Cholera Toxin in Giardia muris Infection and Binding of Cholera

Vol. 50, No. 1INFECTION AND IMMUNITY, OCt. 1985, p. 243-2490019-9567/85/100243-07$02.00/0Copyright © 1985, American Society for Microbiology

Changes in Intestinal Fluid and Mucosal Immune Responses toCholera Toxin in Giardia muris Infection and Binding of Cholera

Toxin to Giardia muris TrophozoitesINGER LJUNGSTROM,1* JAN HOLMGREN,2 ANN-MARI SVENNERHOLM,2 AND ANTONIO FERRANTE'tDepartment of Parasitology, National Bacteriological Laboratory, S-105 21 Stockholm,' and Department of Medical

Microbiology, University of Goteborg, S-413 46 Goteborg, Sweden

Received 20 March 1985/Accepted 8 July 1985

The effect of Giardia muris infection on the diarrheal response and gut mucosal antibody tesponse to choleratoxin was examined in mice. The results obtained showed that the fluid accumulation in intestinal loops exposedto cholera toxin was increased in mice infected with a low number (5 x 104) of G. muds cysts compared withthe response in noninfected mice. This effect was associated with a marked reduction in absorption of oralrehydration fluid from the intestine. In contrast, mice infected with a high dose (2 x 105) of cysts showed amarked decrease in fluid accumulation in response to the toxin. This decrease might be related to the findingthat both G. muris and Giardia lamblia trophozoites can bind significant amounts of cholera toxin. Evidence ispresented which suggests that the gut mucosal antibody response, mainly immunoglobulin A but aIsoimmunoglobulin G, to an immunization course with perorafly administered cholera toxin was depressed in miceinfected with G. muds. The reduction in antibody levels was particularly evident when the priuaryimmunization was made very early after infection. The serum antitoxin antibodies to the oral immunizationwith cholera toxin were, however, not affected. Likewise, the delayed-type hypersensitivity response againstsheep erythrocytes in animals primed subcutaneously with sheep erythrocytes was not modified during thecourse of G. muds infection.

Although enterotoxin-producing bacteria are responsiblefor a high proportion of acute diarrheal diseases, little is stillknown about the host and the environmental factors govern-ing the individual responses to enteric pathogens. A relation-ship between predisposition to cholera and the ABO bloodgroup antigens has been previously noted (9a). However, ofimmense importance is the role of the intestinalmicroecology, an area remaining relatively unexplored. Forinstance, little is known of the effects of intestinal parasiteson the intestinal ecology and intestinal responses to otherenteric pathogens.

Ljungstrom et al. have previously shown (14) that miceinfected with the parasitic helminth Trichinella spiralis showchanges in basal and cholera-toxin-stimulated intestinal fluidtransport processes during the intestinal stage of the disease.The intestinal fluid secretion in response to cholera toxinwas greatly enhanced, probably related to an observedmarked reduction in absorption of fluid from the intestine.There was also a drastic depression of the mucosal immu-noglobulin A (IgA) and IgG antibody response to orallyadministered cholera toxin during the intestinal stage oftrichinosis, which seemed to be related primarily to a de-pression of the development of immunological memory inthe gut (14).

Giardiasis is a common intestinal protozoan infection ofhumans and animals. The interaction between Giardia spp.and other enteropathogens is, however, poorly understood.The availability of a mouse model of giardiasis, with infec-tion with Giardia muris, has provided an opportunity tostudy certain aspects of such interactions. In the presentstudy, we have explored the possible effects of G. muris on

* Corresponding author.t Present address: Department of Paediatrics, University of

Adelaide, Adelaide Children's Hospital, S.A. 5006, Australia.

intestinal fluid transport processes and immune responses tocholera toxin in mice.

MATERIALS AND METHODS

Mice. Inbred mice (C57BL/6J) of both sexes were used. Ineach experiment, the animals tvere closely age and sexmatched. The ages varied between 4 and 6 weeks at the onsetof infection.

