International Journal for Parasitology 40 (2010) 779785

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International Journal for Parasitology

journal homepage: www.elsevier.com/locate/ijpara

Identication of zoonotic Giardia genotypes in sh q

Rongchang Yang a, Anna Reid a, Alan Lymbery b, Una Ryan a,*
a
b

Division of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, WA 6150, Australia
Fish Health Unit, Centre for Fish and Fisheries Research, Murdoch University, Murdoch, WA 6150, Australia

a r t i c l e

i n f o

Article history:
Received 25 September 2009
Received in revised form 30 November 2009
Accepted 2 December 2009

Keywords:
Giardia
Fish
18S rRNA
gdh
tpi
Beta-giardin
Assemblage A2
Assemblage B3
Assemblage B4
Zoonotic

a b s t r a c t
Apart from a single record in a shark, there have been no published studies conducted on Giardia genotypes in sh. The present study investigated the prevalence of Giardia in cultured ngerlings (n = 227),
wild freshwater (n = 227) and wild marine/estuarine species (n = 255) of sh in Western Australia by
PCR amplication at the 18S rRNA, glutamate dehydrogenase (gdh), triose phosphate isomerase (tpi)
and beta-giardin (bg) loci. Results revealed a low prevalence of Giardia, 3.8% (27/709), in sh hosts.
The zoonotic Giardia species, Giardia duodenalis assemblages A, B as well as G. duodenalis assemblage E
and Giardia microti were detected. The identication of zoonotic species of Giardia highlights the public
health importance of investigating parasites within sh host species.
2009 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction
Species of Giardia are protozoan parasites and one of the most
common causes of diarrhoea in humans and other animals (Caccio
and Ryan, 2008). Currently six species of Giardia are recognized,
Giardia duodenalis (syn. Giardia intestinalis and Giardia lamblia) in
humans and many other species of vertebrates, particularly mammals, Giardia muris in rodents, Giardia agilis in amphibians, Giardia
ardeae in herons, Giardia psittaci from psittacine birds and Giardia
microti from voles and muskrats (cf. Caccio and Ryan, 2008).
Giardia duodenalis is the only species found in humans. There is
considerable genetic variation within G. duodenalis and several
major morphologically similar but genetically distinct assemblages/genotypes have been identied. Assemblages A and B are
found in both humans and non-humans; assemblages C and D in
dogs, assemblage E in cattle, sheep and pigs, assemblage F in cats
and assemblage G in rats (Monis et al., 1999; Van der Giessen
et al., 2006; Caccio and Ryan, 2008).
Assemblages A and B can be further subdivided into four subgroups, with the subgroups A1 and A2 within assemblage A and

q
Note: Partial gdh and bg sequences generated as part of this study have been
deposited in the GenBank database under Accession Nos. GQ919295GQ919301
and GQ919292GQ919294.
* Corresponding author. Tel.: +61 08 9360 2482; fax: +61 08 9310 4144.
E-mail address: Una.Ryan@murdoch.edu.au (U. Ryan).

the subgroups 3 and 4 within assemblage B (Monis et al., 1999;

Caccio and Ryan, 2008). However, more recent genetic characterisation studies at the beta-giardin (bg), triosephosphate isomerase
(tpi) and glutamate dehydrogenase gene (gdh) loci have suggested
greater genetic variability exists among a number of assemblages
of G. duodenalis than was rst thought (Caccio and Ryan, 2008).
Very little is known about the prevalence, genetic diversity and
effect of species of Giardia in marine environments and the role
that marine animals play in transmission of these parasites to
humans. Giardia spp. have been reported in California sea lions
(Zalophus californianus), harp seals (Phoca groenlandica), grey seals
(Halichoerus grypus), harbor seals (Phoca vitulina), bowhead whales
(Balaena mysticetus), North Atlantic right whales (Eubalaena glacialis), ringed seals (Phoca hispida), bearded seals (Erignathus barbatus),
and beluga whales (Delphinapterus leucas) (Olson et al., 1997; Measures and Olson, 1999; Deng et al., 2000; Hughes-Hanks et al.,
2005). Giardia duodenalis assemblages A and B have been reported
in the feces of dolphins (Delphinus delphis and Grampus griseus),
porpoises (Phocoena phocoena), seals (E. barbatus, P. groenlandica,
P. hispida and P. vitulina) and a thresher shark (Alopias vulpinus)
(Lasek-Nesselquist et al., 2008; Dixon et al., 2008). Assemblage D
and assemblage C and D-like sequences have been isolated from
harbor seals (Gaydos et al., 2008). Assemblage A has also been
identied in Macoma clams (Macoma balthica and Macoma mitchelli in the Rhode River, USA, Graczyk et al., 1999). Apart from the
single record in a shark referred to above (Lasek-Nesselquist

