1-s2.0-s0304401702003497-main

Veterinary Parasitology 111 (2003) 3146

Giardia duodenalis trophozoites isolated from aparrot (Cacatua galerita) colonize the small

intestinal tracts of domestic kittens and lambsP.A. McDonnell a,, K.G.-E. Scott b, D.A. Teoh b, M.E. Olson b,

J.A. Upcroft c, P. Upcroft c, A.G. Buret ba School of Biomolecular and Biomedical Science, Faculty of Science, Griffith University,

Kessels Road, Nathan 4111, Qld, Australiab Department of Biological Sciences, University of Calgary, Calgary, Alta., Canada T2N 1N4

c Queensland Institute of Medical Research, The Bancroft Centre, 300 Herston Road,Herston 4029, Qld, Australia

Received 3 January 2002; received in revised form 25 September 2002; accepted 15 October 2002

Abstract

This study examines the ability of Giardia duodenalis trophozoites, isolated from a wild bird,to colonize the intestinal tracts of companion animals (kittens) and domestic ruminants (lambs).Trophozoites colonized the intestinal tracts of intraduodenally inoculated animals as demonstratedby increasing parasite burdens within the duodenum and jejunum and by fecal passage of cystswithin 4 days post-inoculation. The pathogenesis of the trophozoites was further investigated inkittens. In these animals, infection significantly reduced jejunal brush border microvillous lengthand density, which resulted in a loss of overall epithelial brush border surface area. This injury wasassociated with the production of diarrhea in four of five infected kittens. These findings indicatethat some bird species may carry G. duodenalis that represent a possible health threat to companionanimals and livestock. Our results describe the first successful colonization of avian-derived G.duodenalis trophozoites in the small intestines of domestic kittens and lambs. 2002 Elsevier Science B.V. All rights reserved.

Keywords: Sheep-protozoa; Cat; Parrot; Companion animals; Giardia duodenalis

1. Introduction

Worldwide, the parasitic protozoan genus, Giardia, represents a major cause of diarrheain numerous vertebrate species including humans. Cross-species transmission studies have

Corresponding author. Tel.: +61-7-3875-3884; fax: +61-7-3875-7656.E-mail address: [email protected] (P.A. McDonnell).

0304-4017/02/$ see front matter 2002 Elsevier Science B.V. All rights reserved.PII: S0 3 0 4 -4017 (02 )00349 -7

32 P.A. McDonnell et al. / Veterinary Parasitology 111 (2003) 3146

assessed both the zoonotic risk of Giardia infection and the role of host specificity as aspecies identification marker for the parasite (Davies and Hibler, 1979; Erlandsen et al.,1991; Erlandsen, 1994). Some human isolates of G. duodenalis (syn. G. lamblia, G. in-testinalis) have successfully infected a variety of experimental hosts, including Mongo-lian gerbils, mice and rats (Belosevic et al., 1983; Hill et al., 1983; Nash et al., 1985;Visvesvara et al., 1988; Byrd et al., 1994; Majewska, 1995). Several anecdotal reports ofzoonotic transmission, as well as the infection of a human volunteer with Giardia derivedfrom a Gambian giant-pouched rat (Majewska, 1994), support the growing evidence that anumber of G. duodenalis isolates are not host-specific and may infect a wide range of hostspecies. Possible infection reservoirs include native fauna, such as beavers, muskrats andsome marsupials; companion animals, in particular cats and dogs; and domestic livestock(Davies and Hibler, 1979; Cribb and Spracklin, 1986; Swan and Thompson, 1986; Pachaet al., 1987; Faubert, 1988; Buret et al., 1990a; Marino and Brown, 1992; Olson et al.,1997; Measures and Olson, 1999; Enriquez et al., 2001; Heitman et al., 2002). Giardiasis inwild birds is well documented and its implication as a source for waterborne and zoonoticdisease remains controversial (Fudge and McEntree, 1986; Georgi et al., 1986; Erlandsenand Bemrick, 1988; Forshaw et al., 1992; McRoberts et al., 1996; Upcroft et al., 1997;Graczyk et al., 1998; Kuhn et al., 2002; Ponce Gordo et al., 2002). Giardia infections havealso been described in aviary birds (Panigrahy et al., 1984; Scholtens et al., 1982; Fudge andMcEntree, 1986; Filippich et al., 1998), but most reports have examined symptomatologyrather than transmission potential.

