206

Identification of a novel assemblage G subgenotype

and a zoonotic assemblage B in rodent isolates of
Giardia duodenalis in the Canary Islands, Spain

ÁNGELA FERNÁNDEZ-ÁLVAREZ 1 , AARÓN MARTÍN-ALONSO 1 ,

NÉSTOR ABREU-ACOSTA 2 , CARLOS FELIU 3 , JEAN-PIERRE HUGOT 4 ,
BASILIO VALLADARES 1 and PILAR FORONDA 1 *
1
University Institute of Tropical Diseases and Public Health of the Canary Islands, University of La Laguna, La Laguna,
Tenerife, Canary Islands, Spain
2
Analytical Biotech, Santa Cruz de Tenerife, Canary Islands, Spain
3
Laboratory of Parasitology, Faculty of Pharmacy, University of Barcelona, Barcelona, Spain
4
Muséum National d’Histoire naturelle, Paris, France

(Received 15 April 2013; revised 11 June 2013; accepted 18 July 2013; first published online 4 September 2013)

SUMMARY

The flagellated parasite Giardia duodenalis is known as one of the most common causes of protozoal diarrhoea in both
humans and animals worldwide. The aim of the present work was to perform the first study of G. duodenalis in rodents in the
Canary Islands (Spain) and analyse the level of genetic variation and the potential zoonotic role of the isolates. Stool samples
were collected from 284 wild rodents and Giardia cysts were detected by light microscopy. The overall prevalence of
giardiasis was 25·4% and ranged from 19·4% in El Hierro to 34% in Gran Canaria. Positive samples were further
characterized by PCR and nucleotide sequencing of the triose phosphate isomerase (TPI), β-giardin (BG) and glutamate
dehydrogenase (GDH) genes. Our study revealed assemblage G as the most frequent genotype and identified two rodent-
infecting G. duodenalis haplotypes of this assemblage, HI and HII. Phylogenetic analysis supported the monophyly of
haplotype HI, which we suggest to be considered as a novel G. duodenalis sub-assemblage GII, due to the high genetic
distances among this sub-genotype and assemblage G. Furthermore, G. duodenalis assemblage B was detected in an
inhabited area in La Palma, a fact that may pose a potential risk of G. duodenalis transmission from rodents to humans.

Key words: Giardia duodenalis, Canary Islands, zoonoses, rodents, assemblage G, sub-assemblage GII.

INTRODUCTION hosts, whereas assemblages C to G are to some extent

host-specific (Cacciò and Ryan, 2008) and assem-
Giardia is a protozoan parasite found in the intestinal
blage G has only been found in rodents (Monis
tract of humans and vertebrate animals (de Souza
et al. 2003). Among others, several genes including
et al. 2009), which presents a broad host range,
the β-giardin (BG), the glutamate dehydrogenase
including livestock, companion animals and wildlife
(GDH) and the triose phosphate isomerase (TPI)
(Paziewska et al. 2007). Of the six recognized species
are commonly used to genetically characterize isolates
of Giardia, Giardia duodenalis is the only one that
of G. duodenalis due to the high degree of genetic poly-
infects humans (Wielinga and Thompson, 2007), but
morphism in these loci (Wielinga and Thompson,
it also parasitizes wild and domestic animals. Isolates
2007). The nucleotide sequence of these genes may
of this species are subdivided into genotypes accord-
contribute to the elucidation of different transmission
ing to host species, morphology and molecular
pathways, including the role of animals as reservoirs
differences (Thompson et al. 2000). In this sense,
for human giardiasis. Since the amplification of a
molecular analysis of isolates from the different host
single gene may not ensure the correct genotyping,
species has revealed the existence of eight different
and due to the preferential amplification of some
G. duodenalis assemblages (A–H), of which assem-
assemblages with the currently available primers, a
blages A and B are responsible for the 99·2% of
multilocus approach is currently recommended
human cases (Sprong et al. 2012). Genotypes A and B
(Cacciò and Ryan, 2008; Covacin et al. 2011).
have been detected in a wide range of mammalian
Two invasive Rattus species, the black rat (Rattus
rattus) and the Norway rat (Rattus norvegicus), and
the house mouse (Mus musculus domesticus) have been
* Corresponding author: University Institute of Tropical introduced to the Canary Islands and nowadays they
Diseases and Public Health of the Canary Islands,
University of La Laguna. Avda. Astrofísico Fco.
are known to be widely spread in all these islands.
Sánchez, s/n 38203 La Laguna, Canary Islands, Spain. Previous studies have demonstrated the introduction
E-mail: pforonda@ull.es of pathogens together with these rodent hosts to the

