ni SaHea

n thtionave

the parasite; (iii) the fusion between cyst nuclei (karyogamy) and the transfer of genetic material (epi-somal plasmids) between them. These results are pivotal for the existence of sexual recombination. How-

testinacausesculiar

culture conditions.Like all diplomonads, Giardia has two diploid nuclei that are

morphologically indistinguishable, replicate at approximately thesame time, and are both transcriptionally active (Adam, 2000). Ineach cell cycle, both nuclei in a trophozoite divide, giving rise to atotal of four daughter nuclei. It has been shown that the two daugh-

that can explain the maintenance of a low level of ASH is geneticrecombination, but direct evidence for this was lacking.

In the following sections, we will rst review the recent exper-imental evidence in favor and against the occurrence of recombi-nation in Giardia and then discuss the implications for taxonomyand epidemiology.

2. Evidence for meiotic genes in Giardia

The question of whether Giardia is potentially capable of sexualreproduction was rst addressed by Ramesh et al. (2005), who

* Corresponding author. Fax: +39 06 49903561.E-mail addresses: simone.caccio@iss.it (S.M. Cacci), hein.sprong@rivm.nl

(H. Sprong).

Experimental Parasitology 124 (2010) 107112

Contents lists availab

Experimental

.e l1 Fax: +31 30 2744434.much interest not only because of itsmedical and veterinary impor-tance (Thompson and Monis, 2004), but also because of its pre-sumed primitive nature, to the point that it has been describedas a biological fossil, namely a true eukaryote with many peculiar-ities that retained some ancestral prokaryotic properties (Upcroftand Upcroft, 1998). Even if this view has been largely disprovedby more recent studies (Embley and Martin, 2006), Giardia remainsan interesting model organism for the study of many cellular pro-cesses (for example cell differentiation and protein trafcking), alsothanks to the possibility to reproduce its simple life cycle, whichcomprises the vegetative trophozoite and the cyst, under axenic

chromosomes, these differenceswould be demonstrated in the formof heterogeneity of homologous chromosomes and allelic sequenceheterozygosity (ASH). Chromosome size heterogeneity is well doc-umented, and ASH of repeat copy number for the vsp genes is com-mon (Adam, 2000).

However, ASH at the sequence level is quite uncommon inG. duodenalis and estimates from theWB strain genome project sug-gest this level to be 0.01% (Morrison et al., 2007). This nding is con-sidered unusual for polyploid organisms like Giardia spp., whichhave been generally assumed to be asexual organisms of ancientorigin, and has puzzled researchers formany years. OnemechanismKeywords:GiardiaProtozoaFlagellatesRecombinationSexTaxonomyMolecular epidemiology

1. Introduction

Giardia duodenalis (syn. Giardia inagellated protozoan parasite thatpets, livestock, and wildlife. This pe0014-4894/$ - see front matter 2009 Elsevier Inc. Adoi:10.1016/j.exppara.2009.02.007ever, many details of the process remain elusive, and experimental data are still scarce. This reviewsummarizes the experimental approaches and the results obtained, and discusses the implications ofrecombination from the standpoint of the taxonomy and molecular epidemiology of this widespreadpathogen.

2009 Elsevier Inc. All rights reserved.

lis, Giardia lamblia) is agiardiasis in humans,

organism has attracted

ters of a single nucleus segregate to different trophozoites, namelythat segregation is equational (Yu et al., 2002; Sagolla et al., 2006).An important prediction results fromequational segregation: differ-ences between the nuclei would be expected to accumulate overtime. If the two nuclei contain the same complement of genes andAccepted 5 February 2009Available online 21 February 2009

homologs of genes specically involved in meiosis in other eukaryotes, and their stage-specic expres-sion; (ii) the exchange of genetic material in different chromosomal regions among human isolates ofGiardia duodenalis: Genetic recombinatioand molecular epidemiology