Although Giardia spp. typically found in birds, such as G. ardeae and G. psittaci,do not appear to cross the host class boundary (Box, 1981; Erlandsen et al., 1991;Filippich et al., 1998), some G. duodenalis-like organisms observed in birds (Gallagheret al., 1995) do warrant further consideration. Upcroft et al. (1997, 1998) reported the de-velopment of chronic giardiasis in neonatal and adult Quackenbush Swiss mice experimen-tally infected with G. duodenalis isolated from a wild-caught, moribund, sulfur-crestedcockatoo (Cacatua galerita), hence describing the first successful inter-class transmis-sion of Giardia infection from an avian source to a mammalian host. Whether this iso-late is infectious and pathogenic in companion animals and/or domestic ruminants hasyet to be established. The aim of the present study was to investigate the capacity oftrophozoites of this isolate to colonize the intestinal tracts of domestic kittens andlambs.

2. Materials and methods

2.1. Parasite culture

The G. duodenalis isolate used in this study is designated BRIS/95/HEPU/2041 (Bris-bane/95/Herston Experimental Parasitology Unit/Sample no. 2041). It was obtained fromthe intestinal washings of a necropsied sulfur-crested cockatoo (Cacatua galerita)(Gallagher et al., 1995; Upcroft et al., 1997, 1998). The trophozoite culture used in these tri-als was established directly from cryopreserved trophozoites of an in vitro culture from theoriginal host. This culture was found to be karyotypically distinct from other G. duodenalis

P.A. McDonnell et al. / Veterinary Parasitology 111 (2003) 3146 33

stocks grown in our laboratory, as reported by Upcroft et al. (1997) through the use ofcontour-clamped homogeneous electric field (CHEF) gel electrophoresis. Trophozoiteswere grown at 37 C in sterile culture tubes (Disposable Products Inc.) containing TYI-S-33medium supplemented with 1 mg/ml bovine bile and 10% heat-inactivated fetal calf serum(FCS) as described previously (Boreham et al., 1986; Upcroft et al., 1997).

2.2. Source of specific pathogen-free (SPF) experimental animals

2.2.1. LambsDorset and Suffolk ewes were supplied by and housed at the AgCanada Agricultural

Research Station, Department of Agriculture, Lethbridge, Alta., Canada. Synchronized es-trous and parturition in ewes were induced following the procedures described by Olsonet al. (1995). Lambs were manually delivered at normal parturition, washed immediately ina 4% BetadineTM solution, dried with sterile towels, sexed, weighed and given 100 mlof bovine colostrum. Over a 3-day birthing period a total of six lambs were obtainedand transferred to an aseptic laboratory isolation room in the Life and EnvironmentalScience Animal Resource Centre (LESARC), Department of Biological Sciences, Uni-versity of Calgary, Alta., Canada. Clean straw bedding was provided and changed daily.Room lighting (12:12 h) was provided by a timed laboratory network system and a sepa-rate air-conditioning conduit regulated ventilation. Room temperature was maintained at25 C. Lambs were fed a milk replacer diet (Browns Feeds, Calgary, Alta., Canada) forthe duration of the trials and given ad libitum access to creep feed from 1 week after birth.At 5 days of age, each lamb received a 1 ml sub-cutaneous injection of combined Clostrid-ium bacterin-toxoid (Covexin 8TM) (Coopers Agripharmaceuticals Inc.). The Giardia-freestatus of all lambs was confirmed prior to the commencement of trials by the daily col-lection of feces for 3 days from each lamb and the screening for the presence of Gia-rdia cysts using sucrose density gradient concentration and immunofluorescent staining(Giardi-a-GloTM, Waterborne Inc.) as described by Olson et al. (1995) and Heitman et al.(2002).