Parasitology (2014), 141, 206–215. © Cambridge University Press 2013

doi:10.1017/S003118201300139X
Giardia in rodents in the Canary Islands
Canary Islands, including potentially zoonotic
species (Foronda et al. 2011).
At present, there is no information about the oc-
currence of Giardia in rodents in the Canary Islands.
Therefore, the aim of this study was to investigate the
prevalence and distribution of giardiasis in rodents
from the Archipelago and to determine the level of
genetic variation of Giardia isolates through the
amplification of the TPI, BG and GDH genes.
MATERIALS AND METHODS
Biological samples and area of study
The study was carried out on the Canary Islands,
located 100 km off the NW coast of Africa, between
13°23′ and 18°8′W and 27°37′ and 29°24′N. A total of
284 wild rodents (116 R. rattus, 3 R. norvegicus and
165 M. m. domesticus) were captured during 2007–
2011 in all the Canary Islands (La Palma, El Hierro,
La Gomera, Tenerife, Gran Canaria, Fuerteventura,
Lanzarote) and in a small islet (La Graciosa). The
animals were captured alive (Sherman traps) at
suburban–rural sites. Traps were set in the afternoon
and evening and checked soon after sunrise. The
animals were euthanased by cervical dislocation or by
CO2 inhalation. Fecal samples were obtained from
the rectum of each rodent and stored at 4 °C in 2·5%
potassium dichromate.
Ethical statement
All the animal procedures were performed according
to the principles of animal welfare in experimental
science. Animal trapping and its use were approved
by the environmental agencies of the governmental
competent entities, the ‘Excmo. Cabildo Insular’ of
all the Canary Islands in accordance with the Laws
151/2001, 33/2003, 42/2007 and 4/2010.
Giardia identification
Fecal cysts were concentrated using a modification of
the Richie’s formaldehyde–ether method (Richie,
1948) in sterile conditions and replacing ether with
ethyl acetate, as described previously (Young et al.
1979). The concentrate was stained with iodine
solution and examined for Giardia cysts by light
microscopy.
DNA isolation
The DNA was extracted directly from aliquots of
positive fecal samples using the Fast Prep (Qbiogene,
Illkirch Cedex, France) procedure, after three wash-
ing steps with PBS–EDTA to remove potassium
dichromate. The DNA released from disrupted cysts
was purified using the Fast DNA® Spin kit for
Soil (Qbiogene, Illkirch Cedex, France) and stored
at 4 °C.
207
TPI genotyping
Giardia-positive samples were genotyped by the am-
plification of a *530 bp fragment of the TPI gene, on
the basis of a nested PCR described previously by
Sulaiman et al. (2003), using the primary primer pair
AL3543 (forward; 5′-AAATTATGCCTGCTCG-
TCG-3′) and AL3546 (reverse; 5′-CAAACCTTTT-
CCGCAAACC-3′), and the secondary pair AL3544
(forward; 5′-CCCTTCATCGGTGGTAACTT-3′)
and AL3545 (reverse; 5′-GTGGCCACCACTCCC-
GTGCC-3′). Identical conditions were used for both
amplifications: 35 cycles (94 °C for 30 s, 50 °C
for 30 s, and 72 °C for 60 s) with an initial hot start
at 94 °C for 5 min and a final extension at 72 °C for
10 min. Both primary and secondary PCR were con-
ducted in a 50 μL reaction mixture containing 200 μM
of each dNTP (Bioline, London, UK), 0·2 uM of
each primer, 2·2 mM MgCl2 (Bioline, London, UK),
0·015 U μL− 1 Diamond DNA polymerase (Bioline,
London, UK) and 1 × PCR buffer (Bioline, London,
UK). 2 μL of DNA template and first PCR products
were used in the primary and in the secondary PCR,
respectively.
BG genotyping
Giardia duodenalis isolates were also genotyped at
the BG locus using a semi-nested PCR which am-
plifies an approximately 292-bp fragment, using
the primary primer pair G7 (forward; 5′-AAGCCC-
GACGACCTCACCCGCAGTGC-3′) and G759
(reverse; 5′-GAGGCCGCCCTGGATCTTCGAG-
ACGAC-3′), and the secondary pair G376 (forward;
5′-CATAACGACGCCATCGCGGCTCTCAGG-
AA-3′) and the reverse primer G759 (Cacciò et al.
2002). Identical conditions were used for both am-
plifications: 35 cycles (95 °C for 45 s, 60 °C for 45 s,
and 72 °C for 60 s) with an initial hot start at 94 °C
for 5 min and a final extension at 72 °C for 10 min.
Both primary and secondary PCR were performed in
a 50 μL reaction mixture containing 200 μM of each
dNTP (Bioline, London, UK), 0·4 μM of each pri-
mer, 1·5 mM MgCl2 (Bioline, London, UK), 1·25
units of Taq DNA polymerase (Bioline, London,
UK), and 1 × PCR buffer (Bioline, London, UK).
2 μL of DNA template and first PCR products were
used in the primary and in the secondary PCR,
respectively.
GDH genotyping
All positive samples were also analysed using a semi-
nested GDH PCR (Read et al. 2004) which amplifies
a 432-bp fragment. Identical conditions were used
for both amplifications: 1 cycle of 94 °C for 2 min,
56 °C for 1 min and 72 for 2 min, followed by
55 cycles of 94 °C for 30 s, 56 °C for 20 s and 72 °C