Simone M. Cacci a,*, Hein Sprong b,1

aDepartment of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore db Laboratory for Zoonoses and Environmental Microbiology, National Institute for PublicP.O. Box 1, 3720 BA Bilthoven, The Netherlands

a r t i c l e i n f o

Article history:Received 20 October 2008Received in revised form 15 December 2008

a b s t r a c t

Traditionally, species withiabsence of sexual reproduca number of studies that h

journal homepage: wwwll rights reserved.and its implications for taxonomy

nit, Viale Regina Elena 299, Rome 00161, Italylth and Environment (RIVM), Mailbox 63, Antonie van Leeuwenhoeklaan 9,

eGiardia genus have been considered as eukaryotic organisms that show anin their simple life cycles. This apparent lack of sex has been challenged bydemonstrated (i) the presence in the Giardia duodenalis genome of true

le at ScienceDirect

Parasitology

sevier .com/ locate/yexpr

Furthermore, Ramesh et al. (2005) provided evidence for the bonade nature of the identied homologs by constructing phylogenies

studies have relied on the analysis of the small subunit ribosomalRNA (ssu-rRNA), the b-giardin (bg), the glutamate dehydrogenase(gdh), the elongation factor 1-alpha (ef-1), the triose phosphateisomerase (tpi), the GLORF-C4 (C4) genes and recently, the inter-genomic rRNA spacer region (Cacci and Ryan, 2008). In early stud-ies, there was a strong bias towards the use of ssu-rRNA that, dueto its multicopy nature and high degree of sequence homology,represented the locus of choice for the genotyping of Giardia iso-lates. As a consequence, the reliability of different genetic loci inthe assignment of isolates to specic G. duodenalis assemblageswas not assessed, the assumption being that these loci will onlydiffer in terms of their polymorphism. Recent studies based on amultilocus approach have shown that a number of isolates, of bothhuman and animal origin, cannot be unequivocally assigned at theassemblage level, because the genotyping data from different lociwere not consistent (Traub et al., 2004; Gelanew et al., 2007;Cacci et al., 2008).

To obtain a more informative picture, we performed an analysisof a dedicated database which combines epidemiological data fromeld isolates (country, year of isolation, source, symptoms, etc.)and sequence data of the ssu-rRNA, bg, gdh and tpi genes. This data-base has been developed in the context of the ZOOnotic ProtozoaNETwork (ZOOPNET), a European network of veterinary and publichealth Institutions working on Cryptosporidium and Giardia, andcurrently includes over 2400 Giardia sequences (including those

tal Parasitology 124 (2010) 107112from the proteins encoded by each of these genes, and showingthat the Giardia proteins group unequivocally with other eukary-otic homologs, usually as a deep branch. A deep branch can be ex-plained in two ways.

1. Phylogenetic analyses places Giardia as a primitive early-branching eukaryote and a pivotal missing link between prokary-otes and eukaryotes. Thus, future studies of meiosis in Giardia willprovide exciting new insights in the origin of meiosis. It should,however, be remember that these genes may have a non-meioticfunction in the last common ancestor of Giardia and other eukary-otes; indeed, many researchers believe that the eukaryotic meiosismachinery originally evolved from genes involved in DNA damagerepair (Vielleneuve and Hillers, 2001).2. Recent developments in evolutionary biology has led to believethat amitochondriate organisms, such as Giardia, are not primitive,but instead highly evolved and specialized for their specic envi-ronments (Dacks et al., 2008). Giardia might therefore utilize thecore meiosis machinery for a process, which might be deviatedfrom meiosis (and sex) found in animals, plants and fungi (Haig,1993). The core meiosis machinery of Giardia might function as aDNA repair machinery between the two nuclei, thereby causinglower ASH than expected.

Recently, Melo et al. (2008) have analysed the expression thetranscription of some of the genes potentially involved in meiosis.They have identied two homologs of the Dcm1 gene (that werenamed Dcm1a and Dcm1b) and one homolog of Spo11 and Hop1genes. Using semi-quantitative RT-PCR, the authors have shownthat Dcm1b, which has the same sequence of Rad51, a gene whoseproduct is involved in DNA repair during mitosis, is transcribed al-most constitutively during the life cycle of Giardia, and suggestedthat it may indeed have a role similar to Rad51. On the other hand,Dcm1a is strongly induced during early encystation and earlyexcystation, and its expression remains high while Spo11 andHop1 are transcribed. The authors concluded that transcription ofthese three genes may facilitate the exchange of genetic materialbetween and within the two nuclei during encystation andexcystation.

