Experimental Parasitology 124 (2010) 107112

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Experimental Parasitology
journal homepage: www.elsevier.com/locate/yexpr

Giardia duodenalis: Genetic recombination and its implications for taxonomy

and molecular epidemiology
Simone M. Cacci a,*, Hein Sprong b,1
a

Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanit, Viale Regina Elena 299, Rome 00161, Italy
Laboratory for Zoonoses and Environmental Microbiology, National Institute for Public Health and Environment (RIVM), Mailbox 63, Antonie van Leeuwenhoeklaan 9,
P.O. Box 1, 3720 BA Bilthoven, The Netherlands
b

a r t i c l e

i n f o

Article history:
Received 20 October 2008
Received in revised form 15 December 2008
Accepted 5 February 2009
Available online 21 February 2009
Keywords:
Giardia
Protozoa
Flagellates
Recombination
Sex
Taxonomy
Molecular epidemiology

a b s t r a c t
Traditionally, species within the Giardia genus have been considered as eukaryotic organisms that show an
absence of sexual reproduction in their simple life cycles. This apparent lack of sex has been challenged by
a number of studies that have demonstrated (i) the presence in the Giardia duodenalis genome of true
homologs of genes specically involved in meiosis in other eukaryotes, and their stage-specic expression; (ii) the exchange of genetic material in different chromosomal regions among human isolates of
the parasite; (iii) the fusion between cyst nuclei (karyogamy) and the transfer of genetic material (episomal plasmids) between them. These results are pivotal for the existence of sexual recombination. However, many details of the process remain elusive, and experimental data are still scarce. This review
summarizes the experimental approaches and the results obtained, and discusses the implications of
recombination from the standpoint of the taxonomy and molecular epidemiology of this widespread
pathogen.
2009 Elsevier Inc. All rights reserved.

1. Introduction
Giardia duodenalis (syn. Giardia intestinalis, Giardia lamblia) is a
agellated protozoan parasite that causes giardiasis in humans,
pets, livestock, and wildlife. This peculiar organism has attracted
much interest not only because of its medical and veterinary importance (Thompson and Monis, 2004), but also because of its presumed primitive nature, to the point that it has been described
as a biological fossil, namely a true eukaryote with many peculiarities that retained some ancestral prokaryotic properties (Upcroft
and Upcroft, 1998). Even if this view has been largely disproved
by more recent studies (Embley and Martin, 2006), Giardia remains
an interesting model organism for the study of many cellular processes (for example cell differentiation and protein trafcking), also
thanks to the possibility to reproduce its simple life cycle, which
comprises the vegetative trophozoite and the cyst, under axenic
culture conditions.
Like all diplomonads, Giardia has two diploid nuclei that are
morphologically indistinguishable, replicate at approximately the
same time, and are both transcriptionally active (Adam, 2000). In
each cell cycle, both nuclei in a trophozoite divide, giving rise to a
total of four daughter nuclei. It has been shown that the two daugh* Corresponding author. Fax: +39 06 49903561.
E-mail addresses: simone.caccio@iss.it (S.M. Cacci), hein.sprong@rivm.nl
(H. Sprong).
1
Fax: +31 30 2744434.
0014-4894/$ - see front matter 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.exppara.2009.02.007

ters of a single nucleus segregate to different trophozoites, namely

that segregation is equational (Yu et al., 2002; Sagolla et al., 2006).
An important prediction results from equational segregation: differences between the nuclei would be expected to accumulate over
time. If the two nuclei contain the same complement of genes and
chromosomes, these differences would be demonstrated in the form
of heterogeneity of homologous chromosomes and allelic sequence
heterozygosity (ASH). Chromosome size heterogeneity is well documented, and ASH of repeat copy number for the vsp genes is common (Adam, 2000).
However, ASH at the sequence level is quite uncommon in
G. duodenalis and estimates from the WB strain genome project suggest this level to be 0.01% (Morrison et al., 2007). This nding is considered unusual for polyploid organisms like Giardia spp., which
have been generally assumed to be asexual organisms of ancient
origin, and has puzzled researchers for many years. One mechanism
that can explain the maintenance of a low level of ASH is genetic
recombination, but direct evidence for this was lacking.
In the following sections, we will rst review the recent experimental evidence in favor and against the occurrence of recombination in Giardia and then discuss the implications for taxonomy
and epidemiology.
2. Evidence for meiotic genes in Giardia
The question of whether Giardia is potentially capable of sexual
reproduction was rst addressed by Ramesh et al. (2005), who