Parasites. The G. muris strain was kindly provided in 1979by Western General Hospital, Edinburgh, Scotland. Sincethen the parasite has been maintained at the National Bac-teriological Laboratory, Stockholm, Sweden, by passage inC57BL/6J mice. The Giardia lamblia strain was kindlyprovided in 1978 by the National Institutes of Health,Bethesda, Md. The trophozoites were cultured in DiamondTPS-1 medium (5) by the modification of Visvesvara (29)with sterile filtered medium.

Reagents. A highly purified preparation of cholera toxinwas purchased from Sigma Chemical Co., St. Louis, Mo.1251I-labeled cholera toxin was prepared by the method ofMarkwell (16). Purified cholera B-subunit was prepared byInstitute Merieux as described by Tayot et al. (28).

G. muds infection. The mice were infected with 104 G.muris cysts when not stated otherwise. The cysts wereisolated from stock animals on the same day they were usedto infect mice. The cysts were given orally in 0.5 ml of salinewith a stomach tube without anesthesia.

Determination of cyst and trophozoite recovery. G. muriscysts in stools were recovered and isolated as described byRoberts-Thomson et al. (23). In brief, the stools from theindividual mice were dispersed in tap water, and the cystswere isolated on a sucrose density gradient. The total cystoutput per 2 h of stool collection was calculated. Cystsisolated 1 week after infection were used for passage of theparasite or for experimental infections. For determination of

243

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trophozoite recovery, mice were killed by cervical disloca-tion, and the intestine from stomach to cecum was carefullyremoved. The small intestine was divided into three equalsegments. Each segment (the anterior, the medial, and theposterior) was flushed with 0.9% saline, and the number ofG. muris trophozoites released from each segment was

determined. Immediately after the flushing, each segmentwas cut longitudinally, the mucosa was carefully scraped off,and the number of trophozoites in the mucosal scraping was

counted with a hemacytometer. The G. lamblia trophozoiteswere cultured in vitro (29). After 72 h of growth, they were

harvested and counted.Determination of intestinal secretion and absorption. Intes-

tinal fluid secretion in response to cholera toxin was studiedin ligated small intestinal loops (11). Two 5- to 7-cm-longloops were prepared in each mouse. One loop was inocu-lated with 0.2 ml of cholera toxin in phosphate-bufferedsaline (PBS), and the other was inoculated with 0.2 ml ofPBS only. After 4 h, the animals were killed, and the lengthand the weight of the loops were determined. Fluid accumu-

lation attributable to the toxin was determined as weightdifference (in milligrams per centimeter) between the testand the control loop in each animal. Intestinal fluid absorp-tion was studied in ligated 8- to 10-cm-long jejunal loopswhich were injected with 0.5 ml of oral rehydration solutioncomposed of (in grams per liter): glucose, 20; NaCl, 3.5;NaHCO3, 2.5; and KCl, 1.5. After 14 or 21 min, the micewere killed, and the loop fluid content was determined (inmilligrams per centimeter) by weighing the loop and an

adjacent portion of the intestine.Binding studies of cholera toxin to G. muris and G. lamblia

trophozoites. (i) Immunological assay. The trophozoites were

washed and incubated in PBS in the presence of 0.1 ,ug ofcholera toxin per ml at 37°C for 1 h. After incubation, thesupernatant was collected by centrifugation, and the contentof residual cholera toxin was determined by the GM1 gan-

glioside enzyme-linked immunosorbent assay (24).(ii) Immunofluorescence. Trophozoites were adjusted to a

concentration of 106 parasites per ml and used to prepare

smears on microscope slides. These were fixed by incuba-tion with 3.5% paraformaldehyde for 5 min at room temper-ature. Cholera toxin (0.1 ,ug/ml) was allowed to react withthe parasites for 30 min at room temperature. This was

followed by incubation with rabbit anti-cholera toxin for 30min at room temperature and a final incubation (30 mmn atroom temperature) with fluorescein isothiocyanate-labeledanti-rabbit conjugate. Each incubation was followed by a

washing step. For the controls, either cholera toxin or rabbitanti-cholera toxin was left out, and PBS was added.

