Veterinary Parasitology 189 (2012) 182188

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Veterinary Parasitology
journal homepage: www.elsevier.com/locate/vetpar

Comparisons of mammalian Giardia duodenalis assemblages based on

the -giardin, glutamate dehydrogenase and triose phosphate
isomerase genes
Andrea V. Scorza , Lora R. Ballweber, Sahatchai Tangtrongsup, Carla Panuska,
Michael R. Lappin
Department of Clinical Sciences, 300 West Drake Road, Fort Collins, CO 80525, United States

a r t i c l e

i n f o

Article history:
Received 24 May 2011
Received in revised form 16 April 2012
Accepted 20 April 2012
Keywords:
Giardia
Dogs
Cats
Genotyping

a b s t r a c t
The objective of this study was to determine and compare the assemblages of Giardia
duodenalis isolated from mammalian fecal samples using the -giardin (bg), glutamate
dehydrogenase (gdh) and triosephosphate isomerase (tpi) genes. A total of 202 samples,
either submitted to the Veterinary Diagnostic Laboratory (Parasitology) at Colorado State
University or part of ongoing research studies, were typed. A subset of 50 dog samples
were also assessed by the tpi-D-specic primers. Of these, 183 were from dogs, 13 were
from cats, two were from llamas, and one each was from a calf, an alpaca, a sheep, and
a horse. The majority of the dogs (171 of 183 isolates) in this study were infected with
only dog-adapted Assemblage C or D. The tpi-D-specic primers conrmed that 28 of the
samples that typed as Assemblage D by the bg and gdh genes were also Assemblage D by
the tpi-D-specic primers. Only 12 isolates were Assemblage A alone or Assemblage A and
Assemblage C or D. Of the 13 cat isolates, seven were Assemblage F, two were Assemblage
D, three were Assemblage A and 1 contained both Assemblages C and D. The calf isolate
was Assemblage E (gdh, tpi) and the alpaca (bg, gdh), llamas (gdh), sheep (bg, gdh, tpi) and
horse (tpi) isolates were all Assemblage A. When the assemblage could be determined for
more than one gene, 91 of 117 dog isolates gave consistent results and 8 of 9 cat isolates
gave consistent results.
2012 Elsevier B.V. All rights reserved.

1. Introduction
Giardia duodenalis is a protozoan parasite that infects
humans as well as a wide variety of domestic animals
and wildlife species. It is estimated that in the United
States (US), two million human cases occur annually
(Yoder et al., 2010). Prevalence estimates among domestic dogs and cats in the US vary widely with estimates
as high as 13.6% for cats and 15.6% for dogs having

Corresponding author at: Veterinary Teaching Hospital, Colorado

State University, 300 West Drake Road, Fort Collins, CO 80523, United
States. Tel.: +1 970 297 4132; fax: +1 970 297 1275.
E-mail address: vscorza@lamar.colostate.edu (A.V. Scorza).
0304-4017/$ see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.vetpar.2012.04.032

been reported (Sulaiman et al., 2003; Vasilopulos et al.,

2007). As with humans, the consistency of feces from
dogs and cats positive for Giardia can vary from normal
to diarrhoic (Ballweber et al., 2010). G. duodenalis is a
species complex comprising at least eight Assemblages
(AH) (Monis and Thompson, 2009; Lasek-Nesselquist
et al., 2010). Assemblages A (subtypes A-I and A-VI) and
B infect humans and have also been detected in some
other species including dogs, cats and livestock (Monis and
Thompson, 2009; Ballweber et al., 2010). Assemblages CH
have a more narrow host range in canids (Assemblages
C and D), cats (Assemblage F), livestock (Assemblage E),
rats (Assemblage G), and marine mammals (Assemblage
H) (Ey et al., 1997; Thompson, 2004; Lasek-Nesselquist
et al., 2010). However, cats and dogs have also been