for 45 s, with a final extension of 72 °C for 7 min.
Giardia in rodents in the Canary Islands
Canary Islands, including potentially zoonotic
species (Foronda et al. 2011).
At present, there is no information about the oc-
currence of Giardia in rodents in the Canary Islands.
Therefore, the aim of this study was to investigate the
prevalence and distribution of giardiasis in rodents
from the Archipelago and to determine the level of
genetic variation of Giardia isolates through the
amplification of the TPI, BG and GDH genes.
MATERIALS AND METHODS
Biological samples and area of study
The study was carried out on the Canary Islands,
located 100 km off the NW coast of Africa, between
13°23′ and 18°8′W and 27°37′ and 29°24′N. A total of
284 wild rodents (116 R. rattus, 3 R. norvegicus and
165 M. m. domesticus) were captured during 2007–
2011 in all the Canary Islands (La Palma, El Hierro,
La Gomera, Tenerife, Gran Canaria, Fuerteventura,
Lanzarote) and in a small islet (La Graciosa). The
animals were captured alive (Sherman traps) at
suburban–rural sites. Traps were set in the afternoon
and evening and checked soon after sunrise. The
animals were euthanased by cervical dislocation or by
CO2 inhalation. Fecal samples were obtained from
the rectum of each rodent and stored at 4 °C in 2·5%
potassium dichromate.
Ethical statement
All the animal procedures were performed according
to the principles of animal welfare in experimental
science. Animal trapping and its use were approved
by the environmental agencies of the governmental
competent entities, the ‘Excmo. Cabildo Insular’ of
all the Canary Islands in accordance with the Laws
151/2001, 33/2003, 42/2007 and 4/2010.
Giardia identification
Fecal cysts were concentrated using a modification of
the Richie’s formaldehyde–ether method (Richie,
1948) in sterile conditions and replacing ether with
ethyl acetate, as described previously (Young et al.
1979). The concentrate was stained with iodine
solution and examined for Giardia cysts by light
microscopy.
DNA isolation
The DNA was extracted directly from aliquots of
positive fecal samples using the Fast Prep (Qbiogene,
Illkirch Cedex, France) procedure, after three wash-
ing steps with PBS–EDTA to remove potassium
dichromate. The DNA released from disrupted cysts
was purified using the Fast DNA® Spin kit for
Soil (Qbiogene, Illkirch Cedex, France) and stored
at 4 °C.
207
TPI genotyping
Giardia-positive samples were genotyped by the am-
plification of a *530 bp fragment of the TPI gene, on
the basis of a nested PCR described previously by
Sulaiman et al. (2003), using the primary primer pair
AL3543 (forward; 5′-AAATTATGCCTGCTCG-
TCG-3′) and AL3546 (reverse; 5′-CAAACCTTTT-
CCGCAAACC-3′), and the secondary pair AL3544
(forward; 5′-CCCTTCATCGGTGGTAACTT-3′)
and AL3545 (reverse; 5′-GTGGCCACCACTCCC-
GTGCC-3′). Identical conditions were used for both
amplifications: 35 cycles (94 °C for 30 s, 50 °C
for 30 s, and 72 °C for 60 s) with an initial hot start
at 94 °C for 5 min and a final extension at 72 °C for
10 min. Both primary and secondary PCR were con-
ducted in a 50 μL reaction mixture containing 200 μM
of each dNTP (Bioline, London, UK), 0·2 uM of
each primer, 2·2 mM MgCl2 (Bioline, London, UK),
0·015 U μL− 1 Diamond DNA polymerase (Bioline,
London, UK) and 1 × PCR buffer (Bioline, London,
UK). 2 μL of DNA template and first PCR products
were used in the primary and in the secondary PCR,
respectively.
BG genotyping
Giardia duodenalis isolates were also genotyped at
the BG locus using a semi-nested PCR which am-
plifies an approximately 292-bp fragment, using
the primary primer pair G7 (forward; 5′-AAGCCC-
GACGACCTCACCCGCAGTGC-3′) and G759
(reverse; 5′-GAGGCCGCCCTGGATCTTCGAG-
ACGAC-3′), and the secondary pair G376 (forward;
5′-CATAACGACGCCATCGCGGCTCTCAGG-
AA-3′) and the reverse primer G759 (Cacciò et al.
2002). Identical conditions were used for both am-
plifications: 35 cycles (95 °C for 45 s, 60 °C for 45 s,
and 72 °C for 60 s) with an initial hot start at 94 °C
for 5 min and a final extension at 72 °C for 10 min.
Both primary and secondary PCR were performed in
a 50 μL reaction mixture containing 200 μM of each
dNTP (Bioline, London, UK), 0·4 μM of each pri-
mer, 1·5 mM MgCl2 (Bioline, London, UK), 1·25
units of Taq DNA polymerase (Bioline, London,
UK), and 1 × PCR buffer (Bioline, London, UK).
2 μL of DNA template and first PCR products were
used in the primary and in the secondary PCR,
respectively.
GDH genotyping
All positive samples were also analysed using a semi-
nested GDH PCR (Read et al. 2004) which amplifies
a 432-bp fragment. Identical conditions were used
for both amplifications: 1 cycle of 94 °C for 2 min,
56 °C for 1 min and 72 for 2 min, followed by
55 cycles of 94 °C for 30 s, 56 °C for 20 s and 72 °C
for 45 s, with a final extension of 72 °C for 7 min.
Ángela Fernández-Álvarez and others 208