Binding of radioiodinated cholera toxin. Trophozoites were

adjusted to a concentration of 5 x 106 parasites per ml, and0.1 ml of the suspension was added to V-formed glass tubesand chilled on ice. To each tube, 0.1 ml of chilled 125I-labeledcholera toxin was added either in buffer alone (PBS contain-ing 0.2% bovine serum albumin) or in buffer supplementedwith 1 ,ug of cholera toxin B-subunit per ml. The tubes were

incubated for 100 min in an ice bath. After incubation, thetrophozoites were extensively washed in PBS containing0.05% Tween 20 by centrifugation at 1,000 x g for 10 min at4°C between each step and harvested. The uptake of iodine-labeled cholera toxin by the trophozoites was estimated in a

gamma-counter. The passive adsorption of radiolabeledcholera toxin to the tube walls was 10% of that of addedspecific-labeled cholera toxin.Immunization with cholera toxin. Groups of G. muris-

infected and -noninfected mice were immunized with cholera

toxin. Four immunizations, each consisting of 5 ,g ofcholera toxin in 0.5 ml of 5% NaHCO3, were given perorallyby a stomach catheter at 6-day intervals. Antibody levelswere measured 4 days after the last (booster) immunization.

Intestinal antibody synthesis. Antibody synthesis by invitro-cultured intestinal tissue was determined as describedpreviously (25). In brief, the small intestine was quicklyexcised, and multiple tissue specimens were taken fromdifferent parts. Equal amounts of tissue (approximately 500mg per animal) were taken from experimental animals andcontrols. The tissue was thoroughly washed, minced intosmall pieces, and then incubated at 37°C for 24 h in Eaglemedium supplemented with 5% heat-inactivated normal rab-bit serum and 200 IU each of penicillin and streptomycin perml (pH 7.2 to 7.4). After incubation, the supernataht wascollected by centrifugation at 3,000 x g for 10 min and frozenat -70°C. The titers of specific IgA and IgG antibodies tocholera toxin were determined by the enzyme-linked im-munosorbent assay (25).

Determination of serum antibodies. Antitoxin of both IgGand IgA classes in serum were determined by the enzyme-linked immunosorbent assay with purified cholera toxin asthe solid-phase antigen (25).

Delayed-type hypersensitivity. The delayed-type hypersen-sitivity test was performed essentially as described by Liew(12). Mice were primed subcutaneously with 10' sheeperythrocytes (SRBC) in 50 RIl of saline and challengedsubcutaneously 5 days later with 108 SRBC in 25 ,ul of salinein the left hind footpad. As a control, 25 [lI of saline wasadministered subcutaneously in the right hind footpad. Theswelling was measured with a caliper 24 h after challenge,and the difference between the left and right footpad wasexpressed as the percent increase in footpad thickness.

RESULTS

Characterization of G. muris infection. (i) Kinetics of cystexcretion. Previous studies have revealed marked straindifferences in the resistance of mice to G. muris infection. Insome mouse strains, the infection is self-limiting and themice are able to eliminate G. muris within 6 to 8 weeks,while in others persistent infection develops (2, 17, 21, 22).To establish the susceptibility to G. muris in the inbredC57BL/6J mouse strain used in this study, mice were given5 x 104 G. muris cysts, and excretion of cysts in stools wasfollowed for 11 weeks (Fig. 1). Cyst excretion was maximal1 to 2 weeks after initiation of the oral infection. In the next2 weeks, a progressive reduction in cyst excretion wasobserved, followed by a gradual decline in cyst numbersthroughout the period of study, with mean cyst counts ofmore than 104/2 h. During the first 2 weeks of infection,doses of 104 and 10 5 showed almost similar cyst excretion asthe 5 x 104 infective dose. The results obtained indicate thatC57BL/6J mice, at the age of 4 to 6 weeks, are susceptible toG. muris infection and develop a mild but chronic infection.

(ii) Presence of trophozoites in the small intestine. Thenumber of trophozoites in the small intestine was determined1 and 4 weeks after oral infection with 104 or 105 G. muriscysts. The total number in each segment was determined byadding the number of trophozoites obtained from the lavageto those obtained from the scraping; 60 to 80% of theparasites were recovered in the scraping. Both 1 and 4 weeksafter infection, somewhat higher recoveries of trophozoiteswere obtained in the segments from mice which had receivedthe higher dose compared with those which had received the

INFECT. IMMUN.

