INFECTION AND IMMUNITY, Nov. 1974, p. 1098-1104Copyright © 1974 American Society for Microbiology

Vol. 10, No. 5Printed in U.S.A.

Experimental Cholera in Chinchillas: the Immune Response inSerum and Intestinal Secretions to Vibrio cholerae and

Cholera ToxinUZY BLACHMAN,' S. R. GRABOFF, G. E. HAAG, E. GOTTFELD, AND M. J. PICKETT

Department of Bacteriology, University of California, Los Angeles, California 90024

Received for publication 12 June 1974

The immune response to Vibrio cholerae and cholera toxin was studied inchinchillas inoculated intra-intestinally with 108 viable V. cholerae 596B. Seraand intestinal fluids were collected from animals on days 1, 2, 3, 5, 7, 10, 14, 30,and 60 postinoculation, and antibodies were quantified by various methods.When radiovibriolytic and radiotoxin precipitin tests were used, a significant(fourfold) rise in both antibacterial and antitoxic titers was detected in intestinalfluids on the 2nd day of infection. In contrast, a significant rise in serumantibodies occurred between days 3 and 5. Since the acute phase of the chinchilladisease lasts 1 to 3 days, recovery coincided with rise in intestinal antibody butnot with rise in serum antibody. Our results indicate that both antibacterial andantitoxic mechanisms are operative in chinchilla cholera and that the initial andcrucial immune response is local in nature.

Recent epidemiological reports (3, 15, 24)have shown that cholera is now present over agreater area than at any time since the turn ofthe century. Of several potential measures forcontrolling cholera, only immunoprophylaxisappears to be attainable in the near future.However, experience with parenterally adminis-tered, whole-cell cholera vaccines has generallybeen disappointing (2, 4, 5, 16), particularlywith young children, in whom the attack rate is10 times higher than in adults (33). The lowefficacy of conventional vaccine may be attrib-uted to its parenteral route of' administrationand/or its inability to effect antitoxic immunity(7, 18).

In recent years considerable information hasaccumulated regarding the immunology of chol-era. Despite this, basic questions concerning therelative role of circulating versus intestinalantibodies and of antibacterial versus antitoxicimmunity in cholera remain largely unan-swered. Understanding the mechanisms of im-munity operating in cholera is important fordeveloping immunoprophylactic measures. In-deed, in 1969 the W.H.O. Scientific Group onCholera Immunology stated that "Success inthe immunological control of cholera wouldtherefore appear to depend very largely on aclear understanding of the nature of local intes-tinal immune mechanisms and not merely on aknowledge of the serologic immunology of the

'Present address: City of Hope National Medical Center,Department of Clinical Pathology, Duarte, Calif. 91010.

disease" (43). Such basic understanding is stilllacking.Recently we described the adult chinchilla as

a suitable animal model for cholera research (U.Blachman, S. J. Goss, and M. J. Pickett. J.Infect. Dis., in press). We studied, using chol-era-inf'ected chinchillas, the kinetics of an-tivibrio and anticholeragen immune responsesin sera and intestinal secretions and determinedthe temporal relationship of those to the prog-ress of the infection. We report here the findingsof these immunological studies.

MATERIALS AND METHODSSmooth Vibrio cholerae 569B Inaba was main-

tained and cultured as described previously. Meatextract agar, pH 7.6, or beef extract-peptone-sucroseagar (Blachman et al., J. Infect. Dis., in press), pH9.0, was used as plating medium. Alkaline peptonewater consisted of 1% peptone (Difco) and 0.5% NaCl,pH 9.0.

Cholera toxin. Choleragen (21) lot 1071 was kindlygiven to us by R. S. Northrup, National Institute ofAllergy and Infectious Diseases (NIAID), Bethesda,Md. The toxin was prepared by R. A. Finkelsteinunder contract with that Institute. (In this publica-tion the following terms are used interchangeably:cholera toxin, toxin, choleragen, and permeabilityfactor. We consider these terms to be synonymous andspecifically refer to the toxic moiety, choleragen, asdescribed by Finkelstein and LoSpalluto [21 ].)

Animals. Chinchillas were donated by local breed-ers. All chinchillas used, both male and female, wereapparently healthy, weighed 350 to 650 g, and were 1to 2 years old. None was previously inoculated with V.