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S.M. Cacci, H. Sprong / Experimental Parasitology 124 (2010) 107112

surveyed the G. duodenalis genome sequence (of the strain WB,

assemblage A) for a common set of genes required for meiotic
recombination (and thus, sex) in other eukaryotes (animals, plants
and fungi). This meiotic gene inventory showed that true homologs
of genes specically required for meiosis in model eukaryotic species are widely distributed among diverse eukaryotes. In particular,
ve genes (Dmc1, Spo11, Mnd1, Hop1, and Hop2) known to function
specically during meiosis in other eukaryotes, are present in Giardia, as further conrmed by cloning and sequencing of PCR products obtained from genomic deoxyribonucleic acid (DNA).
Furthermore, Ramesh et al. (2005) provided evidence for the bona
de nature of the identied homologs by constructing phylogenies
from the proteins encoded by each of these genes, and showing
that the Giardia proteins group unequivocally with other eukaryotic homologs, usually as a deep branch. A deep branch can be explained in two ways.
1. Phylogenetic analyses places Giardia as a primitive earlybranching eukaryote and a pivotal missing link between prokaryotes and eukaryotes. Thus, future studies of meiosis in Giardia will
provide exciting new insights in the origin of meiosis. It should,
however, be remember that these genes may have a non-meiotic
function in the last common ancestor of Giardia and other eukaryotes; indeed, many researchers believe that the eukaryotic meiosis
machinery originally evolved from genes involved in DNA damage
repair (Vielleneuve and Hillers, 2001).
2. Recent developments in evolutionary biology has led to believe
that amitochondriate organisms, such as Giardia, are not primitive,
but instead highly evolved and specialized for their specic environments (Dacks et al., 2008). Giardia might therefore utilize the
core meiosis machinery for a process, which might be deviated
from meiosis (and sex) found in animals, plants and fungi (Haig,
1993). The core meiosis machinery of Giardia might function as a
DNA repair machinery between the two nuclei, thereby causing
lower ASH than expected.
Recently, Melo et al. (2008) have analysed the expression the
transcription of some of the genes potentially involved in meiosis.
They have identied two homologs of the Dcm1 gene (that were
named Dcm1a and Dcm1b) and one homolog of Spo11 and Hop1
genes. Using semi-quantitative RT-PCR, the authors have shown
that Dcm1b, which has the same sequence of Rad51, a gene whose
product is involved in DNA repair during mitosis, is transcribed almost constitutively during the life cycle of Giardia, and suggested
that it may indeed have a role similar to Rad51. On the other hand,
Dcm1a is strongly induced during early encystation and early
excystation, and its expression remains high while Spo11 and
Hop1 are transcribed. The authors concluded that transcription of
these three genes may facilitate the exchange of genetic material
between and within the two nuclei during encystation and
excystation.

3. Indirect evidence from molecular genotyping studies

Giardia duodenalis is considered as a species complex, whose
members show little variation in their morphology, yet can be assigned to seven distinct genetic groups (assemblages AG) based
on protein and DNA polymorphisms (Monis et al., 2003; Cacci
and Ryan, 2008). Among the seven assemblages, only assemblages
A and B have been found in humans and in a wide range of other
mammalian hosts, whereas assemblages CG seem to have a more
restricted host range (Table 1).
The direct characterization of Giardia cysts at the molecular level by the use of PCR techniques is widely used in many laboratories to study the epidemiology of the infection. The vast majority of

