Vol. 58, No. 11

Excystation of In Vitro-Derived Giardia lamblia CystsSHAYNE-EMILE M. BOUCHER AND FRANCES D. GILLIN*

Department ofPathology, H811F, University of California, San Diego Medical Center,225 Dickinson Street, San Diego, California 92103

Received 16 April 1990/Accepted 10 August 1990

This is the first in-depth analysis of the excystation of Giardia lamblia cysts prepared in vitro. Its goals wereboth to achieve efficient excystation and to gain insights into this crucial but poorly understood process. Toidentify the critical elements of excystation, we tested the sequential low-pH induction and protease treatmentswhich had been reported to be important for excystation of fecal cysts. The optimal pH for induction ofexcystation was 4.0. Emergence was greatly (-10-fold) stimulated by subsequent exposure of in vitro-derivedcysts to chymotrypsin, trypsin, or human pancreatic fluid. The stimulatory activity of each was abolished bysoybean trypsin inhibitor, demonstrating that the activity of pancreatic fluid was due to these proteases.Excystation of in vitro-derived cysts was -10 to 38%. Although the walls of in vitro-derived cysts were partiallydigested by protease treatment, trophozoites emerged only from one pole, as observed with fecal cysts. Theconditions of encystation also determined the efficiency of excystation. Specifically, encystation in the presenceof lactic acid, a major metabolite of colonic bacteria, stimulated excystation approximately fourfold, althoughit did not increase the total numbers of cysts. These experiments have shown that excystation of in vitro-derivedcysts reflects that of cysts purified from human feces in that it is dependent upon conditions which simulate thepassage of cysts through the human stomach (low pH) and into the small intestine (pancreatic proteases).

Since excystation of ingested Giardia lamblia cysts isessential to the transmission of disease, it may be considereda virulence factor in human giardiasis. Nonetheless, thisprocess is not well understood, largely because, until re-cently, the only source of G. lamblia cysts was the feces ofinfected humans or experimentally infected animals. Excys-tation of fecal cysts varied from <0.1 to >95% (17; unpub-lished data). Cysts prepared in vitro have the advantages ofbeing free of fecal contaminants and of more consistentphysiologic conditions for studies of excystation.During natural infection, ingested cysts pass through the

stomach of the host, where they are exposed to gastric acid(2). Subsequently, cysts enter the duodenum, where thegastric chyme is rapidly neutralized by influxes of bicarbon-ate (3). It is important that trophozoites not emerge fromcysts in the stomach because they would be killed by theacid. Excystation is presumed to occur in the upper smallintestine, but the exact site is not known. Descent past theentrance of the common bile duct would expose the cysts toa variety of degradative enzymes and bile salts, which havedetergent activity (3, 10).The pioneering studies of fecal cysts by Bingham et al. (1,

2) demonstrated that excystation was dependent upon

"those conditions most closely approximating the organ-ism's in vivo environment" (2). Specifically, they showedthe importance of exposing cysts to a low pH, which mimicspassage through the stomach. Trophozoites emerged whenthe acid-treated cysts were transferred to a neutral medium,reproducing entry into the duodenum (2). Excystation meth-ods published since then have incorporated other physio-logic intestinal factors, including bicarbonate, trypsin (apancreatic protease), thiol-reducing agents (16, 20), and bilesalts (11).

In a previous study (7), we demonstrated the production oflarge numbers (>105/ml) of water-resistant cysts with type Imorphology (smooth, oval, phase bright, with cyst organ-

* Corresponding author.

elles visible in relief by differential interference contrastmicroscopy). Some of the cysts (range, -1 to 9.5% of totalcysts) completed the life cycle by excysting in vitro, butmost of the trophozoites emerged only partially from thecyst wall. Since >90% of the type I cysts were viable (7), asdetermined by the fluorogenic dye method of Schupp andErlandsen (19), we hypothesized that the efficiency of ex-cystation could be increased. Therefore, the goals of thepresent study were to achieve high-efficiency excystation ofin vitro-derived G. lamblia cysts and to gain an increasedunderstanding of this important biologic process.