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cholerae or its toxin. Experimental and control ani-mals were inoculated intra-intestinally with either 108viable, log-phase V. cholerae 569B or with 1 ml ofsterile brain heart infusion broth, respectively. Con-trol animals were sacrificed 7 days after inoculation.Expired chinchillas were excluded from these studies.

Sera. All serum specimens obtained from exsan-guinated animals were inactivated by incubation at56 C for 30 min. Inactivated sera were absorbed withsheep erythrocytes (SRBC). Each serum specimenwas distributed into several small screw-cap vials,quickly frozen in a dry ice-ethanol mixture, andstored at -70 C. None of the vials was frozen andthawed more than once.

Intestinal fluid. Animals were exsanguinatedunder ether anesthesia. The small intestine (SI) wasexposed, ligated at its pyloric and cecal ends, andremoved in toto into a prechilled, sterile petri plate.Unless otherwise stated, all of the following steps werecarried out at 0 to 4 C. SI fluid was asepticallyexpressed into a preweighed tube; particular care wastaken to prevent contamination of intestinal fluidwith blood. The drained lumen was washed with 50 mlof sterile 0.01 M phosphate-buffered saline (PBS), pH7.6, containing 0.1% bovine serum albumin (MilesLaboratories, Kankakee, Ill.) and 0.01% soybean tryp-sin inhibitor (P-L Biochemicals, Inc., Milwaukee,Wisc.). The original SI content and the wash fluidwere pooled and designated intestinal fluid (IF).Intestinal fluid was centrifuged at 10,000 x g for 30min, filtered sequentially through a Whatman no. 1filter paper and 0.45- and 0.22-Mm membrane filters(Millipore Filter Corp., Bedford, Mass.), and dialyzedagainst deionized water for 48 h. The dialyzed IF wasincubated at 56 C for 30 min and immediately lyophi-lized to dryness. The lyophilized material was recon-stituted in 5 ml of 0.01 M PBS, pH 7.2, adsorbed withSRBC, and centrifuged at 20,000 x g for 30 min. Thefluid was filtered through a 0.22-am membrane filter,distributed into several small screw-cap vials, quicklyfrozen in a dry ice-ethanol mixture, and stored at- 70 C. None of the vials was frozen and thawed morethan once.

Assay of anti-V. cholerae antibodies in serumand IF. (i) Hemagglutination. Serum and IF anti-V.cholerae antibodies were quantified by microhemag-glutination. A volume of 2% formalinized SRBC(sheep Formocells, Difco) was sensitized with an equalvolume of a crude preparation of V. cholerae 569Bsomatic antigen containing 2.5 mg of antigen per ml.The antigen was a dialyzed, concentrated super-natant fluid derived from a steamed 18-h culture of569B organisms. The sensitized cells were used at aconcentration of 1%. This cell preparation was stablefor at least 14 months. The titer of our reference rabbitanti-Vibrio serum with this cell preparation was2,560. This and all other immunoassays describedbelow were done in duplicate.

(ii) Radiovibriolytic assay. Vibriolytic antibodiesin serum and intestinal fluid were quantified by the51Cr vibriolytic assay (9). Each reaction tube con-tained 106 labeled vibrios, diluted serum or IF, guineapig complement (Baltimore Biological Laboratories,lot 1091276) diluted 1:20, and 500 gg of hen egg whitelysozyme (Calbiochem, lot 100489). Under these con-

ditions, the radiovibriolytic titer of our rabbit anti-Vibrio serum was 1.8 x 106.

(iii) Vibriocidal test. Vibriocidal antibody wasmeasured by the plate method (19). Each reactiontube contained diluted serum or IF, 101 log-phasevibrios, guinea pig complement diluted 1:20, and 500gg of lysozyme. Under these conditions, the vibrio-cidal titer of our rabbit anti-Vibrio serum was 3.6 x105.Assay of anticholera toxin antibodies in serum

and IF. (i) Hemagglutination. Sheep Formocellswere sensitized with choleragen by a modification ofpreviously described procedures (20, 27). Briefly, avolume of 0.01 M PBS, pH 7.2, containing 20 ug ofcholeragen per ml was added dropwise with continu-ous stirring to an equal volume of 2% washed For-mocells. The mixture was incubated at room tempera-ture for 1 h, after which the cells were washed threetimes and adjusted to a concentration of 1%. Sensi-tized cells were stable for at least 18 months. Thehemagglutination titer of our reference rabbit an-titoxin was 2,048.

