the mucosal surface. Infect.Immun. 2:556-562.

7. Freter, R. 1972. Parameters affecting the association ofvibrios with the intestinal surface in experimentalcholera. Infect. Immun. 6:134-141.

8. Fubara, E. S., and R. Freter. 1973. Protection againstenteric bacterial infection by secretary IgA antibod-ies. J. Immunol. 111:395-403.

9. Guentzel, M. N., and L. J. Berry. 1974. Protection ofsuckling mice from experimental cholera by maternalimmunization: comparison of the efficacy of whole-cell, ribosomal-derived, and enterotoxin immuno-gens. Infect. Immun. 10:167-172.

10. Guentzel, M. N., and L. J. Berry. 1975. Motility as avirulence factor for Vibrio cholerae. Infect. Immun.11:890-897.

11. Hashimoto, N., S. Chandor, W. Mandy, and M. Yokoy-ama, 1970. Atypical IgA with hidden light chain.Clin. Exp. Immunol. 6:941-949.

12. Herbert, G. A., B. Pittman, R. M. McKinney, and W.B. Cherry. 1972. The preparation and physicochemi-cal characterization of fluorescent antibody reagents.Center for Disease Control, Atlanta.

13. Jensen, R., B. Gregory, J. Naylor, and P. Actor. 1972.Isolation of protective somatic antigen from Vibriocholerae (Ogawa) ribosomal preparations. Infect. Im-mun. 6:156-161.

14. Jones, G. W.1975. Adhesive properties of enteropatho-genic bacteria, p. 137-142. In D. Schlessinger (ed.),Microbiology- 1975. American Society for Microbiol-ogy, Washington, D.C.

15. Lankford, E. C., and U. Legsomburana. 1965. Viru-lence factors of the choleragenic vibrios, p. 109-121.In Proceedings of the cholera research symposium.U.S. Public Health Service Publ. no. 1328. Govern-ment Printing Office, Washington, D.C.

16. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-275.

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