3. Indirect evidence from molecular genotyping studies

Giardia duodenalis is considered as a species complex, whosemembers show little variation in their morphology, yet can be as-signed to seven distinct genetic groups (assemblages AG) basedon protein and DNA polymorphisms (Monis et al., 2003; Cacciand Ryan, 2008). Among the seven assemblages, only assemblagesA and B have been found in humans and in a wide range of othermammalian hosts, whereas assemblages CG seem to have a morerestricted host range (Table 1).surveyed the G. duodenalis genome sequence (of the strain WB,assemblage A) for a common set of genes required for meioticrecombination (and thus, sex) in other eukaryotes (animals, plantsand fungi). This meiotic gene inventory showed that true homologsof genes specically required for meiosis in model eukaryotic spe-cies are widely distributed among diverse eukaryotes. In particular,ve genes (Dmc1, Spo11,Mnd1, Hop1, and Hop2) known to functionspecically during meiosis in other eukaryotes, are present in Giar-dia, as further conrmed by cloning and sequencing of PCR prod-ucts obtained from genomic deoxyribonucleic acid (DNA).

108 S.M. Cacci, H. Sprong / ExperimenThe direct characterization of Giardia cysts at the molecular le-vel by the use of PCR techniques is widely used in many laborato-ries to study the epidemiology of the infection. The vast majority offrom Genbank). From 30% of the Giardia isolates in the ZOOPNETdatabase, two or more markers are known (August 2008). The anal-ysis 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). In-tra-assemblage mixing (e.g. AI plus AII) was also observed forassemblages AE (not shown).

Taken at face value, these results are compatible with recombi-nation events occurring between different assemblages and in dif-ferent hosts. However, as genotyping was performed directly onDNA extracted from stool samples, inter-assemblage mixingcan also be explained by PCR bias in the presence of mixed infec-

Table 2Unreliable 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 39 Human, dog, cat, monkeyA and C 2 DogA and D 2 DogA and E 3 CattleB and C 4 DogB and D 2 DogB and E 1 SheepC and D 10 Dog

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

Assemblages Host (s)

A Human, non-human primates, livestock, horses, dogs, cats, guineapigs, fallow deer, white-tailed deer, moose, ferrets

B Human, non-human primates, livestock, horses, dogs, coyotes,muskrats, beavers

C, D Dogs, cats, coyotes, wolvesE Cattle, sheep, goat, water buffaloes, muonsF CatsG RatsC and E 0D and E 1 Sheep

are maintained in vitro, independent conrmation of these impor-tant results would be benecial.