0020-7519/$36.00 2009 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.ijpara.2009.12.001

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R. Yang et al. / International Journal for Parasitology 40 (2010) 779785

et al., 2008), there have been no published studies conducted on

Giardia genotypes in sh. The aim of the present study was to
determine the prevalence of different species and assemblages of
Giardia from wild freshwater and marine shes sampled from
Western Australian waters, and from cultured ngerlings collected
from hatcheries throughout Australia.
2. Materials and methods
2.1. Sample collection
Three different groups of sh, cultured ngerlings (juvenile
sh), wild freshwater species and wild marine/estuarine species
were screened (Table 1). Fingerlings (227 sh belonging to eight
species) were collected from ve commercial aquaculture hatcheries within Australia (Table 1). A total of 255 marine sh from ve
species from estuarine and coastal locations were obtained from
commercial shers. Wild freshwater sh species (227 sh belonging to eight species) were collected from two river systems in the
southwest of Western Australia, the Canning/Swan River and the
Blackwood River.
On arrival in the laboratory, sh were measured for length and
weight, dissected and using a scalpel blade, stomach and intestinal
epithelial cells were scraped off and placed into a 1.5 mL Eppendorf
tube. Remaining stomach and intestinal tissue were stored in 70%
ethanol.
2.2. DNA extraction and PCR amplication
DNA was extracted from 25 mg of intestinal and stomach tissue scrapings from each sh sample using a Qiagen DNeasy tissue

kit (Qiagen, Germany). DNA was eluted in 50 lL of elution buffer to

concentrate the DNA. All extracted DNA samples were stored at
20 C until required for screening.
All samples were screened at the 18S rRNA locus and positive
samples were genotyped by sequencing. Amplication of a fragment of the Giardia 18S rRNA gene was performed as described
(Hopkins et al., 1997; Read et al., 2002) (Table 2). Samples that
were positive at the 18S locus were also amplied at the gdh, tpi
and bg loci (Table 2). A fragment of the gdh locus was amplied
as previously described (Read et al., 2004). For the tpi locus, the primary reaction was amplied as described by Sulaiman et al.
(2003). For the second round reaction, assemblage-specic primers
and conditions for A, B and E were as previously described (Geurden et al., 2008, 2009). A fragment of the bg locus was amplied as
previously described (Sulaiman et al., 2004).
PCR contamination controls were used including negative controls and separation of preparation and amplication areas. The
amplied DNA fragments from the secondary PCR product were
separated by gel electrophoresis and puried using the freeze
squeeze method (Ng et al., 2006). A spike analysis (addition of
0.5 lL of Giardia-positive control into each sample) was conducted
on randomly selected Giardia-negative samples from each group of
DNA extractions to determine whether negative results were due
to PCR inhibition.
2.3. Sequence and phylogenetic analyses
Puried PCR products were sequenced using an ABI Prism Dye
Terminator cycle sequencing kit (Applied Biosystems, Foster City,
California, USA) according to the manufacturers instructions with
the exception that the annealing temperature was raised to 58 C.

Table 1
Cultured ngerlings, wild marine and wild freshwater sh species collected during this study.
Fingerlings

Locations
Hatchery 1

Hatchery 2

Hatchery 3

Hatchery 4

Hatchery 5

Black bream (Acanthopagrus butcheri)

Snapper (Pagrus auratus)
Barramundi (Lates calcarifer)
Yellow tail kingsh (Seriola lalandi)
Mulloway (Argyrosomus japonicus)
Jade perch (Scortum barcoo)
Silver perch (Bidyanus bidyanus)
Murray cod (Maccullochella peelii peelii)

17

11
10
48
9
10

28

20
33

41

11
10
76
9
51
20
33
17

Total

17

88

28

53

41

227

Marine sh

Total

Estuary sampling

Coastal sampling

Total

Peel estuary

Leschenault estuary

Shark Bay

Perth

Sea mullet (Mugil cephalus)