(ii) Radioprecipitin test. Antitoxin antibodies inserum and IF were quantified by a precipitin testusing 125'-labeled chloeragen. Iodination was by amodification of the chloramine-T procedure of Green-wood et al. (25). Two millicuries of Na125I (carrierfree, New England Nuclear Corp., 033H, lot 5734) wasadded to 3.5 ml of 0.15 M PBS, pH 7.2, containing 350gg of choleragen. The mixture was stirred at 0 to 4 C;50 Mliters of distilled water containing 250 Mg ofchloramine-T was added, and the reaction was allowedto proceed for 5 min. The reaction was then stoppedby addition of 250 Mg of sodium metabisulfite and 300MIg of KI. The iodinated toxin was dialyzed at 4 Cagainst PBS until no counts were detectable in thedialysate. The iodinated toxin was stored at 4 C andused within 3 days after preparation. Specific activitywas calculated by assuming an extinction coefficient(1% solution) of 11.42 at 280 nm (30). The effectivespecific activity was 3.22 x 105 counts/min per Mg or0.145 ACi/Mg. (Our study was completed before Cua-trecasas' procedure for iodination was published [Bio-chemistry 12:3547, 1973]. Though our procedure wasquite similar to his, he obtained higher specificactivity, presumably by use of a higher Na'251-to-toxin ratio.) As compared with untreated choleragen,the iodinated toxin fully retained its capacity toinhibit a specific hemagglutination system and toelicit a dermal reaction in the rabbit (1 bluing dosegiving 7-mm bluing lesion [BD7] = 0.0033 Mg).The radioprecipitin test was performed in Beckman

microtubes in the following way. Serum or IF wasdiluted twofold in PBS containing 1% pooled normalchinchilla serum. Amounts (25 Mliters) of seriallydiluted specimens were transferred into microtubeswith an Eppendorf pipette. Each tube then received25 Mliters of the above buffer containing a total of 0.02Mg of iodinated toxin (about 6,000 counts/min pertube). Tubes were incubated for 2 h at 37 C andovernight at 4 C and then centrifuged in a BeckmanMicrofuge model 152 (Beckman Instruments, Inc.) atmaximal speed (10,000 x g) for 2.5 min. The superna-tant fluids were removed by aspiration, the precipi-tates were washed once with cold PBS, and remaining

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BLACHMAN ET AL.

radioactivity was counted in an automatic gamma-counting system (Nuclear-Chicago 1185 series). Thepercentage of toxin precipitated was calculated by thefollowing formula:

counts/minute

%.toxin precipitated in precipitatex 100total counts/

minute put in

No correction for precipitation in normal serum was

necessary since at optimal dilution, pooled or individ-ual normal chinchilla sera precipitated less than 3% oforiginal input counts. The titer was defined as thereciprocal of the highest specimen dilution at which50% of maximal precipitation occurred. This valuewas determined graphically by interpolation from thedescending limb of the precipitin curve. The radio-precipitin titer of our rabbit antitoxin serum was

7,200.(iii) Permeability factor neutralization test. An-

titoxic antibodies were quantified in sera and IFs by a

modification of the permeability factor (PF) neutral-ization test PFNT (7). The PF standard was pure

choleragen. By using Craig's skin test procedure (13,14), we determined that 1 BD7 was equal to 0.0033 tg

of choleragen. The neutralization test was carried outin the following way. Toxin was diluted to a concen-

tration of 0.2 Ag/ml (60 BD7/ml) in a sterile solutioncontaining 0.9% NaCl and 0.1% gelatin, pH 7.2.Volumes (0.5 ml) of this PF preparation were added toequal volumes of twofold serially diluted specimens.The mixtures were incubated at 37 C for 1 h, afterwhich 0.1-ml volumes were injected intradermallyinto the shaved dorsa of rabbits (0.1 ml contained 3BD7). Samples from each dilution tube were injectedin duplicate into two rabbits. Twenty-three hourslater, 2-ml volumes of saline containing 2% Evansblue dye were injected intravenously into the rabbits,and the diameters of the blued lesions were measured1 h later. The titer of a specimen was defined as thereciprocal of the highest specimen dilution which,when mixed with the toxin standard, resulted in a

A B R HEMAGGLUON N B

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000,

lesion with a diameter of <4 mm. The anti-PF titer ofour reference rabbit antitoxin serum was 4,800.

