Variation in Giardia: towards ataxonomic revision of the genusPaul T. Monis1, Simone M. Caccio2 and R.C. Andrew Thompson3

1 Australian Water Quality Centre, South Australian Water Corporation, Adelaide, SA 5000, Australia2 Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanita, Viale Regina Elena 299, 00161

Rome, Italy3 World Health Organization Collaborating Centre for the Molecular Epidemiology of Parasitic Infections, School of Veterinary and

Biomedical Sciences, Murdoch University, Murdoch, WA 6150, Australia

Review

Taxonomic uncertainty has had a negative impact on ourunderstanding of the epidemiology of Giardia infections,particularly the role of wild and domestic animals assources of human infection. The lack of morphologicalcriteria for species identification and the failure of cross-infection experiments to unequivocally determine hostspecificity have largely contributed to this uncertainty.However, over the past ten years, it has been possiblenot only to demonstrate extensive genetic heterogen-eity among Giardia isolates from mammals but also toconfirm levels of host specificity that were recognized byearly taxonomists when they proposed a series of host-related species that we consider should now be re-established.

HistoryThe protozoa that collectively comprise the genus Giardiahave intrigued biologists and clinicians for more than 300years, since Antony van Leeuwenhoek first discovered theorganism [1]. This enigmatic protozoan possesses severalunusual characteristics, including the presence of twosimilar, transcriptionally active diploid nuclei; the absenceof mitochondria and peroxisomes; and a unique attach-ment organelle – the ventral sucking disc [2,3] (Figure 1).Phylogenetic relationships are controversial: one school ofthought suggests that Giardia is a primitive early-branch-ing eukaryote and the other suggests that Giardia com-prises one of many divergent eukaryotic lineages thatadapted to a microaerophilic lifestyle rather than diver-ging before the endosymbiosis of the mitochondrial ances-tor [2,3].

Giardia is themost common enteric protozoan pathogenof humans, domestic animals and wildlife (Figure 2). Chil-dren, particularly those in developing countries and livingin disadvantaged community settings, are most at riskfrom the clinical consequences of Giardia infection. InSeptember 2004, Giardia was included in the ‘NeglectedDiseases Initiative’ of the WHO [4]. However, despite itslong history and ubiquity, our understanding of the patho-genesis of Giardia infections and its relationship with itshost is limited, and we do not know why clinical diseaseoccurs in some individuals but is not apparent in others[4]. There are no known virulence factors or toxins, and

Corresponding author: Thompson, R.C.A. (a.thompson@murdoch.edu.au).

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variable expression of surface proteins might enable eva-sion of host immune responses and adaptation to differentenvironments [3].

Giardia has a simple life cycle comprising rapidly multi-plying non-invasive trophozoites on the mucosal surface ofthe small intestine and the production of environmentallyresistant cysts that are passed in the faeces and can betransmitted directly or indirectly. Giardia has long beenconsidered to reproduce asexually by simple binary fission,but there is increasing evidence from epidemiological andmolecular genetic studies thatGiardia is capable of sexualreproduction [4–6]. However, the frequency of recombina-tion is not known, nor is its impact on the epidemiology ofgiardiasis and the extensive genetic diversity that charac-terizes the forms of Giardia that infect mammals. Thisgenetic diversity undoubtedly has impacted upon the tax-onomy of Giardia and contributed to many years of con-troversy and confusion.

Box 1 summarizes changes in nomenclature and thetaxonomic history of Giardia, Box 2 summarizes our cur-rent knowledge about sex in Giardia, and describedspecies are listed in Table 1 and 2. Apart from those inthe species listed in Table 1, there are no reliable morpho-logical features that can be used to distinguish otherspecies of Giardia or genotypes/assemblages that havebeen described. However, as a consequence of geneticallycharacterizing isolates from many different hosts andbeing able to identify genotypic groupings (Table 2), aclearer picture of host specificity has been obtained (seebelow). In addition, differences have been reported inmetabolism and biochemistry, DNA content, in vitroand in vivo growth rates, drug sensitivity, predilectionsite in vivo and duration of infection, pH preference,infectivity, susceptibility to infection with a dsRNA virus,and clinical features (reviewed in Refs [2,7]). Unfortu-nately, many of the early studies that have investigatedphenotypic differences were conducted before the recog-nition of the current genetic groupings, or assemblages,and so it has been difficult to correlate phenotypic differ-ences with particular assemblages. However, a recentstudy using comparative proteomics has found distinctdifferences in several proteins between Giardia isolatesfrom assemblages A and B [8].