MATERIALS AND METHODS

Materials. Unless otherwise noted, all reagents were ob-tained from Sigma Chemical Co. (St. Louis, Mo.). Bicarbon-ate was purchased from Fisher Scientific, and crude trypsin,from U.S. Biochemicals, was the gift of Judith Sauch. Cystswere isolated from the feces of a patient with symptomaticgiardiasis by the method of Douglas et al. (5).Human pancreatic fluid. An adult male volunteer was

intubated orally with a duodenal tube (AN 20; A. W.Anderson, Santa Monica, Calif.). The tube was allowed topass through the stomach and into the duodenum underfluoroscopic guidance to an area between the ampulla ofVater (common bile duct) and the beginning of the jejunum.Before samples were collected, all residual pancreatic andduodenal fluids were withdrawn through the duodenal tubeand discarded. Basal pancreatic secretions were collectedfor 15 min. Pancreatic secretions were then stimulated byintravenous injection of 75 U of secretin (Ferring Laborato-ries, Sufferin, N.Y.). Two sequential 15-min stimulatedpancreatic fluid samples were then collected.

Parasite cultivation. G. lamblia WB (ATCC 30957) was

routinely cultivated in Diamond TYI-S-33 medium (4) with10% adult bovine serum (Irvine Scientific) and bovine bile(13) but without added iron, vitamins, or antibiotics aspreviously described (7), with subculturing twice weekly.The same lots of Biosate (BBL) and serum were used

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throughout these experiments, since any change which af-fected growth tended to decrease encystation.

Encystation. (i) Pre-encystation cultures. The condition ofthe pre-encystation trophozoite monolayer appeared to playan important role in the efficiency of in vitro encystation (7).Cultures grown to the late log phase in growth medium werechilled, inverted 12 times, and counted in a hemacytometerchamber. Trophozoites (5,000 per ml, final concentration)were added to chilled pre-encystation medium. The pre-encystation medium was freshly prepared TYI-S-33 growthmedium (pH 7.1) containing the antibiotics piperacillin (500Rg/ml; Lederle Laboratories) and amikacin (125 ,ug/ml;Bristol Laboratories) but no bovine bile. Pre-encystationcultures (in 8-ml borosilicate glass screw-capped tubes [13by 100 mm]) were grown for 3 days at 37.5°C and at 5° fromhorizontal. Cultures were still in the log phase, and mono-layers were -50 to 80% confluent. The tubes were invertedeight times, and the unattached trophozoites and mediumwere discarded. The attached trophozoite monolayers wererefed with fresh encystation medium (see below).

(ii) Encystation cultures. Unless otherwise specified, theencystation medium consisted of pre-encystation mediumadjusted to pH 7.8 with 1 M NaOH and supplemented withporcine bile (0.25 mg/ml, final concentration) and lactic acid(hemi-calcium salt, 5 mM, final concentration) (7). Themedium, bile (10-mg/ml stock solution), and lactic acid (100mM stock solution) were all freshly prepared and filtersterilized separately. Encysting trophozoites were incubatedfor 66 h, since initial experiments showed that this length ofincubation was optimal for the biologic activity of cysts(unpublished data). Parasites were harvested and water-resistant cysts were isolated as described previously (7),except that the cultures were not chilled. Tubes were in-verted (eight times), and nonadherent cells, including cysts,were decanted into 15-ml conical centrifuge tubes, pelleted,washed in 15 ml of double-distilled water, and incubated for30 to 45 min at room temperature in 15 ml of double-distilledwater. Trophozoites and cysts with incomplete walls werelysed by this treatment. Type I and type II water-resistantcysts were collected by low-speed centrifugation (5 min, 135x g, 4 to 10°C), suspended in the volume (-200 ,ul) of waterremaining in the tube, and enumerated in a hemacytometerchamber by differential interference contrast microscopy (7).As defined previously (7), type I cysts have a smooth, ovalshape, are somewhat refractile, and have cyst organellesappearing in relief. Cysts not fulfilling these criteria arereferred to as type II.

