Veterinary Parasitology 111 (2003) 3146

Giardia duodenalis trophozoites isolated from a

parrot (Cacatua galerita) colonize the small
intestinal tracts of domestic kittens and lambs
P.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 b
a

School of Biomolecular and Biomedical Science, Faculty of Science, Griffith University,

Kessels Road, Nathan 4111, Qld, Australia
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 demonstrated
by increasing parasite burdens within the duodenum and jejunum and by fecal passage of cysts
within 4 days post-inoculation. The pathogenesis of the trophozoites was further investigated in
kittens. In these animals, infection significantly reduced jejunal brush border microvillous length
and density, which resulted in a loss of overall epithelial brush border surface area. This injury was
associated with the production of diarrhea in four of five infected kittens. These findings indicate
that some bird species may carry G. duodenalis that represent a possible health threat to companion
animals 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 diarrhea
in 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: S 0 3 0 4 - 4 0 1 7 ( 0 2 ) 0 0 3 4 9 - 7

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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 a
species 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. intestinalis) have successfully infected a variety of experimental hosts, including Mongolian 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 of
zoonotic transmission, as well as the infection of a human volunteer with Giardia derived
from a Gambian giant-pouched rat (Majewska, 1994), support the growing evidence that a
number of G. duodenalis isolates are not host-specific and may infect a wide range of host
species. Possible infection reservoirs include native fauna, such as beavers, muskrats and
some marsupials; companion animals, in particular cats and dogs; and domestic livestock
(Davies and Hibler, 1979; Cribb and Spracklin, 1986; Swan and Thompson, 1986; Pacha
et 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 in
wild birds is well documented and its implication as a source for waterborne and zoonotic
disease remains controversial (Fudge and McEntree, 1986; Georgi et al., 1986; Erlandsen
and 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 have
also been described in aviary birds (Panigrahy et al., 1984; Scholtens et al., 1982; Fudge and
McEntree, 1986; Filippich et al., 1998), but most reports have examined symptomatology
rather 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 (Gallagher
et al., 1995) do warrant further consideration. Upcroft et al. (1997, 1998) reported the development of chronic giardiasis in neonatal and adult Quackenbush Swiss mice experimentally infected with G. duodenalis isolated from a wild-caught, moribund, sulfur-crested
cockatoo (Cacatua galerita), hence describing the first successful inter-class transmission of Giardia infection from an avian source to a mammalian host. Whether this isolate is infectious and pathogenic in companion animals and/or domestic ruminants has
yet to be established. The aim of the present study was to investigate the capacity of
trophozoites of this isolate to colonize the intestinal tracts of domestic kittens and
lambs.
2. Materials and methods
2.1. Parasite culture
The G. duodenalis isolate used in this study is designated BRIS/95/HEPU/2041 (Brisbane/95/Herston Experimental Parasitology Unit/Sample no. 2041). It was obtained from
the 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 trials was established directly from cryopreserved trophozoites of an in vitro culture from the
original 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 of
contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Trophozoites
were grown at 37 C in sterile culture tubes (Disposable Products Inc.) containing TYI-S-33
medium 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. Lambs
Dorset and Suffolk ewes were supplied by and housed at the AgCanada Agricultural
Research Station, Department of Agriculture, Lethbridge, Alta., Canada. Synchronized estrous and parturition in ewes were induced following the procedures described by Olson
et al. (1995). Lambs were manually delivered at normal parturition, washed immediately in
a 4% BetadineTM solution, dried with sterile towels, sexed, weighed and given 100 ml
of bovine colostrum. Over a 3-day birthing period a total of six lambs were obtained
and transferred to an aseptic laboratory isolation room in the Life and Environmental
Science Animal Resource Centre (LESARC), Department of Biological Sciences, University 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 separate air-conditioning conduit regulated ventilation. Room temperature was maintained at
25 C. Lambs were fed a milk replacer diet (Browns Feeds, Calgary, Alta., Canada) for
the 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 Clostridium bacterin-toxoid (Covexin 8TM ) (Coopers Agripharmaceuticals Inc.). The Giardia-free
status of all lambs was confirmed prior to the commencement of trials by the daily collection of feces for 3 days from each lamb and the screening for the presence of Giardia 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. Kittens
Two, pregnant, domestic, short-haired cats were supplied by LESARC, University of
Calgary, Alta., Canada and housed in separate isolation cages. Prior to the commencement
of trials, feces from each adult cat were screened daily for 3 days for the presence of Giardia
cysts using the same technique as described for lambs. Subsequently, two litters, one of three
kittens and one of five, were delivered naturally and kept with their respective mothers until
weaning at 68 weeks of age. Kittens were then removed and divided into two groups
of similar weight and sex distribution. Each group was placed in a separate cage isolated
from 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 food
served 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 to
the commencement of experimental trials, feces from all kittens were screened daily for 3
days for the presence of Giardia cysts using the same method as described for lambs and
adult maternal cats.

