cross-species transmission ofgiardia spp.: inoculation ... · (bristol laboratories, syracuse,...

Vol. 54, No. 11APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1988, p. 2777-27850099-2240/88/112777-09$02.00/0Copyright © 1988, American Society for Microbiology

Cross-Species Transmission of Giardia spp.: Inoculation of Beaversand Muskrats with Cysts of Human, Beaver, Mouse,

and Muskrat OriginSTANLEY L. ERLANDSEN,1* LEE ANN SHERLOCK,' MARY JANUSCHKA,1 DANIEL G. SCHUPP,1

FRANK W. SCHAEFER III,2 WALTER JAKUBOWSKI,2 AND WILLIAM J. BEMRICK3Department of Cell Biology and Neuroanatomyl and Department of Veterinary Pathobiology, University of Minnesota,

Minneapolis, Minnesota 55455, and Toxicology and Microbiology Division, Health Effects Research Laboratory,U.S. Environmental Protection Agency, Cincinnati, Ohio 452682

Received 7 July 1988/Accepted 29 August 1988

Giardia cysts isolated from humans, beavers, mice, and muskrats were tested in cross-species transmissionexperiments for their ability to infect either beavers or muskrats. Giardia cysts, derived from multiplesymptomatic human donors and used for inoculation of beavers or muskrats, were shown to be viable byincorporation of fluorogenic dyes, excystation, and their ability to produce infections in the Mongolian gerbilmodel. Inoculation of beavers with 5 x 105 Giardia lamblia cysts resulted in the infection of 75% of the animals(n = 8), as judged by the presence of fecal cysts or intestinal trophozoites at necropsy. The mean prepatentperiod was 13.1 days. An infective dose experiment, using 5 x 10' to 5 x 105 viable G. lamblia cysts collectedby fluorescence-activated cell sorting, demonstrated that doses of between, less than 50, and less than 500 viablecysts were required to produce infection in beavers. Scanning electron microscopy of beaver small intestinerevealed that attachment of G. lamblia trophozoites produced lesions in the microvillous border. Inoculation ofmuskrats with G. lamblia cysts produced infections when the dose of cysts was equal to or greater than 1.25 x105. The inoculation of beavers with Giardia ondatrae or Giardia muris cysts did not produce any infection;however, the administration to muskrats of Giardia cysts of beaver origin resulted in the infection of 62% ofthe animals (n = 8), with a prepatent period of 5 days. Our results clearly demonstrated that beavers andmuskrats could be infected with Giardia cysts derived from humans, but only by using large numbers of cysts.Muskrats could be infected with Giardia cysts derived from beavers, but the latter cannot be infected with thebinary cysts characteristic of G. ondatrae. Because of their association with waterborne outbreaks of giardiasis,the beaver and muskrat must be considered as possible intermediate reservoirs for Giardia spp. capable ofinfecting humans. However, because of the evidence for their harboring different Giardia species than has beendescribed for humans, plus the potential contributions from other unevaluated sources, such as birds and even

humans, it is not possible at this time to assign a major role in waterborne transmission to these animals.

The intestinal disease, giardiasis, caused by the protozoanGiardia lamblia (syn. Giardia duodenalis and Giardia in-testinalis) has been reported as a major cause of diarrhea (46,47) and may be transmitted between hosts by several dif-ferent methods. Common modes of transmission have in-cluded fecal-oral contact due to poor sanitary habits (partic-ularly among children, institutionalized patients, orhomosexuals) and the ingestion of contaminated food ordrinking water (2, 26, 29, 39, 49). From 1965 to 1985, 95outbreaks of waterborne giardiasis were reported in theUnited States, with the majority of them attributed tocontaminated community water systems (8). On the basis oflimited morphological information (R. Davies, Wildl. Dis.Assoc. Newsl., p. 3, 1978) and a pilot experiment using threebeavers, this species of aquatic mammal was implicated as amajor source of Giardia cysts in surface water used forhuman consumption (9, 22, 25, 45). Other aquatic mammals,including the water vole and the muskrat, also were sug-gested as reservoir hosts for Giardia cysts capable of infect-ing humans (28, 31), but no experiments using these animalsin cross-species transmission of Giardia spp. have beenreported.To determine whether beavers or muskrats have any

* Corresponding author.

involvement in cross-species transmission of human giardi-asis, we investigated the responses of these animals whenexposed under controlled laboratory conditions to Giardiacysts derived from symptomatic humans. Colonies of bea-vers and muskrats, free of endogenous Giardia infections,were established and inoculated with G. lamblia cysts ob-tained from multiple donors. The presence of Giardia cystsin the feces was monitored on a daily basis and correlatedwith the occurrence of trophozoites within the intestine ofeach animal at necropsy.