A.V. Scorza et al. / Veterinary Parasitology 189 (2012) 182188

reported to harbor Assemblages A-I, A-II, A-III, A-IV, and

B (Sprong et al., 2009; Ballweber et al., 2010; Covacin et al.,
2011).
Few studies have characterized the Giardia assemblages
of dogs and cats within the US. A study of 17 isolates from
cats, characterized 6 as Assemblage A-I and 11 as Assemblage F (Vasilopulos et al., 2007), whereas 15 isolates from
dogs were characterized as Assemblage D (Sulaiman et al.,
2003). In a recent study, genotyping of 128 dog samples
showed that 83% had multiple assemblages including A and
B in addition to C and D (Covacin et al., 2011). To date,
Assemblages C, D, and F have not been found in feces of
humans in the US. The potential zoonotic risk from dogs
and cats, therefore, arises from the detection of Assemblage
A in dogs and cats, and Assemblage B in dogs.
Several genes including the -giardin (bg), the glutamate dehydrogenase (gdh) and the triose phosphate isomerase (tpi) among others are used to amplify Giardia DNA
by PCR. Most of these are sufcient for typing at the level of
assemblage; however, only bg, tpi, and gdh are sufciently
discriminatory to type at the level of sub-assemblage
(Cacci and Ryan, 2008; Sprong et al., 2009; Covacin et al.,
2011). Additionally, the currently available primers may
preferentially amplify some assemblages (Cacci and Ryan,
2008; Covacin et al., 2011). Since the amplication of a
single gene may not provide enough information to adequately type isolates, a multilocus approach is currently
recommended (Cacci and Ryan, 2008; Covacin et al.,
2011). The objective of this study was to genetically characterize isolates of G. duodenalis from domestic animals
(mainly dogs) in the US using the bg, tpi, and gdh loci.

183

EDTA-PBS. 10 l of the fecal solution were pipetted onto

microscope slides supplied in an in vitro direct immunouorescence assay (DFA) capable of simultaneous detection
of Giardia sp. cysts and Cryptosporidium sp. oocysts (Meriuor Crypto/Giardia kit, Meridian Diagnostic Corporation,
Cincinnati, OH). The assay was then performed and interpreted following the manufacturers instructions.
DNA isolation. Giardia spp. cysts were concentrated and
processed as previously described (Scorza et al., 2003;
Vasilopulos et al., 2007). The samples were stored at 20 C
until molecular analysis was performed.
Molecular methods. The PCR assays were performed
following published protocols with several modications
(Sulaiman et al., 2003; Read et al., 2004; Lalle et al., 2005;
Vasilopulos et al., 2007). In the gdh and the bg protocols,
24 l/reaction of a commercial master mix (Hot Start Qiagen Master Mix) and 24 l of water were used instead
of the master mix described in the publication and 2 l
of DNA. Additionally, for the second amplication of the
bg, 10 l of the rst PCR amplicons was diluted into 90 l
water and 2 l of that dilution was used as template for the
second amplication. In the tpi PCR, the annealing temperatures for the rst and second amplication reactions
were 51 C and 55 C, respectively. Because the tpi protocol
of Sulaiman et al., 2003 does not amplify Assemblage D, a
subset of 50 canine samples were also tested using the tpiD-specic primers which detects Assemblage D (Lebbad
et al., 2010). Not all cases had sufcient material to determine the assemblages by all three genes. The nucleotide
sequences generated in this study were placed in GenBank under the accession numbers (JF958083JF958121
and JQ688275JQ688298).

2. Materials and methods

Source of isolates. Fecal samples determined to contain
Giardia spp. cysts were used in this study. The samples were
either submitted to the Veterinary Diagnostic Laboratory
(Parasitology) at Colorado State University or were part of
ongoing research studies. Samples primarily came from the
southeastern and western US and were collected between
March 2005 and November 2010. The presence of G. duodenalis was assessed by fecal otation or immunouorescent
assay.
Fecal otation. Between 2 and 3 g of feces were washed
with 4 ml of phosphate buffered saline solution containing
ethylenediaminetetraacetic acid (PBS-EDTA) and ltered
through gauze. The ltrate was mixed with Sheathers
sugar solution ( = 1.26) in a 15 ml centrifuge tube to give a
positive meniscus, a coverslip was placed on top of the solution, and the tube was centrifuged at 1500 rcf for 10 min.
The coverslip was placed on a microscope slide and the
slide was examined microscopically for Giardia cysts.
Immunouorescent assay. When adequate sample was
available, 3 g of feces were also ltered through gauze,
placed on top of 7 ml of Sheathers sugar solution ( = 1.26)
and centrifuged at 800 g for 10 min. The interface and
the upper layer of the ltrate were carefully pipetted
into a separate conical tube and centrifuged for 10 min at
1200 g. The supernatant was discarded and the washes
were repeated for two more times as described. After
the nal wash, the pellet was resuspended in 1 ml of