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

2.3. Animal inoculation

2.3.1. Lambs
The 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; Yanke
et al., 1998). At 14 days of age, five SPF, Giardia-free, lambs were inoculated by the
standard 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 was
induced with 2% halothane (MTC Pharmaceuticals) and lambs were positioned in dorsal
recumbency. A ventral midline abdominal incision was made and the duodenum isolated. A
suspension of 1 106 trophozoites of G. duodenalis (BRIS/95/HEPU/2041) in 1 ml of cold
TYI-S-33 medium was inoculated intraduodenally through a 20-gauge needle attached to
a 1 ml syringe and the abdomen closed with absorbable sutures. One control lamb received
an intraduodenal injection of sterile TYI-S-33 medium only. Post-operatively, each lamb
was administered a 6 ml intramuscular (IM) injection of PenlongTM (benzathine penicillin
G 150,000 Units/ml and procaine penicillin G 150,000 Units/ml) (Rogar/STB Inc.), and
infected and control lambs were housed in separate isolation rooms. At 8 days pi, lambs
were euthanased with an overdose of sodium pentobarbital (NembutalTM ) and segments
of the small intestine were removed and prepared for trophozoite and cyst enumeration, as
described below.
2.3.2. Kittens
Following 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) received inoculations of cold TYI-S-33 medium only. Each kitten was administered a 0.5 ml
IM injection of PenlongTM after surgery. At 7 days pi, kittens were euthanased with an
overdose of sodium pentobarbital and segments of the small intestine were removed and
prepared for trophozoite and cyst enumeration.
2.4. Clinical observations
Lambs and kittens were observed twice daily for clinical signs of disease, such as
decreased activity level, abnormal eating behavior or altered respiration rate. In
addition, fecal consistency of each animal was scored using the following ratings: (1)
normal, formed stool; (2) soft, unformed stool; and (3) loose, diarrheal
stool.
Lambs were weighed at birth, 1 day prior to inoculation, and daily thereafter before
their morning feed using industrial, platform scales (Mettler Toledo Inc., Columbus, OH).
Kittens were also weighed prior to inoculation and daily thereafter before their morning meal using MC-1 1200S weighing scales (Sartorius Pty Ltd., Mississauga, Ont.).
Percentage weight gains or losses from baseline weights were calculated for all recordings.

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

35

2.5. Parasite enumeration

2.5.1. Fecal cysts
Fecal samples were collected from lambs and kittens at the same time each day (immediately after morning feeding). Two grams of feces were collected per animal, placed in
sterile, 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 and
detected using a fluorescein isothiocyanate (FITC)-labeled monoclonal antibody preparation against Giardia spp. cysts (Giardi-a-GloTM , Waterborne Inc.) and an epifluorescence
microscope (Leica DMRB). Counts of fluorescent cysts were performed at 250 and 400
magnifications and calculated as cysts per gram of feces. Positive and negative control
samples (provided by Waterborne Inc.) were run with test samples.
2.5.2. Luminal parasites
The 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 the
lambs included the duodenum (2 cm distal to the pyloric sphincter and proximal to the ligament 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 proximal to the ileo-caecal valve). The three intestinal segments collected from the kittens were
the duodenum (2 cm distal to the pyloric sphincter), jejunum (approximately 15 cm distal
to 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 of
ice-cold, sterile PBS for at least 30 min, then vortexed to dislodge attached trophozoites from
the intestinal mucosa (Buret et al., 1991). Numbers of trophozoites or cysts per centimeter
of gut section were calculated from the mean of at least five haemocytometer counts on the
intestinal 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 using
transmission electron microscopy as described by Scott et al. (2000). Briefly, jejunal segments (1 cm) of control and infected kittens were removed 10 cm distal to the ligament
of 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 examined
at 75 kV using a Hitachi H7000 transmission electron microscope and micrographs of
midvillous enterocyte apical brush border membranes were taken at the same magnification
(12,000). Microvillous height, width and density were measured and overall brush border
surface 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,