2.2.2. KittensTwo, pregnant, domestic, short-haired cats were supplied by LESARC, University of

Calgary, Alta., Canada and housed in separate isolation cages. Prior to the commencementof trials, feces from each adult cat were screened daily for 3 days for the presence of Giardiacysts using the same technique as described for lambs. Subsequently, two litters, one of threekittens and one of five, were delivered naturally and kept with their respective mothers untilweaning at 68 weeks of age. Kittens were then removed and divided into two groupsof similar weight and sex distribution. Each group was placed in a separate cage isolatedfrom other groups and supplied with individual feeding and drinking bowls and litter trays.Throughout the study, kittens were fed twice daily with commercial, canned kitten foodserved in sterilized bowls and had ad libitum access to water (chlorinated and filtered).Litter trays were removed twice daily and replenished with fresh granules. Again, prior tothe commencement of experimental trials, feces from all kittens were screened daily for 3days for the presence of Giardia cysts using the same method as described for lambs andadult maternal cats.


2.3. Animal inoculation

2.3.1. LambsThe SPF lamb model was chosen as it represents a domestic ruminant of commercial

importance, and has been utilized successfully in similar studies (Olson et al., 1995; Yankeet al., 1998). At 14 days of age, five SPF, Giardia-free, lambs were inoculated by thestandard procedure of direct introduction of live Giardia trophozoites into the duodenum.This inoculation procedure has been employed in several studies examining the infection,colonization and pathogenesis of Giardia in experimental animals (Olson et al., 1995;Yanke et al., 1998; Scott et al., 2000; McAllister et al., 2001). In brief, anaesthesia wasinduced with 2% halothane (MTC Pharmaceuticals) and lambs were positioned in dorsalrecumbency. A ventral midline abdominal incision was made and the duodenum isolated. Asuspension of 1106 trophozoites of G. duodenalis (BRIS/95/HEPU/2041) in 1 ml of coldTYI-S-33 medium was inoculated intraduodenally through a 20-gauge needle attached toa 1 ml syringe and the abdomen closed with absorbable sutures. One control lamb receivedan intraduodenal injection of sterile TYI-S-33 medium only. Post-operatively, each lambwas administered a 6 ml intramuscular (IM) injection of PenlongTM (benzathine penicillinG 150,000 Units/ml and procaine penicillin G 150,000 Units/ml) (Rogar/STB Inc.), andinfected and control lambs were housed in separate isolation rooms. At 8 days pi, lambswere euthanased with an overdose of sodium pentobarbital (NembutalTM) and segmentsof the small intestine were removed and prepared for trophozoite and cyst enumeration, asdescribed below.

2.3.2. KittensFollowing the same procedure as for lambs, one group of Giardia-free kittens (n = 5) was

anaesthetized and intraduodenally inoculated with 1 106 trophozoites of G. duodenalis(BRIS/95/HEPU/2041) in 1 ml of cold TYI-S-33 medium. The other group (n = 3) re-ceived inoculations of cold TYI-S-33 medium only. Each kitten was administered a 0.5 mlIM injection of PenlongTM after surgery. At 7 days pi, kittens were euthanased with anoverdose of sodium pentobarbital and segments of the small intestine were removed andprepared for trophozoite and cyst enumeration.

2.4. Clinical observations

Lambs and kittens were observed twice daily for clinical signs of disease, such asdecreased activity level, abnormal eating behavior or altered respiration rate. Inaddition, fecal consistency of each animal was scored using the following ratings: (1)normal, formed stool; (2) soft, unformed stool; and (3) loose, diarrhealstool.

Lambs were weighed at birth, 1 day prior to inoculation, and daily thereafter beforetheir morning feed using industrial, platform scales (Mettler Toledo Inc., Columbus, OH).Kittens were also weighed prior to inoculation and daily thereafter before their morn-ing meal using MC-1 1200S weighing scales (Sartorius Pty Ltd., Mississauga, Ont.).Percentage weight gains or losses from baseline weights were calculated for all recor-dings.