For the purpose of calculations, specimens havinghemagglutination, anti-PF, or antitoxin precipitintiters of <4 were assumed to have titers of 2.Specimens having vibriocidal or radiovibriolytic titersof <20 were assumed to have titers of 10. Theantibody titer of a specimen was considered signifi-cantly elevated if it was at least fourfold higher thanthat of control specimens.

RESULTS

Assay of hemagglutinins directed against thevibrio somatic antigens revealed no such anti-bodies in either serum or IF on the 1st and 2nddays of infection (Fig. 1A). However, the IFhemagglutinin titer rose 16-fold on day 3,reached peak levels on day 5, and declinedrapidly thereafter, reaching base-line levels 1month after inoculation. The kinetics of serumhemagglutinin was quite different. A significant(fourfold) rise in titer was not reached until the4th or 5th day, and peak titer was reached bythe end of the 2nd week. A significantly highserum titer was maintained for at least 2months.

Vibriocidal antibody was also detected inIF early in the infection (Fig. iB). On the 3rdday, IF vibriocidal antibody titer was fivefoldhigher than that of control animals. At thattime, serum antibody level was insignificantly(threefold) elevated. Peak IF titer was reachedon day 5, after which the titer declined rapidly;at 2 weeks after inoculation, the vibriocidal titerin SI fluid was elevated only twofold. Thekinetics of appearance of serum vibriocidinparalleled that of serum hemagglutinin. How-ever, peak titer of the former was considerablyhigher. Vibriocidal activity in all sera and IF

VIBR OCIDIN

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DAY -2) NI-TC AT IN

FIG. 1. Kinetics of appearance of anti-Vibrio cholerae 569B antibodies in sera and intestinal fluids of

intra-intestinally inoculated chinchillas. Bars represent standard error of mean. Radiovibriolysin was assayed

in pooled sera and intestinal fluid specimens.

1100 INFECT. IMMUNITY

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EXPERIMENTAL CHOLERA IN CHINCHILLAS

specimens was complement dependent.When antibody was titrated by the radiovi-

briolytic assay, a significant (fourfold) rise in IFvibriolysin was detected as early as the 2nd dayof infection (Fig. 1C). At that time, serumantibody titer was elevated twofold. Except forthis difference, the kinetics of appearance of IFand serum vibriolysins paralleled that of vibri-ocidins, but peak titers of the former werehigher. Like vibriocidal activity, vibriolysis wascomplement dependent.As determined from results obtained by the

vibriocidal test, all inoculated animals re-sponded with at least a fourfold rise in serumand IF antibody titers by the 5th day ofinfection (Fig. 2). However, on day 3 only 25% ofthe animals showed a rise in serum antibodytiters, whereas 75% of the animals already hadresponded with a significant rise in IF antibodytiters. At the end of the 2nd week the situationwas reversed. Only 20% of animals still hadsignificantly high IF antibody titers, but allanimals had high serum titers.

Levels of vibrio hemagglutinins and vibri-ocidins in sera and IF specimens of control,asymptomatic, and morbid chinchillas areshown in Table 1. (Animals were consideredmorbid if the amount of fluid accumulated intheir SI was >50 mg/cm. Although these ani-mals were never diarrheic, they were shown tobe physiologically dehydrated and acidotic tovarying degrees.) Data presented are those ofdays 2 and 3. There was no detectable antibodyon day 1, and after the day 3 all survivinganimals had convalesced. Antivibrio antibodytiters were essentially similar in sera of asymp-

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FIG. 2. Elevated vibriocidins in sera and intestinalfluids of intra-intestinally inoculated chinchillas. Afourfold or greater rise in titer (as compared withcontrols) was considered significant.

tomatic and morbid animals. However, titers ofantivibrio antibody in IF of asymptomatic chin-chillas were 2.8- to 3.3-fold higher than those inIF of morbid animals.