Common blow sh (Torguigener pleurogramma)
King George whiting (Sillaginodes punctata)
School whiting (Sillago vittata)
Yellow eyed mullet (Aldrichetta forsteri)

55

26

30

40
55
40

111
9
40
55
40

Total

55

26

165

255

Freshwater sh

Canning/Swan River

Blackwood River

Total

Western minnow (Galaxias occidentalis)

Western hardyhead (Leptatherina wallacei)
Mosquito sh (Gambusia holbrooki)
Nightsh (Bostockia porosa)
Western pigmy perch (Edelia vittata)
One spot live bearer (Phalloceros caudimaculatus)
Goldsh (Carassius auratus)
Freshwater cobbler (Tandanus bostocki)

30
5
10

91
6
16
15
3
1
1
48

121
11
26
15
4
1
1
48

Total

46

181

227

Location of Hatcheries: Hatchery 1 was located in Alexander, Victoria, hatchery 2 in Fremantle, Western Australia, hatchery 3 in West Beach, South Australia, hatchery 4 in
Childers, Queensland and hatchery 5 was located in West Beach, South Australia.

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R. Yang et al. / International Journal for Parasitology 40 (2010) 779785

Table 2
PCR primers for the various loci examined in the present study. I = inosine (modied adenine). I can base pair with C, A or T. Y = C or T, R = A or G.
Target

Primer name

Primer sequence

Product (bp)

Reference

18S rRNA

RH11 external F
RH4LM external R
GIAR18SER
GIAR18SIR

CATCCGGTCGATCCTGCC
GTCGAACCCTGATTCTCCG
GACGCTCTCCCCAAGGAC
CTGCGTCACGCTGCTCG

292

Hopkins et al. (1997)

150

Read et al. (2002)

AL3543
Al3546
AF
AR
EF
ER
BF
BR

AAATIATGCCTGCTCGTCG
CAAACCTTITCCGCAAACC
CGCCGTACACCTGTCA
AGCAATGACAACCTCCTTCC
CCCCTTTCTGCCGTACATTTAT
GGCTCGTAAGCAATAACGACTT
GTTGTTGTTGCTCCCTCCTTT
CCGGCTCATAGGCAATTACA

gdh

GDHeF
GDHiF
GDHiR

TCAACGTYAATCGYGGYTTCCGT
CAGTACAACTCYGCTCTCGG
GTTRTCCTTGCACATCTC

450

Read et al., 2004

bg

G7 external F
G759 external R
G internal F
G internal R

AAGCCCGACGACCTCACCCGCAGTGC
GAGGCCGCCCTGGATCTTCGAGACGAC
GAACGAACGAGATCGAGGTCCG
CTCGACGAGCTTCGTGTT

753

Sulaiman et al. (2004)

tpi

Nucleotide sequences were analysed using Chromas lite version

2.0 (http://www.technelysium.com.au) and aligned with reference
genotypes from GenBank using Clustal W (http://www.clustalw.
genome.jp).
Phylogenetic trees were constructed using additional isolates
from GenBank. Distance estimation was conducted using TREECON
(Van de Peer and De Wachter, 1997), based on evolutionary distances calculated with the Tamura-Nei model and grouped using
the neighbour-joining (NJ) method. Parsimony analyses were conducted using MEGA version 3.1 (MEGA3.1: Molecular Evolutionary
Genetics Analysis software, Arizona State University, Tempe,
Arizona, USA). Bootstrap analyses were conducted using 1000
replicates to assess the reliability of inferred tree topologies. Maximum likelihood (ML) analyses were conducted using the program
PhyML (Dereeper et al., 2008) and the reliability of the inferred
trees was assessed by the approximate likelihood ratio test (aLRT)
(Anisimova and Gascuel, 2006). Trees were rooted using sequences
from G. ardeae and G. muris as outgroups.
2.4. Microscopy
Tissue samples that were PCR-positive were embedded in parafn and histological sections were cut at 5 mm and stained with
H&E. Parasites were examined with the aid of an ocular micrometer in a Zeiss Axioskop microscope at 1000 magnication.
2.5. Statistical analysis
Prevalences were expressed as percentages of positive samples,
with 95% condence intervals (CIs) calculated assuming a binomial
distribution, using the software Quantitative Parasitology 3.0 (Rozsa et al., 2000).
3. Results
3.1. Prevalence of Giardia in sh
Twenty-seven of the 709 sh (3.8%) analysed were positive for
species of Giardia by PCR at the 18S locus (Table 3). Hatchery
reared ngerlings had the highest prevalence at 8.4% (19/227),
while wild marine sh had a prevalence of 2.7% (7/255) and wild
freshwater sh had a prevalence of 0.4% (1/227). The 19 positive