studies have relied on the analysis of the small subunit ribosomal

RNA (ssu-rRNA), the b-giardin (bg), the glutamate dehydrogenase
(gdh), the elongation factor 1-alpha (ef-1), the triose phosphate
isomerase (tpi), the GLORF-C4 (C4) genes and recently, the intergenomic rRNA spacer region (Cacci and Ryan, 2008). In early studies, there was a strong bias towards the use of ssu-rRNA that, due
to its multicopy nature and high degree of sequence homology,
represented the locus of choice for the genotyping of Giardia isolates. As a consequence, the reliability of different genetic loci in
the assignment of isolates to specic G. duodenalis assemblages
was not assessed, the assumption being that these loci will only
differ in terms of their polymorphism. Recent studies based on a
multilocus approach have shown that a number of isolates, of both
human and animal origin, cannot be unequivocally assigned at the
assemblage level, because the genotyping data from different loci
were not consistent (Traub et al., 2004; Gelanew et al., 2007;
Cacci et al., 2008).
To obtain a more informative picture, we performed an analysis
of a dedicated database which combines epidemiological data from
eld isolates (country, year of isolation, source, symptoms, etc.)
and sequence data of the ssu-rRNA, bg, gdh and tpi genes. This database has been developed in the context of the ZOOnotic Protozoa
NETwork (ZOOPNET), a European network of veterinary and public
health Institutions working on Cryptosporidium and Giardia, and
currently includes over 2400 Giardia sequences (including those
from Genbank). From 30% of the Giardia isolates in the ZOOPNET
database, two or more markers are known (August 2008). The analysis of these isolates for the presence of inter-assemblage mixing
(in other words, inconsistent typing between two markers)
showed that this phenomenon occurred in 15% of the isolates,
and was predominantly observed in humans and dogs (Table 2). Intra-assemblage mixing (e.g. AI plus AII) was also observed for
assemblages AE (not shown).
Taken at face value, these results are compatible with recombination events occurring between different assemblages and in different hosts. However, as genotyping was performed directly on
DNA extracted from stool samples, inter-assemblage mixing
can also be explained by PCR bias in the presence of mixed infec-

Table 1
Giardia duodenalis assemblages and their distribution in mammalian hosts.
Assemblages

Host (s)

Human, non-human primates, livestock, horses, dogs, cats, guinea

pigs, fallow deer, white-tailed deer, moose, ferrets
Human, non-human primates, livestock, horses, dogs, coyotes,
muskrats, beavers
Dogs, cats, coyotes, wolves
Cattle, sheep, goat, water buffaloes, muons
Cats
Rats

B
C, D
E
F
G

Table 2
Unreliable assignment of individual isolates to specic G. duodenalis assemblages.
Data were taken from the ZOOPNET database (August 2008).
Mixed assemblages

Occurrence (n)

Host

A and B
A and C
A and D
A and E
B and C
B and D
B and E
C and D
C and E
D and E

39
2
2
3
4
2
1
10
0
1

Human, dog, cat, monkey

Dog
Dog
Cattle
Dog
Dog
Sheep
Dog
Sheep

S.M. Cacci, H. Sprong / Experimental Parasitology 124 (2010) 107112

tions. The frequency of inter-assemblage mixing appears higher

in isolates collected from endemic regions (data not shown), suggesting that the contribution of mixed infections should not be
underestimated (Traub et al., 2004; Gelanew et al., 2007).