MATERIALS AND METHODSAnimals. Permits were obtained from the Minnesota De-

partment of Natural Resources to trap, transport, and main-tain colonies of both beavers and muskrats. Beavers rangingin weight from 9 to 44 lb (1 lb = 453.592 g) were livetrappedin the metropolitan area of Minneapolis and St. Paul by usingHancock live traps (Hancock Trap Co., Buffalo Gap, S.Dak.). The animals were transported to our animal facilitiesand housed individually in modified dog cages. Metal con-tainers were used for both food and water in each cage. Thebeavers were fed a high-protein diet (Purina high proteinchow 5045), occasionally supplemented with fresh vegeta-bles and apples. Muskrats (1 to 2 lb) were livetrapped andmaintained in rabbit cages containing metal water baths.They were fed standard rodent laboratory pellets (Purina

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2778 ERLANDSEN ET AL.

rodent chow), also supplemented with fresh vegetables andapples. Two-week-old litters of neonatal mice were pur-chased from Harland Sprague-Dawley (Madison, Wis.). Allanimals were given food and water, ad libitum, and main-tained in alternating 12-h cycles of light and darkness.Beavers and their cages were washed daily with water. Thedrainage of each cage was controlled through use of metalsplash guards to prevent contamination of adjacent cages bycysts suspended in fecal material. The distribution of ani-mals, cyst inoculated versus controls, was random withinthe cage facilities; thus, the control animals served assentinels for possible transmission between cages.Animal anesthesia and surgery. Beavers or muskrats were

immobilized with 10 to 13 mg of ketamine hydrochloride(Bristol Laboratories, Syracuse, N.Y.) per kg in combina-tion with 2.5 mg of acepromazine (Aveco Co., Inc., FortDodge, Iowa), as recommended by Lancia et al. (24) byusing a pole-syringe (Ideal Instruments, Chicago, Ill.), foradministration. These drugs facilitated animal handling,stomach intubation for administering either giardiacidaldrugs or Giardia cysts, and preparation for surgical proce-dures. Rompun (Xylazine; Miles Laboratories, Inc., Shaw-nee, Kans.), at a dose of approximately 0.5 to 1.0 mg/kg, wassubstituted for acepromazine and proved to be more effec-tive for surgical preparation. Laparotomies were performedon immobilized beavers and muskrats by using Fluothane(halothane USP, Ayerst Laboratories, Div. American HomeProducts Corp., New York, N.Y.) inhalation for the induc-tion of surgical anesthesia. The tails of the anesthetizedbeavers were cooled with cold water as necessary, since thisappendage has been recognized as important for thermalregulation (7). By aseptic surgical techniques, segments ofupperjejunum, 0.5 to 1.0 m in length, were exteriorized on asterile field. An intestinal loop was created by clamping withfingers, and 20 to 30 ml of sterile saline was inoculated intothe intestinal lumen. The loop of intestine was gently mas-saged, and the saline rinse was aspirated by syringe forsubsequent microscopic examination. After removal of theintestinal rinse, the segment of intestine was returned to theabdominal cavity. Simple interrupted sutures, either Softgut(2-0; American Cyanamid Co., Davis & Geck Medical De-vice Div., Wayne, N.J.) or coated Vicryl (0; Ethicon, Inc.,Somerville, N.J.), were used to close the abdominal muscu-lature opening, while interrupted mattress sutures of surgi-cal steel monofilament (3-0; Ethicon) were used for closureof the skin incision. The closed skin incision was coated withan antibiotic ointment (nitrofurazone dressing; Vedco), andeach animal was given an injection of 1 ml of penicillin-streptomycin (10,000 U/ml and 10,000 p.g/ml, respectively;GIBCO Laboratories, Grand Island, N.Y.). Upon comple-tion of surgical procedures (or intubation), the animals werereturned to their respective cages and monitored for recov-ery from anesthesia. For 2 to 3 days following surgery, theincisions were inspected for signs of bacterial infection andthe antibiotic ointment was reapplied.Drug treatment for endogenous Giardia infection. The pro-

tocol of Smith et al. (41) was modified for use in thetreatment of either beavers or muskrats for endogenousGiardia infections. Lightly anesthetized beavers were immo-bilized within a conical-shaped confinement bag made ofribstock nylon, and their stomachs were intubated via poly-ethylene (p.e.) tubing (beavers, p.e. 160; muskrats, p.e. 50)during drug administration. For three successive days, asuspension of 500 mg of metronidazole (Flagyl; G. D. Searle& Co., Skokie, Ill.) and 200 mg of quinacrine hydrochloride(Atabrine; Sterling Drug, Inc., New York, N.Y.) was admin-

istered to each animal in 5 to 10 ml of distilled water,followed by an additional 5 ml of water to purge the stomachtube of any residual drug content. The drug treatment formuskrats was administered daily for two consecutive daysand consisted of 125 mg of metronidazole and 50 mg ofquinacrine hydrochloride suspended in 2 to 3 ml of distilledwater followed by a rinse of similar volume. By the lightmicroscopic procedure described below, no Giardia cystswere detected in daily examinations of fecal samples fromeither beavers or muskrats following this cycle of drugtreatment nor were there any trophozoites detected in intes-tinal contents obtained surgically.Endogenous Giardia infection in beavers. Livetrapped bea-