Sulaiman et al. (2003)

332

Geurden et al. (2008, 2009)

388
400

511

samples from ngerlings came from barramundi (11/76; 14.5%,

95% CI 7.924.2%), black bream (4/11; 36.4%, 95% CI 13.566.7%)
mulloway (3/51; 5.9%, 95% CI 1.616.4%) and snapper (1/10;
10.0%, 95 % CI 0.544.6%). Of the freshwater sh, the single positive
sample was from a western minnow from the Blackwood River. All
seven positive samples from marine/estuarine sh were from mullet (7/111; 6.3%, 95% CI 3.012.5%). Six of the seven positive mullet
samples were from the Peel estuary (n = 55; 10.9%, 95% CI 4.9
22.5%) and one was from coastal waters off Shark Bay (n = 26;
3.8%, 95% CI 0.218.8%).

3.2. Identication of Giardia spp. in sh

At the 18S locus, only 22 of the 27 isolates were successfully sequenced (Table 4). At the gdh and bg loci, 23 and 13 isolates were
successfully sequenced, respectively. At the tpi locus 23 isolates
were typed (Table 4). Three species/assemblages were identied
at the 18S locus; assemblage B in 14 isolates, assemblage A in seven isolates and G. microti in one isolate (Table 4). Typing at the
tpi locus agreed largely with the typing at the 18S locus with the
exception of two isolates; isolate MW151 which was a mixed A
and E infection at the tpi locus and was assemblage A at the 18S locus, and isolate ML138 which was assemblage A at the tpi locus but
assemblage B at the 18S locus (Table 4). Three isolates (BAR37,
SNAP147 and ML312), which were unable to be sequenced at the
18S locus, were all identied as assemblage A at the tpi locus. Typing at the gdh locus identied nine isolates as assemblage A2, six as
assemblage B3, ve as assemblage B4 and two as assemblage E (Table 4). Typing at the gdh locus was also largely in agreement with
the 18S locus, with the exception of the same two isolates (ML138
and MW151) that did not concur with the tpi locus; ML138 was
assemblage A2 and MW151 was assemblage E at the gdh locus.

Table 3
Prevalence of Giardia in cultured, wild freshwater and wild marine sh. Upper and
lower 95% condence intervals are given in parentheses.
Number positive

Prevalence

Cultured sh
Freshwater sh
Marine sh

19/227
1/227
7/255

8.4 (4.8, 12.0)

0.4 (0, 1.3)
2.7 (0.7, 4.8)

Total

27/709

3.8 (2.4, 5.2)

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R. Yang et al. / International Journal for Parasitology 40 (2010) 779785

Table 4
Giardia spp./assemblages identied in ngerlings, wild freshwater and wild marine host species at the 18S rRNA, tpi, gdh and bg loci.
Sample

Fish species

Microscopy positive (Y/N)

18S

tpi

gdh

bg

Fingerlings
BAR26
BAR37
BAR158
BAR162
BAR164
BAR165
BAR166
BAR168
BAR172
BAR177
BAR205
BB3
BB12
BB157
BB161
MW151
MW153
MW154
SNAP147

Barramundi
Barramundi
Barramundi
Barramundi
Barramundi
Barramundi
Barramundi
Barramundi
Barramundi
Barramundi
Barramundi
Black bream
Black bream
Black bream
Black bream
Mulloway
Mulloway
Mulloway
Snapper

N
N
Y
Y
Y
N
Y
N
N
N
Y
Y
N
N
N
Y
N
N
N

Positive no seq.
Positive no seq.
A
A
B
Giardia microti
A
A
B
B
Positive no seq.
B
B
A
A
A
B
B
Positive no seq.