2.5. Parasite enumeration

2.5.1. Fecal cystsFecal samples were collected from lambs and kittens at the same time each day (imme-

diately after morning feeding). Two grams of feces were collected per animal, placed insterile, 50 ml, screw-top tubes and suspended in 10 ml of cold phosphate-buffered saline(PBS), pH 7.2. Fecal cysts were collected by sucrose density gradient concentration anddetected using a fluorescein isothiocyanate (FITC)-labeled monoclonal antibody prepara-tion against Giardia spp. cysts (Giardi-a-GloTM, Waterborne Inc.) and an epifluorescencemicroscope (Leica DMRB). Counts of fluorescent cysts were performed at 250 and 400magnifications and calculated as cysts per gram of feces. Positive and negative controlsamples (provided by Waterborne Inc.) were run with test samples.

2.5.2. Luminal parasitesThe entire small intestine of each euthanased lamb or kitten was removed and divided

into five or three sections, respectively. The five small intestine segments collected from thelambs included the duodenum (2 cm distal to the pyloric sphincter and proximal to the lig-ament of Treitz), three sections from the jejunum (proximal jejunum, 35 cm; mid-jejunum,70 cm; and distal jejunum, 170 cm distal to the pyloric sphincter) and the ileum (5 cm prox-imal to the ileo-caecal valve). The three intestinal segments collected from the kittens werethe duodenum (2 cm distal to the pyloric sphincter), jejunum (approximately 15 cm distalto the ligament of Treitz), and ileum (10 cm proximal to the ileo-caecal valve).

The first 1 cm of each intestinal segment was placed in a separate tube containing 2 ml ofice-cold, sterile PBS for at least 30 min, then vortexed to dislodge attached trophozoites fromthe intestinal mucosa (Buret et al., 1991). Numbers of trophozoites or cysts per centimeterof gut section were calculated from the mean of at least five haemocytometer counts on theintestinal washes. Cyst and trophozoite viabilities were assessed by Trypan Blue (0.1%)dye exclusion (ICN Biomedicals) (Shaio et al., 1987) and observations of parasite motility,adherence and flagellar movement using light microscopy.

2.6. Transmission electron microscopy

Jejunal tissue samples were collected and epithelial ultrastructure was assessed usingtransmission electron microscopy as described by Scott et al. (2000). Briefly, jejunal seg-ments (1 cm) of control and infected kittens were removed 10 cm distal to the ligamentof Treitz and fixed in 5% glutaraldehyde. Samples were dissected into 1 mm squares,post-fixed in 1% OsO4, and embedded in Spurr low-viscosity medium (Sigma, St. Louis,MO). Semi-thin sections were stained with toluidine blue and midvillous sections (80 nm)were double stained with saturated uranyl acetate in 50% ethanol and 0.4% lead citrate(aqueous) (Venable and Coggeshall, 1965; Buret et al., 1992). Sections were examinedat 75 kV using a Hitachi H7000 transmission electron microscope and micrographs ofmidvillous enterocyte apical brush border membranes were taken at the same magnification(12,000). Microvillous height, width and density were measured and overall brush bordersurface area was calculated as described by Buret et al. (1991). To eliminate observer bias,micrographs were coded and observations were recorded in a blind fashion. For each group,


2729 micrographs were obtained from 3 to 4 animals and microvillous brush border surfacearea was calculated. Previous findings have established that Giardia-induced alterations inmicrovillous ultrastructure in the midvillous region are representative of changes along theentire villous axis (Buret et al., 1991, 1992; Scott et al., 2000).

3. Results

3.1. Trophozoite colonization and cyst development

Prior to the commencement of trials, Giardia cysts were not detected in the feces ofmaternal ewes and lambs nor domestic cats and their kittens. However, within 4 daysafter inoculation with trophozoites of G. duodenalis BRIS/95/HEPU/2041, 14-day-oldlambs and 68-week-old weaned kittens were excreting viable cysts in their feces. Incontrast, no Giardia cysts were ever detected in the feces of the control (non-inoculated)animals.