vers used in infectivity experiments were monitored daily forthe presence of Giardia cysts in the feces for a period of 30days. The prevalance of endogenous Giardia infection in thebeavers used in this study was 4.9% (n = 41). Because somebeavers had endogenous Giardia infections, all beavers weretreated orally by using the giardiacidal regimen describedabove. To determine the efficacy of treatment, each of thebeavers in the first experiment (n = 18) was subjected to anintestinal laparotomy for the purpose of collecting a salineirrigation sample from the upper small intestine to confirmthe presence or absence of Giardia trophozoites. By usingthe light microscopic procedure described below, none ofthese beavers were found to contain any trophozoites.Control studies in experiments had shown that this methodwas capable of detecting as few as 2.5 x 103 trophozoiteswhich had been inoculated into a 0.5- to 1.0-m segment ofsmall bowel (unpublished data). In the second experiment,only one-half of the animals (n = 11) were subjected tolaparotomies and intestinal irrigation. None of the beaverswere found to harbor Giardia infection, as measured by thepresence of trophozoites within the intestinal rinses. Follow-ing surgical intervention, the beavers were allowed 10 daysto recover before inoculation with any exogenous Giardiacysts. During the 4-week period between chemotherapytreatment for endogenous Giardia infection and inoculationwith exogenous Giardia cysts, daily fecal examinationsrevealed that all of the beavers were negative for cysts.Endogenous Giardia infection in muskrats. Wild muskrats

trapped for use in infectivity experiments were found to be100% infected with Giardia ondatrae. All of these animalswere treated chemotherapeutically for Giardia for two suc-cessive days, rather than for three as described above forbeavers. This treatment resulted in the elimination of theGiardia infections in most animals; however, occasionally asecond course of treatment was necessary. Muskrats used ininfectivity experiments were monitored for 14 to 30 daysprior to the administration of G. lamblia cysts to ensure thatno infection with G. ondatrae existed.

Giardia cysts. Human fecal samples containing cysts of G.lamblia were obtained and the cysts were isolated by flota-tion on 1 M sucrose by a modification of the method ofSheffield and Bjorvatn (40). After isolation, the cysts wererinsed free of sucrose and stored at 4°C in distilled water asa stock cyst suspension, containing penicillin (10,000 U/ml)-streptomycin (10,000 ,ug/ml), for 1 week or less prior touse. Cysts from humans were used for animal inoculationwithin 21 days of stool sample collection (see Table 1).Giardia muris cysts were obtained from the feces of infectedneonatal CF-1 mice by the method of Roberts-Thomson etal. (34). Giardia cysts were obtained from infected beaversand muskrats and isolated from the feces by flotation on asucrose gradient (40). The viability of Giardia cysts wasdetermined by using the uptake of the fluorogenic dyes,

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CROSS-SPECIES TRANSMISSION OF GIARDIA SPP. 2779

fluorescein diacetate (FDA) and propidium iodide (PI), as

described by Schupp and Erlandsen (38), or the excystationmethod of Bingham and Meyer (6) as modified by Rice andSchaefer (33). Inocula containing greater than 100,000 Giar-dia cysts were prepared by dilution and counted with a

hemacytometer. A fluorescence-activated cell sorter (FACSIV; Becton Dickinson and Co., Paramus, N.J.) was used toselect viable, PI-negative Giardia cysts for cross-speciestransmission experiments, in which doses of 5,000 cysts or

less per animal were used. Sorting by FACS was accom-

plished by using laser excitation at 488 nm with a 530-nmband-pass filter for fluorescein excitation and a 630-nmband-pass filter for PI. The sorted cysts were collecteddirectly into the syringes used for oral inoculation. At thetime of inoculation, all syringes were flushed postinoculationwith 3 to 10 ml of water to ensure complete delivery of thecyst-containing suspension.

Light microscopy. Light microscopic detection of Giardiacysts in fecal samples was accomplished by using a zincsulfate flotation method. Approximately 2 g of feces was

suspended in a zinc sulfate solution (specific gravity, 1.180)and centrifuged at 600 x g for 10 min. Material from theair-zinc sulfate interface was transferred with a wire loop toa slide and covered with a cover slip, and the entire 18-mm2cover slip area was examined for the presence of Giardiacysts. A modification of the criteria of Schaefer and Rice (37)was used for cyst identification. In addition to correct sizeand shape and the following two morphological features, twoto four nuclei and presence of axonemes, we also observedpaired stained inclusions, representing either median bodiesor portions of the adhesive disc. The light microscopicdetection of trophozoites was performed in the following twoways. (i) Scrapings of intestinal mucosa were mixed with a

drop of saline on a slide, covered with a cover slip, andexamined directly for the presence of motile trophozoites.(ii) The rinse solution obtained from the irrigation of upperjejunum was centrifuged at 275 x g for 10 min at 4°C, and thepellet was examined for the presence of motile trophozoites.Samples of Giardia trophozoites were air dried on slides andstained with Diff-Quick (Dade Diagnostics, Inc., Aguada,P.R.) to determine the morphology of the median body.Scanning electron microscopy (SEM). Samples of beaver

and muskrat intestine were fixed in 2 to 3% glutaraldehydebuffered with 0.1 M cacodylate hydrochloride containing 5%sucrose. The tissue was postfixed in 1 to 2% osmiumtetroxide in the same buffer and then treated with thiocar-bohydrazide (19). The samples were dehydrated in an as-cending ethanol series and critical point dried by the CO2method of Anderson (1). After coating with gold-palladiumby cold-cathode discharge sputtering, the tissue sampleswere examined in a Hitachi S-450 SEM and photographedwith Polaroid type 55 P/N film.

