Excystation and Culturing of Human and Animal Giardia spp. byUsing Gerbils and TYI-S-33 Medium
PETER M. WALLIS'* AND HUGH M. WALLIS2
Kananaskis Centre fkr Environmental Researchlc, University of Calgary, CailgaryNI, Albertta, Candla T2N IN4,1 anidDowns viewv Rehabilitation Centre, Downsview, Onta.ljo, Caincada M3L IG82
Received 25 March 1985/Accepted 18 November 1985
Mongolian gerbils were used as an animal model to excyst and host Giardia spp. isolated from meadow voles,dogs, beavers, and humans. Both cysts and trophozoites were used to establish infections. Gerbils were infectedwith Giardia duodenalis from beaver, dog, and human sources, and the trophozoites were extracted andcultured in Diamond TYI-S-33 medium. The use of gentamicin and ampicillin in the medium, coupled withtreatment of gerbils with gentamicin before they were sacrificed, permitted the elimination of trophozoitepurification techniques before culturing. An extract of whole bovine calf blood, CLEX, was substituted for fetalbovine serum in TYI-S-33 medium and was found to be both adequate and less expensive.
Formerly, the study of various species of the parasitegenus Giaidia was limited owing to difficulty in obtainingcultured stocks for study. This situation has changed over
the past 20 years, and a number of methods for the excysta-tion and culturing of Giardia spp. have been described. Inthis paper we describe a refinement of some of thesemethods for use in the isolation and propagation of Giari-diatrophozoites for experimental purposes.
Giardia mniis has been extensively studied by using themouse model, but an equivalent model for the study ofGiar-dia diuodenalis has not yet been generally accepted. Forthis reason, we tested gerbils (Men-ionies mmnguicildatis) as an
animal model for the excystment of Giardia from humans,wild meadow voles (Microtus pennsylv'anicils), domesticdogs (Canis familiaris), and beavers (Castor- catndenisis) byusing the methods of Belosevic et al. (4). We were able toculture trophozoites obtained from gerbils infected withhuman, dog, and beaver Giairdia isolates, but not voleGiairdia isolates, by using Diamond TYI-S-33 medium. TheGiardia isolates extracted from voles were assumed to be G.
maniis, and the Giardia isolates extracted from humans,dogs, and beavers were assumed to be G. diuodenalis, usingthe terminology of Filice (9).
MATERIALS AND METHODS
Sources of Giardia spp. Cysts were obtained from beaver,dog, and human feces. The beaver feces were obtained froma small pond 3 km north of the Kananaskis Field Station insouthwestern Alberta, Canada. Infested dog feces were
obtained from the Calgary Animal Shelter, Calgary, Alberta,and human stools containing Giairdia cysts were obtainedfrom the Alberta Provincial Health Laboratory and FoothillsGeneral Hospital in Calgary, Alberta, or from the Universityof Alberta Hospital, Edmonton. All fecal specimens obtainedin Calgary were shipped directly to our laboratory over ice
within 6 h of excretion and were processed immediately. G.manis trophozoites were obtained by stripping the entire smallintestines of naturally infected meadow voles that were
trapped live in the vicinity of the Kananaskis Field Station.Cyst concentration. Beaver feces consisted mostly of large
particulate matter that was easily separated from the finersediment and cysts by straining and rinsing with phosphate-
* Corresponding author.
647
buffered saline through four layers of cheesecloth. Thecentrifuge-concentrated pellet was usually pure enough to beused without further treatment. If the concentrate requiredclarification, the sucrose centrifugation method was used, as
described below.Good cyst recovery rates (about 17% of the cysts were
recovered during seeding trials) were obtained by using thesucrose centrifugation method of Roberts-Thomson et al.(15). In order to obtain approximately 100,000 cysts, 25 g(wet weight) of feces was placed in 50 ml of phosphate-buffered saline and thoroughly mixed with a spatula. Thefecal suspension was then strained through four layers ofcheesecloth, and about 5 ml of the mixture was carefullylayered over 5 ml of 1.0 M sucrose in each of 12 centrifugetubes (capacity, 15 ml). The tubes were centrifuged at 500 x
g for 5 min, and the clarified upper layer was removed witha pipette. The samples were then pooled and centrifugeconcentrated to a volume of 2 ml. In the case of humanfeces, fat was removed by mixing 8 ml of a fecal suspensionwith 1 ml of diethyl ether and centrifuging the preparation at500 x g for 5 min. The emulsified fat and liquid were pouredoff, and the pellet was suspended in phosphate-bufferedsaline for further clarification over sucrose.
