Professional Documents
Culture Documents
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).
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
780
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
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
55
26
30
40
55
40
111
9
40
55
40
Total
55
26
165
255
Freshwater sh
Canning/Swan River
Blackwood River
Total
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.
781
Primer name
Primer sequence
Product (bp)
Reference
18S rRNA
RH11 external F
RH4LM external R
GIAR18SER
GIAR18SIR
CATCCGGTCGATCCTGCC
GTCGAACCCTGATTCTCCG
GACGCTCTCCCCAAGGAC
CTGCGTCACGCTGCTCG
292
150
AL3543
Al3546
AF
AR
EF
ER
BF
BR
AAATIATGCCTGCTCGTCG
CAAACCTTITCCGCAAACC
CGCCGTACACCTGTCA
AGCAATGACAACCTCCTTCC
CCCCTTTCTGCCGTACATTTAT
GGCTCGTAAGCAATAACGACTT
GTTGTTGTTGCTCCCTCCTTT
CCGGCTCATAGGCAATTACA
gdh
GDHeF
GDHiF
GDHiR
TCAACGTYAATCGYGGYTTCCGT
CAGTACAACTCYGCTCTCGG
GTTRTCCTTGCACATCTC
450
bg
G7 external F
G759 external R
G internal F
G internal R
AAGCCCGACGACCTCACCCGCAGTGC
GAGGCCGCCCTGGATCTTCGAGACGAC
GAACGAACGAGATCGAGGTCCG
CTCGACGAGCTTCGTGTT
753
tpi
388
400
511
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
Total
27/709
782
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
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
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
64
Assemblage E Ad133-AY178740
BAR205 (Barramundi, Assemblage E)
MW151 (Mulloway, Assemblage E)
MW153 (Mulloway, Assemblage E)
100
79
91
100
63
77
99
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.
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.
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
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
64
B4-AY07272
100
50
51
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
Fig. 3. Section from the intestine of a sea mullet (Mugil cephalus), showing Giardia trophozoites and cysts (stained with H&E).
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,
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