4 SNPs compared to A1. Therefore, the genomic regions from chro-mosome 4 are much more conserved than those from chromo-

tal PThe second study (Cooper et al., 2007) focussed on 5 G. duode-nalis 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 sequencedtions. The frequency of inter-assemblage mixing appears higherin isolates collected from endemic regions (data not shown), sug-gesting that the contribution of mixed infections should not beunderestimated (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. duodenalisbelonging to assemblage A (4 strains from genotype AI and 2 fromgenotype AII), and assemblage B (3 strains), which they treatedeach as a distinct population. The experimentswere designed to de-tect a low amount of allelic sequence heterozygosity (ASH) withinand among isolates, therefore PCR products from six coding andfour non-coding (2 introns and 2 intergenic) regions were cloned,and 20 independent clones were sequenced, for a total number of652,729 bp analysed. The ASH estimates were exceedingly low,both at the intra- and inter-isolates level, conrming data fromthe genome sequencing project (ASH is less than 0.002% in theWB genome, Morrison et al., 2007). However, the most striking re-sult was the complex haplotype structure that specically charac-terized the assemblage B strains. Indeed, at ve loci (CPN60,ferredoxin gene, ferredoxin intron, and two intergenic regions), allB sequences grouped with AI sequences (that is, plasmids derivedfrom the B strains contained only AI sequences). Thus, phylogeneticanalysis 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 dichoto-mous structure, represented by two divergent haplotypes. A por-tion of sequences grouped again with AI sequences, but additionalsequences formed a B-specic clade, representing an independentlong branch on the tree. Thus, three main clades were detected atthese ve loci: AI/B, AII, and a B-specic haplotype. The authorsinterpreted these results as the product of genetic exchanges be-tween AI and B, therefore supporting the existence of a sexual cyclein G. duodenalis. As this study was based on PCR, and primers weredesigned based on the reference AI genome sequence, some biasesin the results obtained were evident, particularly the lack of ampli-cation of several markers from assemblage B strains pointed tosub-optimal binding of primers to non-A genomic templates. Someresults were suspicious: indeed, the fact that the actin gene of G. ar-deae (a bird parasite) had a sequence very closely related to AI wassurprising, in view of the large genetic distance observed at othergenetic loci when this species was compared to G. duodenalis(Monis et al., 1999). Similarly, the rare allele at the actin gene locusfrom isolate CM (assemblage B) displayed 14 SNPs in the rst180 bp, and no SNP at all in the remaining 434 bp, a very unusualdistribution not observed at other loci. Comparison at the CPN60locus was possible for only 1 of the 3 assemblage B strains, and thissingle case supported genetic recombination by showing an assem-blage A sequence. Therefore, a bias towards amplication of assem-blage A sequences in non-A isolates seems to affect at least some ofthe data. The authors commented on this aspect saying that thefailure to detect a group B-specic haplotype at ve loci is a not aconsequence of their absence but rather of their low frequency inthe population. As the isolates used by Teodorovic et al. (2007)

S.M. Cacci, H. Sprong / Experimenrelatively large (several kb) regions of chromosomes 3, 4, and 5,to identify single-nucleotide polymorphisms (SNPs). The SNP den-sity was low, around 0.9%, and the majority of mutations were syn-somes 3 and 5, and show no sign of recombination.Thus, this work showed evidence for recombination among iso-

lates of assemblage A, genotype AII, in some regions of the genome,but non in others. Certainly, the data do not support that such re-combinant genotypes could only arise from exchange between A2genotypes and another genotypically distinct parent (likely B) ascommented by Logsdon (2008).

5. Cytological evidence for nuclear fusion and transfer ofgenetic material

Poxleitner et al. (2008) performed uorescent in situ hybridiza-tion (FISH) on trophozoites and cysts to determine whether thecyst nuclei can exchange genetic material or remain physicallyautonomous, as they do in trophozoites. In Giardia, plasmids canbe stably transfected into trophozoites as episomes; importantly,episomes are found in only one of the nuclei, and this pattern per-sists through cell division and cytokinesis (Sagolla et al., 2006). Incontrast, two distinct patterns were observed after FISH detectionof episomes in cysts, a prevalent one (in about 70% of cysts) show-ing several episomes in two of the four nuclei, and a minor one (inabout 30% of cyst) showing episomes in three of the four nuclei(Poxleitner et al., 2008). This suggests plasmid transfer betweenthe nuclei during encystation. The authors then used transmissionelectron microscopy (TEM) to demonstrate fusion of the nuclearenvelopes (karyogamy), a process which can facilitate plasmidtransfer, 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 trophozoitenuclei adds weigh to the notion that genetic exchanges occur inGiardia. In the model proposed by Poxleitner et al. (2008), nuclearfusion appears to be typically restricted to only one set of non-daughter nuclei, albeit the FISH experiments they used did not al-low to detect plasmid transfer between daughter nuclei. Theonymous, as expected from isolates belonging to a single genotype.Through a multilocus comparison, the authors concluded that locifrom different chromosomes yielded signicantly different phylo-genetic trees, indicating that they do not share the same evolution-ary history. For example, isolates JH, 55, and 335 were nearlyidentical at the chromosome 5 locus, but all three were substan-tially different at the chromosome 3 locus. The apparently differentinheritance pattern for loci on different chromosomes providedqualitative evidence for recombination. The authors then per-formed a quantitative test designed to detect gene conversionevents (GENECONV) to determine whether the within-chromo-some regions showed evidence of recombination, and obtained sig-nicant statistical support for recombination.