Hemagglutinating antitoxic antibody was de-tected in neither IF nor serum of any control orinoculated chinchillas. However, when precipi-tin and neutralization assays were used, an-titoxic antibodies were detected in both serumand IF specimens (Fig. 3). The appearance ofantitoxic antibodies resembled that of an-tivibrio antibodies in that they appeared earlierin intestinal secretions then in serum. Therewas a fivefold increase in titers as early as day 2,and peak titers were reached on day 5. Thedecline of IF antibodies was rapid; on day 10 theantitoxin titers had dropped to insignificantlevels. In contrast, serum antitoxin titers roseslowly and were maintained longer. Peak serumtiters were reached by the end of the 2nd weekand were still significantly elevated 2 monthsafter inoculation. In comparison with antivibrioantibody titers, antitoxin levels in sera and IFspecimens of convalescing chinchillas were defi-

TABLE 1. Hemagglutinins and vibriocidins in seraand intestinal fluids of control, asymptomatic, and

morbid chinchillas

Control Asymp- MorbidSubstances tomatic

Serum IF Serum IF Serum IF

Hemagglutinins 2a 2 8 44 4 16Vibriocidins .... 15b 10 40 50 30 15

a Reciprocal of geometric mean titer. Data are thoseof day 2 (IO/,'6 asymptomatic; 6/6 morbid).

bReciprocal of geometric mean titer. Cumulativedata of days 2 and 3 (6/24 asymptomatic; 8h4 morbid).

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DAY POST-INOCULATION

FIG. 3. Kinetics of appearance of antitoxin anti-bodies in sera and intestinal fluids of intra-intesti-nally inoculated chinchillas. Antitoxin was assayed inpooled sera and intestinal fluid specimens.

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BLACHMAN ET AL.

nitely elevated but were very low. Peak an-titoxin titer by radioprecipitin assay was only 28in IF and 90 in serum.

DISCUSSIONCholera-infected chinchillas responded with

formation of antivibrio and antitoxic antibodiesin both serum and intestinal secretions. Serumantivibrio response was similar in magnitudeand duration to that seen in human (1, 6, 8, 11,17, 31, 39) and canine (40) cholera. Regardlessof the assay used, antivibrio antibodies weredetected in IF considerably earlier than inserum. Whereas serum antibody appeared afteranimals recovered, a rise in intestinal antivibrioantibody temporally coincided with convales-cence. Freter and co-workers reported similarobservations in human cholera (23). That theassociation between recovery and appearance ofintestinal antivibrio antibody is indicative ofthe role of local antibacterial immunity issupported by the observation that intestinalhemagglutinins and vibriocidin titers werelower in morbid than in asymptomatic animals.The latter may indicate an increased suscepti-bility to cholera in individuals who fail tomount a vigorous immune response. Waldmanand co-workers (42) suggested that in humansthis phenomenon may also represent a "soakingup" effect of antibodies by the abundant vibrioantigen. However, we favor the former hypothe-sis since it was shown that cholera infectionpredominantly occurs in individuals with lowserum vibriocidal titers (34) and that childrenunder the age of five, in whom the attack rate ishigher than in adults (33), do not respond tocholera immunization as well as older individu-als (31). An intestinal antibacterial mechanismwas implicated in both human cholera (36, 42)and in the experimental disease in guinea pigs(10, 22), infant rabbits (32), and infant mice(12).Immunoassays varied in their sensitivities for

measuring antivibrio antibodies in intestinalsecretions. A comparison of the ratios of peak IFtiters with peak serum titers (Fig. 1) indicatesthat the ratio was much higher in the hemag-glutination assay than in the vibriocidal andvibriolytic assays. This indicates that, relativeto IF antibody, serum antibody was more activein complement-dependent assays. These resultsmay be expected if the predominant class ofantibody in IF was immunoglobulin A, an

immunoglobulin that does not fix complementin the classic manner (28). Immunoglobulin Awas shown to be the predominant immunoglob-ulin in intestinal secretions of human patients(35, 41).