RESPONSE TO CHOLERA TOXIN DURING GIARDIA INFECTION

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FIG. 1. The number of cysts excreted per 2 h + standard error of the mean in C57BL/6J mice at various times after G. muris infection.Number of mice examined on each occasion is indicated.

lower dose (Fig. 2). Independent of infectious dose, therewas a 10-fold reduction in trophozoite recovery between 1and 4 weeks. The trophozoites were distributed along thewhole intestine, although maximal recovery was consis-

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tently observed in the midsection. This part of the smallintestine was selected for the studies of fluid transportprocesses.

Effect of G. muris infection on intestinal secretion andabsorption. Intestinal fluid secretion in response to choleratoxin was estimated in G. muris-infected mice during theearly phase of infection. One group of mice was infectedwith 5 x 104 cysts and challenged with 5 ,ug of cholera toxin.Both 7 and 18 days after infection, these mice respondedwith an increased fluid accumulation as compared with thatin noninfected mice (P < 0.05) (Fig. 3). Other groups of micewere given a fourfold higher dose of cysts and challengedwith 1.5 or 5 ,ug of cholera toxin. A significantly decreasedfluid accumulation in relation to noninfected controls (P <0.01) in response to both doses of cholera toxin was recordedon both days 7 and 12 after infection (Fig. 3).To determine the effects on intestinal fluid absorption

FIG. 2. Geometric mean + standard error of the mean of tropho-zoite recovery 1 and 4 weeks after G. muris infection in proximal(P), middle (M), and distal (D) segments of the small intestine.

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VOL. 50, 1985 245

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246 LJUNGSTROM ET AL.

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FIG. 3. Fluid secretion ± standard error of the mean in intestinalloops in response to cholera toxin at various times after G. murisinfection. In noninfected animals (n = 9), the fluid accumulation was151 ± 16 and 46 ± 13 mg/cm in response to 5 and 1.5 ,ug of choleratoxin, respectively. These values represent 100% fluid secretion inthis figure. Each point represents data from three animals. Micewere infected with either 5 x 104 (0-----0) or 2 x 105 (N *) G.muris cysts.

during the first 2 weeks after G. muris infection, the ligatedloops were injected with oral rehydration solution containingglucose. The weight of the loops (in milligrams per centime-ter) was determined 14 and 21 min after injection of oralrehydration solution containing glucose and compared withthat of an adjacent portion of the intestine. The absorption oforal rehydration solution containing glucose was signifi-cantly decreased in the infected mice compared with thenoninfected animals (P < 0.001) (Fig. 4).Uptake of cholera toxin by G. muris and G. lamblia

trophozoites. Incubation of 105 G. muris or G. lambliatrophozoites with 0.1 ,ug of cholera toxin resulted in 41 or36%, respectively, reduction of cholera toxin from theincubation mixture as measured with the GM1 gangliosideenzyme-linked immunosorbent assay. When the number oftrophozoites was decreased (103 and 104), the reduction inbinding was about 10%. Immunofluorescence studies sug-gested that this reduction was due to binding of cholera toxinto the trophozoites (Fig. 5).To prove further that Giardia spp. trophozoites are able to

bind cholera toxin, G. lamblia trophozoites were incubatedwith iodine-labeled cholera toxin. The results revealed thatthere was an uptake of cholera toxin by the trophozoites. Itwas found that 45% of the added specific-labeled toxin wasbound to the trophozoites. This uptake was nearly com-pletely inhibited when 1 jig of B-subunit was added alongwith the radiolabeled toxin (97.5% inhibition).

Influence of G. muris on immune responses to unrelated

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antigens. The ability of the small intestine to respond immu-nologically to cholera toxin antigen during the parasiteinfection was tested in mice orally immunized with choleratoxin at various times after infection. An immunizationschedule known to induce optimal protection against exper-imental cholera infection was used (11). The degree ofmucosal immune response was assessed by measuring anti-toxin IgA and IgG antibodies synthesized in vitro by tissue-cultured intestines. When immunization was started 4 daysafter G. muris infection, there was a marked decrease in thelocal formation of both antitoxin IgA and IgG as comparedwith that in noninfected controls (P < 0.01 and P < 0.02,

FIG. 5. G. lamblia trophozoites incubated with cholera toxin,followed by rabbit anti-cholera toxin and fluorescein isothiocyanate-labeled anti-rabbit conjugate.