Excystation. Unless otherwise specified, excystation wascarried out on the same day that in vitro-derived cysts wereharvested. Our standard excystation procedure incorporatesvariations of the two-step method of Rice and Schaefer forfecal cysts (16), based on the results of the experimentspresented here. The variables tested are described in eachexperiment. A low-pH induction solution was prepared freshby mixing 6.8 ml of Hanks balanced salt solution containingL-cysteine hydrochloride (57 mM) and reduced glutathione(32.5 mM) with 6.8 ml 0.1 M NaHCO3 and 11.3 ml ofdouble-distilled water. The pH of the solution was adjustedto 4.0 with 0.01 N HCl. In the first, or induction step, 50 ,ulof cyst suspension (5 x 103 to 5 x 104 cysts) and 0.55 ml ofthe low-pH induction solution were mixed in a 1.5-mlEppendorf tube. The tube was capped, vortexed, and incu-bated for 30 min in a 37.5°C water bath. The cysts weresedimented for 3 min at 135 x g and room temperature, andthe supernatant was aspirated and discarded.

In the second, or excystation, step, 1 ml of a prewarmed

mixture containing 1 mg of chymotrypsin (from bovinepancreas, 3X crystallized, 50 U/mg of protein) in Tyrode saltsolution (16), with the pH raised to 8.0 with freshly prepared7.5% sodium bicarbonate, was added to the cyst pellet. Thetube was capped, vortexed, and incubated for 30 min in a37.5°C water bath. The cysts were sedimented for 3 min at135 x g and room temperature, and the supernatant wasremoved to avoid prolonged exposure to chymotrypsin,which was detrimental to the emerging trophozoites.

After the excystation step, 100 ,ul of prewarmed TYI-S-33growth medium was added to the pellet, which was sus-pended with a Pipetman, to promote trophozoite motilityand viability. Twenty microliters of each sample was loadedinto a hemacytometer chamber, which was placed in a 37°Chumidified chamber. Partially and totally excysted tropho-zoites and intact cysts were enumerated by differentialinterference contract microscopy with a 20X objective.Early experiments showed that the percent excystation didnot change significantly during the time required to count allthe samples (-4 h).The formula of Bingham et al. (1) was used to determine

percent excystation. The original formula was as follows:percent excystation = {[(TET/2) + PET]/[(TET/2) + PET +IC]} x 100, where TET is the number of totally excystedtrophozoites, PET is the number of partially excysted tro-phozoites, and IC is the number of intact cysts. PET aremotile and active but not completely emerged from the cystwall. This formula included a twofold factor based on theobservation that TET from fecal cysts divided immediatelyupon emergence, producing two trophozoites (1). However,on the basis of morphologic observations, the division ofstrain WB trophozoites emerging from cysts prepared invitro was delayed by several hours. Therefore, for in vitrocysts, we did not divide TET by two, unless the trophozoiteshad already divided, as determined microscopically.Each figure shown is from a single experiment repre-

sentative of two to six repetitions with different cyst prepa-rations. Each determination represents an average of fourfields with -200 cells per field. The results of each repetitionwere consistent with those shown in the figures. Significancewas determined by Student's t test.

RESULTS

Roles of acid and protease treatments in excystation. In aninitial experiment, we assessed two published methods ofexcystation. With the method of Rice and Schaefer (16), weobserved 13.5% + 0.8% excystation. Moreover, >97% ofthe excysted trophozoites emerged completely from the cystwall. In contrast, with the method of Schupp et al. (20),which does not include protease treatment, we observedonly 4.3% + 0.8% excystation. Moreover, most (90.1%) ofthe trophozoites were only partially excysted, meaning thatthe trophozoites did not emerge completely from the cystwall or remained partially adherent to the cyst shell (P <0.0005). Therefore, we investigated the two major parame-ters of the method of Rice and Schaefer: pH in the first, orinduction, step and treatment of cysts with protease in thesecond, or emergence, step.The studies of Bingham et al. (1, 2) demonstrated the

importance of the exposure of fecal cysts to a low pH forsubsequent excystation. Therefore, we assessed the effect ofexposure to an acidic pH on excystation of in vitro-preparedcysts (Fig. 1). The pH curve was quite broad, with 10 to>35% excystation after exposure to pH 2 to 8, with anoptimum at pH 4.0, which was used in subsequent experi-

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FIG. 1. Effect of various pHs on the excystation of in vitro-derived cysts. Water-resistant cysts were incubated in cysteine-glutathione-bicarbonate solution, the pH of which was adjusted withdilute HCI or NaOH. Otherwise, the procedure was as described inMaterials and Methods. S.D., Standard deviation of the totalpercent excystation. The P values are for exposure to the indicatedpH, compared with pH 4.0.

ments. In other experiments, excystation did not differgreatly at pHs between 2 and 4 but always decreasedgradually with increasing pH above pH 4. Although theproportion of totally excysted trophozoites decreasedslightly with increased pH, it remained >80%.