Gerbil infection. Mongolian gerbils that were 5 weeks oldwere obtained from Tumblebrook Farm, West Brookfield,Mass., transferred to separate cages, and cleared of intesti-nal protozoa with metronidazole as described by Belosevicet al. (4). Before the animals were infected, stool specimenswere collected from each gerbil on 3 consecutive days andexamined for Giar-dia cysts. After at least 1 week of rest, thegerbils were inoculated by gastric intubation with cystsconcentrated from feces or with trophozoites obtained fromintestinal scrapings. After 72 h, stool samples were collectedon a daily basis for examination. This practice was eventu-ally discontinued because of poor correlation between thepresence of cysts in the feces and the presence oftrophozoites in the gut. In all, 9 gerbils were infected withtrophozoites from meadow voles, 13 gerbils were infectedwith human Giardia cysts, 12 gerbils were infected withcysts from dogs, and 11 gerbils were infected with cysts frombeavers. Gerbils were sacrificed 4 to 20 days after infection.
In some cases, infected gerbils were given two 25-mgdoses of gentamicin (GIBCO Laboratories, Grand Island,N.Y.) by gastric intubation 48 and 24 h before they were
sacrificed in order to reduce the number of intestinal bacte-ria. The animals were anesthetized with diethyl ether andkilled by cervical dislocation, and the peritoneal cavity was
opened. A small portion of the upper duodenum 2 cm fromthe pylorus of the stomach was removed, mounted in phos-phate-buffered saline on a slide, and examined at x400 byusing dark phase-contrast microscopy. If no trophozoiteswere detected, a second sample was taken approximately 10cm from the pylorus. If trophozoites were detected in eithersample, the small intestine was removed and placed in RPMI1640 medium. If trophozoites were not detected in eithersample, the gerbil was judged to be uninfected.
Concentration of trophozoites. The entire small intestinewas opened longitudinally by using scissors, and the gutcontents were scraped with a scalpel into a disposable petridish containing RPMI 1640 medium. The entire contents ofthe petri dish were rinsed into six 15-ml centrifuge tubesafter passage through a quadruple layer of cheesecloth. Thepetri dish was examined by using a Zeiss inverted micro-scope to make sure that most of the trophozoites had beenrinsed off. If large numbers of trophozoites were still adher-ing to the bottom of the dish, the dish was placed in a
refrigerator at 4°C for 5 min to detach the trophozoites andrinsed again. All of the trophozoites were then concentratedby centrifugation at 500 x g for 5 min. Attempts were madeto separate the trophozoites from intestinal bacteria by usingthe nylon fiber column method of Andrews et al. (1) or byallowing the trophozoites to adhere to clean petri dishes,using the method of Feely and Erlandsen (8).