INFECT. IMMUN.

RESPONSE TO CHOLERA TOXIN DURING GIARDIA INFECTION

respectively) (Fig. 6). A slight but not significant effect (P >0.05) on the local IgA and IgG antitoxin responses wasobserved when the immunization was started 8 days afterinfection (Fig. 6).The serum antibody response against cholera toxin in

infected mice given oral immunizations was also deter-mined. No differences in the serum antitoxin responses ofeither class were observed between G. muris-infected and-noninfected mice (data not shown).The effect of G. muris infection on the development of

delayed-type hypersensitivity response to SRBC was inves-tigated during the course of the infection. Animals wereprimed with SRBC 2, 4, 6, 14, and 21 days after infection. Nosignificant effect on the delayed-type hypersensitivity re-sponse was observed during this period (data not shown).

DISCUSSIONGiardiasis is a common intestinal protozoan infection of

humans and animals. The infection can be classified intothree categories: self-limiting, recurrent, and persistent. Inthe present study, we show that C57BL/6J mice infected atthe age of 4 to 6 weeks developed a mild but chronic G.muris infection. Although our finding is not in agreementwith an earlier report (2) showing a self-limiting infection inC57BL/6J mice, the difference could be because the authorsof the previous study infected the mice at the age of 8 weeks.Indeed, MacDonald et al. (15) have suggested a relationshipbetween the development of chronic infection and the age ofmice. Our data showed that trophozoites were distributedthroughout the small intestine, an observation similar tofindings in Swiss albino (CF-1) mice (18).The interaction between Giardia spp. and other

enteropathogens is poorly understood. We present datashowing that infection with G. muris has a profound effect onintestinal fluid transport processes as well as on the estab-lishment of a gut mucosal immune response to orally admin-istered cholera toxin. When mice were given a high inoculumof parasites, fluid accumulation in response to cholera toxinwas greatly decreased during the early phase of infection. Toinitiate its striking effects on fluid transport processes in thesmall intestine, cholera toxin has to bind to GM1 gangliosidereceptors on the epithelial cells (10). There are severalpossible means by which the parasites could reduce toxin-induced secretion. They may bind the toxin and thus reducethe concentration of toxin available for binding to theepithelial target cells, they may provide a mechanical barrierwhich prevents binding to epithelial cells by blocking toxinreceptor sites, or they may directly or indirectly, via theimmune system, modulate intestinal epithelial functions.Our results do not allow definite conclusions about therelative role of any of these possible mechanisms, althoughthey clearly show that both G. muris and G. lambliatrophozoites are able to bind cholera toxin, which mightexplain the decreased fluid accumulation observed in re-sponse to cholera toxin. The mechanical barrier hypothesisis less likely, especially since the trophozoites mainly adhereto the microvilli near the bases of the villi (20), and activeelectrolyte and water secretion is thought to be mediated bythe cells in the crypts (7).

In contrast, the net fluid response to cholera toxin wasincreased when the mice were infected by a lower number ofG. muris cysts. Intestinal fluid accumulation is the net resultof absorption and secretion by both passive diffusion andactive transport mechanisms. Our results suggest that infec-tion with G. muris profoundly affects (inhibits) absorption,and this could explain increased fluid accumulation to a

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FIG. 6. Effect of G. muris infection on in vitro antibody synthe-sis in the small intestine after four oral immunizations with choleratoxin, starting 4 or 8 days after infection. Shaded bars representnoninfected animals. The titers are expressed as geometric mean +standard error of the mean. Number of mice examined in each groupis indicated in the bars. N.S, Not significant; *, P < 0.02; **, P <0.01.