Following exposure to gastric acid, ingested cysts passinto the small intestine, where they are exposed to pancre-atic proteases at a slightly alkaline pH. This process wasmodeled by Rice and Schaefer (16), who treated fecal cystswith a crude preparation of trypsin. To elucidate the role ofproteases in excystation, we exposed in vitro-derived cyststo crude and purified trypsin preparations and to purifiedchymotrypsin. The highest level of excystation (19.2%7.7%) was observed following exposure of in vitro-derivedcysts to purified chymotrypsin, as compared with 15.7% +

5.1% with crude trypsin at 5 mg/ml or 6.8% ± 1.6% withpurified trypsin at 1 or 5 mglml (data not shown). Sincechymotrypsin was effective at lower concentrations, weused it in all subsequent experiments.We next compared the effects of a low pH and proteolytic

treatment on the excystation of cysts isolated (5) from thefeces of a patient with symptomatic giardiasis (Fig. 2).Excystation was maximal after exposure to pH 2.0 andalmost absent after exposure to pH 6.0. Moreover, chymo-trypsin stimulated the excystation of fecal cysts by approx-imately twofold. The maximal excystation (-38%) of fecalcysts in this experiment was directly comparable to that ofthe in vitro-derived cysts in Fig. 1, since these experimentswere performed at the same time.

Since trypsin and chymotrypsin have different substratespecificities, we next tested whether other endoproteaseswould also stimulate excystation of in vitro-derived cysts.Four relatively nonspecific proteases, proteinase K, subtili-sin, thermolysin, and elastase, stimulated excystation asefficiently (14.0% ± 3.3% to 19.7% ± 1.6%) as did chymo-trypsin (16.5% ± 1.2%; P > 0.05), but neither the exopro-

teases carboxypeptidase A and leucine aminopeptidase nor

pepsin (incorporated into the low-pH induction step) stimu-lated excystation (data not shown).

Stimulation of excystation of in vitro-derived cysts by pan-creatic fluid. Since trypsin and chymotrypsin are secretedinto the small intestine by the pancreas, we tested the effects

0O2.0 4.0 6.0

pH

FIG. 2. Roles of low pH and chymotrypsin in the excystation offecal cysts. The percent excystation of fecal cysts can be directlycompared with that of in vitro-derived cysts in Fig. 1, since theseexperiments were carried out at the same time. The second step ofexcystation was carried out in the presence of a 1-mg/ml concentra-tion of active chymotrypsin (CT) or chymotrypsin which had beeninactivated by boiling for 20 min and cooled prior to the experiment.Excystation was significantly decreased following exposure to pH 4or 6 (P s 0.001) or to boiled chymotrypsin (P < 0.001).

of pancreatic secretions from a healthy human volunteer onexcystation. Pancreatic fluids collected from the same sub-ject before and after stimulation with secretin (3) greatlyincreased excystation (P s 0.002) (Fig. 3). Stimulated pan-creatic secretions were more effective at lower concentra-tions and consistently yielded higher levels of excystationthan unstimulated pancreatic secretions. Both stimulatedpancreatic fluid and chymotrypsin increased excystationover a >100-fold concentration range. Concentrations ofstimulated pancreatic fluid above 2% or of chymotrypsinabove 1 mg/ml led to decreased excystation (data notshown).To determine whether the stimulation of excystation by

pancreatic fluid was due to chymotrypsin and/or trypsinactivity, we assessed the effect of soybean trypsin inhibitor,an inhibitor of both proteases (Fig. 4). Stimulation of excys-tation by both chymotrypsin and pancreatic fluid was virtu-ally eliminated by soybean trypsin inhibitor (P < 0.01).