Interestingly, however, the results from the two shorter regionson chromosome 4 (coding region of the glutamate dehydrogenasegene, and coding regions for the beta-giardin gene and an hypo-thetical protein) showed a different pattern, which does not sup-port the authors claim that at the chromosome 4 locus, 55 and335 were identical to each other but substantially different fromJH. First, no SNPs were observed in the gdh coding region amongthe 6 isolates, so no comparison can be made. Second, at the betagiardin coding region, isolates 55 and 355 have exactly the se-quence of the previously reported A3 b-giardin subtype (GenbankAY072724 and DQ116612) with 2 SNPs from the JH (A2) but alsofrom the A1 subtype. At the hypothetical protein coding region,isolates 55 and 355 have 8 SNPs compared to JH (A2), but only have

arasitology 124 (2010) 107112 109authors proposed to call this process diplomixis, and commentedthat, unlike automixis, diplomixis is not accompanied by meioticgenome reduction and the subsequent fusion of gametes from

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 chromosomenumbers indicate that at least for some chromosome(s), eitherone 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 bemonosomic for one chromosome pair. Therefore, the authors con-

tal Parasitology 124 (2010) 107112the same parent, as is found in the sexual or parasexual life cycle ofother organisms. This unique process is thought to be shared byother members of the order Diplomonadida, albeit direct evidencesfor this are lacking.

6. Other potential drivers: Mobile elements and the Giardia-specic virus

Othermechanisms that could enhance genetic exchanges withinand between trophozoites should also be considered, such as thosemediated by genetic mobile elements and a Giardia-specic virus.G. duodenalis harbors non-LTR (Non-Long Terminal Repeats) ret-roelements of the LINEs family (Long Interspersed Nuclear Ele-ments), that are transposed by reverse transcription of mRNAdirectly into the site of integration.MostG. duodenalis subtelomeresconsist of tandem copies of active LINE retroposons (either GilM orGilT elements), which directly abut the telomeric repeats and areoriented such that reverse transcription would have run towardthe chromosome end (Wickstead et al., 2003). Repeats play integralparts in ongoing genomic evolution and can play diverse roles atdifferent times, imparting a greater changeability to genomes.When an organism faces a changeable environment, the advantagesof genomic exibility (particularly if it can be contained at specicloci) may outweigh the extra cost of replication and of mutageniceffect exerted on other genomic regions. It is currently unknownwhat role the retroposons are playing (or have played) in the evolu-tion of the G. duodenalis genome.

Giardia-specic virus (GLV) is a double-stranded (ds) RNA virusof the Totiviridae family, constituted by a 36-nm nonenvelopedicosahedron comprising one dsRNA of about 7 kb (Wang andWang, 1991). GLV infects many G. duodenalis from assemblagesA, B, C/D and E, albeit no correlation between the presence or ab-sence of the virus and the specic assemblage has been found(Chen et al., 2007), and cohabitation of multiple GLV species inthe same parasite has been also demonstrated (Tai et al., 1996).In G. duodenalis Portland I strain, which is chronically infected bythis virus, viral RNA was detected in the cytoplasm as well as inthe twin nuclei (Tai et al., 1991). Recombinant GLV cDNA has beensuccessfully introduced into GLV-infected trophozoites to expressa heterologous gene in G. duodenalis. Moreover, the chimeric RNAcould be replicated as double-stranded RNA and packaged intovirus-like particles and the recombinant virions, by themselves,can superinfect G. duodenalis trophozoites and start new roundsof expression (Yu et al., 1996). All these observations are compat-ible with fragment(s) of the G. duodenalis genome being inserted inthe GLV and then shuttled between trophozoites of different G.duodenalis assemblages.

Intriguingly, plasmid DNA transfected into trophozoites ofassemblage A is maintained as a multimeric episome, whereas inassemblage B is always integrated into the genome by homologousrecombination, with insertion of multiple copies (Singer et al.,1998). This opposite behavior could reect isolate-specic differ-ences in the factors determining whether exogenous DNA is inte-grated or maintained episomally. If plasmid- or virus-mediatedlateral exchange of DNA between two trophozoites can occur,homologous recombination could explain, at least in part, thehigher level of ASH observed in assemblage B compared to assem-blage A.