N/A
A
A
A
B
N/A
A
A
B
B
E
B
B
A
A
A+E
B
A
A

N/A
A2
A2
A2
B3
N/A
A2
A2
B3
B3
E
B3
B4
N/A
A2
E
B4
B3
A2

N/A
A
A
A
N/A
N/A
A
A
N/A
N/A
E
N/A
N/A
B
A
E
N/A
A
B

Freshwater
BW156

Western minnow

B4

N/A

Marine
ML134
ML135
ML138
ML148
ML155
ML156
ML313

Sea
Sea
Sea
Sea
Sea
Sea
Sea

N
Y
N
N
Y
N
N

B
B
B
B
B
B
Positive no seq.

B
B
A
B
N/A
N/A
A

B4
B4
A2
B3
N/A
N/A
A2

N/A
N/A
A
N/A
N/A
N/A
A

mullet
mullet
mullet
mullet
mullet
mullet
mullet

N/A, no amplication; seq., sequence.

0.1
BAR166 (Barramundi, Assemblage A2)
BAR168 (Barramundi, Assemblage A2)
BAR158 (Barramundi, Assemblage A2)
BAR161 (Barramundi, Assemblage A2)
BB161 (Barramundi, Assemblage A2)
88

Assemblage A2-Ad2-L40510
SNP147 (Snapper, Assemblage A2)

100

ML313 (Sea mullet, Assemblage A2)

BAR37 (Barramundi, Assemblage A2)

64

Assemblage A1 Ad1 L40509

100

Assemblage E Ad133-AY178740
BAR205 (Barramundi, Assemblage E)
MW151 (Mulloway, Assemblage E)
MW153 (Mulloway, Assemblage E)

100

BB12 (Black bream, Assemblage B4 - GQ919297)

75
ML135 (Sea mullet, Assemblage B4)
89
BB157 (Black bream, Assemblage B4 - GQ919298)
63
ML134 (Sea mullet, Assemblage B4 - GQ919295)

79

91
100

Assemblage B4 Ad-45 AY178739

BW156 (Western minnow, Assemblage B4 - GQ919296)
Assemblage B3 BAH12 AF069059

MW154 (Mulloway, Assemblage B3)

63

BAR172 (Barramundi, Assemblage B3 - GQ919301)

82

BAR177 (Barramundi, Assemblage B3)

77

BAR3 (Barramundi, Assemblage B3 - GQ919299)

99

BAR164 (Barramundi, Assemblage B3 - GQ919300)

ML148 (Sea mullet, Assemblage B3)

G. ardeae
Fig. 1. Phylogenetic relationships of sh-derived Giardia isolates inferred by neighbour-joining analysis of Tamura-Neis distances calculated from pair-wise comparisons of
glutamate dehydrogenase (gdh) sequences. Percentage bootstrap support (>70%) from 1000 replicate samples is indicated at the left of the supported node.

R. Yang et al. / International Journal for Parasitology 40 (2010) 779785

Table 5
Polymorphisms in sh-derived Giardia duodenalis isolates compared with B3 reference isolate BAH12 (AF069059) at the glutamate dehydrogenase (gdh) locus.
Polymorphic sites are numbered with reference to the full-length gene.
Isolate

Nucleotide positions
243

339

438

465

483

585

B3-BAH12
Bar3
MW154
ML135

T
T
T
C

C
T
C
C

C
T
T
T

C
T
T
T

G
A
A
A

T
C
C
C

B4
BB157
ML134
ML135
BW156

Nucleotide positions
354

357

429

489

552

606

612

666

671

G
G
G
G
A

T
C
C
C
T

C
T
C
C
C

T
T
T
C
T

G
A
A
A
G

C
T
T
T
C

A
G
G
G
A

T
C
C
C
C

A
G
G
G
A

18S and tpi loci but assemblage B at the bg locus. The isolate
BAR165 identied as G. microti at the 18S locus was unable to be
amplied at the other three loci.

3.3. Phylogenetic analysis of Giardia spp. in sh

Table 6
Polymorphisms in sh-derived Giardia duodenalis B4 isolates compared with B4
reference isolate Ad45 (AY178739) at the glutamate dehydrogenase (gdh) locus.
Polymorphic sites are numbered with reference to the full-length gene.
Isolate

783

At the bg locus, most of the assemblage B isolates did not amplify.