4. Evidence for genetic exchanges among G. duodenalis isolates

Two recent papers have dealt with this aspect. In the rst,
Teodorovic et al. (2007) analysed 9 axenic strains of G. duodenalis
belonging to assemblage A (4 strains from genotype AI and 2 from
genotype AII), and assemblage B (3 strains), which they treated
each as a distinct population. The experiments were designed to detect a low amount of allelic sequence heterozygosity (ASH) within
and among isolates, therefore PCR products from six coding and
four non-coding (2 introns and 2 intergenic) regions were cloned,
and 20 independent clones were sequenced, for a total number of
652,729 bp analysed. The ASH estimates were exceedingly low,
both at the intra- and inter-isolates level, conrming data from
the genome sequencing project (ASH is less than 0.002% in the
WB genome, Morrison et al., 2007). However, the most striking result was the complex haplotype structure that specically characterized the assemblage B strains. Indeed, at ve loci (CPN60,
ferredoxin gene, ferredoxin intron, and two intergenic regions), all
B sequences grouped with AI sequences (that is, plasmids derived
from the B strains contained only AI sequences). Thus, phylogenetic
analysis detected two main clades at these ve loci: AI/B and AII.
However, at the remaining ve loci (actin, RPL gene, RPL intron,
TPI, and beta giardin) sequences of B strains exhibited a dichotomous structure, represented by two divergent haplotypes. A portion of sequences grouped again with AI sequences, but additional
sequences formed a B-specic clade, representing an independent
long branch on the tree. Thus, three main clades were detected at
these ve loci: AI/B, AII, and a B-specic haplotype. The authors
interpreted these results as the product of genetic exchanges between AI and B, therefore supporting the existence of a sexual cycle
in G. duodenalis. As this study was based on PCR, and primers were
designed based on the reference AI genome sequence, some biases
in the results obtained were evident, particularly the lack of amplication of several markers from assemblage B strains pointed to
sub-optimal binding of primers to non-A genomic templates. Some
results were suspicious: indeed, the fact that the actin gene of G. ardeae (a bird parasite) had a sequence very closely related to AI was
surprising, in view of the large genetic distance observed at other
genetic loci when this species was compared to G. duodenalis
(Monis et al., 1999). Similarly, the rare allele at the actin gene locus
from isolate CM (assemblage B) displayed 14 SNPs in the rst
180 bp, and no SNP at all in the remaining 434 bp, a very unusual
distribution not observed at other loci. Comparison at the CPN60
locus was possible for only 1 of the 3 assemblage B strains, and this
single case supported genetic recombination by showing an assemblage A sequence. Therefore, a bias towards amplication of assemblage A sequences in non-A isolates seems to affect at least some of
the data. The authors commented on this aspect saying that the
failure to detect a group B-specic haplotype at ve loci is a not a
consequence of their absence but rather of their low frequency in
the population. As the isolates used by Teodorovic et al. (2007)
are maintained in vitro, independent conrmation of these important results would be benecial.
The second study (Cooper et al., 2007) focussed on 5 G. duodenalis human isolates collected from an endemic region in Peru,
all belonging to genotype AII, and included a reference AII strain
(JH) for comparison. The authors have amplied and sequenced
relatively large (several kb) regions of chromosomes 3, 4, and 5,
to identify single-nucleotide polymorphisms (SNPs). The SNP density was low, around 0.9%, and the majority of mutations were syn-

109

onymous, as expected from isolates belonging to a single genotype.

Through a multilocus comparison, the authors concluded that loci
from different chromosomes yielded signicantly different phylogenetic trees, indicating that they do not share the same evolutionary history. For example, isolates JH, 55, and 335 were nearly
identical at the chromosome 5 locus, but all three were substantially different at the chromosome 3 locus. The apparently different
inheritance pattern for loci on different chromosomes provided
qualitative evidence for recombination. The authors then performed a quantitative test designed to detect gene conversion
events (GENECONV) to determine whether the within-chromosome regions showed evidence of recombination, and obtained signicant statistical support for recombination.
Interestingly, however, the results from the two shorter regions
on chromosome 4 (coding region of the glutamate dehydrogenase
gene, and coding regions for the beta-giardin gene and an hypothetical protein) showed a different pattern, which does not support the authors claim that at the chromosome 4 locus, 55 and
335 were identical to each other but substantially different from
JH. First, no SNPs were observed in the gdh coding region among
the 6 isolates, so no comparison can be made. Second, at the beta
giardin coding region, isolates 55 and 355 have exactly the sequence of the previously reported A3 b-giardin subtype (Genbank
AY072724 and DQ116612) with 2 SNPs from the JH (A2) but also
from the A1 subtype. At the hypothetical protein coding region,
isolates 55 and 355 have 8 SNPs compared to JH (A2), but only have
4 SNPs compared to A1. Therefore, the genomic regions from chromosome 4 are much more conserved than those from chromosomes 3 and 5, and show no sign of recombination.
Thus, this work showed evidence for recombination among isolates of assemblage A, genotype AII, in some regions of the genome,
but non in others. Certainly, the data do not support that such recombinant genotypes could only arise from exchange between A2
genotypes and another genotypically distinct parent (likely B) as
commented by Logsdon (2008).