In a comprehensive evaluation of described species,Filice [9] recognised the inherent variability within

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Figure 1. Trophozoite of Giardia duodenalis, with characteristic duplication of

organelles, nuclei, median bodies and four pairs of flagella, and ventral disc.

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Giardia affecting mammals, but without the tools avail-able to discriminate reliably between variants, he cre-ated a ‘holding position’ by placing many describedspecies under the Giardia duodenalis ‘umbrella’.

Figure 2. Major cycles of transmission of Giardia duodenalis. Some assemblages/specie

have low host specificity and are capable of infecting humans and other animals (red).

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Genetic basis for phenotypic variationTwo main techniques have been used to characterizeGiardia isolates: enzyme electrophoresis and DNA-basedanalyses. Early enzyme electrophoretic studies revealedextensive genetic heterogeneity within G. duodenalis(reviewed in Ref. [2]), prompting the proposition that itcomprises cryptic species [10]. Mayrhofer et al. [11] demon-strated that all human-derived isolates belonged to one oftwo genetic assemblages, designated assemblage A andassemblage B. Assemblage B isolates seemed to be highlyheterogeneous, with only two isolates exhibiting the sameenzyme profiles, whereas assemblage A isolates displayedless variation and all clustered within one of two groups[11,12]. Enzyme electrophoretic studies also provided thefirst evidence that some assemblages of isolates seemed tobe associated with particular host species (reviewed in Ref.[2]) and suggested sub-structuring within some assem-blages (particularly assemblages A and E) [13]. Impor-tantly, the levels of enzyme variation observed withinthe assemblages ofG. duodenaliswere similar to or greaterthan that observed among isolates ofGiardiamuris [13,14](Figure 3).

DNA analyses using a variety of fingerprinting tech-niques have confirmed the genetic polymorphism amongisolates (reviewed in Ref. [2]), and DNA-based diagnosticassays have confirmed the widespread distribution of theassemblages, particularly A and B (see, for example, Refs[15–19]), as well as the host association of particularassemblages [19]. DNA sequencing and phylogeneticanalyses of several genes have confirmed the enzymeelectrophoresis groupings [20,21] and shown that the

s are host specific and cycle between their respective hosts (blue), whereas others

Box 1. A brief history of nomenclature.

Generic names

The generic name Giardia was established by Kunstler in 1882 [55]

for a flagellate found in the intestine of tadpoles. Six years later,

Blanchard [56] suggested that Lamblia be used in commemoration

of the first accurate description of the parasite by Lambl [57]. It

took 55 years until, in 1914, Alexeieff [58] pointed out the error and

synonymized Lamblia Blanchard, 1888 and Giardia Kunstler, 1882,

which was accepted by the majority of early workers [50,59–61].

Species names

As stated above, the first detailed description of Giardia was given by

Lambl [57] for a flagellate, which he named Cercomonas intestinalis,

in the human intestine. However, this name was pre-empted in toto

by the transfer of Bodo intestinalis Ehrenberg into the genus

Cercomonas Dujardin by Diesing [9,62]. According to the Interna-

tional Code of Zoological Nomenclature (before 1961), both the

generic and specific names given by Lambl fall into homonymy (i.e.

both names already established for other taxa). Workers obviously

accepted this with regard to the incorrectness of the generic name,

Cercomonas. As such, the subsequent description of the same

flagellate in tadpoles by Kunstler [55], Giardia agilis, settled the

correct generic name. Seven years before Kunstler’s finding,

Davaine [63] described a form of Giardia in the rabbit, which he

called Hexamita duodenalis. Although the generic name ascribed to

this parasite was not correct, Filice [9] proposed that the specific

name used by Davaine [63] should remain as a valid name for the

form in the rabbit. This is an important observation because if a

single specific name is to be used for forms of Giardia in humans and

other mammals, then duodenalis has priority over intestinalis,

according to the Rules of Zoological Nomenclature. Indeed, Stiles

(quoted by Filice in Ref. [9]) stated that ‘If you look upon the form in

the rabbit as identical with that in man, duodenalis would be the

correct name. If you consider the various forms in man, rabbits, rats,

etc as distinct, then in all probability a new name should be

suggested for the form that occurs in man’.