Typing at the bg locus was largely in agreement with the other
three loci with the exception of three isolates, BB157, SNAP147
and MW153. The latter two were assemblage A and B at the tpi
and gdh loci, respectively, but assemblage B and E, respectively,
at the bg locus (Table 4). Isolate BB157 was assemblage A at the

Phylogenetic analyses of the partial nucleotide sequences of the

two most variable loci (gdh and bg) examined in the present study
using distance, parsimony and ML analyses produced similar results (data not shown). Analysis at the gdh locus identied that
all of the eight assemblage A2 isolates were 100% identical to the
A2 reference isolate AD-2 (L40510), the three assemblage E isolates
were also 100% identical to reference isolate Ad133 (AY178740)
and there was variation within the assemblage B3 and B4 isolates
(Fig. 1 NJ distance tree shown). Within B3, no isolates matched
100% with the reference B3 isolate BAH12 (AF069059) but were
different from BAH12 by four and ve single nucleotide polymorphisms (SNPs) (Table 5).
Within B4, again no isolates matched 100% with the B4 reference isolate Ad45 (AY178739), but differed by two and ve SNPs
(Table 6). All of the variations in both B3 and B4 isolates were synonymous changes and therefore resulted in no amino acid differences, with the exception of a SNP at position 671 which
resulted in an amino change from asparagine to serine. Both are
polar and neutral amino acids.
At the bg locus, the sh isolates BAR37, BAR158, BAR162,
BAR168, BB161, ML313, ML156 and MW154 had multiple SNPs
and did not match 100% with A1 (X85958), A2 (AY072723) or A3
(AY072724) but still grouped with assemblage A (Fig. 2). Similarly,
SNAP147 did not match 100% with either B3 or B4. Isolates MW151

0.05
ML156 (Sea mullet, Assemblage A)
BB161 (Black bream, Assemblage A)
MW154 (Mulloway, Assemblage A)
BAR162 (Barramundi, Assemblage A)
BAR158 (Barramundi, Assemblage A)
BAR168 (Barramundi, Assemblage A)
56 BAR37 (Barramundi, Assemblage A)
59 ML313 (Sea mullet, Assemblage A - GQ919293)
A1-X85958
99
A3-AY07272
63
A2-AY0727234
BAR166 (Barramundi, Assemblage A - GQ919292)
BAR205 (Barramundi, Assemblage E)

100

E-AY072729
100

MW151 (Mulloway, Assemblage E)

MW153 (Mulloway, Assemblage E)

64

B4-AY07272
100
50
51

SNAP147 (Snapper, Assemblage B - GQ919294)

B3-AY072726
EU274391
B3-BAH 8-AY072727

G. muris
Fig. 2. Phylogenetic relationships of sh-derived Giardia isolates inferred by neighbour joining analysis of Tamura-Neis distances calculated from pair-wise comparisons of
beta-giardin (bg) sequences. Percentage bootstrap support (>70%) from 1000 replicate samples is indicated at the left of the supported node.

784

R. Yang et al. / International Journal for Parasitology 40 (2010) 779785

Fig. 3. Section from the intestine of a sea mullet (Mugil cephalus), showing Giardia trophozoites and cysts (stained with H&E).

and MW153 were identical to the assemblage E reference isolate

P15 (AY072729). Isolate BAR205 differed from P15 by one SNP.
3.4. Nucleotide sequence accession numbers
Partial gdh and bg sequences generated as part of this study
have been deposited in the GenBank database under accession
numbers GQ919295 GQ919301 and GQ919292GQ919294.
3.5. Microscopy
Giardia was detected in tissue samples by microscopy in 10 isolates (Table 4) and several isolates had large numbers (>100 parasites per eld of view) of Giardia trophozoites and cysts (Fig. 3).
Cysts were elliptically shaped and ranged in size from 812  7
10 lm in size. Trophozoites were between 920  712 lm in size.
However, accurate measurements were not possible because sh
tissue autolyses very quickly which can distort morphological features (Woo, 2006).
4. Discussion
In the present study, we believe Giardia was identied for the
rst time in cultured, wild freshwater and wild marine sh hosts
in Australia. Prevalences ranged from 0.4% in freshwater sh to
8.4% in cultured sh species. Analysis at four different loci identied a total of four species/assemblages, G. duodenalis assemblages
A, B and E and G. microti.
Giardia microti has been reported from voles and muskrats (Van
Keulen et al., 1998) and microti-like sequences have been identied
in a dog sample and a deer mouse (Van Keulen et al., 2002). The
nding of G. microti in a barramundi ngerling in the present study
suggests that this species might not be as host-specic as previously thought.
Giardia duodenalis assemblage E was identied in two isolates
from cultured ngerlings; isolate BAR205 from a barramundi and
MW151 from a mulloway. Isolate MW151 also appeared to have
an assemblage A infection at the tpi locus. Assemblage E is usually
reported in cattle, sheep and pigs and is thought to be moderately
host-specic (Caccio and Ryan, 2008), although a recent study reported that 15% of positive Giardia isolates from humans in Egypt
belonged to assemblage E (Foronda et al., 2008). This is the rst
published report of assemblage E in sh hosts and calls into question the presumed host-specicity of the livestock genotype
(assemblage E).
Giardia duodenalis assemblages A and B are both zoonotic,
infecting a variety of human and non-human mammal hosts
including dogs, cats, livestock and wildlife (Caccio and Ryan,
2008). Potentially zoonotic G. duodenalis assemblages A and B com-