5. Cytological evidence for nuclear fusion and transfer of

genetic material
Poxleitner et al. (2008) performed uorescent in situ hybridization (FISH) on trophozoites and cysts to determine whether the
cyst nuclei can exchange genetic material or remain physically
autonomous, as they do in trophozoites. In Giardia, plasmids can
be stably transfected into trophozoites as episomes; importantly,
episomes are found in only one of the nuclei, and this pattern persists through cell division and cytokinesis (Sagolla et al., 2006). In
contrast, two distinct patterns were observed after FISH detection
of episomes in cysts, a prevalent one (in about 70% of cysts) showing several episomes in two of the four nuclei, and a minor one (in
about 30% of cyst) showing episomes in three of the four nuclei
(Poxleitner et al., 2008). This suggests plasmid transfer between
the nuclei during encystation. The authors then used transmission
electron microscopy (TEM) to demonstrate fusion of the nuclear
envelopes (karyogamy), a process which can facilitate plasmid
transfer, and, more generally, genetic exchanges between nuclei.
The specic expression of giardial homologs of meiotic-specic
(recombination) genes in the cyst nuclei but not in the trophozoite
nuclei adds weigh to the notion that genetic exchanges occur in
Giardia. In the model proposed by Poxleitner et al. (2008), nuclear
fusion appears to be typically restricted to only one set of nondaughter nuclei, albeit the FISH experiments they used did not allow to detect plasmid transfer between daughter nuclei. The
authors proposed to call this process diplomixis, and commented
that, unlike automixis, diplomixis is not accompanied by meiotic
genome reduction and the subsequent fusion of gametes from

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S.M. Cacci, H. Sprong / Experimental Parasitology 124 (2010) 107112

the same parent, as is found in the sexual or parasexual life cycle of

other organisms. This unique process is thought to be shared by
other members of the order Diplomonadida, albeit direct evidences
for this are lacking.
6. Other potential drivers: Mobile elements and the Giardiaspecic virus
Other mechanisms that could enhance genetic exchanges within
and between trophozoites should also be considered, such as those
mediated by genetic mobile elements and a Giardia-specic virus.
G. duodenalis harbors non-LTR (Non-Long Terminal Repeats) retroelements of the LINEs family (Long Interspersed Nuclear Elements), that are transposed by reverse transcription of mRNA
directly into the site of integration. Most G. duodenalis subtelomeres
consist of tandem copies of active LINE retroposons (either GilM or
GilT elements), which directly abut the telomeric repeats and are
oriented such that reverse transcription would have run toward
the chromosome end (Wickstead et al., 2003). Repeats play integral
parts in ongoing genomic evolution and can play diverse roles at
different times, imparting a greater changeability to genomes.
When an organism faces a changeable environment, the advantages
of genomic exibility (particularly if it can be contained at specic
loci) may outweigh the extra cost of replication and of mutagenic
effect exerted on other genomic regions. It is currently unknown
what role the retroposons are playing (or have played) in the evolution of the G. duodenalis genome.
Giardia-specic virus (GLV) is a double-stranded (ds) RNA virus
of the Totiviridae family, constituted by a 36-nm nonenveloped
icosahedron comprising one dsRNA of about 7 kb (Wang and
Wang, 1991). GLV infects many G. duodenalis from assemblages
A, B, C/D and E, albeit no correlation between the presence or absence of the virus and the specic assemblage has been found
(Chen et al., 2007), and cohabitation of multiple GLV species in
the same parasite has been also demonstrated (Tai et al., 1996).
In G. duodenalis Portland I strain, which is chronically infected by
this virus, viral RNA was detected in the cytoplasm as well as in
the twin nuclei (Tai et al., 1991). Recombinant GLV cDNA has been
successfully introduced into GLV-infected trophozoites to express
a heterologous gene in G. duodenalis. Moreover, the chimeric RNA
could be replicated as double-stranded RNA and packaged into
virus-like particles and the recombinant virions, by themselves,
can superinfect G. duodenalis trophozoites and start new rounds
of expression (Yu et al., 1996). All these observations are compatible with fragment(s) of the G. duodenalis genome being inserted in
the GLV and then shuttled between trophozoites of different G.
duodenalis assemblages.
Intriguingly, plasmid DNA transfected into trophozoites of
assemblage A is maintained as a multimeric episome, whereas in
assemblage B is always integrated into the genome by homologous
recombination, with insertion of multiple copies (Singer et al.,
1998). This opposite behavior could reect isolate-specic differences in the factors determining whether exogenous DNA is integrated or maintained episomally. If plasmid- or virus-mediated
lateral exchange of DNA between two trophozoites can occur,
homologous recombination could explain, at least in part, the
higher level of ASH observed in assemblage B compared to assemblage A.
7. Evidence against sex
In a study of single Giardia cells, Tumova et al. (2006) studied 4
isolates (WB and HP-1 from assemblage A, and HH and CH-105
from assemblage B) and showed that chromosomes were not distributed symmetrically between nuclei. Indeed, considering the
ve chromosome linkage groups and a ploidy of two for each nu-