secretagogue like cholera toxin in an infection with too fewparasites to bind significant amounts of the added choleratoxin. Decreased absorptive capacity in giardiasis is inagreement with earlier findings, which showed that Giardiainfection causes reduced brush border enzyme activity (8)and that the small intestine may have decreased capability toabsorb sugars (1, 9), vitamins (3, 4, 31), and fat (4, 31) whensubjected to Giardia infection. The precise mechanismwhereby the absorption is reduced is not clear. One possi-bility may be damage of microvilli covering the luminal cellsurface accompanied by the presence of Giardia spp. adja-cent to the epithelial cells (6, 20, 26, 27). The inhibition couldalso be due to the increased turnover of epithelial cells,which is observed during giardiasis (15), because activeabsorption is mainly mediated by mature villus cells (7).Proliferation of the intestinal epithelial cells mainly takesplace in the crypts, and the new cells are continuouslymoved upwards during progressive differentiation. En-hanced epithelial cell turnover could therefore result in agreater proportion of functionally immature cells on the vililacking absorptive capacity.

In addition to the changes in intestinal fluid processes,mice infected with G. muris demonstrated some clearchanges in the local immune response similar to thosepreviously shown for murine trichinosis (13). When oral

VOL. 50, 1985 247

248 LJUNGSTROM ET AL.

immunization with cholera toxin was started 4 days afterinfection, the intestinal synthesis of both antitoxin IgA andIgG was decreased; this was most pronounced for the IgAantibodies. However, when the immunizations were initi-ated 8 days after infection, no statistically significant effecton the local antibody response was observed. This effect ofgiardiasis on mucosal antitoxin immunity seems to be mostpronounced during the period when the number of parasitesin the intestine is very high. When the immunization wasstarted 4 days after infection, the mice received two antigenstimuli before the rapid diminution in parasite number oc-curred. When the immunization was started on day 8, onlyone antigen dose was given before the reduction in parasitenumber took place.

Earlier studies have shown that the antitoxin response inthe intestines of mice is transient and dose dependent. Theactual antibody response to the immunization regimen em-ployed reaches its maximum by day 4 to 8 after oralimmunization and declines thereafter (25). However, con-comitantly, immunological memory of a longer durationdevelops which is boosted by renewed immunizations andwith a lower antigen dose than that needed for priming (11).One interpretation of our results could thus be based oneffects on the development of immunological memory for thelocal antibody response to cholera toxin. This could resultfrom a combination of two events. First, it is known thatpriming of the gut mucosal immune system is much moredependent on the dose of antigen than the boosting phase ofthe response. This would make it more sensitive to uptake ofcholera toxin by parasites. Second, modification of theresponse to cholera toxin may be attributable in part to thechanges in the mucosal lymphoid-macrophage system thatoccurs during G. muris infection (9, 19). However, it must beviewed with some speculation that the decreased antibodyresponse to cholera toxin was due to effects on the develop-ment of immunological memory since this was not studied.Thus, it could still be speculated that T- and B-cell memoryresponses were normal but that the parasites evoked specificor nonspecific suppressive mechanisms that prevented thefull manifestation of memory.

In this study, we could not demonstrate any influence ofG. muris infection on either the systemic IgG and IgAantibody responses to orally administered cholera toxin orthe delayed-type hypersensitivity response to subcutane-ously injected SRBC. The discrepancy observed in thisstudy between the local and the systemic immune responsesto orally administered cholera toxin suggests that a nonin-vasive intestinal parasite only affects the local immunity. Incontrast, studies with T. spiralis, a parasite which is invasiveduring the intestinal stage (30), showed a depression of bothlocal and systemic immune responses during the intestinalstage (13, 13a).A high frequency of asymptomatic and symptomatic

giardiasis occurs in people living in countries in which manyother intestinal pathogens are also common. It is clearlydifficult to speculate how closely the findings reported herefor experimental giardiasis in mice are paralleled in naturalgiardiasis in humans. Also, the significant effects on intesti-nal fluid transport processes and mucosal immune responsein the murine model were only seen during the first week(s)after infection. Nonetheless, these effects were striking and,as mentioned, similar findings, although perhaps mediatedby other mechanisms, were also made in murine trichinosis(14). This warrants studies of this kind to be undertaken inhuman parasitic infections. If they extend to human infec-tions, effects such as those observed in the experimental

system could have considerable implications for the under-standing of both aspects of the pathogenesis of entericinfections and disease- or vaccine-induced mucosal immuneresponses.