PCR reaction mixtures consisted of 12·5 ρmol of each Table 1. Frequency of Giardia in rodents in the
primer, 200 μM of each dNTP, 1·5 mM MgCl2, 1 unit Canary Islands
of Taq DNA polymerase (Bioline, London, UK),
and 1 × PCR buffer (Bioline, London, UK). GDHeF Island and Analysed Positive samples (%)
host species samples (n) [CI 95%]
(5′-TCAACGTYAAYCGYGGYTTCCGT-3′) and
GDHiR (5′-GTTRTCCTTGCACATCTCC-3′) Tenerife 48 15 (31·3) [20–45·3]
were used in the primary PCR reaction with 1 μL of R. rattus 25 9 (36) [20·3–55·5]
genomic DNA. 1 μL of PCR product from the R. norvegicus 1 0 (0) [0–79·4]
M. m. domesticus 22 6 (27·3) [13·2–48·2]
primary reaction was added to the secondary PCR
containing primers GDHiF (5′-CAGTACAACTC- La Palma 20 5 (25) [11·2–46·9]
R. rattus 3 2 (66·7) [20·8–93·9]
YGCTCTCGG-3′) and GDHiR. R. norvegicus 1 1 (100) [20·7–100]
All the amplifications were carried out in a Labnet M. m. domesticus 16 2 (12·5) [3·5–36]
thermocycler (Labnet International, Berkshire, UK) La Gomera 32 7 (21·9) [11–38·8]
and amplification products were analysed on 1·7% R. rattus 12 2 (16·7) [4·7–44·8]
agarose gel. PCR products were purified using M. m. domesticus 20 5 (25) [11·2–46·9]
UltraClean PCR Clean-up kit (MO BIO, Carlsbad, El Hierro 31 6 (19·4) [9·2–36·3]
CA). PCR products were sequenced at the Genomic R. rattus 14 3 (21·4) [7·6–47·6]
Service of the University of La Laguna and at M. m. domesticus 17 3 (17·6) [6·2–41]
Macrogen Inc. (Korea). In order to elucidate any Gran Canaria 50 18 (36) [22·7–49·3]
R. rattus 19 11 (57·9) [36·3–76·9]
similarities in sequences with those previously pub-
R. norvegicus 1 1 (100) [20·7–100]
lished in GenBank, a BLAST search was performed. M. m. domesticus 30 6 (20) [9·5–37·3]
Fuerteventura 44 11 (25) [14·6–39·4]
Phylogenetic analysis R. rattus 23 8 (34·8) [18·8–55·1]
M. m. domesticus 21 3 (14·3) [5–34·6]
New and previously published sequences were Lanzarote 39 10 (25·6) [14·6–41·1]
aligned with the multiple alignment program R. rattus 20 7 (35) [18·1–56·7]
ClustalW as implemented in Mega 5.0 (Tamura M. m. domesticus 19 3 (15·8) [5·5–37·6]
et al. 2007). Nucleotide substitutions were indicated La Graciosa 20 1 (5) [0·9–23·6]
from the ATG codon of each gene. Isolates were R. rattus – –
M. m. domesticus 20 1 (5) [0·9–23·6]
grouped into assemblage B sub-assemblages based on
Total 284 74 (26·1) [021–31·2]
the substitution patterns proposed by Wielinga and
R. rattus 116 42 (36·2) [30·6–41·8]
Thompson (2007). The existence of novel substi- R. norvegicus 3 2 (66·7) [22·8–95·6]
tution positions was examined. Phylogenetic analysis M. m. domesticus 165 29 (17·6) [11·8–23·4]
was performed on the aligned TPI, BG and GDH
sequences to assess the extent of genetic diversity
among Giardia isolates detected in this study and
previously published sequences. Neighbour-joining by comparative analysis against three reference se-
(NJ) was performed with distances calculated with quences of G. duodenalis genotype G: EU781013
the Kimura 2-parameter (Kimura, 1980) for the three (TPI), EU769221 (BG) and AY178748 (GDH).
loci. To evaluate the support for inferred topologies,
bootstrapping (Felsenstein, 1985) was done with Data analysis
1000 replicates. The quartet-puzzling tree and the
intra-assemblages and inter-assemblages distances An SPSS/PC package was used to perform a
were determined for both TPI and GDH loci by statistical analysis of the influence of extrinsic host
the analysis of the number of substitutions per nucleo- factors (qualitative values) on the prevalence. Chi-
tide site, which is estimated by using the HKY model square test was used to evaluate parasitological results
of substitution (Hasegawa et al. 1985) with gamma relative to host, island and season. 95% confidence
correction (eight categories) as implemented by intervals were measured by the Wilson method. A
PUZZLE version 5.2 (Strimmer and Haeseler, probability value less than <0·05 was considered as
1996). Parameter estimation was done using the NJ statistically significant.
tree. Support for internal nodes was assessed using
1000 puzzling steps. Dendrograms were drawn using RESULTS
TREEVIEW (Page, 1996).
Prevalence of Giardia in rodents
In 72 out of 284 animals (25·4%) Giardia cysts were
Protein analysis
found by microscopy. Positive samples were detected
Using the amino acid sequences inferred by trans- in all the Canary Islands with a prevalence ranging
lation of the open reading frames (ORFs) of in- from 5% to 34% (Table 1), without significant differ-
dividual sequences, substitutions were identified ences in the prevalence among islands. According
Giardia in rodents in the Canary Islands 209