Culturing. Concentrated trophozoites were inoculatedinto Diamond TYI-S-33 medium (5). Glass tubes (15 ml)were used routinely, although 50-ml tissue culture flaskswere useful for growing large numbers of trophozoites.Culture tubes were subcultured every 3 to 4 days. Themedium was prepared as described by Diamond et al. (5).The vitamin solution of Diamond et al. was initially pur-
chased from North American Biologicals, Miami, Fla., butwas later made in our laboratory by using the formulationdescribed by Diamond et al. (5) and Evans et al. (6). Bothfetal bovine serum and CLEX (Dextran Products Ltd.,Scarborough, Ontario, Canada) were used as medium sup-
plements at concentrations of 10%. TYI-S-33 medium rap-
idly inactivated antibiotics, so the medium was filter steril-ized and stored for no more than 7 days at 4°C. Antibioticswere added to each culture tube when the medium was
changed (see below).Cultures were stored by freezing. Mature culture tubes
were placed in the refrigerator for 30 min, centrifuged at 500x g for S min, and decanted. The resulting pellet was mixedin 1 ml of fresh TYI-S-33 medium in a fresh disposableculture tube and diluted with 1 ml of 10% dimethyl sulfoxide(final concentration, 5%). The culture tube was frozen at-25°C for 60 min and then transferred to a Revco Ultra Lowfreezer and held at -60°C. Cultures were recovered bythawing them quickly in a 37°C water bath and filling thetubes with fresh medium. When the majority of trophozoiteshad become attached to the sides of a culture tube (about 60min), the medium was changed in order to avoid toxicityproblems from the dimethyl sulfoxide (14). All strains ofGitir-dita isolated during this study have been maintained in
the Kananaskis Centre for Environmental Reseairch labora-tory and are available for study.
Control of bacterial overgrowth. Initially, we used strepto-
mycin (50 jig/mI) and penicillin G (50 jg/ml) to suppress
bacterial overgrowth in the medium. These antibiotics were
not very effective, even at concentrations of 200 jig/ml. In
order to find more efficient antibiotics, complete TYI-S-33medium containing serum was solidified with 1.5% agar, andantibiotic susceptibility disks (BBL Microbiology Systems,Cockeysville, Md.) were added. The resulting plates wereinoculated with 0.1 ml of medium that had been overgrownwith bacteria the night before. The following antibiotics weretested: penicillin G (2 U), kanamycin (30 jig), streptomycin(2 pg), trimethoprim (23.75 [Lg)-sulfamethoxazole (1.25 [ig),erythromycin (15 jig), gentamicin (10 [Lg), ampicillin (10 Vig),tetracycline (5 VLg), and nalidixic acid (30 [Lg). The results ofthis test showed that overgrown bacteria were susceptible tonalidixic acid, gentamicin, and ampicillin. Nalidixic acidinhibited the growth of human Giardia trophozoites so it wasnot tested further. A similar trial with gentamicin andampicillin showed that the growth of human Giao1dio isolateswas not inhibited by these antibiotics at concentrations up to200 jLg/ml. In order to determine the best concentrationrange for routine culturing, we prepared replicate tubes ofTYI-S-33 medium containing concentrations of gentamicinand ampicillin ranging from 50 to 200 jLg/ml each in 25-[tg/mlincrements. In addition, one set of tubes contained 400 jig ofgentamicin per ml and 400 jig of ampicillin per ml. Threereplicate tubes were prepared for each concentration. Eachtube was inoculated with approximately 106 human Giardiatrophozoites and was incubated for 4 days without changingthe medium.
RESULTS
Gerbil infection. Gerbils were successfully infected withGitii-clia isolates from meadow voles, humans, dogs, andbeavers. Both cysts and trophozoites were capable of infect-ing gerbils.The results of the infection experiments with meadow vole
Gitar-dia isolates are shown in Table 1. All of the gerbils, withthe possible exception of gerbil 6, became infected, asdemonstrated by either gut or fecal examination. Negativeresults in gut analyses seemed to be correlated with longerincubation times. The overall infection rate was at least 89%when both fecal and gut analyses were considered.
Table 2 shows the results of infection of gerbils withhuman Gitirclia isolates. The overall rate of infection withhuman parasites (46%) was lower than the rate with voleparasites, and there was only one case in which both fecaland gut specimens were positive. Trophozoites were foundin the small intestines of animals after incubation timesranging from 4 to 18 days.
TABLE 1. Infection of gerbils with Giardia isolates frommeadow voles
Incubation Results of:Animal InoCLILnlti time Fcal Gutrno.Fea Gu
a The fecal samples used to infect gerbils 1 through 5 were from 7 to 10 daysold, and gerbils 6 through 10 were contaminated with Trichomoonas sp.