cleus, then the chromosome number per nucleus should be 10,

which falls in the range (911) observed by Tumova et al. (2006).
However, asymmetrical distribution as well as odd chromosome
numbers indicate that at least for some chromosome(s), either
one or both nuclei are aneuploid (Tumova et al., 2006). Moreover,
if each nucleus contain one copy of each of the ve linkage groups
(Yu et al., 2002), then the nucleus with 9 chromosomes should be
monosomic for one chromosome pair. Therefore, the authors concluded that a major requirement for meiosis, namely the presence
of just two homologous copies of each chromosome, is not met,
and that the stable transmission of an aneuploid pattern implies
absence of meiosis. As a consequence, each nucleus is believed to
evolve independently as a clonal lineage during the life cycle of
Giardia. This model predicts that the nuclei will accumulate differences during evolution by rearrangements and nondisjunctions,
but cannot explain how low levels of ASH are maintained.
As already mentioned, a low level of ASH appears to characterize G. duodenalis isolates. This is largely based on results from the
analysis of few isolates and genetic loci, with a bias towards
assemblages A and B, often on axenized strains (Baruch et al.,
1996; Teodorovic et al., 2007). The genome sequence of the WB
strain conrmed a low ASH level in this particular strain of assemblage A (Morrison et al., 2007). However, as sequence data from
isolates collected worldwide are continuously generated, it is possible to evaluate levels of ASH from eld isolates. Albeit limited by
the small number of loci investigated, and by the difculty in distinguishing allelic sequence heterozygosity from mixed infections,
the data presented in Table 3 clearly shows that heterogeneous
sequencing proles (characterized by two overlapping nucleotide
peaks at specic positions in the electropherograms) occur much
more often in isolates of assemblages B and C than in those from
assemblage A. This has been reported in several papers from different research groups (Hopkins et al., 1997; Gelanew et al., 2007;
Lebbad et al., 2008; Cacci et al., 2008). Therefore, levels of ASH
vary in different G. duodenalis assemblages, and are lower in
assemblage A. More data are needed to robustly conrm this nding which, if proven, will support the independent accumulation of
mutations in the nuclei, and thus, lack of recombination between
them.
8. Implications for taxonomy and epidemiology
An appropriate classication for Giardia spp. is critical to an
understanding of the pathogenesis and epidemiology of infection,
as well as the biology of the organism. Historically, Giardia species
have been named on the basis of host occurrence with more than
40 species described in the literature, mainly from mammals. This
criterion was criticized by Filice (1952) who recognized the inherent variability within Giardia affecting mammals and created a
holding position in placing many described species under the G.
duodenalis umbrella. His proposal of only three valid Giardia species (G. duodenalis, G. muris and G. agilis), dened by differences in
the overall shape of the trophozoites and in median bodies, was
largely accepted and forms the basis of the current taxonomy.
More recently, two species from birds (G. ardeae from herons and

Table 3
Occurrence of heterogeneous positions in the beta-giardin (BG), glutamate dehydrogenase (GDH) and triose phosphate isomerase (TPI) genes as found in isolates of
assemblage AC. Data were taken from the ZOOPNET database (August 2008).
Assemblage

BG (%)

GDH (%)

TPI (%)