Observations on antitoxin response in chol-era-infected chinchillas indicate that resultsdepended greatly on the immunoassay used.Hemagglutinating antitoxic antibody was neverdetected in either serum or IF. In contrast,precipitating and neutralizing antibodies weredetected in both specimens relatively early inthe infection. The difference in findings wasprobably not so much a reflection of the relativesensitivities of the assays as an indication of thelow efficiency of chinchilla serum and intestinalantitoxic antibodies in agglutinating cholera-gen-sensitized SRBC. In titrating our stockrabbit antitoxic sera, we found the PF neutral-ization test to be only slightly more sensitivethan the hemagglutination test; other workersreported similar observations (20, 27).Our studies of serum antitoxin corroborate

and extend observations made on paired acuteand convalescent human sera (7, 20, 26, 38).Pierce and co-workers (38) reported that inhuman cholera no serum antitoxic antibody wasdetected on day 1, that titer was maximal onday 15 (the next interval tested), and that it wasstill significantly elevated a year or longer afteronset of symptoms. Cash et al. (11) recentlydemonstrated a rise in serum hemagglutinatingantitoxin titers in human volunteers infectedwith toxigenic V. cholerae. Peak titers, thoughlow, were detected at the end of the 2nd weekafter inoculation; by the 6th week titers ap-proached base-line levels. In chinchillas, a sig-nificant rise in neutralizing and precipitatingserum antitoxin occurred between the 4th and5th days of infection, and peak titers werereached on day 14 and were still significantlyelevated 2 months after inoculation. Kinetics ofappearance of intestinal antitoxin was similarto that of intestinal antivibrio antibody in thata significant rise in titer occurred 48 h afterinoculation. The appearance of IF antitoxinearlier than serum antitoxin might be expectedsince it was shown that cholera toxin does notpenetrate the intestinal wall beyond the mucousepithelium (29, 37), in which case it is likely tostimulate a local immune response.Data presented here are consistent with the

hypothesis that antitoxic and antivibrio anti-bodies were produced, at least in the initialstages of the infection, in the intestine, and thatboth types of antibodies were instrumental inrecovery. It is not possible, however, to definefrom these data the relative roles of antivibrioand antitoxic antibodies in recovery of chinchil-las from cholera.

ACKNOWLEDGMENTSWe wish to thank R. S. Northrup for the kind gift of

choleragen, and Michael Cecka for performing the radioiodi-

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nation of choleragen. The generous donation of chinchillas bythe many Southern California breeders is gratefully acknowl-edged.

LITERATURE CITED

1. Ahmed, A., A. K. Bhattacharjee, and W. H. Mosley.1970. Characteristics of the serum vibriocidal andagglutinating antibodies in cholera cases and in normalresidents of the endemic and non-endemic choleraareas. J. Immunol. 105:431-441.

2. Azurin, J. C., A. Cruz, T. P. Pesigan, M. Alvero, T.Camena, R. Suplido, L. Ledesma, and C. Z. Gomez.1967. A controlled field trial of the effectiveness ofcholera and cholera El Tor vaccines in the Philippines.Bull. W.H.O. 37:703-727.

3. Barua, D. 1972. The global epidemiology of cholera inrecent years. Proc. Roy. Soc. Med. 65:423-428.

4. Benenson, A. S., P. R. Joseph, and R. 0. Oseasohn. 1968.Cholera vaccine field trials in East Pakistan. 1. Reac-tion and antigenicity studies. Bull. W.H.O. 38:347-358.

5. Benenson, A. S., W. H. Mosley, M. Fahimuddin, and R.0. Oseasohn. 1968. Cholera vaccine field trials in EastPakistan. 2. Effectiveness in the field. Bull. W.H.O.38:359-372.

6. Benenson, A. S., A. Saad, and W. H. Mosley. 1968.Serological studies in cholera. 2. The vibriocidal anti-body response of cholera patients determined by a

microtechnique. Bull. W.H.O. 38:277-285.7. Benenson, A. S., A. Saad, W. H. Mosley, and A. Ahmed.

1968. Serological studies in cholera. 3. Serum toxinneutralization-rise in titre in response to infectionwith Vibrio cholerae, and the level in the "normal"population of East Pakistan. Bull. W.H.O. 38:287-295.