I An analysis was considered positive if cysts were found in at least onespecimen.
' ND, No data.
The results of infections with dog Giardia isolates areshown in Table 3. In this case, no fecal specimens werepositive, although the overall infection rate was 50%. Ahigher incidence of trophozoites in the small intestinesappeared to be correlated with shorter incubation times.
Table 4 shows the results from the beaver Giardia infec-tion experiments. As with the dog parasites, no positive fecalsamples were found, although the overall infection rate was91%. No trend was apparent between positive gut specimensand incubation times.A number of gerbils were found to be contaminated with
Trichomonas sp. or Hexamita spp. (from voles) or both,which may have influenced the results of the experimentalinfections.
Culturing. Despite a few unsuccessful attempts, tropho-zoites from gerbils infected with dog, human, and beaverGiardia isolates were cultured in TYI-S-33 medium. VoleGiardia isolates could not be cultured. Trophozoites grew aswell in medium prepared with CLEX (which is about one-half as expensive as fetal bovine serum) as in mediumprepared with fetal bovine serum. Similarly, we found nodifference between the vitamin supplement purchased from
TABLE 3. Infection of gerbils with Giacrdia isolates from dogs
AnimalnoculumIncubation
Results of:Animal Inoculum ncubtimeno. (no.ofcyst ime Fecal Gut
(days) analysis examination
1 18,000 13 - -
2 18,000 13 - -
3 250,000 16 - -
4 250,000 16 - -
5 50,000 6 - +
6 40,000 7 - +
7 40,000 7 - +
8 100,000 8 ND" +9 24,000 9 ND +
10 25,000 10 ND -
11 50,000 18 ND -
12 50,000 7 ND +
" ND, No data.
TABLE 4. Infection of gerbils with Giairdia isolates from beavers
AnimalIncubation Results of:no. Inoculum time Fecal Gut(days) analysis examination
North American Biologicals and that prepared in our labo-ratory.Attempts to purify inocula of trophozoites scraped from
small intestines by using the nylon fiber method (1) or theattachment method (8) met with only limited success. Ingeneral, we found that separation of bacteria and tro-phozoites could be obtained only at the cost of losing most ofthe parasites. The resulting inoculum still contained enoughbacteria to contaminate cultures overnight in the absence ofantibiotics. Gerbils that had been treated with gentamicinbefore being sacrificed were found to be almost completelyfree of bacteria, and no deleterious effects on trophozoiterecovery were observed.We found that a combination of gentamicin and ampicillin
in equal quantities was very effective in suppressing bacte-ria. When human Giardia isolates were cultured in TYI-S-33medium containing a range of concentrations of these twoantibiotics, we found that complete inhibition of bacteriaoccurred at concentrations of 175 ,ug of each antibiotic perml with no inhibition of the growth of trophozoites. After 4days, all of the tubes were overgrown with either Giardiatrophozoites or bacteria. A combination of 200 ,ug of genta-micin per ml and 200 ,ug of ampicillin per ml was usedthereafter for routine culturing.Attempts to culture trophozoites from intestinal scrapings
were not always successful initially. Although up to 500,000trophozoites were inoculated into each tube, most of thesedied within 72 h. If trophozoites were still alive after thistime, they began to replicate and slowly build up theirnumbers. In a typical successful run, a tube was not ready tobe subcultured for about 2 weeks. Sometimes the initialsubculture was unsuccessful, but if cultures survived to thispoint, usually very little trouble was encountered thereafter.Repeated attempts to culture Giardia spp. from meadow
voles were unsuccessful. The trophozoites did not stick wellto the culture tubes and did not survive for more than 72 h.Boosting the concentrations of cysteine and ascorbic acidand adding glutathione had no effect. Cultures of dog,human, and beaver Giacrdia trophozoites frozen in dimethylsulfoxide have been successfully recovered after up to 6months of freezing.