Although, on the grounds of zoological nomenclature, the specific

name duodenalis would seem to be correct, the names intestinalis and

even lamblia are often used, particularly for isolates of human origin,

even though the workers might accept Filice’s scheme of only three

morphologically distinct species. There is, thus, no justification for

using the name intestinalis and as Meyer [64] concluded, it would be

beneficial to adopt Filice’s nomenclature because the use of other

names for the ‘duodenalis’ group (i.e. G. intestinalis or G. lamblia)

‘suggests that there is something unique about the human parasite,

which seems on present evidence not to be the case’.

Many species subsequently were described on the basis of host

occurrence and/or minor morphological differences but, in 1952, Filice

[9] evaluated available differential criteria and concluded that on the

experimental proof available at that time, ‘it would be valueless to

name species on the basis of host differences’. After rejecting host

specificity, he undertook a thorough re-appraisal of which morpho-

logical characters could be used as reliable means for differentiating

species. He concluded that described species of Giardia could be

divided into only three morphologically distinct groups, differentiated

primarily on the shape of the median bodies, body shape and length

(Figure 1). He also concluded that, within these three groups, there

might well be morphologically similar forms exhibiting distinct

physiological characteristics but that their taxonomic status awaited

the advent of more refined and discriminatory methodology [9]. The

soundly based, reproducible, and logical scheme proposed by Filice

[9] found widespread favour and forms the basis of the widely

accepted current taxonomy.

Box 2. Sex in Giardia.

More than a decade ago, population genetic studies of Giardia in

endemic communities, where the frequency of transmission is very

high, found evidence of occasional bouts of genetic exchange in the

parasite [12]. These authors demonstrated multiple banding pat-

terns in several isolates of Giardia by allozyme electrophoresis,

which – if a true reflection of the underlying genotypes of the

isolates – would seem to indicate that G. duodenalis is functionally

diploid and that recombination or sexual reproduction must have

occurred at some stage to produce the apparent heterozygotes [12].

These observations recently have been supported by genomic

studies that indicate the existence of genetic exchange and a sexual

phase in the parasite [5,6,65]. These studies demonstrated that

Giardia has maintained at least part of the meiotic machinery and

the ability of chromosomes to cross over, as well as providing

evidence of recombination events.

The evolutionary advantage of recombination is the capacity of

Giardia to respond to adversity, such as selection pressures

imposed by regular exposure to antigiardial drugs or competition

with co-habiting ‘strains’ in circumstances in which the likelihood of

mixed infections is common [66]. As such, it might be a rare event,

and further population genetic studies are required in foci of

infection where the frequency of infection is high. The fact that

available data indicate that the genetic assemblages of Giardia are

conserved in terms of geographic location and host occurrence

suggests that any recombination is not reflected at the assemblage

and species level.

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assemblages represent distinct evolutionary lineages. Insome cases, the divergence among the lineages is of asimilar order of magnitude to that separating the recog-nized species of Giardia [21].

Correlation of genotype with phenotype

Correlations between particular genotypes and pheno-types were first reported in the early to mid-1990 s, withthe observation that different culture conditions selectedfor a particular genotype from a mixture of genotypes. Inparticular, assemblage A isolates seem to have a selectiveadvantage compared with assemblage B isolates underaxenic in vitro culture conditions and vice versa for passagein suckling mice [22–24]. Infectivity and the developmentof clinical disease could also be related to genotype orinteractions between genotype and environmental factors.A study by Geurden et al. [25], found the prevalence ofassemblages A and E in dairy calves (59% and 41%,respectively) to be different to that in beef calves (16%and 84%, respectively). Assemblage E was more frequentlydetected (74% of cases) in calves with clinical diseasecompared to assemblage A (26% of cases).