8. Benenson, A. S., A. Saad, and M. Paul. 1968. Serologicalstudies in cholera. 1. Vibrio agglutinin response ofcholera patients determined by a microtechnique. Bull.W.H.O. 38:267-276.

9. Blachman, U., W. R. Clark, and M. J. Pickett. 1973.Radiobacteriolysis: a new technique using chromi-um-51 for assaying anti-Vibrio cholerae antibodies.Infect. Immunity 7:53-61.

10. Burrows, W., and L. L. Ware. 1953. Studies on immunityto Asiatic cholera. VII. Prophylactic immunity toexperimental enteric cholera. J. Infect. Dis.92:164-174.

11. Cash, R. A., S. I. Music, J. P. Libonati, M. J. Snyder, R.P. Wenzel, and R. B. Hornick. 1974. Response of manto infection with Vibrio cholerae. I. Clinical, serologic,and bacteriologic responses to a known inoculum. J.Infect. Dis. 129:45-52.

12. Chaicumpa, W., and D. Rowley. 1973. Mechanisms ofantibacterial immunity against Vibrio cholerae in theintestinal tracts of baby mice. J. Infect. Dis. 128:56-62.

13. Craig, J. P. 1965. The effect of cholera stool and culturefiltrates on the skin of guinea pigs and rabbits, p.

153-158. In Proc. Cholera Res. Symp., Public HealthService publication 1328. U.S. Government PrintingOffice, Washington, D.C.

14. Craig, J. P. 1965. A permeability factor (toxin) found incholera stools and culture filtrates and its neutraliza-tion by convalescent cholera sera. Nature (London)207:614-616.

15. Cvjetanovic, B., and D. Barua. 1972. The seventh pan-demic of cholera. Nature (London) 239:137-138.

16. Das Gupta, A., R. Sinha, D. L. Shrivastava, S. P. De, B.

L. Taneja, M. S. Rao, and A. H. Abou-Gareeb. 1967.Controlled field trial of the effectiveness of cholera andcholera El Tor vaccines in Calcutta. Bull. W.H.O.37:371-385.

17. Feeley, J. C. 1965. Comparison of vibriocidal and aggluti-nating antibody responses in cholera patients, p.220-222. In Proc. Cholera Res. Symp., Public Health

Service publication 1328. U.S. Government PrintingOffice, Washington, D.C.

18. Feeley, J. C., and C. 0. Roberts. 1969. Immunologicalresponses of laboratory animals to cholera vaccines,toxin, and toxoid. Tex. Rep. Biol. Med. 27(Suppl.1):213-226.

19. Finkelstein, R. A. 1962. Vibriocidal antibody inhibition(VAI) analysis: a technique for the identification of thepredominant vibriocidal antibodies in serum and forthe detection and identification of Vibrio choleraeantigens. J. Immunol. 89:264-271.

20. Finkelstein, R. A., and J. W. Peterson. 1970. In vitrodetection of antibody to cholera enterotoxin in cholerapatients and laboratory animals. Infect. Immunity1:21-29.

21. Finkelstein, R. A., and J. J. LoSpalluto. 1970. Productionof highly purified choleragen and choleragenoid. J.Infect. Dis. 121(Suppl.):S63-S72.

22. Freter, R. 1956. Coproantibody and bacterial antagonismas protective factors in experimental enteric cholera. J.Exp. Med. 104:419-426.

23. Freter, R., S. P. De, A. Mondal, D. L. Shrivastava, and F.W. Sunderman, Jr. 1965. Coproantibody and serum

antibody in cholera patients. J. Infect. Dis. 115:83-87.24. Gangarosa, E. J. 1971. The epidemiology of cholera: past

and present. Bull. N.Y. Acad. Med. 47:1140-1151.25. Greenwood, F. C., W. M. Hunter, and J. S. Glover. 1963.

The preparation of 13II-labeled human growth hormoneof high specific radioactivity. Biochem. J. 89:114-123.

26. Hochstein, H. D., J. C. Feeley, and W. E. DeWitt. 1970.Titration of cholera antitoxin in human sera by mi-crohemagglutination with formalinized erythrocytes.Appl. Microbiol. 19:742-745.