Table 2. Giardia duodenalis haplotypes detected in rodents from the Canary Islands for the TPI, BG and
GDH loci (SNPs: number of substitutions identified by comparing against G. duodenalis genotype G
EU781013 for TPI, EU769221 for BG and AY178748 for GDH)

GenBank
Locus Haplotype accession no. SNPs Isolates Island Host
TPI HI KC855112 28 G9 El Hierro R. rattus
KC855113 G29 El Hierro M. m. domesticus
KC855114 G82 La Gomera R. rattus
KC855115 G114 Lanzarote R. rattus
KC855116 G128 Lanzarote R. rattus
KC855117 G162 Fuerteventura M. m. domesticus
KC855118 G243 Gran Canaria R. rattus
KC855119 G247 Gran Canaria R. rattus
KC855120 G250 Gran Canaria R. rattus
KC855121 G253 Gran Canaria M. m. domesticus
KC855122 G283 Gran Canaria R. rattus
HII KC855123 1 G159 Fuerteventura R. rattus
KC855124 G184 Fuerteventura R. rattus
KC855125 G314 Tenerife R. rattus
HIII KC855111 0 G111 La Palma R. rattus
BG HI KC855129 8 G243 Gran Canaria R. rattus
KC855130 G250 Gran Canaria R. rattus
HII KC855127 2 G184 Fuerteventura R. rattus
KC855128 G314 Tenerife R. rattus
HIII KC855126 0 G111 La Palma R. rattus
GDH HI KC855133 7 G243 Gran Canaria R. rattus
HII KC855132 0 G314 Tenerife R. rattus
HIII KC855131 3 G111 La Palma R. rattus

to the host species, the prevalence was higher in
R. rattus than in M. m. domesticus (P-value <0·05),
when rodents from Gran Canaria were analysed.
No significant differences were observed between
R. rattus and M. m. domesticus in the rest of the
islands.
TPI genotyping and estimation of genetic distances
TPI sequences were obtained for 15 samples and
submitted to GenBank under the Accession numbers
KC855111-KC855125. Three different TPI haplo-
types, which we named as HI–HIII, were detected
and included in a 433-bp alignment with previously
published sequences. Similarity search using
BLAST demonstrated that all isolates showed the
highest similarity with G. duodenalis assemblage G
(GenBank Accession number EU781013) (Table 2)
with one (n = 3) (HII) and 28 (n = 11) (HI) SNPs
(Table 3), except G111 (HIII), which was identical to
G. duodenalis assemblage B (GenBank Accession
number JN587446). This isolate was characterized
as BIV at the TPI gene in accordance with the
substitution patterns proposed by Wielinga and
Thompson (2007).
According to the haplotype HI, five SNPs caused
a conversion of amino acid (P34L, S90N, G146A,
K152N and E160T). The evolutionary distance
between haplotype HI and assemblage G was 0·078.
Considering this high genetic distance, we propose
the haplotype HI to be considered as the novel
sub-assemblage GII. It was detected in five out of the
seven islands in both M. m. domesticus and R. rattus.
On the other hand, G159, G184 and G314 (HII)
showed one synonymous SNP at position 282 (T to
G), which was shared with the proposed novel sub-
assemblage GII detected in this study.
The NJ tree based on an alignment of the TPI
sequences is presented in Fig. 1. A similar topology
was found using the quartet puzzling method (data
not shown). Both methods grouped the 11 isolates
that belonged to sub-assemblage GII together in one
cluster. These trees clustered these isolates into a
well-supported monophyletic clade that was closely
related to assemblage G. On the other hand, G159,
G184 and G314 (HII) were clustered with previously
published sequences of G. duodenalis assemblage
G. A third node was composed of G111 and
assemblage B sequences.
BG genotyping
We further genotyped at the BG locus those isolates
for which TPI sequences were obtained and we used
them together with previously published BG se-
quences to build a 178-bp alignment. The sequences
obtained were submitted to GenBank under the
Accession numbers KC855126–KC855130. In this
case, G111 was also typed as assemblage B, showing
100% identity to a sequence obtained from children
(GenBank Accession number FJ971486) (Kosuwin
et al. 2010). At present, polymorphisms are not
Ángela Fernández-Álvarez and others 210

available to link the BG locus heterogeneity to the

Table 3. Variable positions of TPI, BG and GDH genes in the new assemblage G sub-genotype (position reference following EU781013 for TPI, EU769221 for