Effect of cyst storage on biological activity. Since Binghamet al. (1) showed that fecal cysts were able to excyst after>30 days of storage in distilled water at 8°C, we monitoredthe biologic activity of two preparations of in vitro-derivedcysts from the day they were harvested (day 1 in Fig. 5).Biologic activity was detected for up to 26 days of incubationat 4°C, after which excystation dropped below 0.1%. Al-though the initial percent excystation of the two cyst prep-arations differed by a factor of approximately three, it washighest on the day of harvest and declined with incubation at40C.

Effect of encystation conditions on the biologic activity ofcysts. Using the optimized excystation procedure, we testedthe hypothesis that the conditions of encystation determinecyst biologic activity and the idea that metabolites of colonicbacteria may be important for this process. To do this, weassessed the effects of organic acids produced by the colonicbacterial flora (3) on both the numbers of cysts and theefficiency of excystation. None of the organic acids testedincreased the numbers of total cysts as compared withporcine bile alone (at pH 7.8), although lactic acid led toslightly increased numbers of type I cysts (Fig. 6A) (7). Incontrast, the percent excystation was increased more thanfourfold by the inclusion of 5 mM lactic acid in the encysta-tion medium (P = 0.001) (Fig. 6B).

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EXCYSTATION OF G. LAMBLIA 3519

Unstimulated

tStimulated

*

*

*FT

*

None 2 5 10 25 0.02 0.05 0.5 2.0 1 0 50 250 1000

Pancreatic Fluid, % Chymotrypsin,pg/ml

FIG. 3. Stimulation of the excystation of in vitro-derived cysts by pancreatic fluid. Unstimulated or stimulated pancreatic fluid or

chymotrypsin at the concentrations shown was substituted for the standard 1-mg/ml concentration of chymotrypsin in the second step ofexcystation. *, Significant increase in percent excystation (P - 0.002), compared with the control with no pancreatic fluid or chymotrypsin.

DISCUSSION

Taken together, data from our laboratory (6-9, 24) andother laboratories (e.g., 1, 2, 11, 13, 16, 23) show thatconditions encountered by G. lamblia during each step of itsnatural life cycle are crucial to reproducing that step in vitro.By varying the conditions of encystation (7) and excystation,we have consistently obtained in vitro-derived cysts of G.lamblia with levels of biologic activity comparable to thoseof fecal cysts.Our experiments, based on earlier work with fecal cysts

(2, 16), have permitted us to identify two parameters neces-

sary for the excystation of in vitro-derived cysts. The first,exposure of in vitro-derived cysts to a low pH, which mimicsthe passage of ingested cysts through the stomach, wasshown by Bingham et al. (1, 2) to be crucial for theexcystation of fecal cysts. The second, exposure of invitro-derived cysts to trypsin, as reported by Rice andSchaefer (16) for fecal cysts, or to chymotrypsin, mimics the

20-

10-

0

passage of cysts into the lower duodenum, where they are

bathed in pancreatic secretions containing these proteases(3).At the optimal pH for each, we observed equal excysta-

tion of fecal and in vitro-derived cysts. The optimal pH (2.0)for triggering the excystation of fecal cysts was slightlylower than that for triggering the excystation of in vitro-derived cysts (4.0). A second difference was that excystationof in vitro-derived cysts was totally dependent upon expo-sure to proteases, whereas excystation of fecal cysts was

depressed only by 50 to 60% in the absence of proteases.Differences between the excystation of in vitro-derived

and fecal cysts may simply be related to differences betweenstrains. The WB strain, which typifies the most commongroup of G. lamblia isolates (14), was isolated in 1979 from a

patient infected in Afghanistan, whereas the fecal cysts usedin this study were isolated from a patient infected in Kenya.Alternatively, the physical state of the wall of in vitro-

*

* No Addition* 1 mg/ml ChymotrypsinE3 2% Pancreatic Fluid, stim.

25% Pancreatic Fluid, unstim.

*

*

n~ ~I* *

5

Soybean Trypsin Inhibitor, mg/mlFIG. 4. Inhibition by soybean trypsin inhibitor of stimulation of the excystation of in vitro-derived cysts by pancreatic fluid or

chymotrypsin. Chymotrypsin or pancreatic fluid was preincubated with soybean trypsin inhibitor at the indicated concentrations for 15 minat 37°C prior to being used in the second step of excystation. *, Significant inhibition of excystation by soybean trypsin inhibitor (P < 0.001).**, Significant inhibition (P = 0.006); stim., stimulated; unstim., unstimulated.