All lambs inoculated with trophozoites passed cysts in their feces until necropsy (8 dayspi). Cyst excretion peaked in two infected lambs at 56 days pi with 501767 cysts passedper gram of feces (Table 1a). This was followed by a decline in cyst numbers and theselambs were passing an average of 51 cysts per gram of feces at time of death. The main siteof trophozoite colonization in one of these lambs was the distal third of the jejunum with amean count of 6.7 103 trophozoites per centimeter of gut section (Table 2a). Intermittentcyst excretion was observed in the three, other inoculated lambs with the highest mean

Table 1(a) Daily mean cyst counts per gram of feces in the control, uninfected lamb and lambs intraduodenally inoculatedwith trophozoites of G. duodenalis (BRIS/95/HEPU/2041)a; (b) daily mean cyst counts per gram of feces incontrol, uninfected kittens and inoculated kittens

(a) Lambs (b) KittensDayspi

Control, uninfectedlamb, no detectablecyst excretion(n = 1)

Inoculated lambs Dayspi

Control, uninfectedkittens (n = 3) nodetectable cystexcretion

Inoculatedkittens(n = 5)Single peak

cyst excretion(n = 2)

Intermittentcyst excretion(n = 3)

0 0 0 0 0 0 01 0 0 0 1 0 02 0 0 0 2 0 03 0 0 0 3 0 101 344 0 184 17 111 16 4 0 187 915 0 501 167b 300 125b 6 0 687 6736 0 767 567b 78 16 7 07 0 67 0 1889 1031b8 0 51 17 867 576Values are mean S.D.

a Two lambs displayed a single peak in cyst excretion, while the three remaining lambs excreted cysts inter-mittently.

b Peaks of cyst excretion.

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Table 2Mean trophozoite (a) and cyst (b) burdens (105) per centimeter of small intestinal section from and the control, uninfected lamb and lambs intraduodenally inoculatedwith trophozoites of G. duodenalis (BRIS/95/HEPU/2041) at 8 days piTreatment Duodenum Jejunal region Ileum

Proximal Mid Distal

(a) Mean trophozoite burdens (105) per centimeter of small intestinal sectionControl, uninfected lamb (n = 1) 0 0 0 0 0Inoculated lambs (single peak) 0 0 0.017 0.017 0.067 0.067 0.042 0.042Inoculated lambs (intermittent) (n = 3) 1.22 1.62 2.78 2.11 69.43 42.20 2.89 1.03 2.78 0.75Overall mean for inoculated lambs 0.73 1.39 1.67 2.13 41.67 47.17 1.76 1.60 1.69 1.46

(b) Mean cyst burdens (105) per centimeter of small intestinal sectionControl, uninfected lamb (n = 1) 0 0 0 0 0Inoculated lambs (single peak) (n = 2) 0 0 0 0.20 0.04 1.23 0.50Inoculated lambs (intermittent) (n = 3) 0 0 0 0.12 0.07 3.72 1.39Overall mean for inoculated lambs 0 0 0 0.15 0.07 2.73 1.66

Mean trophozoite and cyst counts of the two inoculated lambs that displayed single peaks in fecal cyst excretion are shown separately from the three remaining inoculatedlambs that displayed intermittent peaks of cyst excretion. Values are mean S.D.


Table 3Mean parasite burdens (105) per centimeter of small intestinal section at 7 days pi in control, uninfected kittensand kittens intraduodenally inoculated with trophozoites of G. duodenalis (BRIS/95/HEPU/2041)Treatment Duodenum Jejunum Ileum

Trophozoites Cysts Trophozoites Cysts Trophozoites Cysts

Control, uninfectedkittens (n = 3)

0 0 0 0 0 0

Inoculated kittens(n = 5)

2.10 0.73 0 13.50 7.10 0.32 0.52 2.70 1.80 0.07 0.075

Values are mean S.D.

count of 1889 cysts per gram of feces at 7 days pi (Table 1a). The main site of trophozoitecolonization in these lambs was the mid-jejunum with a maximum mean count of 6.94106trophozoites per centimeter of gut section (Table 2a). Cysts were found predominantly inthe ileum (mean = 3.72 105) of these animals, with small numbers also present in thedistal jejunum (Table 2b). No trophozoites or cysts were observed in the intestinal samplesor feces of the uninfected control lamb.