2.1. Phylogenetic analysis

Phylogenetic analysis was performed in alignments of
approximately 405 nucleotides at the bg gene, 289 at
the gdh gene, and 439 at the tpi gene, respectively. The
phylogenetic analysis was performed based on multiple
alignments of the sequences of the three genes of this
study and reference sequences obtained from the GenBank.
The analysis was carried out using the Neighbor-Joining
method (Saitou and Nei, 1987) using MEGA 5.0 (Tamura
et al., 2011). Bootstrap proportions were assessed by the
analysis of 1000 replicates. The nucleotide data were translated into amino acids and phylogenetic analysis was also
performed on the amino acid data.
3. Results
3.1. PCR results and sequencing
A total of 202 samples were evaluated in the bg, gdh and
tpi gene PCR assays. Of these, 183 were from dogs, 13 were
from cats, two were from llamas, and one each was from
a calf, an alpaca, a domestic sheep, and a horse. One hundred and forty-ve of 171 (84.79%), 172 of 189 (91%) and
34 of 172 (19%) of the samples were positive by the bg, gdh
and tpi assays, respectively. Interpretable sequence data
for assemblage determination were generated for all three

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A.V. Scorza et al. / Veterinary Parasitology 189 (2012) 182188

Table 1
G. duodenalis assemblages in animals in the United States by -giardin,
gdh and tpi genes.
Species

-Giardin

gdh

tpi

Dogs (183)
29
13
1
15
6
1
1
80
7
5
2
1
1
1
2
1
7
8
1
1

D
C

D
C
C
D

C
AII

A
D
D
D
C

D
C
AI

D
C
D

AI

C
D
AI
D
D
C
C

AI
C

C
AI
C

C
AI
AI
C
C
C

Cats (13)
3
1
1
2
1
1
2
1
1

F
D
F
A
D
F

F
D
A
F
D

AI
C
F

AI

F
AI
C
F

3 (1)

Sheep (1)
1

1(1)

Equine (1)
1

2 (1)

Calf (1)
1

2 (1)

Alpaca (1)
1

1 (2)a

Llamas (2)
2

# of genes amplied
1 (66)a

2 (99)a

3 (18)a

1 (4)a
2 (5)a

3 (4)a

Number of animals that tested positive for that number of gene tested.

genes from 23 samples, for two genes from 106 samples,

and for one gene from 72 samples (Table 1).
In dogs, Assemblages C and D were most common; only
ve of 183 isolates were Assemblage A (2.7%). Assemblage
A was identied from 3 of 13 cats (21.3%) and 4 of the 6
(66.6%) food animal species by at least one gene (Table 1).
Of the 106 samples with assemblage determination for 2
genes, the results were discordant for 10 (9.4%). The most
common discordant result was Assemblage D by one gene
and Assemblage C for the alternate gene (9 of 10 samples). Only one sample was Assemblage A by one gene but
Assemblage C by the other gene. Of the 23 samples with
assemblage determination for 3 genes, the results were
discordant for 17 samples (77.2%) but for each of these
discordant results, 2 of the gene results were in agreement. For 8 samples, Assemblage A was identied using one

gene with Assemblage D being identied by the alternate

2 genes.
The tpi-D-specic primers conrmed that 28 of the samples that typed as Assemblage D by the bg and gdh genes
were also Assemblage D by the tpi-D-specic primers.
Twelve of the samples were typed as either Assemblage C
or D by different genes, two samples typed as Assemblage
A-I by the tpi generic primers and as Assemblage D by the
other genes. Four of the samples typed as Assemblage C or
D by only one gene and four samples typed as Assemblage
C or D by two genes (Table 2).
3.2. Phylogenetic analysis
One hundred and seven of 172 isolates at the gdh
locus, 71 of 145 of isolates at the bg locus, and 31 of 71
isolates at the tpi locus were included in the phylogenetic analysis. The sequences included were interpretable
and those with heterogeneous sequencing proles were
conrmed with the sequences in the GenBank. These heterogeneous sequences were included in the phylogenetic
analysis if both nucleotides present at any polymorphic site
had been previously reported in other GenBank sequences.
Heterogeneous sequencing proles were observed in
Assemblages A, C and D at the gdh gene, in Assemblage C in
the tpi gene and in Assemblages A, C, D and F at the bg gene.
Phylogenetic analysis of the bg, gdh and tpi genes placed
the isolates from Assemblages A, C, D and F separated from
each other with strong bootstrap support. The clustering
generated is similar among the genes particularly among
the gdh and bg genes. In the bg and gdh analyses substructuring was observed in the Assemblages C and D, but with
low bootstrap values. Similar results were reported by previous researchers (Beck et al., 2011). Subgrouping was also
observed among Assemblages C and D in the tpi analysis,
in the amino acid analysis the isolates from Assemblage
C cluster together while the ones from Assemblage D
remained separated in subgroups with low bootstrap values. Results of the amino acid tree of the bg, gdh and tpi
genes were similar to the ones of the nucleotide analysis.
As previously reported, no correlation with the epidemiology information regarding country of origin of the isolates
or year of collection was observed (Beck et al., 2011). Dog
isolates from different regions of the US (Colorado, New
York, South Dakota) were grouped together with reference
isolates from Sweden and Nicaragua. Several isolates were
identical to each other, so only one isolate of each group
was included in the nal phylogenetic analysis (Figs. 13).
4. Discussion
The majority of the dogs in this study were infected
with the dog-adapted assemblages which is in agreement
with most previous studies. In a recent review, 1049 of
1563 (67%) isolates from dogs worldwide were either
Assemblage C or D (Feng and Xiao, 2011). Additionally,
Assemblages C and D mostly prevail in kennel and household dogs from Croatia (Beck et al., 2011). However, these
results are in contrast to a study from the western US in
which multiple infections with zoonotic assemblages were
most commonly detected, followed by mixed infections