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3520 BOUCHER AND GILLIN

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..- -- cyst prep. B

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DayFIG. 5. Effect of storage at 4°C on the excystation of in vitro-derived cysts. Water-resistant cysts were harvested on day 1 and kept in an

ice water bath in double-distilled water with the antibiotics piperacillin and amikacin. At the times indicated, samples were removed andexcysted by the standard procedure. A and B are two different cyst preparations (cyst prep.). Percent excystation was significantly decreasedon and after day 5 (P - 0.002) for preparation A and day 9 (P < 0.002) for preparation B.

derived cysts mof fecal cysts.the latter withiisage. For exanbacterial or hos

8 - A

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FIG. 6. Effectnumbers of wateistandard 5 mM lacand replaced as inacetic acid (AA),(B) Percent excysstandard assay.0.001) following e

lay differ in subtle ways from that of the wall Exposure of cysts to an acidic pH has been a hallmark ofThis difference may be due to incubation of published excystation procedures (1, 2, 11, 17, 18). The pHn the fecal mass both before and after pas- curve for excystation of in vitro-derived cysts was broad.nple, the cyst wall may be acted upon by The percent excystation was maximal at pH 2 to 4 but,t enzymes or metabolites. always declined steadily at pHs above 5. Even so, substan-

tial excystation (25 to 35% of maximal) was observed fol-lowing exposure to pH 7 to 8. This result may have been due

* Type cysts to prior exposure of cysts during harvesting, incubating, and* Type 11 cysts washing in double-distilled water at a pH of -5.7. Such

exposure may trigger low levels of excystation which can besubsequently increased by exposure to a lower pH. Thisprocess may explain the incidence of giardiasis in patientswith reduced gastric acidity.

In the present study, excystation of in vitro-derived cystswas >90% dependent on exposure to proteases in the second

*3-step. The stimulatory activity of human pancreatic fluid was__ _ - _ ~~~~~~~~dueto trypsin and chymotrypsin, since it was virtually

abolished by soybean trypsin inhibitor, an inhibitor of bothmM5mM1mM5M 1 mM5mM 1 M 5mM proteases. This observation shows that other components in

+ LA + SA + BA + AA or collected with the pancreatic secretions, such as bile salts* or other enzymes, are not required, although they may have

a contributory role (11). The requirement for proteases was

* 1 mM not specific, since all endoproteases tested were effective.* 5mM The idea that proteins are important components of the cystwall IS consistent with our earlier observation that cyst

antigens separated by polyacrylamide gel electrophoresisand transferred to nitrocellulose were digested by trypsin

_ ~~~~~~~~~~~~(15).Compared with the walls of fecal cysts, the walls of in

vitro-derived cysts appeared by light microscopy to be moreMTNygtransparent after protease treatment. However, trophozoites

_T L always emerged from a pole, as reported earlier for fecal+ LA + SA + BA +M cysts (2), suggesting the presence of a protease-sensitiveOrganic Acid Addition component or area at the pole of the cyst, possibly produced

or unmasked by prior exposure of the cyst to acid and/ors of organic acids in the encystation medium upon thiol-reducing agents. In contrast, treatment of cysts withr-resistant cysts and subsequent excystation. The purified chitinase did not stimulate excystation (data notCtic acid was omitted from the encystation mediumdicated with succinic acid (SA), butyric acid (BA), shown), suggesting that chitin, which was previously re-or lactic acid (LA). (A) Numbers of cysts (105/ml). ported to be a cyst wall constituent by one group (22) but notstation of each cyst preparation determined by the by another (12), may not be located at the site of trophozoite*, Excystation was significantly increased (P = emergence.ncystation in the presence of 5 mM lactic acid. Excystation is the most stringent criterion of cyst biologic