A
B
C

8
20
18

2
40
18

5
19
40

S.M. Cacci, H. Sprong / Experimental Parasitology 124 (2010) 107112

G. psittaci from psittacine birds) and one from rodents (G. microti)
have been described based on morphological features of the trophozoites and of the cysts (Thompson and Monis, 2004).
The characterization of G. duodenalis by isoenzyme electrophoresis rst revealed extensive genetic polymorphism among isolates, and prompted the proposition that it comprises cryptic
species (Andrews et al., 1989). The existence of genetic groups,
or assemblages, within G. duodenalis was later conrmed by further enzyme electrophoretic studies (Monis et al., 2003) which
clearly showed the host association of particular assemblages. Sequence and phylogenetic analyses of a number of genes have conrmed the enzyme electrophoresis groupings and shown that the
assemblages represent distinct evolutionary lineages and that a degree of host specicity exists (reviewed by Thompson and Monis,
2004). In addition, differences have been reported in metabolism
and biochemistry, DNA content, in vitro and in vivo growth rates,
drug sensitivity, predilection site in vivo and duration of infection,
pH preference, infectivity, susceptibility to infection with a dsRNA
virus, and clinical features (reviewed in Cacci et al., 2005).
Thus, it appears that the taxonomic status afforded previously
to Giardia described in dogs, cats, rats and cattle as separate species
(namely G. canis, G. cati, G. simondi, and G. bovis) has to be re-evaluated to give appropriate recognition to these original taxonomic
descriptions. Similarly, since separate species names for assemblages A and B are needed, a proposal for G. duodenalis and G. enterica has been put forward (Thompson and Monis, 2004).
The occurrence of recombination, if proven, will have an important impact on the taxonomy of G. duodenalis. Admittedly, there
are problems to dene when a parasite species should be considered as such (e.g., Kunz, 2002). The biological species concept
states that species are groups of actual or potentially interbreeding natural populations, which are reproductively isolated from
other such groups (Mayr, 1942). While the applicability of this
concept to protozoa is debatable, the data of Teodorovic et al.
(2007) will indicate that assemblages A and B are able to exchange
genetic material, and will not support them as separate species. On
the contrary, the data of Cooper et al. (2007) will not invalidate this
proposal.
It is therefore crucial to demonstrate if recombination can occur
between different G. duodenalis assemblages. At the molecular level, this can be evaluated by testing the genetic material from single cysts, and by applying a method that allow to trace specically
the presence of each assemblage. We are currently developing an
assay based on the use of assemblage-specic primers coupled
with sensitive detection in a real-time PCR platform (Almeida, A.,
Pozio E., Cacci S.M., unpublished data). The application of this assay to G. duodenalis cysts puried by immunomagnetic separation
allows to detect DNA of assemblage A and/or B at different loci
using known number of cysts, thereby permitting to distinguish
between recombinants and mixed infections. Preliminary data
support the existence of true recombinants between assemblages
A and B, but more data should be collected by exploring other genetic loci to conrm present results.
From the perspective of molecular epidemiological studies, it is
of particular relevance the fact that animal isolates can be typed as
potentially zoonotic with one marker, but as host-specic with
another. For, therefore, this has very important implications, as totally different conclusions may be inferred depending on the way
genotyping data are obtained and interpreted.

9. Conclusions
The occurrence of recombination among G. duodenalis isolates
has been recently supported by several studies. A number of
important aspects remain, however, unresolved (see Table 4),

111

Table 4
Open questions regarding genetic recombination in Giardia.
How often does recombination events occur in the wild?
Are all G. duodenalis assemblages involved in recombination?
At what stage does it happen: cyst or trophozoite?
What purpose does recombination serve: sex or repair?
Are there specic hot-spots of recombination along the chromosomes?
What are the consequences of recombination for the etiology/pathogenesis of
giardiasis?
What is the impact of recombination in shaping the population structure of the
parasite?
What processes underlie the low ASH in Giardia isolates?

and conicting results have also been reported. Future studies

are needed to understand if, how often, and under which conditions, recombination occurs in the wild. This will be instrumental
for a critical re-evaluation of debated issues on the taxonomy
and the epidemiology of Giardia, and will also shed light on the origin of meiosis and sexual recombination in eukaryotes.
Acknowledgments
Work in the laboratories of S.M. Cacci and H. Sprong were partially supported by the European MED-VET-NET project, contract
FOOD-CT-2004-506122.
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