A
C
4
7
9
GDH and TPI gene sub-assemblage classifications,
but position 354 seems to be a promising candidate to

G
A
4
7
8
distinguish between the sub-assemblage BIII (cyto-
sine) from BIV (thymine) (Levecke et al. 2009). How-

G
T
4
6
8
ever, G111 could not be assigned to a sub-assemblage

A
C
at the BG gene due to our alignment starting at posi-
4
5
6
tion 452. On the other hand, while G184 and G314
G
A
showed again the highest similarity with G. duode-
4
4
1

nalis assemblage G (99% BLAST identity) (GenBank

G
C
4
3
7

Accession number EU769221), G243 and G250,

which belonged to haplotype HI, shared more iden-
T
C
4
3
5

tity with G. duodenalis assemblage F (98%) (GenBank

Accession number AY647264) than with G. duode-
G
A
4
2
6

nalis assemblage G (96%). The isolate G243 carried

eight synonymous SNPs when compared with G.
T
C
3
9
9

duodenalis genotype G, whereas G250 showed those

SNPs plus one non-synonymous mutation at this
G
A
3
9
6

locus (V227A).
The NJ method grouped G184 and G314 together
G
A
3
8
7

with G. duodenalis assemblage G in one cluster with

T
C

high support (99% bootstrap support), whereas G243

3
8
1

and G250 were clustered together in another well-

T
C
3
5
1

supported group (97% bootstrap support) (Fig. 2).

BG and AY178748 for GDH). Shaded base numbers indicate non-synonymous substitution sites

Finally, G111 was grouped with 11 sequences of

G
C
3
1
8

G. duodenalis assemblage B from humans (97%

bootstrap support).
G
A
2
9
7

G
A

GDH genotyping
2
9
4

Our results were further confirmed by amplification

T
C
2
9
1

and sequencing of the GDH gene. Amplicons

representing the three haplotypes were sequenced
G
T
2
8
2

and submitted to GenBank under the Accession num-

bers KC855131–KC855133. They were used to build
G
A
2
6
9

a 215-bp alignment with previously published GDH

T

T
C

sequences in order to carry out the phylogenetic

2
4
3

6
8
0

analysis. The BLAST search revealed that G111

G

G
A

exhibited 99% identity with G. duodenalis assemblage

2
3
4

6
2
1

B (GenBank Accession number EU594663) obtained

G
T
C

A
1
3
2

6
1
8

from children, and three nucleotide substitutions

were found between G111 and this sequence. This
T

T
C

C
1
1
7

5
9
1

6
3
3

isolate consisted of a combination of BIII (p 447:

thymine, 540: cytosine and 561: cytosine) and BIV-
G
T

T
C

C
1
0
8

5
6
4

6
2
4

like (p 429: cytosine) substitution patterns and it also

showed a novel synonymous substitution at position
T

T
C

C
1
0
1

4
8
9

5
9
7

564 (adenosine) at the GDH gene.