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EXCYSTATION OF G. LAMBLIA 3521

activity and is extremely sensitive to the conditions ofencystation. For example, although encystation in the pres-ence of lactic acid led to relatively small (-20 to 100%) (Fig.6) (7) increases in type I cysts but not in total cysts, itstimulated excystation strongly (more than fourfold). Theidea that lactic acid may have a role in the terminal stages ofencystation is consistent with the fact that it is a majormetabolite of bacteria in the large intestine. Moreover, theaddition of lactic acid to cultures after 24 h in encystationmedium did not decrease excystation (data not shown).Our earlier study (7) showed that viability, defined by the

fluorogenic dye assay of Schupp and Erlandsen (19), was aless stringent criterion for cyst quality, since viable cystswere not necessarily able to excyst (20). Similarly, fecalcysts retained the ability to exclude eosin (viability) afterthey lost the ability to excyst (1).

In the present study, excystation of different preparationsof in vitro-derived cysts using the same encystation andexcystation procedures varied from -8 to 38%. This value iswell within the broad range (1 to 95%) reported for cystsisolated from human patients (17). Nonetheless, the appar-ent efficiency of excystation of in vitro-derived cysts may below because the calculation is based on total numbers ofwater-resistant cysts, a heterogeneous population of whichonly -10 to 30% has type I morphology. While -90% of typeI cysts are viable (7) and therefore are theoretically capableof excystation, the remaining type II cysts include cysts witha shrunken cytoplasm as well as cysts which appear mor-phologically less mature. Only -33% of type II cysts werefound viable (unpublished data) by the fluorogenic dyemethod (19). We have not been able to determine whethertype I or type II cysts or both excyst, because the excysta-tion procedure obscures cyst morphology. However, on thebasis of morphology and viability, type I cysts may be morecompetent at excystation.The variability in cyst morphology is not an artifact of

encystation in vitro, since both type I and type II cysts areisolated from feces (7, 19). Moreover, the numbers of cystspassed by infected humans fluctuate greatly. We have ob-served cyst numbers from >106/g of stool to barely detect-able in the same untreated patient (unpublished data; 21),suggesting that the formation and/or shedding of cysts invivo is highly sensitive to variations in conditions whichhave not yet been identified. Our in vitro encystation condi-tions yield more consistent numbers of biologically activecysts.Our experiments have shown that a low pH and pancreatic

proteases which are important for the excystation of fecalcysts also induce high-efficiency excystation of in vitro-derived cysts. These observations again underscore thevalue of mimicking human gastrointestinal tract conditionsfor completing the life cycle of G. lamblia in vitro. Theavailability of all stages of the life cycle of G. lamblia willpermit new cellular, molecular, and immunologic studies ofthis important pathogen.

ACKNOWLEDGMENTS

We are grateful to our colleagues at the Environmental ProtectionAgency for suggesting the method of Rice and Schaefer, to D.Reiner for stimulating discussions, to Dan Hogan (Division ofGastroenterology, University of California, San Diego) for thepancreatic fluid, to C. Davis and J. Sauch for critiquing the manu-script, to W. Strum (Scripps Clinic) for patients, and to S. McFarlinfor preparing the manuscript.

This study was supported by U.S. Environmental ProtectionAgency cooperative agreement CR 814537 and by Public Health

Service grants AM 35108, AI 24285, and Al 19863 from the NationalInstitutes of Health.

LITERATURE CITED

1. Bingham, A. K., E. L. Jarroll, E. A. Meyer, and S. Radulescu.1979. Giardia sp.: physical factors of excystation in vitro andexcystation vs eosin exclusion as determinants of viability. Exp.Parasitol. 47:284-291.

2. Bingham, A. K., and E. A. Meyer. 1979. Giardia excystation canbe induced in vitro in acidic solutions. Nature (London) 227:301-302.

3. Davenport, H. W. 1977. Physiology of the digestive tract. YearBook Medical Publishers, Chicago.

4. Diamond, L. S., D. Harlow, and C. C. Cunnick. 1978. A newmedium for the axenic cultivation of Entamoeba histolytica andother Entamoeba. Trans. R. Soc. Trop. Med. Hyg. 72:431-432.

5. Douglas, H., D. S. Reiner, and F. D. Gilln. 1987. A new methodfor purification of G. lamblia cysts. Trans. R. Soc. Trop. Med.Hyg. 81:315-316.

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