7. Evidence against sex

In a study of single Giardia cells, Tumova et al. (2006) studied 4isolates (WB and HP-1 from assemblage A, and HH and CH-105

110 S.M. Cacci, H. Sprong / Experimenfrom assemblage B) and showed that chromosomes were not dis-tributed symmetrically between nuclei. Indeed, considering theve chromosome linkage groups and a ploidy of two for each nu-cluded that a major requirement for meiosis, namely the presenceof just two homologous copies of each chromosome, is not met,and that the stable transmission of an aneuploid pattern impliesabsence of meiosis. As a consequence, each nucleus is believed toevolve independently as a clonal lineage during the life cycle ofGiardia. This model predicts that the nuclei will accumulate differ-ences 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 character-ize G. duodenalis isolates. This is largely based on results from theanalysis of few isolates and genetic loci, with a bias towardsassemblages A and B, often on axenized strains (Baruch et al.,1996; Teodorovic et al., 2007). The genome sequence of the WBstrain conrmed a low ASH level in this particular strain of assem-blage A (Morrison et al., 2007). However, as sequence data fromisolates collected worldwide are continuously generated, it is pos-sible to evaluate levels of ASH from eld isolates. Albeit limited bythe small number of loci investigated, and by the difculty in dis-tinguishing allelic sequence heterozygosity from mixed infections,the data presented in Table 3 clearly shows that heterogeneoussequencing proles (characterized by two overlapping nucleotidepeaks at specic positions in the electropherograms) occur muchmore often in isolates of assemblages B and C than in those fromassemblage A. This has been reported in several papers from differ-ent research groups (Hopkins et al., 1997; Gelanew et al., 2007;Lebbad et al., 2008; Cacci et al., 2008). Therefore, levels of ASHvary in different G. duodenalis assemblages, and are lower inassemblage A. More data are needed to robustly conrm this nd-ing which, if proven, will support the independent accumulation ofmutations in the nuclei, and thus, lack of recombination betweenthem.

8. Implications for taxonomy and epidemiology

An appropriate classication for Giardia spp. is critical to anunderstanding of the pathogenesis and epidemiology of infection,as well as the biology of the organism. Historically, Giardia specieshave been named on the basis of host occurrence with more than40 species described in the literature, mainly from mammals. Thiscriterion was criticized by Filice (1952) who recognized the inher-ent variability within Giardia affecting mammals and created aholding position in placing many described species under the G.duodenalis umbrella. His proposal of only three valid Giardia spe-cies (G. duodenalis, G. muris and G. agilis), dened by differences inthe overall shape of the trophozoites and in median bodies, waslargely accepted and forms the basis of the current taxonomy.More recently, two species from birds (G. ardeae from herons and

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

Assemblage BG (%) GDH (%) TPI (%)

A 8 2 5

B 20 40 19C 18 18 40

phozoites and of the cysts (Thompson and Monis, 2004).The characterization of G. duodenalis by isoenzyme electropho-

R.C., 1997. Ribosomal RNA sequencing reveals differences between thegenotypes of Giardia isolates recovered from humans and dogs living in thesame locality. Journal of Parasitology 83, 4451.

Kunz, W., 2002. When is a parasite species a species? Trends in Parasitology 3, 121124.

Lebbad, M., Ankarklev, J., Tellez, A., Leiva, B., Andersson, J.O., Svrd, S., 2008.Dominance of Giardia assemblage B in Leon, Nicarauga. Acta Tropica 106, 4453.

Logsdon Jr., J.M., 2008. Evolutionary genetics: sex happens in Giardia. CurrentBiology 18, R66R68.

Mayr, E., 1942. Systematics and the Origin of Species. Columbia University Press.Melo, S.P., Gmez, V., Castellanos, I.C., Alvarado, M.E., Hernndez, P.C., Gallego, A.,

Wasserman, M., 2008. Transcription of meiotic-like-pathway genes in Giardiaintestinalis. Memorias Instituto Oswaldo Cruz 103, 347350.