On the other hand, G243 showed the highest simi-
G
SNP position

T
C

A
8
7

4
8
6

5
7
6

larity with G. duodenalis assemblage G (97% BLAST

identity) (GenBank Accession number AY178748),
G
T
A
C

A
C
8
4

4
8
3

5
2
8

whereas G314 was identical to two assemblage G se-

G

quences (GenBank Accession numbers AY178748

A

C
7
8

4
5
6

5
0
1

and AF069058). Isolate G243 carried seven synon-

Genotype

ymous SNPs when compared with assemblage G

sequences.
GII

GII

GII

The phylogenetic analysis showed that G243 was

G

clustered alone whereas G314 was grouped with the

Locus

GDH

rest of the assemblage G sequences, being supported

TPI

BG

by a high bootstrap value (99%) (Fig. 3). Estimates

Giardia in rodents in the Canary Islands
Fig. 1. Phylogenetic relationship among assemblages (A to G) of Giardia duodenalis at the TPI locus as assessed by a
neighbour-joining analysis of the nucleotide sequence covering a 433-bp region (position 70 to 503 of EU781013) of the
gene, using the distance calculated by the Kimura 2-parameter model. Bootstrap values were calculated by the analysis
of 1000 replicates. Only bootstraps values >70 are shown.
for the evolutionary distances among G243, which
belongs to haplotype HI, and assemblage G se-
quences were carried out in order to support the
results obtained with the TPI gene (data not shown).
In this sense, the number of substitutions per nucleo-
tide site, estimated by the HKY model, was 0·038.
The isolate G243 was nearly as divergent from as-
semblage F as it was from assemblage G (0·046),
whilst the genetic distance found between assemblage
G and F was nearly twice the distance observed
between assemblage G and assemblage GII (0·092).
DISCUSSION
To our knowledge, this work constitutes the
first extensive study on the presence of Giardia
in rodents in the Canary Islands. These results
indicate that both mouse and rat infection with
G. duodenalis is common and widespread throughout
the Archipelago.
The weather in the Canary Islands is strongly
influenced by the humid trade winds from the north-
east which, in combination with the altitude of the
volcanoes and the drier north-west winds blowing at
higher levels, produce an inversion zone and marked
vegetation belts. Several authors have demonstrated
211
the influence of these variables on the existence and
distribution of some parasites of mammal species in
the Archipelago (e.g. Foronda et al. 2003). However,
this effect was not detected for G. duodenalis in our
study, since it was found in all the studied micro-
habitats, probably due to the high ability to support
varying environmental conditions of this parasite.
Previous studies of Giardia in rodents have found
overall point prevalences between 22·2% and 100%.
In this sense, the overall prevalence of Giardia spp.
found in the present study was lower than that
observed in rodents from other regions, as Belgium
(66·3% in Chinchilla lanigera) (Levecke et al. 2011),
Poland (58·3% and 93·9% in Myodes glareolus
and 48·3%, 74·2% and 87% in Apodemus flavicollis,
Microtus arvalis and Ondatra zibethicus, respectively)
(Bajer et al. 2002, 2008; Bednarska et al. 2007; Bajer,
2008) and USA (65·9%, 73·3%, 100% and 100% in
O. zibethicus, Myocastor coypus, M. glareolus and
M. arvalis, respectively) (Dunlap and Thies, 2002;
Bitto and Aldras, 2009). On the other hand, the mean
prevalence observed in our study was similar to those
observed in other cases, such as A. flavicollis and
Castor canadensis in the USA (33·3% and 33%,
respectively) (Dunlap and Thies, 2002; Bednarska
et al. 2007) and Mesocricetus auratus, Phodopus
Ángela Fernández-Álvarez and others 212

Fig. 2. Phylogenetic relationship among assemblages (A to G) of Giardia duodenalis at the BG locus as assessed by a
neighbour-joining analysis of the nucleotide sequence covering a 178-bp region (position 422 to 600 of FJ971423) of the
gene, using the distance calculated by the Kimura 2-parameter model. Bootstrap values were calculated by the analysis
of 1000 replicates. Only bootstraps values >70 are shown.