Statistical analysis. The chi-square criterion was used totest for significant differences between various cyst dosetreatment groups (see Table 3), testing the hypothesisagainst two-sided alternatives with a correction for continu-ity (42).

RESULTS

Inoculation of beavers with G. lamblia cysts from humansand G. muris cysts from mice. Fecal samples from multiplesymptomatic human donors, having Giardia cysts exhibitinggreater than 85% viability by FDA-PI, were selected andpooled for use (Table 1). Of the pooled G. lamblia cysts usedfor inoculation in experiment 1, 90% were viable on the basis

TABLE 1. Viability of G. lamblia and G. muris cysts used forinfection of beavers and muskrats

No. and Viability (%) ofAnimal CstyeCyst agea typ of donor cysts

(expt no.) (days) donor FDA-PI Excystation

Beaver1 G. lamblia 7 4 patients 90 41

G. muris 3 10 mice 77 +b2 G. lamblia 21 3 patients 91C NDd

Muskrat1 G. lamblia 21 3 patients 91C ND2 G. lamblia 12 3 patients 88C ND

a Length of time between specimen collection, or cyst isolation, andadministration to experimental animals.

b Excystation was observed but not expressed numerically.c Sorted by FACS.d ND, Not done.

of the FDA-PI test, and 41% were viable by using thecriterion of excystation. A pool of G. muris cysts, obtainedfrom 10 mice, displayed a viability of 77% by FDA-PIincorporation and was observed to excyst in vitro, but theexcystation percentage was not determined. Beavers weregiven an oral inoculum containing 5 x 105 cysts of either G.lamblia or G. muris. The inoculation of beavers with G.lamblia cysts resulted in the infection of six of the eightanimals (Table 2). The first appearance of cysts in the fecesoccurred at 4 days postinoculation, and the mean prepatentperiod (the time between inoculation and demonstration ofthe parasite infective form by recovery from the host) was13.1 days (range, 4 to 23 days). Three of the infected beaversshed cysts continuously in their feces for periods of 34 to 41days, while the other three beavers intermittently releasedcysts. Trophozoites were isolated from the intestines of fiveof the six infected beavers and were shown to have theclawhammer median body morphology described by Filice(15). A systematic examination of the small intestine of theinfected beavers showed Giardia trophozoites to be presentfrom the mid-duodenal region to the lower ileum, with theupper jejunal region being the most common site of infec-tion. SEM examination of the mucosa from infected beaversrevealed Giardia trophozoites (Fig. la) attached to theintestinal mucosa. Lesions, resembling the mirror image ofthe ventral adhesive disc (the attachment organelle of thetrophozoite; see Fig. lb through d), were detected in themicrovillous border of intestinal epithelial cells.

Five beavers were not inoculated with Giardia cysts andserved as controls for the possibility of cage-to-cage trans-mission by cyst-containing feces. No Giardia cysts weredetected in the feces of these animals throughout the exper-imental period of 54 to 57 days, and no sign of intestinaltrophozoites was encountered at necropsy.A group of four beavers was inoculated with G. muris

cysts. Two of the beavers did not show any infection, asdetermined by the lack of either cysts in the feces orintestinal trophozoites at necropsy. One beaver (beaver 6[Table 2]) became infected on day 40 postinoculation, butexamination of the median body morphology by light micros-copy revealed the presence of a clawhammer type of medianbody typically found in G. lamblia, not the small roundedtype characteristic of G. muris (15). Also, the immunoreac-tivity of the cysts isolated from this animal, as determinedwith antisera provided by John Riggs, California Departmentof Public Health, was specific for the G. lamblia type, notthe G. muris type (S. L. Erlandsen and W. J. Bemrick, in

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TABLE 2. Infectivity of Giardia cysts in beavers

Inoculum treatment Beaver Day of cyst Antibody Type of Duration (days) of cyst sheddinggroup

Beaver no. appearance staininga median bodyb postinoculationCgroup ~~~~~~~~~postinoculationG. lambliad (n = 8) 12 16 +H Clawhammer +(16-57)

26 9 Clawhammer +(9), 0(10, 11), +(12-14) autopsy29 23 +H Clawhammer +(23-57)33 16 +H Clawhammer +(16-57)34 4 +(4), 0(5-23), +(24), 0(25-57)35 11 +H Clawhammer +(11-15), 0(16-20), +(21-53), 0(54)16, 39 0 0

Controls (n = 5) 4, 8, 25, 28, 30 0 0G. muris (n = 4) 6e 40 +H Clawhammer +(40-54)

36 39 +(39), 0(40-54)19,41 0 0

a Giardia cysts recovered in feces were immunostained with antisera for G. lamblia (H), or G. muris (M) kindly provided by John Riggs, Department of HealthServices, Berkeley, California.

b Demonstrated by using Diff-Quick stain.Days postinoculation in which stools were negative (0) or positive (+) for Giardia cysts.

d For infected beavers, the mean prepatent period was 13.1 days.eAnimal contaminated with human G. lamblia, as indicated by long prepatent period and median body morphology.