DISCUSSIONThe use of gerbils to excyst Giarcdia isolates obtained from
different animal sources was generally effective, althoughproblems were encountered. The first of these was the age ofthe cysts that were obtained for infection. The experimentswere begun with human cysts obtained from the Provincial
Laboratory of Public Health in Calgary. Samples submittedfor parasite analysis are invariably preserved, and it ispossible to obtain nonpreserved material only if a sample forbacteriological testing is also submitted. By the time apositive diagnosis was made and the corresponding bacteri-ology sample was located, up to 7 days had passed before wereceived the sample. It is probable that this was the reasonfor the low success rate of infection in the first five attemptsshown in Table 2. If these data had not been included, thesuccess rate would have been much higher. Another prob-lem may have resulted from the use of diethyl ether in theextraction of cysts. Young et al. (19) recommended substi-tution of ethyl acetate for diethyl ether, although theyconcluded that neither solvent caused any visible distortionor morphological change in cysts. In order to test for cystinactivation by diethyl ether, five additional gerbils wereinfected with approximately 50,000 Giardia cysts that hadbeen extracted from dog feces by using diethyl ether, andfive gerbils were infected with the same number of cystsextracted without diethyl ether from the same fecal sample.In both cases four of the five gerbils were found to beinfected after 7 days. It is possible that the use of diethylether was detrimental to cyst viability, but enough viablecysts survived to cause infection. However, we noticed thatin the case of dog feces the yield of cysts was lower whendiethyl ether was used.
Similar problems were experienced with dog and beaverfeces. The dog feces were less than 6 h old when they werecollected, but they had been deposited in an open, shadedrunway and were subject to some desiccation. Even lesscontrol was possible with beaver scats because sampleswere taken from the bottom of a local pond and it wasdifficult to ensure that the samples were fresh. The highinfection rate (91%) of these cysts was probably the result ofthe beneficial effects of storage in cold (6°C), alkaline water.The infection of gerbils with trophozoites suggests that
this parasite stage is less fragile than previously thought.Hewlett et al. (11) were able to infect dogs with culturedhuman Giardia trophozoites. Belosevic et al. (4) reportedsuccessful infection with cultured human trophozoites (Port-land 1 strain) in gerbils, and Schleinitz et al. (16) obtainedsimilar results with cultured trophozoites that were origi-nally obtained from rabbit and human small intestine con-tents.As discussed by Faubert et al. (7), the output of G.
duodenalis type cysts from gerbils is irregular. For thisreason, a very poor correlation was found between cystoutput in the feces and the ultimate discovery of tropho-zoites in the guts of infected gerbils. Therefore, the use ofstool examination to demonstrate infection in animals infec-ted with G. duodenalis may result in false-negative findings.We concluded that the gerbil model is efficient for
excysting G. duodenalis from dogs, beavers, and humans ifthe cysts are viable. These results confirm the results ofFaubert et al. (7), who infected gerbils with cysts fromhumans, beavers, and mice. In addition, Belosevic et al. (3)reported that they infected gerbils with cysts from cats (Felisdomesticus).
During this study we encountered problems with contam-ination by Trichomonas spp. in gerbils. When Trichomonaswas found in gerbils, its presence usually meant that Giardiacysts and trophozoites were either reduced or absent aftermore than 1 week of incubation. If cysts were detected in thefeces but not in the gut, it may have been because Giardiafailed to establish itself in the presence of Trichomonas spp.This may also be the reason why more positive gut samples
were found after shorter incubation times. The source ofTrichomonas was eventually tracked down to the AnimalSciences Vivarium at the University of Calgary, where thegerbils were held for from a few hours to 1 day before beingshipped on to the Kananaskis Centre for EnvironmentalResearch laboratory. The problem was solved by sacrificingall of the animals in the laboratory, sterilizing the animalroom, and arranging for all shipments to come directly to ourlaboratory.When gerbils were infected with intestinal contents re-
moved from meadow voles, Hexamita spp. were also oftenpresent, but these organisms did not appear to influence thepopulation of Giardia, suggesting that the two parasites cancoexist. We noted that meadow voles were often naturallyinfected with Giardia, Hexamita, and Trichomonas isolateswithout any deleterious effects on either the parasites (com-mensals?) or the hosts. It is possible that these organismshave evolved to coexist with G. muris but outcompete G.duodenalis in unnatural hosts.