All kittens inoculated with trophozoites were positive for fecal cysts from 4 days pi(Table 1b). Cyst excretion continued to increase daily until animals were killed at 7 dayspi, at which time two kittens were passing greater than 1300 cysts per gram of feces (meancount = 687 cysts per gram of feces). No cysts were detected in the feces of uninfectedcontrol kittens.

Upon necropsy, trophozoites were present in the small intestinal sections of all kittensinoculated with trophozoites. The main parasite colonization site was the jejunum, with amean count of 1.35106 trophozoites per centimeter of gut section (Table 3). Cysts were alsodetected in the jejunal and ileal sections with mean counts of 3.2104 and 7103 cysts percentimeter of gut section, respectively; cysts were not detected in the duodenum (Table 3).All enumerated cysts and trophozoites in intestinal washes were viable by exclusion ofTrypan Blue dye and the presence of parasite motility, adherence and flagellar movement.No trophozoites or cysts were observed in intestinal sections of any of the uninfected controlkittens.

3.2. Clinical manifestations

No significant differences in weight gain were observed between lambs inoculated withtrophozoites and the uninfected control over the 8-day trial period (data not shown). Dailyfluctuations in fecal consistency were seen in all lambs, irrespective of treatment, and mayhave been attributed to adaptation to the milk replacer and creep feed diets (Table 4a).Lambs did not exhibit any other clinical signs of giardiasis and appeared active with normalappetite.

Similarly, infected kittens did not show reduced weight gain compared to uninfectedcontrols (data not shown), but four of five infected kittens did pass soft, unformed stoolsat 7 days pi (Table 4b). In contrast, uninfected control kittens passed normal formed stoolsfor the duration of the trial.

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Table 4Fecal consistency ratings of the control, uninfected lamb and lambs intraduodenally inoculated with trophozoites of G. duodenalis (BRIS/95/HEPU/2041) (a), and thecontrol (uninfected) and infected kittens (b)(a) Lambs (b) KittensDays pi Control, uninfected lamb (n = 1) Inoculated lambs (n = 5) Days pi Control, uninfected kittens (n = 3) Inoculated kittens (n = 5)

Rating 1 Rating 2 Rating 3 Rating 1 Rating 2 Rating 3 Rating 1 Rating 2 Rating 3 Rating 1 Rating 2 Rating 3

0 1 5 0 3 5 2 1 2 3 2 3 5 3 1 5 3 3 5 4 1 1 4 4 3 4 1 5 1 2 3 6 3 3 2 6 1 1 4 7 3 1 4 7 1 3 2 8 1 2 3

Fecal consistency scale: Rating 1:= normal, formed stool; Rating 2= soft, unformed stool; Rating 3= loose, diarrheal stool. The number of animals per group displayinga specific fecal consistency rating.


Fig. 1. Representative transmission electron micrographs, obtained at the same magnification, of the apical brushborder membrane of midvillous enterocytes from the jejunum of control, uninfected kittens (A) and kittens inoc-ulated with trophozoites of G. duodenalis (BRIS/95/HEPU/2041) (B) at 7 days pi. Microvillous attributes wereconsistent over the entire epithelium, not only at parasite attachment sites. Bar, 1m.

3.3. Brush border ultrastructure of intestinal sections from kittens

Representative transmission electron micrographs illustrating the apical brush bordermembrane of midvillous enterocytes of each experimental group (control, uninfected kittenand infected kitten) are shown in Fig. 1. Infection with trophozoites caused shorteningof the brush border microvilli over the entire epithelium, at sites of parasite attachmentas well as in other areas (Fig. 1). Height, width, density and overall surface area of thebrush border microvilli are described in Table 5. Infection of kittens with trophozoites

Table 5Brush border microvillous characteristics of midvillous enterocytes from the jejunum of control, uninfected kittensand kittens intraduodenally inoculated with trophozoites of G. duodenalis (BRIS/95/HEPU/2041)

Height (m) Width (m) Density(number/m2of epithelium)

Surface area(m2/m2 ofepithelial surface)

Control, uninfectedsections (n = 27)

1.242 0.050 0.116 0.0023 72.18 3.19 33.16 1.94

Inoculated sections(n = 29)

0.751 0.057 0.114 0.0052 60.65 4.67 17.99 1.75

Measurements were performed at 7 days pi. Values are mean S.E. P < 0.05 vs. control. P < 0.001 vs. control.