P. M. Wallis and B. Hammond, ed., Advances in GiardiaResearch, in press). The other beaver (beaver 6) was posi-tive for cysts in the feces (only a few cysts were detectedmicroscopically) on day 39 postinoculation, and no tropho-zoites were observed in the intestine at necropsy.

Determination of infective dose of G. lamblia cysts inbeavers. The G. lamblia cysts used in this experiment wereobtained from three symptomatic patients from a giardiasisoutbreak in Pittsfield, Mass., in December of 1985 (20). Theviability of the cysts (mean of three donors) by FDA-PI was91%. Cysts from two of the three donors used in experiment2 (Table 1) were shipped in overnight mail to the Environ-mental Protection Agency in Cincinnati, Ohio, and each wasinoculated into two Mongolian gerbils (104 cysts per animal).The third human donor was not tested because of aninsufficient number of cysts. Three of the four gerbils inoc-ulated were positive for cysts in the feces within 1 weekpostinoculation and for trophozoites in the small intestine atnecropsy.A FACS IV was used to collect only the FDA-positive G.

lamblia cysts from the three human donors. G. lamblia cystswere first detected by forward and 900 light scattering andthen sorted on the basis of FDA fluorescence into separatesyringes for oral administration to each beaver (Fig. 2).Approximately equal numbers of cysts were collected byFACS from the three donors for the preparation of eachinoculating dose. Sorting by FACS of FDA-positive G.muris cysts produced infections in 10 of 10 mice at 4 dayspostinoculation (1,000 FDA-positive cysts administeredorally).The doses of G. lamblia cysts administered to beavers

were <50 (mean, 48 cysts), <500 (mean, 454 cysts), and<5,000 (mean, 4,460 cysts), all of which were prepared byFACS of FDA-positive cysts (Table 3). A higher dose wasprepared by dilution of the stock cyst suspension, and itcontained 5.5 x 105 cysts. None of the six beavers receivingdoses of <50 Giardia cysts showed the presence of eitherfecal cysts during the experiment or intestinal trophozoitesat necropsy (Table 3). Two of the six beavers receiving <500Giardia cysts and one of the three beavers receiving <5,000Giardia cysts were infected as indicated by the presence ofcysts in their feces and the detection of intestinal trophozo-ites at necropsy. The prepatent period appeared to be similarfor all groups, regardless of the number of cysts adminis-

tered, and the mean was 15.4 days (range, 10 to 30 days).Intestinal trophozoites recovered at necropsy from each ofthe infected beavers had the clawhammer-type median body.In a systematic microscopic examination of mucosal scrap-ings from the small intestine of infected beavers, trophozo-ites were usually found within the upper small intestine, butin one animal they were detected only in the terminal regionof the distal ileum.Examination of the body weights (data not shown) of the

11 beavers infected with G. lamblia cysts derived fromhumans (Tables 2 and 3) indicated no obvious associationwith body weight (a reflection of animal age). Infectedbeavers were distributed in the following weight ranges: 10to 20 lb (four beavers), 20 to 30 lb (four beavers), and 30 to40 lb (three beavers).

Infection of muskrats with G. lamblia cysts for humans. G.lamblia cysts obtained from multiple human donors wereused in two different infection experiments. The first exper-iment was a pilot experiment to determine if muskrats couldbe infected. Each of three muskrats was given a dose of 5 x105 cysts via stomach tube (Table 1). All three muskratsreceiving this high dose of cysts became infected, as indi-cated by the presence of cysts in their feces after a prepatentperiod of 5 days. These animals continued to shed cysts untilthe conclusion of the experiment (one animal died at day 17,and the other two were necropsied at day 40 postinocula-tion), and all were positive for intestinal trophozoites. Onemuskrat, housed in the same facility, served as an uninocu-lated control in this pilot experiment and remained negativefor Giardia infection throughout the course of the experi-ment.

In the second experiment, a total of 21 muskrats wereinoculated by stomach tube with three different doses of G.lamblia cysts: 3 x 101 (n = 8), 3 x 102 (n = 7), and 3 x 10'(n = 6) per animal (Table 1). Six muskrats were not inocu-lated with cysts and were housed in the same cage facility assentinel controls. Regardless of the dose of Giardia cystsadministered, none of the muskrats released detectablelevels of cysts in their feces during the course of theexperiment, nor were any of the animals positive for intes-tinal trophozoites at necropsy (animals sacrificed at 6 [n =2], 13 [n = 2], 20 to 22 [n = 3], 27 [n = 2], 36 [n = 4], and 42[n = 4] days postinoculation). All six control animals werenegative for Giardia infection.