Conflicting results have been reported for the corre-lation between disease and genotype in humans. A recentsurvey conducted in Ethiopia found a significant corre-lation between symptomatic infection and the presenceof assemblage B [26]. A similar correlation was reportedby Homan and Mank [27], with assemblage B isolatesassociated with persistent diarrhoea, whereas assemblageA infections were associated with intermittent diarrhoea.However, in a case-control study in Bangladesh, Haqueet al. [28] reported that, although assemblage B was the

most prevalent and had the highest parasite burden,patients infected with assemblage A (genotype A2) hadthe highest probability of developing diarrhoea. Similarly,Sahagun et al. [29] also found a strong correlation betweensymptomatic infection and assemblage A2 in patients fromSpain. Interestingly, the proportion of asymptomatic:-symptomatic infections with assemblage A was similar

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Table 2. Genotypic groupings (assemblages) of Giardia duodenalis and speciesa

Species (= assemblage) Host

G. duodenalis (= assemblage A) Humans and other primates, dogs, cats, livestock, rodents and other wild mammals

G. enterica (= assemblage B) Humans and other primates, dogs, some species of wild mammals

G. agilis Amphibians

G. muris Rodents

G. psittaci Birds

G. ardeae Birds

G. microti Rodents

G. canis (= assemblages C/D) Dogs, other canids

G. cati (= assemblage F) Cats

G. bovis (= assemblage E) Cattle and other hoofed livestock

G. simondi (= assemblage G) RatsaDesignation based on original taxonomic descriptions.

Table 1. Recognized species in the genus Giardia 1952–2007

Species Hosts Morphological characteristics Trophozoite dimensionsLength Width

G. duodenalis Wide range of domestic and wild

mammals, including humans

Pear-shaped trophozoites with claw-shaped median bodies 12–15 mm 6–8 mm

G. agilis Amphibians Long, narrow trophozoites with club-shaped median bodies 20–30 mm 4–5 mm

G. muris Rodents Rounded trophozoites with small round median bodies 9–12 mm 5–7 mm

G. ardeae Birds Rounded trophozoites, with prominent notch in ventral disc and

rudimentary caudal flagellum. Median bodies round-oval to claw

shaped.

�10 mm �6.5 mm

G. psittaci Birds Pear-shaped trophozoites, with no ventro-lateral flange.

Claw-shaped median bodies.

�14 mm �6 mm

G. microti Rodents Trophozoites similar to G. duodenalis. Mature cysts contain fully

differentiated trophozoites.

12–15 mm 6–8 mm

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for all three studies (62% [26], 57% [28] and 67% [29]symptomatic). The key difference was that 100% ofdetected assemblage B infections in Ref. [26] were associ-ated with diarrhoea, compared with 16% of infectionsresulting in diarrhoea in Ref. [28] and 42% in Ref. [29].One factor that was not considered was the degree ofgenetic variation within assemblage B, which couldpossibly account for the differences between the studies.It is also likely that the outcome of infection is a complexphenotype and that host factors will also affect the de-velopment of disease.

Molecular epidemiology and host specificity

The question of host specificity has dominated debate onthe taxonomy ofGiardia for nearly 100 years. Indeed, untilFilice’s revision [9], the majority of species had beendescribed principally on the basis of host occurrence. Aswell as taxonomy, a major driver in studies on host speci-ficity has been the question of zoonotic potential. To thisend, numerous cross-transmission experiments have beenundertaken for both taxonomic and epidemiologicalreasons to determine whether G. duodenalis is strictlyhost specific and to elucidate whether humans might besusceptible to infection with isolates of G. duodenalis fromother animals. The majority of experiments have involvedtrying to establish infectionwith human isolates ofGiardiain a variety of animal species, and very few experimentshave involved the attempted infection of humans withisolates from other animals (reviewed in Refs [2,30]). Therehas been great variability in results among differentlaboratories, and the accurate interpretation of data hasbeen difficult, largely because of procedural factors (forexample, differences in the number of cysts dosed and the

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use of isolates that have not been characterized geneti-cally) and the unknown contribution of host and/or para-site factors to the results. Suffice to say, such cross-infection experiments have contributed little to elucidatingtaxonomic issues, although they have questioned thenotion of host-adapted species as a tenable criterion forspecies recognition.