In this study we demonstrated that G. duodenalis isolatesfrom several hosts can be cultured in Diamond TYI-S-33medium. Giardia isolates from beavers and humans havebeen cultured previously (2, 7, 13, 17), but we have found noprevious reference to the culturing of dog Giardia isolates inthe literature. Once established, these strains were amenableto culturing even when CLEX was substituted for fetalbovine serum. The trophozoites grow rapidly and can with-stand being held at room temperature for several days andeven refrigeration (4°C) for up to 48 h.A major problem encountered in culturing Giardia
trophozoites was overgrowth by bacteria. Early in the studywe found that TYI-S-33 medium can inactivate antibioticswithin 24 to 48 h. A great many overgrowth problems wereavoided by adding the antibiotics when fresh medium wasadded to the cultures and by occasionally supplementing thetubes. Attempts to remove bacteria from culture inocula byusing nylon fiber columns or adherence methods were onlypartially successful and resulted in a large loss oftrophozoites. Much better results were obtained when thegerbils were treated with gentamicin and cleared of intestinalflora before being sacrificed. This method, combined withthe use of gentamicin and ampicillin in the medium, allowedthe use of concentrated gut scrapings as culture inocula, thusavoiding the use of any further purification technique. Aconsiderable saving in medium also resulted as the mediumneeded to be changed only twice per day at most for freshinocula. After 2 to 3 days, the medium was changed onlywhen the trophozoites were subcultured.We have not yet succeeded in producing axenic cultures.
All of our cultures are contaminated with Pseudomonascepacia, and one (human strain WB) also containsAlcaligenes faecalis; both of these contaminants are resist-ant to gentamicin and ampicillin. These organisms are motileso we have not been able to separate them fromtrophozoites. Additional antibiotic susceptibility tests havebeen carried out by using Mueller-Hinton medium, but so farwe have discovered only that these bacteria are resistant tothe following antibiotic disks supplied by Difco Laborato-ries, Detroit, Mich.: colistin (10 ,ug), carbenicillin (100 ,ug),amikacin (30 ,ug), cefoxitin (30 ,ug), and tobramycin (10 ,g).In the meantime, we have been able to control bacterialovergrowth in our cultures with gentamicin and ampicillinbut have not yet succeeded in eliminating the bacteria.The culturing methods described in this paper are similar
to those used by Gordts et al. (10; B. Gordts et al., Abstr.Annu. Meet. Am. Soc. Microbiol. 1985, C13, p. 302), who
cultured Giardia lamblia isolated from 10 human patients inmodified TPS-1 medium. These authors obtained theirinocula from duodenal aspirates and used streptomycin (100,ug/ml), penicillin (100 IU/ml), vancomycin (20 ,ug/ml), andclindamycin (20 ,uglml) to control bacteria. All of thesestrains were subcultured in media that contained only strep-tomycin and penicillin. The use of these antibiotics avoidedthe need for purification of trophozoites.The survival rate of frozen trophozoites after recovery
was low, but enough viable trophozoites were present torecolonize tubes within 72 h, as found by Meyer and Chadd(14). The final concentration of dimethyl sulfoxide which weused (5%) was close to the concentration recommended(6.5%) by Lyman and Marchin (12), but we did not store ourcultures in liquid nitrogen as recommended by these authorsor by Warhurst and Wright (18). The cooling rate which weused was close to -1°C/min, as recommended by Lymanand Marchin (12) and Warhurst and Wright (18). If we hadused the Dewar flask and ethanol cooling method describedby Lyman and Marchin (12) and liquid nitrogen storage, ourrecovery method could have been improved.
ACKNOWLEDGMENTS
This work was supported by a contract from Alberta Environmentand by grants from the Alberta Environmental Research Trust andthe Natural Sciences and Engineering Research Council of Canada.We thank A. Jones, C. Anand, and B. Stewart for providing stool
specimens.
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