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FIG. 1. (a) SEM of an intestinal villus from the small intestine of a beaver infected with G. lamblia. Trophozoites can be seen adherentto the microvillous border of epithelial cells on the apex of the villus. Bar, 25 pLm. (b) SEM of the ventral surface of a trophozoite of G.lamblia. The ventral disk or attachment organelle (arrowheads) displays an asymmetry resembling the number 6 (*) due to the overlappingof cytoplasmic microtubular arrays. The ventrolateral flange (arrow) forms the lateral cytoplasmic border of the trophozoite. VF, Ventralflagella. Bar, 1 p.m. (c) SEM of the microvillous border of an intestinal villus from a beaver infected with G. lamblia, revealing portions oftwo trophozoites, one of which has a clearly recognizable ventral disk (*). Also seen in a characteristic lesion in the microvillous border ofthe epithelium (arrowheads) produced by attachment of the ventral disk of the trophozoite. Bar, 2 p.m. (d) SEM showing higher magnificationof the lesion in the microvillous border (see panel c) produced by attachment of a G. lamblia trophozoite. The rod-shaped microvilli weredisplaced by the edge of the ventral disk, presumably through a change in diameter of the disk produced by contraction of contractile proteinsfound within its cytoplasmic margin. Bar, 0.5 p.m.

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FDA FluorescenceFIG. 2. FACS analysis of G. lamblia cysts labeled with FDA and

PI. (a) Contour plot depicting a population of cysts by size. Theabscissa shows light scatter at 900; the ordinate shows forward lightscatter. (b) The same population of cysts shown in gated area (box)in panel a were sorted by using fluorescence. The abscissa showsFDA fluorescence, and the ordi'nate shows PI fluorescence. ThePI-positive cysts (gated box) in quadrant I represented less than 2%of the total sample, whereas the FDA-positive cysts (gated box inquadrant III to IV) constituted 74% of the sample. Fluorescencemicroscopy was used to confirm the identity of each of these cystfractions before samples of FDA-positive cysts in the gated box inquadrant III to IV were collected for inoculation of animals.

As a result of the lack of infection in muskrats in thesecond experiment with low dose levels of cysts (3 x 101 to3 x 104), the infective potential of the G. lamblia cyst

inoculum used was tested by using greater numbers of cysts.The control group of muskrats (n = 6) was divided into twogroups; one was not inoculated with cysts, while the other

TABLE 3. Determination of infective dose of Giardia lambliacysts in beavers

Cyst No. ofdose' beavers Day of cyst Detection of Type ofinfected/no, appearance intestinal dtreatment inoculated postinoculationb trophozoitesC median bodygroup (%)

<50 0/6 (0) No<500 2/6 (33)e 10, 13 Yes Clawhammer<5,000 1/3 (33)' 10 Yes Clawhammer550,000 2/3 (66)e 14, 30 Yes ClawhammerControls 0/3 (0) No

a Doses of <50, <500, and <5,000 prepared by FACS using the fluorogenicdye, FDA, as a marker for cell viability.

b Cysts detected in fecal sample by using zinc sulfate flotation. For infectedbeavers, the mean prepatent period was 15.4 days.

c Determined by light microscopy of intestinal scrapings at necropsy.d Demonstrated by using Diff-Quick stain.Significantly greater (P < 0.01) compared with controls or <50 cyst dose

treatment group.

muskrats received doses of either 1.25 x 105 or 5.0 xcysts per animal. The muskrats were necropsied at 13 or 21days postinoculation, and two of the three muskrats receiv-ing cysts (1.25 x 105 [sacrificed after 20 days] and one of twoanimals receiving 5 x 105 [sacrificed after 13 days]) werepositive for Giardia spp., as indicated by the presence oftrophozoites at necropsy. Neither of these two animals waspositive for the presence of cysts in the feces at any time.The control group of muskrats (n = 3), which had not beeninoculated with Giardia cysts, remained free of infection.

Cross-species infectivity of Gwardia cysts in beavers andmuskrats. Nine beavers were inoculated with 1.25 x i05 G.ondatrae cysts isolated from muskrats (n = 5). Daily micro-scopic examination of feces throughout the experiment (31days) did not reveal any Giardia cysts, nor did the exami-nation of mucosal scrapings from the small intestine atnecropsy (14, 20, 27, and 31 days) show any trophozoites.A total of 12 muskrats were inoculated with 5 x 105

Giardia cysts isolated from infected beaver feces (n = 3).Four of the muskrats died within 1 to 3 days of inoculationand were not necropsied. Five of the remaining eight musk-rats (62%) became infected as indicated by the presence ofcysts in the feces, with the prepatent period being approxi-mately 5 days. Three animals were necropsied at 21 dayspostinoculation, and trophozoites were detected in lightmicroscopic examination of mucosal scrapings. Infectedmuskrats shed cysts throughout the 30-day duration of theexperiment and were still releasing cysts 4 months later.Immunocytochemical studies (results not shown) using anantiserum which has been shown to immunologically stainbeaver Giardia cysts, but not G. ondatrae cysts (Erlandsenand Bemrick, in press), produced positive immunostainingof cysts isolated from these infected muskrats, thus provid-ing evidence that the experimentally infected muskrats wereshedding beaver Giardia cysts.