ACKNOWLEDGMENTS

We thank Maria Karpinska and Britt Hammarberg at the NationalBacteriological Laboratory, Stockholm, Sweden, for excellenttechnical assistance. The instructive information on the isotopetechnique by Marianne Lindblad, University of Goteborg, Sweden,is gratefully acknowledged.The work was supported by a World Health Organization-

Diarrhoeal Diseases Control Programme and Swedish Medical Re-search Council grant 16 X 3382.

LITERATURE CITED

1. Anand, B. S., A. Mahmood, N. K. Ganguly, M. M. Rehani, J. B.Dilawari, and R. C. Mahajan. 1982. Transport studies andenzyme assays in mice infected with human Giardia lamblia.Trans. R. Soc. Trop. Med. Hyg. 76:616-619.

2. Belosevic, M., G. M. Faubert, E. Skamene, and J. D. MacLean.1984. Susceptibility and resistance of inbred mice to Giardiamuris. Infect. Immun. 44:282-286.

3. Deka, N. C., A. K. Sehgal, and P. N. Chhuttani. 1981. Absorp-tion and transport of radioactive cobalt(57) vitamin B12 inexperimental giardiasis in rats. Indian J. Med. Res. 74:675-679.

4. Desai, H. G., and R. H. Kalro. 1983. Functional and histologicalabnormalities of small bowel mucosa in giardiasis. Indian J.Med. Res. 77:239-244.

5. Diamond, L. S. 1968. Techniques of axenic cultivation ofEntamoeba histolytica Schaudinn 1903 and E. histolytica-likeamoebae. J. Parasitol. 54:1047-1056.

6. Erlandsen, S. L., and D. G. Chase. 1974. Morphological alter-ations in the microvillous border of villous epithelial cellsproduced by microorganisms. Am. J. Clin. Nutr. 27:1277-1286.

7. Field, M. 1980. Intestinal secretion and its stimulation byenterotoxins. Nobel Symp. 43:46-52.

8. Ganguly, N. K., R. C. Mahajan, V. Vasudeva, A. Mahmood,P. K. Dudeja, V. Radhakrishna, J. B. Dilawari, and B. S.Anand. 1982. Intestinal uptake of nutrients and brush borderenzymes in normal and thymectomised Giardia lamblia infectedmice. Indian J. Med. Res. 75:33-39.

9. Gillon, J., D. Al Thamery, and A. Ferguson. 1982. Features ofsmall intestine pathology (epithelial cell kinetics, intraepitheliallymphocytes, disaccharidases) in a primary Giardia muris infec-tion. Gut 23:498-506.

9a.Glass, R. I., J. Holmgren, C. E. Harley, M. R. Khan, A.-M.Svennerholm, B. J. Stoll, K. M. B. Houssain, M. Yunus, andR. E. Black. 1985. Predisposition for cholera of individuals with0-blood group: possible evolutionary significance. Am. J. Epi-demiol. 121:791-796.

10. Holmgren, J. 1981. Actions of cholera toxin and the preventionand treatment of cholera. Nature (London) 292:413-417.

11. Lange, S., and J. Holmgren. 1978. Protective antitoxic choleraimmunity in mice: influence of route and number of immuniza-tions and mode of action of protective antibodies. Acta Pathol.Microbiol. Scand. Sect. C 86:145-152.

12. Liew, F. Y. 1977. Regulation of delayed-type hypersensitivity. I.T suppressor cells for delayed-type hypersensitivity to sheeperythrocytes in mice. Eur. J. Immunol. 7:714-718.

13. Ljungstrom, I. 1983. Immunodepression in Trichinella spiralisinfection. Wiad. Parazytol. 29:401-412.

13a.Ljungstrom, I., and A. Ferrante. 1985. The in vitro and in vivoimmunodepression by Trichinella spiralis, a preliminary report,p. 63-70. In C. W. Kim (ed.), Trichinellosis. State University ofNew York Press, Albany, N.Y.