prised 37% and 52%, respectively, of all positive isolates in the present study. Assemblage B was identied in all three sample groups
(wild marine, wild freshwater and cultured ngerlings) with the
highest prevalence in marine mullet (6/7). Assemblage A was identied in cultured ngerlings and in one marine mullet.
As analysis was conducted on scrapings from intestinal and
stomach contents and not from faecal samples, this demonstrated
that the sh were likely infected with these zoonotic genotypes
and not acting as mechanical vectors. In addition, histological studies identied large numbers of Giardia trophozoites and cysts in the
intestine of a number of host individuals, which suggests infection
and growth of zoonotic Giardia assemblages in sh. Giardia assemblage B was originally thought to be specic to humans, but has
more recently been identied within a variety of other animal
hosts (Caccio and Ryan, 2008). It is often the most prevalent Giardia
assemblage reported in studies of human infection and is the most
prevalent assemblage in Australians, recently identied in 75% of
sporadic human cases of giardiasis in Western Australia (Caccio
and Ryan, 2008; Yang et al., in press). As assemblage B is very prevalent amongst human populations, it is possible that the source of
infections in sh in the present study could be due to exposure to
human sewage and urban and agricultural run-off. The identication of assemblages A and B and well as E and G. microti in sh supports this hypothesis. It is possible that the disease may then pass
back to humans through exposure to contaminated faecal material
and gut contents from sh. There have been a number of reports of
potential transmission of Giardia from sh to humans, one of which
involved an intestinal infection in Thai laborers in Taipei resulting
from the consumption of uncooked freshwater sh (Cheng and
Shieh, 2000). Infections have also been linked to home-canned salmon in the USA (Osterholm et al., 1981).
All of the assemblage A isolates were typed as A2 at the gdh locus and were genetically identical. At the bg locus it was not possible to assign isolates to A1 or A2. This is because at this locus, and
using the current primers, there is only one substitution clearly differentiating the A1/A2 subgroups. In the present study, a number
of other SNPs were detected, resulting in the isolates not grouping
with either A1 or A2. Similarly with isolate SNAP147, which typed
with assemblage A at the tpi and gdh loci and grouped with assemblage B at the bg locus, it was not possible to subtype it as B3 or B4.
Previous studies have shown that the bg gene consistently demonstrates numerous subgroups within all assemblages represented
by sufcient samples (Wielinga and Thompson, 2007), but that
these subgroups do not necessarily match the groupings determined at other loci (Wielinga and Thompson, 2007).
In the present study, Giardia isolates from sh were analysed at
four different loci. Analysis of Giardia isolates using at least two loci
is essential due to assemblage swapping or the assignment of
Giardia isolates to different assemblages using different markers,
which has been frequently reported with Giardia (Caccio and Ryan,

R. Yang et al. / International Journal for Parasitology 40 (2010) 779785

2008; Traub et al., 2004). There are various explanations for

assemblage swapping such as mixed infections and/or meiotic
recombination (Caccio and Ryan, 2008). The use of assemblagespecic primers at the tpi locus conrmed the existence of mixed
infections with different assemblages in the same host and highlights the importance of using assemblage-specic primers for
analysis of variation in Giardia.
In conclusion, zoonotic Giardia assemblages were identied for
the rst time in ve different species of sh from freshwater, marine and cultured environments. The nding of genetically identical
isolates in different sh species suggests that infections with Giardia are transmitted between these species, and that such transmission may contribute to the rapid spread of this parasite within and
between aquatic and terrestrial environments. This highlights the
public health importance of further investigations of this parasite
in sh hosts.

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