Monis, P.T., Andrews, R.H., Mayrhofer, G., Ey, P.L., 1999. Molecular systematics ofthe parasitic protozoan Giardia intestinalis. Molecular Biology and Evolution 16,11351144.

tal Presis rst revealed extensive genetic polymorphism among iso-lates, and prompted the proposition that it comprises crypticspecies (Andrews et al., 1989). The existence of genetic groups,or assemblages, within G. duodenalis was later conrmed by fur-ther enzyme electrophoretic studies (Monis et al., 2003) whichclearly showed the host association of particular assemblages. Se-quence and phylogenetic analyses of a number of genes have con-rmed the enzyme electrophoresis groupings and shown that theassemblages represent distinct evolutionary lineages and that a de-gree of host specicity exists (reviewed by Thompson and Monis,2004). In addition, differences have been reported in metabolismand 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 dsRNAvirus, and clinical features (reviewed in Cacci et al., 2005).

Thus, it appears that the taxonomic status afforded previouslyto 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-eval-uated to give appropriate recognition to these original taxonomicdescriptions. Similarly, since separate species names for assem-blages A and B are needed, a proposal for G. duodenalis and G. ent-erica has been put forward (Thompson and Monis, 2004).

The occurrence of recombination, if proven, will have an impor-tant impact on the taxonomy of G. duodenalis. Admittedly, thereare problems to dene when a parasite species should be consid-ered as such (e.g., Kunz, 2002). The biological species conceptstates that species are groups of actual or potentially interbreed-ing natural populations, which are reproductively isolated fromother such groups (Mayr, 1942). While the applicability of thisconcept to protozoa is debatable, the data of Teodorovic et al.(2007) will indicate that assemblages A and B are able to exchangegenetic material, and will not support them as separate species. Onthe contrary, the data of Cooper et al. (2007) will not invalidate thisproposal.

It is therefore crucial to demonstrate if recombination can occurbetween different G. duodenalis assemblages. At the molecular le-vel, this can be evaluated by testing the genetic material from sin-gle cysts, and by applying a method that allow to trace specicallythe presence of each assemblage. We are currently developing anassay based on the use of assemblage-specic primers coupledwith sensitive detection in a real-time PCR platform (Almeida, A.,Pozio E., Cacci S.M., unpublished data). The application of this as-say to G. duodenalis cysts puried by immunomagnetic separationallows to detect DNA of assemblage A and/or B at different lociusing known number of cysts, thereby permitting to distinguishbetween recombinants and mixed infections. Preliminary datasupport the existence of true recombinants between assemblagesA and B, but more data should be collected by exploring other ge-netic loci to conrm present results.

From the perspective of molecular epidemiological studies, it isof particular relevance the fact that animal isolates can be typed aspotentially zoonotic with one marker, but as host-specic withanother. For, therefore, this has very important implications, as to-tally different conclusions may be inferred depending on the waygenotyping data are obtained and interpreted.

9. ConclusionsG. psittaci from psittacine birds) and one from rodents (G. microti)have been described based on morphological features of the tro-

S.M. Cacci, H. Sprong / ExperimenThe occurrence of recombination among G. duodenalis isolateshas been recently supported by several studies. A number ofimportant aspects remain, however, unresolved (see Table 4),and conicting results have also been reported. Future studiesare needed to understand if, how often, and under which condi-tions, recombination occurs in the wild. This will be instrumentalfor a critical re-evaluation of debated issues on the taxonomyand the epidemiology of Giardia, and will also shed light on the ori-gin of meiosis and sexual recombination in eukaryotes.

Acknowledgments

Work in the laboratories of S.M. Cacci and H. Sprong were par-tially supported by the European MED-VET-NET project, contractFOOD-CT-2004-506122.

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Table 4Open questions regarding genetic recombination in Giardia.

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

Giardia duodenalis: Genetic recombination and its implications for taxonomy and molecular epidemiologyIntroductionEvidence for meiotic genes in GiardiaIndirect evidence from molecular genotyping studiesEvidence for genetic exchanges among G. duodenalis isolatesCytological evidence for nuclear fusion and transfer of genetic materialOther potential drivers: Mobile elements and the Giardia-specific virusEvidence against sexImplications for taxonomy and epidemiologyConclusionsAcknowledgmentsReferences