Fig. 2. Transmission electron micrographs of jejunal enterocyte apical membranes from kittens infected withtrophozoites of G. duodenalis (BRSI/95/HEPU/2041) at 7 days pi. Microvillous effacement (arrowheads) occursin areas of trophozoite (T) attachment (A) as well as in other areas (B). Bars, 1m.

caused a significant decrease in microvillous height (60% of control, P < 0.001) anddensity (84% of control, P < 0.05). Microvillous width did not differ between groups.Combined, these ultrastructural changes lead to a 46% reduction in overall microvillousbrush border surface area per square micrometer of epithelial surface compared with tissuefrom control animals (P < 0.001) (Table 5). Interestingly, in addition to diffuse brushborder shortening, localized microvillous effacement was observed in some areas of theinfected jejunum independent of trophozoite attachment (Fig. 2).

4. Discussion

Some previous studies have suggested that rigid host-specificity between major host phy-logenetic groups may prevent transmission of giardiasis between birds and mammals. Theavian Giardia spp., G. ardeae and G. psittaci, are restricted to infecting avian hosts only(Box, 1981; Erlandsen et al., 1991; McRoberts et al., 1996; Filippich et al., 1998), but therehave been limited studies on the infectious potential of avian-derived G. duodenalis-likeorganisms in mammals. Observations from the present study indicate that G. duodenalistrophozoites derived from a wild, native parrot colonized the intestinal tracts of lambs andkittens and were pathogenic to the latter mammal species. These results are consistentwith those of Upcroft et al. (1997, 1998), who reported infections of the same G. duode-nalis isolate in neonatal mice were more intense with longer patency periods than those


of two human-derived isolates. In the present study, this isolate produced heavy infectionsin 68-week-old weaned kittens and 14-day-old lambs, and produced a diffuse loss ofepithelial brush border surface area in the jejunum of kittens.

Numerous studies have examined the transmission and zoonotic potential of G. duode-nalis derived from mammalian companion animals (Swan and Thompson, 1986; Thompsonet al., 1988; Thompson and Boreham, 1994), yet investigations on trophozoite coloniza-tion of G. duodenalis derived from birds in domestic pets have been lacking. Moreover,there have been many reports of Giardia infections in farm animals and domestic ruminants(Kirkpatrick, 1989; Buret et al., 1990a; Xiao, 1994; Xiao et al., 1994; Ruest and Faubert,1995; Heitman et al., 2002; van Keulen et al., 2002), and some investigators have examinedthe effects of experimental infections on health and productivity in lambs and goat kids,but again, these earlier trials used mammalian-derived Giardia only (Olson et al., 1995;Koudela and Vitovec, 1998; Yanke et al., 1998). In our experiments, all lambs inoculatedwith trophozoites of avian-derived G. duodenalis (BRIS/95/HEPU/2041) excreted viablefecal cysts after 4 days pi, with most lambs shedding cysts intermittently. Intermittent ex-cretion and similar infection patterns have been noted in calves (Xiao et al., 1993), foals(Xiao, 1994) and lambs (Xiao et al., 1994; Yanke et al., 1998) infected with G. duodenalis.Our findings also suggest that the main site of trophozoite colonization in infected lambsis the mid-to-distal jejunum, while cysts are most predominant in the distal jejunum andileum.

Past studies have implicated Giardia infection as a cause of weight gain impairment,retarded growth and productivity losses in calves (Xiao et al., 1993), goat kids (Koudela andVitovec, 1998), foals (Kirkpatrick and Skand, 1985) and lambs (Kiorpes et al., 1987; Olsonet al., 1995). As our experiments sought to characterize trophozoite colonization ratherthan long-term pathophysiological effects, animals were killed at 7 or 8 days pi. Hence,whether longer-term infections may affect weight gain and body development in lambs andkittens requires further investigation. In sheep, cattle, as well as in other mammals, it iswell established that giardiasis may cause diarrhea while in other instances the infectionmay remain asymptomatic (Kiorpes et al., 1987, Olson et al., 1995; Buret et al., 1990a;Xiao, 1994). In our studies, infected lambs did not develop diarrhea and this low visibilityof symptoms may contribute to the failure to recognize the infection in some livestockspecies. The results shown here indicate that G. duodenalis trophozoites derived from anavian source can colonize, replicate and become viable cysts within the intestinal tractof lambs, and subsequently, these animals can excrete large numbers of cysts into theenvironment.