14. Ljungstrom, I., J. Holmgren, G. Huldt, S. Lange, and A.-M.Svennerholm. 1980. Changes in intestinal fluid transport andimmune responses to enterotoxins due to concomitant parasiticinfection. Infect. Immun. 30:734-740.

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RESPONSE TO CHOLERA TOXIN DURING GIARDIA INFECTION

15. MacDonald, T. T., A. Ferguson, and M. R. C. Path. 1978. Smallintestinal epithelial cell kinetics and protozoal infection in mice.Gastroenterology 74:496-500.

16. Markwell, M. A. K. 1982. A new solid-state reagent to iodinateproteins. Anal. Biochem. 125:427-432.

17. Mitchell, G. F., R. F. Anders, G. V. Brown, E. Handmnan, I. C.Roberts-Thomson, C. B. Chapman, K. P. Forsyth, L. P. Kahl,and K. M. Cruise. 1982. Analysis of infection characteristicsand antiparasite immune responses in resistant compared withsusceptible hosts. Immunol. Rev. 61:137-188.

18. Olveda, R. K., J. S. Andrews, Jr., and E. L. Hewlett. 1982.Murine giardiasis: localization of trophozoites and small bowelhistopathology during the course of infection. Am. J. Trop.Med. Hyg. 31:60-66.

19. Owen, R. L., C. L. Allen, and D. P. Stevens. 1981. Phagocytosisof Giardia muris by macrophages in Peyer's patch epithelium inmice. Infect. Immun. 33:591-601.

20. Owen, R. L., P. C. Nemanic, and D. P. Stevens. 1979.Ultrastructural observations on giardiasis in a murine model. I.Intestinal distribution, attachment, and relationship to the im-mune system of Giardia muris. Gastroenterology 76:757-769.

21. Roberts-Thomson, I. C., and G. F. Mitchell. 1978. Giardiasis inmice. I. Prolonged infections in certain mouse strains andhypothymic (nude) mice. Gastroenterology 75:42-46.

22. Roberts-Thomson, I. C., G. F. Mitchell, R. F. Anders, B. D.Tait, P. Kerlin, A, Kerr-Grant, and P. Cavanagh. 1980. Geneticstudies in human and murine giardiasis. Gut 21:397-401.

23. Roberts-Thomson, I. C., D. P. Stevens, A. A. F. Mahmoud, and

K. S. Warren. 1976. Giardiasis in the mouse: an animal model.Gastroenterology 71:57-61.

24. Svennerholm, A.-M., and J. Holmgren. 1978. Identification ofEscherichia coli heat-labile enterotoxin by means of a ganglio-side immunosorbent assay (GM1-ELISA). Curr. Microbiol.1:19-23.

25. Svennerholm, A.-M., S. Lange, and J. Holmgren. 1978. Corre-lation between intestinal synthesis of specific immunoglobulin Aand protection against experimental cholera in mice. Infect.Immun. 21:1-6.

26. Takano, J., and J. H. Yardley. 1965. Jejunal lesions in patientswith giardiasis and malabsorption. An electron microscopicstudy. Bull. Johns Hopkins Hosp. 116:413-429.

27. Tandon, B. N., B. K. Puri, P. C. Gandhi, and S. G. Tewari.1974. Mucosal surface injury ofjejunal mucosa in patients withgiardiasis: an electron microscope study. Indian J. Med. Res.62:1838-1842.

28. Tayot, Y.-L., J. Holmgren, L. Svennerholm, M. Lindblad, andM. Tardy. 1981. Receptor-specific large scale purification ofcholera toxin on silica beads derivatized with lyso-GM1 gangli-oside. Eur. J. Biochem. 113:249-258.

29. Visvesvara, G. S. 1980. Axenic growth of Giardia lamblia inDiamond's TPS-1 medium. Trans. R. Soc. Trop. Med. Hyg.74:213-215.

30. Wright, K. A. 1979. Trichinella spiralis: an intracellular parasitein the intestinal phase. J. Parasitol. 65:441-445.

31. Wright, S. G., A. M. Tomkins, and D. S. Ridley. 1977.Giardiasis: clinical and therapeutic aspects. Gut 18:343-350.

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