A.V. Scorza et al. / Veterinary Parasitology 189 (2012) 182188

185

JQ688281
JQ688286
JF958111
JQ688284
JF958114
JF958104
JQ688282
JF958113
JQ688285
JQ688287

Assemblage D

JF958106
JF958108
94

65

JF95109

65 JF958110

JF958112

99

JF958105
64 EF455597-D
65

JQ688283
73 JF958117

EU769212-C
99

JF958116

Assemblage C

66 JF958115

EU769210-B
EU769221-G
78 EU769204-A
99
62

76

EU642897-A2

Assemblage A

JF958121
EU769215-E

70

68 JF958120

EU769220-F
99
0.005

67

JF958107

Assemblage F

JF958119
JF958118

Fig. 1. Phylogenetic relationships of Giardia duodenalis assemblages inferred by the neighbor joining analysis of the -giardin nucleotide sequences.

(zoonotic and dog assemblages) and lastly by infections

with either Assemblage B, C, or D (Covacin et al., 2011).
Although the reason for this difference cannot be stated
with certainty it probably relates to the housing environments of the dogs tested. When animals tend to be grouped
together, as in kennels or catteries, it is thought that the
host-adapted strain will predominate. However, in environments contaminated with cysts from humans and other
animals, the zoonotic assemblage prevails (Hopkins et al.,
1997; Bugg et al., 1999; Traub et al., 2002; Ballweber et al.,
2010; Covacin et al., 2011). Because we do not have adequate histories on many of the animals in our study, we
cannot add to this discussion. However, our results do
show the presence of potentially zoonotic (as dened by
Sprong et al., 2009) assemblages in some dogs and cats in
the US, a nding that was consistent with previous studies
(Vasilopulos et al., 2007; Covacin et al., 2011).

Even though we interpreted the results in the light of

a multilocus analysis and considering that the tpi generic
primers do not detect Assemblage D, gdh was the gene
with a higher amplication rate. Gdh analysis generated
the larger number of amplications (159 of 203) and in the
majority of the isolates the results were consistent with the
results from a second gene. Other researchers worldwide
reported similar ndings (Abe et al., 2003; Leonhard et al.,
2007; Lebbad et al., 2010). The bg PCR yielded the higher
amplication rate on the subset of samples tested by the tpi
assemblage-specic PCR. The majority of the samples that
typed as Assemblage D by other genes typed as Assemblage D by the tpi-D-specic primers. It is likely that the
discordant samples in this subgroup were mixed infections
since Assemblage D was conrmed with the tpi-D-specic
primers. However, as previously stated, genetic exchange
can also explain these ndings (Cooper et al., 2007).

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A.V. Scorza et al. / Veterinary Parasitology 189 (2012) 182188

Table 2
G. duodenalis assemblages in a subset of dogs isolates in the United States by -giardin, gdh and tpi genes.
Number of positives

-Giardin

gdh

tpi

tpi-Dog specic primers

28
2
3
1
1
1
1
2
2
1
1
1
1
1
1
1
1
1

D
C
C
C
C
Neg
Neg
D
D
D
D
Neg
D
C
C
D
C
Neg

D
C
D
Neg
Neg
C
D
D
D
D
D
D
Neg
Neg
Neg
Neg
C
D

Neg
Neg
Neg
Neg
Neg
Neg
C
Neg
C
A1
A1
Neg
Neg
Neg
Neg
Neg
Neg
C

D
D
Neg
Neg
D
D
Neg
Neg
D
D
Neg
Neg
D
Neg
D
Neg
Neg
Neg

The tpi generic primers typed an isolate as Assemblage

A-I while the other genes typed it as Assemblage D, we
believed that this a mixed infection and the zoonotic
component would not have been observed if only one gene
have been used. As previously reported, Giardia genotyping
results from dogs should be interpreted carefully specically if one gene was analyzed (Beck et al., 2011). The use

of both the generic and D-specic tpi primers appeared

to be a good combination in genotyping dog isolates. The
study from Croatia found that this primer combination
yielded the highest amplication rate (Beck et al., 2011).
In our study, heterogeneous sequencing proles were
detected in Assemblages A, C and D and F as previously
reported (Sprong et al., 2009; Lebbad et al., 2010). The
JF958092
EF685691-AI