Fig. 3. Phylogenetic relationship among assemblages (A to G) of Giardia duodenalis at the GDH locus as assessed by a
neighbour-joining analysis of the nucleotide sequence covering a 215-bp region (position 179 to 393 of AY178745) of
the gene, using the distance calculated by the Kimura 2-parameter model. Bootstrap values were calculated by the
analysis of 1000 replicates. Only bootstraps values >70 are shown.
Giardia in rodents in the Canary Islands
sungorus, Phodopus campbelli and Phodopus roborovs-
kii in China (22·2%) (Lv et al. 2009).
The genotyping analyses performed in the present
study were consistent among the three loci assessed
(TPI, BG and GDH) and revealed that rodents from
six Canary Islands were infected with G. duodenalis
assemblage G and B. The proposed novel sub-
genotype GII was not identified in Tenerife and
La Palma. However and considering the wide-
spread distribution of this sub-assemblage we believe
that it could also be present in these islands. The
TPI sequences of these isolates clustered into a well-
supported monophyletic clade that was closely rela-
ted to assemblage G. Taking into account that
this haplotype was being shared by R. rattus and
M. m. domesticus, it can be concluded that it is not
species-specific. Previous studies based on the TPI
gene have shown that intra-assemblage distances
among Giardia sub-assemblages range between 0·004
and 0·01, while inter-assemblage distances among
Giardia genotypes range between 0·11 and 0·41,
based on the number of substitutions per nucleotide
site (estimated using the HKY model of substi-
tution). In our study, the evolutionary genetic dis-
tance observed between assemblage G and the novel
sub-assemblage GII was eight times superior to the
highest genetic distance found between two sub-
assemblages, and it was close to the minimum genetic
distance found between two assemblages. It must also
be taken into account that the SNPs responsible for
this genetic variation were found to cause five mu-
tations at the primary structure level of the TPI
protein. Our finding was also confirmed by the high
genetic distance found between sub-assemblage GII
and assemblage G through the use of the sequences
obtained from the GDH locus and the grouping of
the sequences belonging to sub-assemblage GII in a
well-supported and separate clade when we analysed
the BG gene. Both GDH and TPI genotyping
of G243 and G250 ratified that these isolates were
more similar to previously published assemblage G
sequences, although they showed more similarity
with assemblage F than with assemblage G when the
BG gene was studied. However, assemblage F has a
strong host specificity and a narrow host range, since
it has only been found in cats, one pig (Armson et al.
2009) and seven humans (Gelanew et al. 2007). In
this sense this BLAST analysis could be limited
by the use of different primers that amplify different
portions of the BG gene, with only a partial overlap
(178 bp) among our and previously published se-
quences. Previous studies have highlighted the neces-
sity of a multi-locus approach, due to the occurrence
of heterogeneous templates with the consequent
difficulty in assigning some isolates to a specific
assemblage (Cacciò and Ryan, 2008).
The species-specificity of G. duodenalis assemblage
G and the high degree of isolation of both hosts and
parasite in this Archipelago could have led to the deep
213
separation between genotype G and sub-assemblage
GII. This event has been found in other parasites of
rodents, such as Hymenolepis diminuta in the Canary
Islands (Foronda et al. 2011). Nevertheless, another
hypothesis could indicate a colonization of murid
species carrying G. duodenalis sub-assemblage GII
from the source population, which remains un-
known. This possibility is supported by the wide
distribution of this haplotype, which was found in
five out of the seven islands analysed.
In the past, G. duodenalis assemblage B was
thought to be largely restricted to humans, and it
represents the most frequent Giardia assemblage in
human studies (Amar et al. 2002). Recently, this geno-
type has been reported in beavers (Fayer et al. 2006),
cattle (Coklin et al. 2007; Mendonça et al. 2007),
dogs (Read et al. 2004; Traub et al. 2004; Lalle et al.
2005), horses (Traub et al. 2005), monkeys (Itagaki
et al. 2005), muskrats (Sulaiman et al. 2003), rabbits
(Sulaiman et al. 2003) and sheep (Castro-Hermida
et al. 2007). This study constitutes the first descrip-
tion of G. duodenalis assemblage B in R. rattus, which
is not surprising since it has been found previously
in other rodent species (beavers and muskrats) by
characterization of TPI and BG loci (Sulaiman et al.
2003). Nowadays, in order to increase the accuracy of
genotyping of isolates at this level, subtypes from two
or three loci are combined to define multi-locus
genotypes (MLGs) (Sprong et al. 2012). However, a
previous work has demonstrated that isolates geno-
typed with BG, GDH and TPI genes can be con-
sidered as potentially zoonotic when all three markers
were found to be zoonotic individually (Sprong et al.
2012). These authors consider that an alternative
approach for the identification of potential zoonotic
MLGs is to combine the zoonotic information of
subtypes of individual markers. MLG tools are
further being used to answer the question of the infec-
tivity of some host-adapted assemblages (such as
assemblage G) in humans. In our study, all three loci
confirmed that G111 belonged to assemblage B.
Thus, this finding could be relevant from a zoonotic
point of view, especially due to the presence of this
rodent in a rural and inhabited area. The available
data about giardiasis in humans demonstrated that
4–12 year-old children from all the Canary Islands
were infected with G. duodenalis with prevalences
that ranged from 11·6% to 24·8% among the islands
(Valladares et al. 1983).
Subgrouping within this zoonotic assemblage is of
interest as there may be a link between specific assem-
blage B subgroups and their hosts, with particular
attention on those that may affect humans. It is there-
fore important to establish the specific subgroups,
based on their intra-genotypic substitution patterns
(Wielinga and Thompson, 2007). The isolate G111
was classified as BIV at the TPI gene, but consisted of
a combination of BIII and BIV-like substitution
patterns at the GDH gene. However, it displayed
Ángela Fernández-Álvarez and others
a novel intra-assemblage B substitution site at this
locus, although this substitution pattern found at the
GDH gene must be confirmed with other assemblage
B isolates.
In conclusion, our results establish a basis to iden-
tify the sub-genotype GII as a novel sub-genotype
in the process of becoming a new genotype, which
has evolved from G. duodenalis assemblage G. The
further finding of an assemblage B isolate in an inhabi-
ted area poses a risk for transmission of giardiasis and
should be used by Canarian public health authorities
to build an effective control strategy.
ACKNOWLEDGEMENTS
We thank ‘Excmos. Cabildos Insulares’ of Tenerife,
La Palma, La Gomera, El Hierro, Gran Canaria,
Fuerteventura and Lanzarote. We also thank
M. Hernández-Ferrer for his help in the phylogenetic
analysis.
FINANCIAL SUPPORT
This work was supported by projects ‘Programa Nacional
de Biodiversidad, Ciencias de la Tierra y Cambio Global:
Subprograma de Biología de Organismos y Sistemas’
(CGL 2006-04937 and CGL2009-07759BOS) of the
‘Ministerio de Ciencia y Educación’ and Canary Govern-
ment (A. M. A., grant number TESIS20100083).
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