27. Hochstein, H. D., J. C. Feeley, and S. H. Richardson.1970. Titration of cholera antitoxin levels by passivehemagglutination tests using fresh and formalinizedsheep erythrocytes. Proc. Soc. Expt. Biol. Med.133:120-124.

28. Ishizaka, T., K. Ishizaka, T. Borsos, and H. Rapp. 1966.C'l fixation by human isoagglutinins: fixation of C'l by-yG and yM but not by -yA antibody. J. Immunol.97:716-726.

29. Kao, V. C. Y., H. Sprinz, and W. Burrows. 1972.Localization of cholera toxin in rabbit intestine. Animmunohistochemical study. Lab. Invest. 26:148-153.

30. LoSpalluto, J. J., and R. A. Finkelstein. 1972. Chemicaland physical properties of cholera exoenterotoxin (cho-leragen) and its spontaneously formed toxoid (choler-agenoid). Biochim. Biophys. Acta 257:158-166.

31. McCormack, W. M., J. Chakraborty, A. S. M. MizanurRahman, and W. H. Mosley. 1969. Vibriocidal anti-body in clinical cholera. J. Infect. Dis. 120:192-201.

32. McIntyre, 0. R., and J. C. Feeley. 1964. Passive serum

protection of the infant rabbit against experimentalcholera. J. Infect. Dis. 114:468-475.

33. Mosley, W. H. 1969. The role of immunity in cholera. Areview of epidemiological and serological studies. Tex.Rep. Biol. Med. 27(Suppl. 1):227-241.

34. Mosley, W. H., S. Ahmad, A. S. Benenson, and A.Ahmed. 1968. The relationship of vibriocidal antibodytitre to susceptibility to cholera in family contacts of

cholera patients. Bull. W.H.O. 38:777-785.35. Northrup, R. S., J. Bienenstock, and T. B. Tomasi, Jr.

1970. Immunoglobulins and antibody activity in the

intestine and serum in cholera. I. Analysis of immuno-

globulins in cholera stool. J. Infect. Dis.121(Suppl.):S137-S141.

36. Northrup, R. S., and S. A. Hossain. 1970. Immunoglobu-lins and antibody activity in the intestine and serum in

cholera. II. Measurement of antibody activity in jejunalaspirates and sera of cholera patients by radioim-

munodiffusion. J. Infect. Dis. 121(Suppl.):S142-146.37. Peterson, J. W., J. J. LoSpalluto, and R. A. Finkelstein.

VOL. 10, 1974 1103

on January 28, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

1104 BLACHMAN ET AL.

1972. Localization of cholera toxin in vivo. J. Infect.Dis. 126:617-628.

38. Pierce, N. F., J. G. Banwell, R. B. Sack, R. C. Mitra, andA. Mondal. 1970. Magnitude and duration of antitoxicresponse to human infection with Vibrio cholerae. J.Infect. Dis. 121(Suppl.):S31-S35.

39. Sack, R. B., D. Barua, R. Saxena, and C. C. J. Carpenter.1966. Vibriocidal and agglutinating antibody patternsin cholera patients. J. Infect. Dis. 116:630-640.

40. Sack, R. B., and C. C. J. Carpenter. 1969. Experimentalcanine cholera. III. Serologic studies and re-challengeexperiments. J. Infect. Dis. 119:158-164.

41. Waldman, R. H., Z. Bencic, R. Sakazaki, R. Sinha, R.

INFECT. IMMUNITY

Ganguly, B. C. Deb, and S. Mukerjee. 1971. Choleraimmunology. I. Immunoglobulin levels in serum, fluidfrom the small intestine, and feces from patients withcholera and noncholeraic diarrhea during illness andconvalescence. J. Infect. Dis. 123:579-586.

42. Waldman, R. H., Z. Bencic, R. Sinha, B. C. Deb, R.Sakazaki, K. Tamura, S. Mukerjee, and R. Ganguly.1972. Cholera immunology. II. Serum and intestinalsecretion antibody response after naturally occurringcholera. J. Infect. Dis. 126:401-407.

43. W. H. 0. Scientific Group on Cholera Immunology. 1969.Cholera immunology. W.H.O. Tech. Rep. Ser., no. 414,p. 5-21.

on January 28, 2020 by guesthttp://iai.asm

.org/D

ownloaded from