66 JF958094

EF685689-AII
89

Assemblage A

JF958095
L40510-A-polish
JF958093

74

EU769223-A
54

EU769234-F

72

Assemblage F

JF958091

99

EU769231-E
AY178748-G
HM136891-B
79
60

EF507637-C
Assemblage C

JF958090
JF958089

JF958083

91
99
55
52

EU769228-D
JF958086
JF958085

Assemblage D

JF958088
0.01

JF958084
JF958087

Fig. 2. Phylogenetic relationships of Giardia duodenalis assemblages inferred by the neighbor joining analysis of the glutamate dehydrogenase nucleotide
sequences.

A.V. Scorza et al. / Veterinary Parasitology 189 (2012) 182188

presence of heterogeneous sequencing proles has been

attributed to either mixed infections or allelic sequence
heterozygosity (ASH) (Sprong et al., 2009; Lebbad et al.,
2010). Lately, it has been demonstrated that G. duodenalis may undergo sexual reproduction and consequently,
may inuence ASH levels; however, its impact on the
epidemiology of giardiasis is unknown (Sprong et al.,
2009).
Worldwide, 162 of 270 (60.0%) Giardia isolates
were characterized as the cat-adapted Assemblage F
(Sprong et al., 2009; Ballweber et al., 2010; Suzuki et al.,
2011; Paoletti et al., 2010; Yoshiuchi et al., 2010), which
was also the predominant isolate in the present study.
Not expected, however, was the identication of the dog
Assemblages C and D in three of the cats. For two cats,

187

the results were conrmed using multiple genes. These

ndings are not considered to be a result of accidental contamination during the molecular process as the negative
controls used throughout did not show contamination.
Whether these represent ingestion and pass-through of
cysts originating from dogs, contamination of the sample
during collection, or actual infection of cats by these
assemblages remains to be determined. However, given
that both Assemblages C and D have been previously
identied in cats (Sprong et al., 2009), it is likely that cats
can become infected with these assemblages under certain
circumstances.
Complete clinical evaluations are not available from
the majority of dogs and cats with Giardia assemblages
reported in the literature. Thus, denitive statements

JQ688292
JQ688288
JF958103
JQ688295
JQ688294

Assemblage C

JF958101
57
100

JQ688296
JF958102
JQ688293

71

EU781005-C
EU781014-B
68 JQ688276
56 JQ688277

79

JQ688275
Assemblage D

EU781028-D

100

JQ688280
JQ688278
69 JQ688279

EU781013-G
100 JF958099

EU781021-E

67

EU781003-F

59

100 JF958100

Assemblage E

Assemblage F

GL50803 93938
100

86 JQ688291

JF958098
JQ688289
99

56
81

EU781000-A
JQ688290

Assemblage A

JQ688298
JF958097

0.02

JQ688297
JF958096

Fig. 3. Phylogenetic relationships of Giardia duodenalis assemblages inferred by the neighbor joining analysis of the triose phosphate isomerase nucleotide
sequences.

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A.V. Scorza et al. / Veterinary Parasitology 189 (2012) 182188

concerning whether individual assemblages are more

or less likely to be associated with clinical disease is
unknown. However, it is clear from the work published to
date that all of the Giardia assemblages can be amplied
from feces of subclinical animals.
The horse, alpaca, sheep and cattle isolates in this study
harbored expected assemblages as previously described
(Traub et al., 2005; Giangaspero et al., 2005; Trout et al.,
2006, 2008; Mendonca et al., 2007; Veronesi et al., 2009;
Yang et al., 2009).
As a conclusion of our study most of the dogs we examined were infected with dog-adapted assemblages of G.
duodenalis with few potentially zoonotic types detected.
Mixed infections with multiple assemblages did occur;
however, this was relatively uncommon. It is clear that
the choice of primers can inuence the results, particularly
with regards to Assemblage D. Thus, assemblage specic
primers should be included for the analysis of dog isolates
and single gene results should be interpreted carefully.
Acknowledgements
The authors would like to thank Dr. Simone Cacci for
his expertise with the bg PCR and for donating positive controls for the study. The authors also thank Dr. Mariel Aguilar
Dominguez for her help processing the samples with the
tpi-D-specic primers.
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