It has been the ability to apply PCR-based tools directlyto faecal or environmental samples, without a reliance onsubsequent laboratory amplification, that has helped toaddress the question of host specificity between isolates ofGiardia [27,31,32]. Suchmolecular epidemiological studieshave demonstrated that there are four main cycles oftransmission in which host-specific and zoonotic assem-blages of Giardia can be maintained in nature (Figure 2).Thus, assemblages A and B can be maintained by directtransmission between humans (e.g. between infants in aday-care centre), assemblage E between livestock (e.g.dairy cattle in the enclosed environment of a barn), assem-blage C/D between dogs (e.g. puppies in a breeding kennel)and novel wildlife genotypes between various wildlifespecies. However, assemblage A and, to a lesser extent,assemblage B, can infect all host populations shown inFigure 2. For example, several studies have shown thatzoonotic genotypes of Giardia can occur frequently inindividual pet dogs living in urban areas (reviewed inRef. [33]), highlighting their potential role as reservoirsof human infection. However, although such studies on theoccurrence of the different assemblages of Giardia indifferent host species serve to emphasize the potentialpublic health risk from domestic dogs, cats and livestock,and the potential for wildlife to act as reservoirs of humaninfection, data on the frequency of zoonotic Giardia trans-

Figure 3. Dendrogram depicting the genetic relationships of isolates of G. duodenalis determined by NJ analysis of Roger’s distances calculated from enzyme

electrophoretic data. The host origin of each isolate is in parentheses. Modified, with permission, from Ref. [14].

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mission is lacking [31,33]. Such information can beobtained from molecular epidemiological studies that gen-otype parasite isolates of the parasites from susceptiblehosts in localized endemic foci of transmission or as a resultof longitudinal surveillance and genotyping of positivecases. Recent research in localized endemic foci of trans-mission has provided evidence in support of the role of dogsin cycles of zoonotic Giardia transmission involvinghumans and domestic dogs from communities in tea-grow-ing areas of Assam in India, and in temple communities in

Bangkok, Thailand [34,35]. In both these studies, somedogs and their owners sharing the same living area wereshown to harbour isolates of G. duodenalis from the sameassemblage.

Phylogenetic relationshipsThe phylogenetic position of the genus Giardia has beenstudied since the late 1980 s, examining the position ofGiardia both within the ‘tree of life’ (see, for example, Refs[36,37]) and within the Diplomonadida [38]. Interestingly,

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Figure 4. Phylogeny of G. duodenalis isolates and Giardia ardeae, inferred from gdh nucleotide sequence data using maximum likelihood analysis. Modified, with

permission, from Ref. [15].

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an early phylogenetic analysis using morphological char-acters from members of the Diplomonadida, includingGiardia, was the first to propose that Giardia is not aprimitive protozoan and does not hold a pivotal position inthe evolution of eukaryotes [39]. This has been supportedby molecular data, which show: (i) that Giardia is from alineage of early diverging eukaryotes but, like many otherprotistan parasites, it is highly evolved; and (ii) that theabsence of organelles and biochemical pathways is due tosecondary loss [40].

A detailed phylogenetic analysis of G. duodenalis wasnot conducted until the late 1990 s, when Monis et al. [15]used four loci (fragments of the genes encoding glutamatedehydrogenase, triose phosphate isomerase, elongationfactor 1 a and small-subunit rRNA) to examine the phy-logenetic relationships of the major genotypes comprisingG. duodenalis. This study provided comprehensive evi-dence that the assemblages of isolates identified by enzymeelectrophoretic analysis (reviewed in Ref. [2]) representdistinct evolutionary lineages. Furthermore, there wasgeneral agreement between the relationships inferredfrom the enzyme electrophoresis data and the DNAsequence data (Figures 3 and 4). Neighbour-joininganalysis of enzyme electrophoretic data from a larger setof isolates from diverse hosts provided evidence of furthersub-structuring within the recognized assemblages, someof which seemed to indicate host specificity or host restric-tion [13]. The cluster of assemblage A isolates from non-

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human mammalian hosts identified in this study to beexternal to the known AI and AII groups might be equiv-alent to the novel assemblage A subtype described fromdeer [41,42] in which (in both cases) the novel genotypesare external to the clustering of AI and AII.

The host restriction exhibited by some of the assem-blages has been supported further by phylogeneticanalyses or molecular typing (e.g. assemblage E and live-stock [43,44], assemblage F and cats [45], assemblages C/Dand dogs [45], and assemblages A and B and humans [45–

47]). The phylogenetic relationship of the assemblages doesnot reflect that of their mammalian hosts (for example,dogs and cats are more closely related to each other than toartiodactylids, and all three are more closely related toeach other than to other mammalian lineages such asrodents and primates, but such a pattern is not apparentfor the assemblages). This indicates host switching and/orhost adaptation rather than co-evolution as the basis forhost specificity.