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

2729 micrographs were obtained from 3 to 4 animals and microvillous brush border surface
area was calculated. Previous findings have established that Giardia-induced alterations in
microvillous ultrastructure in the midvillous region are representative of changes along the
entire 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 of
maternal ewes and lambs nor domestic cats and their kittens. However, within 4 days
after inoculation with trophozoites of G. duodenalis BRIS/95/HEPU/2041, 14-day-old
lambs and 68-week-old weaned kittens were excreting viable cysts in their feces. In
contrast, 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 days
pi). Cyst excretion peaked in two infected lambs at 56 days pi with 501767 cysts passed
per gram of feces (Table 1a). This was followed by a decline in cyst numbers and these
lambs were passing an average of 51 cysts per gram of feces at time of death. The main site
of trophozoite colonization in one of these lambs was the distal third of the jejunum with a
mean count of 6.7 103 trophozoites per centimeter of gut section (Table 2a). Intermittent
cyst 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 inoculated
with trophozoites of G. duodenalis (BRIS/95/HEPU/2041)a ; (b) daily mean cyst counts per gram of feces in
control, uninfected kittens and inoculated kittens
(a) Lambs

(b) Kittens

Days
pi

Control, uninfected
lamb, no detectable
cyst excretion
(n = 1)

Inoculated lambs
Single peak
cyst excretion
(n = 2)

Intermittent
cyst excretion
(n = 3)

0
1
2
3
4
5
6
7
8

0
0
0
0
0
0
0
0
0

0
0
0
0
184 17
501 167b
767 567b
67 0
51 17

0
0
0
0
111 16
300 125b
78 16
1889 1031b
867 576

Days
pi

Control, uninfected
kittens (n = 3) no
detectable cyst
excretion

Inoculated
kittens
(n = 5)

0
1
2
3
4
6
7

0
0
0
0
0
0
0

0
0
0
101 34
187 91
687 673

Values are mean S.D.

a Two lambs displayed a single peak in cyst excretion, while the three remaining lambs excreted cysts intermittently.
b Peaks of cyst excretion.

Treatment

Duodenum

Jejunal region
Proximal

Ileum
Mid

Distal

(a) Mean trophozoite burdens (105 ) per centimeter of small intestinal section
Control, uninfected lamb (n = 1)
0
0
Inoculated lambs (single peak)
0
0
Inoculated lambs (intermittent) (n = 3)
1.22 1.62
2.78 2.11
Overall mean for inoculated lambs
0.73 1.39
1.67 2.13

0
0.017 0.017
69.43 42.20
41.67 47.17

0
0.067 0.067
2.89 1.03
1.76 1.60

0
0.042 0.042
2.78 0.75
1.69 1.46

(b) Mean cyst burdens (105 ) per centimeter of small intestinal section
Control, uninfected lamb (n = 1)
0
Inoculated lambs (single peak) (n = 2)
0
Inoculated lambs (intermittent) (n = 3)
0
Overall mean for inoculated lambs
0

0
0
0
0

0
0.20 0.04
0.12 0.07
0.15 0.07

0
1.23 0.50
3.72 1.39
2.73 1.66

0
0
0
0

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 inoculated
lambs that displayed intermittent peaks of cyst excretion. Values are mean S.D.