DISCUSSION

The results of the experiments on the cross-species trans-mission of G. lamblia cysts clearly demonstrated that bea-vers and muskrats can be infected with cysts derived fromsymptomatic human patients. However, it should be empha-sized that despite our use of FACS methods for selectingpresumably viable Giardia cysts for inoculation, the doserequired for establishing human infections in beavers andmuskrats was large, particularly compared with that re-

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ported to be necessary for homologous host infection ofanimal models (3, 5). The infection of beavers with G.lamblia cysts was accomplished in 73% (8 of 11) of theanimals at doses exceeding 5 x 105 cysts per inoculum,whereas the administration of approximately 500 G. lambliacysts resulted in only 33% (2 of 6) of the beavers becominginfected. The smallest dose tested, approximately 50 cystsper animal, did not produce infections in any of six beaversinoculated. The infection of muskrats with G. lamblia cystswas successful only at doses greater than 1.25 x 10' cysts.The seemingly large number of G. lamblia cysts required toinfect beavers and muskrats has introduced the question asto whether these animals serve as secondary reservoir hostfor human-derived Giardia spp., rather than being a primaryhost as is the case with humans. Support for the idea thatlarge numbers of cysts may be needed for the transmission ofG. lamblia between heterologous hosts can be found in thestudies by Visvesvara et al. (43) on the cross-species trans-mission of G. lamblia from multiple human donors intogerbils, in which a dose of 1,000 cysts (the lowest dosetested) was required for establishment of an infection ap-proximately 55% of the time, and some human cyst isolateswere noninfective. Hibler et al. (16) have reported that only2 to 12 G. duodenalis cysts of human origin were required toinfect Mongolian gerbils in a cross-species transmissionexperiment, whereas in a similar study, Visvesvara et al. (43)obtained infections with a minimum of 100 cysts. However,it should be noted that in both of these reports, the cystswere originally of human origin but had been physiologicallyadapted to passage in gerbils. Thus, their results usingadapted G. duodenalis cysts should be considered morerepresentative of homologous host (intraspecies) transmis-sion since these cysts were passed experimentally fromgerbil to gerbil and did not represent the use of freshlyderived human cysts into an animal model. Contrary to theneed for large numbers of cysts for transmission betweenheterologous hosts, experiments on the intraspecies trans-missions of G. muris in murine hosts (17) and on G. lambliain humans (32) have shown that only 5 cysts per host wererequired to establish an infection 50% of the time in mice,while 10 cysts were able to produce infections in humanstudies.Previous experiments on the transmission of Giardia

cysts, both within and between various hosts, have reliedsolely on the use of dilution techniques to prepare inoculat-ing doses of cysts at various concentration levels; therefore,each inoculum contained both viable and nonviable cysts inunknown proportions (3, 21, 32, 43). In some instances,Giardia cyst inocula were totally ineffective in producinginfections (27, 32, 43, 48), thus raising the question as towhether the cysts were in fact viable or if the lack ofinfection was due to host factors. In these experiments withbeavers and muskrats, the viability of Giardia cysts wasmeasured in two ways. First, the incorporation of fluoro-genic dyes was used as a metabolic indicator of individualcyst viability. Second, the infectivity of the same cystinoculum used in beavers or muskrats was measured, whenpossible, in a second animal host (gerbils or mice). Inaddition, the use of the fluorogenic dyes, FDA and PI,enabled us to select, by FACS methods, only viable Giardiacysts for inoculation. Control experiments using G. muriscysts sorted by FACS resulted in the infection of 100% ofmice inoculated with a dose of 1,000 cysts per animal, thusdemonstrating that the sorting methodology and use offluorogenic dyes were compatible with cyst viability. How-ever, the interpretation of viability, as measured by fluoro-

genic dye incorporation, must be made carefully since in ourattempts to measure the dose response for G. lamblia cystinfectivity in muskrats, we observed a total lack of infectionin muskrats each given 30 to 30,000 human cysts, eventhough the viability was determined to be 88% by fluorogenicdye incorporation. Administration of the same cyst inoculumat higher doses (1.25 x 10' or greater) produced infections inmuskrats, thus demonstrating that the inocula containedviable infective cysts. The lack of infectivity in muskratsinoculated with low numbers of cysts was not likely relatedto undefined host factors which were protective againstinfection unless the host was inoculated with large numbersof cysts. Such host factors might include immunologicalsuppression of infection since the muskrats all had preexist-ing Giardia infections. Resistance to reinfection has beenreported for both G. muris and G. lamblia (13); however, onthe basis of cyst morphology, these Giardia spp. should beconsidered to be different species of Giardia than thosefound in muskrats. To be an important host factor, resis-tance would require that common antigens, such as GS65 introphozoites (35, 36), exist between these different Giardiastrains (species?) and more importantly, that they would beimmunologically protective.