Case for a revised taxonomyThere are two broad reasons for revising the taxonomy.First, the taxonomy needs to recognize and reflect thebiological and evolutionary differences within G duodena-lis, particularly host specificity. Poulin and Keeney [48]emphasized the now-routine use of DNA sequences toidentify and discriminate morphologically similar speciesand also emphasized that in many cases, we have pre-

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viously underestimated the levels of host specificity shownby parasites in nature. We would argue that early workerson Giardia recognised such host specificity, as reflected inthe largely host-related nomenclature they proposed, andthat subsequent molecular studies have validated theirproposals. Second, a formal nomenclature is essential foreffective communication at all levels. Furthermore, asBowman [49] suggests, the taxonomy can affect the waypolicy is made. Recognising the different G. duodenalisassemblages as distinct species can affect policy and waysof thinking in terms of zoonotic potential and humanhealth threats.

The fact that the genetic characteristics of the assem-blages aremaintained in sympatry in endemic areas wherethe cycles of transmission might overlap (Figure 2)reinforces the argument that the assemblages representseparate species.

NomenclatureIn 1952, Filice was at pains to emphasise that his ration-alization of the species taxonomy of Giardia was only atemporary solution in the absence of valid discriminatorycriteria other than morphology. We now have appropriatediscriminatory tools, and molecular characterization ofGiardia isolated from different host species has revealedthe existence of several distinct genotypic assemblages,some of which seem to have distinct host preferences (e.g.assemblages C/D, F and G, for dogs, cats and rats, respect-ively) or have a limited host range (e.g. assemblage E forhoofed livestock, particularly cattle). There is, thus, amplejustification to reconsider the taxonomic status previouslyafforded to Giardia described in dogs, cats, rats and cattleas separate species, namelyGiardia canis [50]Giardia cati(Deschiens, 1925, in Ref. [51]) Giardia simondi [52] andGiardia bovis (Fantham, 1921, in Ref. [51]) and, thus, giveappropriate recognition to these original taxonomicdescriptions.

The genetic distance separating assemblages A and B isat the same level as that separating the other proposedspecies (see above), strongly suggesting that separatespecies names for each of these assemblages is warranted.This case is further strengthened considering the differ-ences in in vitro and in vivo growth rates [22,23] andpossible differences in clinical disease outcomes [27,53](and see above). The most appropriate name requiresfurther consideration, but Giardia enterica [54] might bea logical choice in view of its previous use to describe a formof Giardia in humans subsequent to Lambl’s description ofGiardia in humans that was eventually named G. duode-nalis (see Box 1).

Table 2 summarizes the eleven species that we thinkshould be recognized in the genus Giardia at the presenttime. The choice of species names reflects those affordedoriginally by the authors who proposed them. Although thedescriptions provided varied in their detail, it is of littleconsequence given the lack of any useful morphologicalfeatures to discriminate between variants of the G. duo-denalis morphological group (reviewed in Ref. [2]).

We hope that subsequent discussion of the argumentsand evidence presented here will result in consensus and anew nomenclature for the assemblages of G. duodenalis.

Concluding remarksIt has been more than 50 years since Filice’s landmarkpaper on the taxonomy of Giardia, but it is only within thepast decade that appropriate tools have been developed toaddress outstanding questions on the taxonomic and epi-demiological significance of variation in the G. duodenalismorphological group. However, this is not the end of thestory. Increasing recognition of genetic subgroupingswithin assemblages and species will be a focus of futureresearch, and it is likely that some of the underlyingsubstructure within assemblages A and B will accountfor the apparently conflicting reports of different assem-blages with different clinical outcomes. Genetic studiesand sequencing of theGiardia genome have laid an import-ant foundation for understanding this parasite. However,the complexity of any biological system, includingGiardia,lies at the protein level and genomics alone cannot be usedto understand these complexities. The phenotypic differ-ences referred to above underline the need to obtain infor-mation from the entire proteome of Giardia to identifyproteins associated with different phenotypic character-istics, particularly those associated with particular diseasetraits, and host infectivity.

AcknowledgementsWe thank Mark Preston from Murdoch Design for the production ofFigures 1 and 2.

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