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

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

37

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

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

Control, uninfected
kittens (n = 3)
Inoculated kittens
(n = 5)

Duodenum

Jejunum

Ileum

Trophozoites

Cysts

Trophozoites

Cysts

Trophozoites

Cysts

2.10 0.73

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 trophozoite
colonization in these lambs was the mid-jejunum with a maximum mean count of 6.94106
trophozoites per centimeter of gut section (Table 2a). Cysts were found predominantly in
the ileum (mean = 3.72 105 ) of these animals, with small numbers also present in the
distal jejunum (Table 2b). No trophozoites or cysts were observed in the intestinal samples
or 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 days
pi, at which time two kittens were passing greater than 1300 cysts per gram of feces (mean
count = 687 cysts per gram of feces). No cysts were detected in the feces of uninfected
control kittens.
Upon necropsy, trophozoites were present in the small intestinal sections of all kittens
inoculated with trophozoites. The main parasite colonization site was the jejunum, with a
mean count of 1.35106 trophozoites per centimeter of gut section (Table 3). Cysts were also
detected in the jejunal and ileal sections with mean counts of 3.2 104 and 7 103 cysts per
centimeter 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 of
Trypan 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 control
kittens.
3.2. Clinical manifestations
No significant differences in weight gain were observed between lambs inoculated with
trophozoites and the uninfected control over the 8-day trial period (data not shown). Daily
fluctuations in fecal consistency were seen in all lambs, irrespective of treatment, and may
have 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 normal
appetite.
Similarly, infected kittens did not show reduced weight gain compared to uninfected
controls (data not shown), but four of five infected kittens did pass soft, unformed stools
at 7 days pi (Table 4b). In contrast, uninfected control kittens passed normal formed stools
for the duration of the trial.

(a) Lambs
Days pi

0
2
3
4
5
6
7
8

(b) Kittens
Control, uninfected lamb (n = 1)

Inoculated lambs (n = 5)

Rating 1

Rating 2

Rating 3

Rating 1

Rating 2

Rating 3

1
1

1
1
1

5
2
5
1
2
1
3
2

4
3
4
2
3

Days pi

0
2
3
4
6
7

Control, uninfected kittens (n = 3)

Inoculated kittens (n = 5)

Rating 1

Rating 2

Rating 3

Rating 1

Rating 2

Rating 3

3
3
3
3
3
3

5
5
5
4
3
1

1
2
4

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

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

Table 4
Fecal consistency ratings of the control, uninfected lamb and lambs intraduodenally inoculated with trophozoites of G. duodenalis (BRIS/95/HEPU/2041) (a), and the
control (uninfected) and infected kittens (b)

39

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

3.3. Brush border ultrastructure of intestinal sections from kittens

Representative transmission electron micrographs illustrating the apical brush border
membrane of midvillous enterocytes of each experimental group (control, uninfected kitten
and infected kitten) are shown in Fig. 1. Infection with trophozoites caused shortening
of the brush border microvilli over the entire epithelium, at sites of parasite attachment
as well as in other areas (Fig. 1). Height, width, density and overall surface area of the
brush border microvilli are described in Table 5. Infection of kittens with trophozoites
Table 5
Brush border microvillous characteristics of midvillous enterocytes from the jejunum of control, uninfected kittens
and kittens intraduodenally inoculated with trophozoites of G. duodenalis (BRIS/95/HEPU/2041)

Control, uninfected
sections (n = 27)
Inoculated sections
(n = 29)

Height (m)

Width (m)

Density
(number/m2
of epithelium)

Surface area
(m2 /m2 of
epithelial surface)

1.242 0.050

0.116 0.0023

72.18 3.19

33.16 1.94

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.