G. lamblia infections produced in beavers and muskratswere characterized by a prepatent period of 13 to 15 days forbeavers and 3 to 5 days in muskrats. In the infection of otheranimal hosts with Giardia cysts, the prepatent period inMongolian gerbils has been reported to be 5 days for G.muris (13), 7 to 8 days for G. lamblia (5, 13), and 11 to 13days for G. duodenalis from the cat (4). The small timedifferences observed in the prepatent periods in these exper-imental infections were reasonably similar in length, despitethe fact that the cyst dosage in the inocula were different.Also, the onset of G. muris infections in mice has beenshown to correlate with increased numbers of cysts in theinoculum (3).On the basis of ultrastructural observations in rats (10-12)

and later in mice (30), it was demonstrated that G. muristrophozoites attached and produced morphological lesions inthe microvillous border of the intestinal epithelial cells.These physical lesions have been postulated as a mechanismfor explaining the pathogenesis of Giardia infections result-ing in malabsorption (12). Our SEM observations on theintestinal mucosa of beavers infected with G. lamblia hasdemonstrated clearly, for the first time, that human Giardiatrophozoites were capable of producing similar lesions in themicrovillous border of the intestinal epithelium, albeit in ananimal host.The beaver and muskrat are both aquatic mammals and

share the same ecological habitat; therefore, it is surprisingto find that cross-species transmission of Giardia cystsproduces infection in muskrats with beaver-derived cysts,but not in beavers with cysts derived from muskrats. Ourexamination of endogenous Giardia infections in beaversand muskrats livetrapped in five different states reveals thateach aquatic animal appears to possess a strain of Giardiaunique to each individual host (Erlandsen and Bemrick, inpress). The muskrat possesses a binary type of Giardia cystthat is characteristic of microtine rodents (7, 14) and whichwe do not observe in beavers. Also, we do not detectbeaver-type Giardia cysts in livetrapped muskrats; thereforehow does one explain the infection of muskrats with beaver-derived cysts in captivity? It is feasible that muskrats innature may be continuously ingesting beaver-type Giardiacysts suspended in the water. Although these cysts areingested and undergo excystation within the muskrat, the

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endogenous Giardia strain normally found within theseanimals (infection rate equals 95 to 100% (S. L. Erlandsen,W. J. Bemrick, and L. A. F. Sherlock, manuscript inpreparation]) may be favored in competition for some un-known reason. As a result, the beaver-type Giardia cystwould not be competitive in establishing a patent infection inthe muskrat, which might explain the dominance of themuskrat type of Giardia cyst in the wild host. While thishypothetical explanation is not proven, it is not inconsistentwith the data obtained, and more importantly, it raises thequestion about the extrapolation of successful laboratorytransmission of Giardia cysts with what is observed in thenatural habitat.The epidemiological role of the beaver and muskrat in

serving as an intermediate reservoir for G. lamblia, perhapsthrough their infection from water polluted by human fecalwaste, must be considered (28). Although human-derivedcysts do not appear to be highly infectious for beavers ormuskrats, this does not rule out the possibility that Giardiacysts from humans could transiently infect these (or similar)animals and undergo adaptation to a strain that might beinfectious to humans, such as has occurred with humanGiardia cysts adapting to the gerbil (16, 43). In addition, theactual importance of the role of these aquatic mammals inwaterborne giardiasis is extremely difficult to assess, sinceone must consider not only the presence of human-derivedcysts in the water from other sources but also the fact thatconsiderable variation exists in the ability of different G.lamblia isolates to infect either volunteer human subjects(27) or animal hosts (43). The suggestion that these aquaticmammals play a major role in waterborne giardiasis wasoriginally based on the detection of a few infected animals insites where outbreaks of giardiasis had occurred (Erlandsenand Bemrick, in press). The ubiquitous geographic distribu-tion of beavers in North American obviously correlated withsites of waterborne giardiasis outbreaks. The contributionsof Giardia cysts from a number of previously unassessedsources, including the large numbers of cysts that have beencalculated or reported in raw human waste (18; J. L. Sykora,W. D. Bancroft, A. H. Brunwasser, S. L. States, M. A.Shapiro, S. N. Boutros, and L. F. Conley, in P. M. Wallisand B. Hammond, ed., Advances in Giardia Research, inpress) and the considerable number of bird species reportedto harbor this parasite (23), would seem to make an exactinterpretation, using existing methods, as to the animalorigin of waterborne cysts difficult, if not impossible, at thistime. Thus, based on this information, it does not appear thatbeaver, muskrat, or any animal other than humans canpresently be assigned a role as a major reservoir for the cystsresponsible for waterborne giardiasis outbreaks.

ACKNOWLEDGMENTS

We thank the following individuals for their assistance in trappingand maintaining beavers and muskrats in captivity for these studies:Richard Buech, Clarence and Daniel Rademacher, Susan Spence,Robert Tanaka, and Kevin Voller. We also thank Jack Korlath,Minnesota Department of Public Health; Thomas Eng, Massachu-setts Department of Public Health; and George Kent, Centers forDisease Control, Atlanta, Ga., for their assistance in providingaccess to human Giardia cysts. We also acknowledge the assistanceof H. K. Ghobrial in statistical analysis. This document has beenreviewed in accordance with U.S. Environmental ProtectionAgency policy through cooperative agreement no. CR-811834 to theUniversity of Minnesota and approved for publication.

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