Epithelial microvillous integrity correlates with enterocyte function in health and disease.Previous studies have shown that intestinal malabsorption in giardiasis was caused, at leastin part, by a diffuse loss of brush border surface area (Buret et al., 1992). Loss of epithe-lial brush border surface area resulting from diffuse microvillous shortening represents theprimary injury responsible for impaired disaccharidase activities and malabsorption of elec-trolytes, nutrients and water in giardiasis (Buret et al., 1992; Farthing, 1997; Koudela andVitovec, 1998; Scott et al., 2000). Conversely, a recent study demonstrated that successfulpharmacotherapy of giardiasis in cattle was associated with significant lengthening of ep-ithelial microvilli (OHandley et al., 2001). Moreover, a number of reports have shown thatdiffuse loss of microvillous length may cause malabsorptive diarrhea in disorders other than


giardiasis, including bacterial enteritis, chronic intestinal anaphylaxis, Crohns disease, andcoeliac disease (Rubin et al., 1966; Dvorak, 1988; Buret et al., 1990b, 1998; Curtis et al.,1990; Buret, 1994). Together, these observations indicate that diffuse reduction of brush bor-der surface area represents a reliable marker for small intestinal malfunction. Findings fromthis present study establish that the avian-derived G. duodenalis used in our investigationscould induce a significant loss of brush border surface area in the jejunum of kittens, andhence may have the potential to cause disease in these and possibly other mammals. Indeed,this epithelial injury was associated with diarrhea in four out of five of the experimentallyinfected kittens.

In summary, our investigations using G. duodenalis trophozoites isolated from a wildparrot demonstrate that some bird species can harbor Giardia that may have the potentialto be infectious to domestic ruminants and companion animals such as sheep and cats.Additional experiments in kittens also indicate that trophozoite colonization by this isolateis pathogenic, as illustrated by the diffuse injury it induces to the epithelial brush bordermicrovilli, the cause of malabsorptive diarrhea common to a number of disorders of thesmall intestine, including giardiasis. Although further studies involving cyst inoculationare necessary to verify the true infectious potential of this isolate, our findings do lendweight to the growing evidence that, unlike G. ardeae and G. psittaci, G. duodenalis mayinfect a variety of host species. From an alternative perspective, it is speculated whether themoribund birds that were the original hosts of this G. duodenalis (Gallagher et al., 1995)may have acquired it from a mammalian source. As waterborne transmission of giardiasisis common, these results raise important implications for water resource and livestockmanagement, especially in regions where surface waters are open to fecal contamination.Future avenues of research should therefore include the monitoring and characterization ofwaterborne Giardia cysts and isolates derived from avian, livestock and other host species.

Acknowledgements

We thank the staff and students of the Department of Biological Sciences, University ofCalgary for their technical expertise and advice throughout this study. The Canadian Inter-national Fellowship Scheme, University of Calgary, Alta., Canada and the Land and WaterResources Research and Development Corporation, Canberra, ACT, Australia, providedfunding for P.A. McDonnell. The Natural Sciences and Engineering Research Council ofCanada funded this research.

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Giardia duodenalis trophozoites isolated from a parrot (Cacatua galerita) colonize the small intestinal tracts of domestic kittens and lambsIntroductionMaterials and methodsParasite cultureSource of specific pathogen-free (SPF) experimental animalsLambsKittens

Animal inoculationLambsKittens

Clinical observationsParasite enumerationFecal cystsLuminal parasites

Transmission electron microscopy

ResultsTrophozoite colonization and cyst developmentClinical manifestationsBrush border ultrastructure of intestinal sections from kittens

DiscussionAcknowledgementsReferences

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