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

41

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

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

4. Discussion
Some previous studies have suggested that rigid host-specificity between major host phylogenetic groups may prevent transmission of giardiasis between birds and mammals. The
avian 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 there
have been limited studies on the infectious potential of avian-derived G. duodenalis-like
organisms in mammals. Observations from the present study indicate that G. duodenalis
trophozoites derived from a wild, native parrot colonized the intestinal tracts of lambs and
kittens and were pathogenic to the latter mammal species. These results are consistent
with those of Upcroft et al. (1997, 1998), who reported infections of the same G. duodenalis isolate in neonatal mice were more intense with longer patency periods than those

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

of two human-derived isolates. In the present study, this isolate produced heavy infections
in 68-week-old weaned kittens and 14-day-old lambs, and produced a diffuse loss of
epithelial brush border surface area in the jejunum of kittens.
Numerous studies have examined the transmission and zoonotic potential of G. duodenalis derived from mammalian companion animals (Swan and Thompson, 1986; Thompson
et al., 1988; Thompson and Boreham, 1994), yet investigations on trophozoite colonization 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 examined
the 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 inoculated
with trophozoites of avian-derived G. duodenalis (BRIS/95/HEPU/2041) excreted viable
fecal cysts after 4 days pi, with most lambs shedding cysts intermittently. Intermittent excretion 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 lambs
is the mid-to-distal jejunum, while cysts are most predominant in the distal jejunum and
ileum.
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 and
Vitovec, 1998), foals (Kirkpatrick and Skand, 1985) and lambs (Kiorpes et al., 1987; Olson
et al., 1995). As our experiments sought to characterize trophozoite colonization rather
than 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 and
kittens requires further investigation. In sheep, cattle, as well as in other mammals, it is
well established that giardiasis may cause diarrhea while in other instances the infection
may 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 visibility
of symptoms may contribute to the failure to recognize the infection in some livestock
species. The results shown here indicate that G. duodenalis trophozoites derived from an
avian source can colonize, replicate and become viable cysts within the intestinal tract
of lambs, and subsequently, these animals can excrete large numbers of cysts into the
environment.
Epithelial microvillous integrity correlates with enterocyte function in health and disease.
Previous studies have shown that intestinal malabsorption in giardiasis was caused, at least
in part, by a diffuse loss of brush border surface area (Buret et al., 1992). Loss of epithelial brush border surface area resulting from diffuse microvillous shortening represents the
primary injury responsible for impaired disaccharidase activities and malabsorption of electrolytes, nutrients and water in giardiasis (Buret et al., 1992; Farthing, 1997; Koudela and
Vitovec, 1998; Scott et al., 2000). Conversely, a recent study demonstrated that successful
pharmacotherapy of giardiasis in cattle was associated with significant lengthening of epithelial microvilli (OHandley et al., 2001). Moreover, a number of reports have shown that
diffuse loss of microvillous length may cause malabsorptive diarrhea in disorders other than

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43

giardiasis, including bacterial enteritis, chronic intestinal anaphylaxis, Crohns disease, and
coeliac 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 border surface area represents a reliable marker for small intestinal malfunction. Findings from
this present study establish that the avian-derived G. duodenalis used in our investigations
could induce a significant loss of brush border surface area in the jejunum of kittens, and
hence 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 experimentally
infected kittens.
In summary, our investigations using G. duodenalis trophozoites isolated from a wild
parrot demonstrate that some bird species can harbor Giardia that may have the potential
to be infectious to domestic ruminants and companion animals such as sheep and cats.
Additional experiments in kittens also indicate that trophozoite colonization by this isolate
is pathogenic, as illustrated by the diffuse injury it induces to the epithelial brush border
microvilli, the cause of malabsorptive diarrhea common to a number of disorders of the
small intestine, including giardiasis. Although further studies involving cyst inoculation
are necessary to verify the true infectious potential of this isolate, our findings do lend
weight to the growing evidence that, unlike G. ardeae and G. psittaci, G. duodenalis may
infect a variety of host species. From an alternative perspective, it is speculated whether the
moribund 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 giardiasis
is common, these results raise important implications for water resource and livestock
management, especially in regions where surface waters are open to fecal contamination.
Future avenues of research should therefore include the monitoring and characterization of
waterborne 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 of
Calgary for their technical expertise and advice throughout this study. The Canadian International Fellowship Scheme, University of Calgary, Alta., Canada and the Land and Water
Resources Research and Development Corporation, Canberra, ACT, Australia, provided
funding for P.A. McDonnell. The Natural Sciences and Engineering Research Council of
Canada funded this research.
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