Variation in Giardia: towards a taxonomic revision of the genus

Review

Variation in Giardia: towards a taxonomic revision of the genus Paul T. Monis1, Simone M. Caccio2 and R.C. Andrew Thompson3 1

Australian Water Quality Centre, South Australian Water Corporation, Adelaide, SA 5000, Australia Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanita`, Viale Regina Elena 299, 00161 Rome, Italy 3 World Health Organization Collaborating Centre for the Molecular Epidemiology of Parasitic Infections, School of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, WA 6150, Australia 2

Taxonomic uncertainty has had a negative impact on our understanding of the epidemiology of Giardia infections, particularly the role of wild and domestic animals as sources of human infection. The lack of morphological criteria for species identification and the failure of crossinfection experiments to unequivocally determine host specificity have largely contributed to this uncertainty. However, over the past ten years, it has been possible not only to demonstrate extensive genetic heterogeneity among Giardia isolates from mammals but also to confirm levels of host specificity that were recognized by early taxonomists when they proposed a series of hostrelated species that we consider should now be reestablished. History The protozoa that collectively comprise the genus Giardia have intrigued biologists and clinicians for more than 300 years, since Antony van Leeuwenhoek first discovered the organism [1]. This enigmatic protozoan possesses several unusual characteristics, including the presence of two similar, transcriptionally active diploid nuclei; the absence of mitochondria and peroxisomes; and a unique attachment organelle – the ventral sucking disc [2,3] (Figure 1). Phylogenetic relationships are controversial: one school of thought suggests that Giardia is a primitive early-branching eukaryote and the other suggests that Giardia comprises one of many divergent eukaryotic lineages that adapted to a microaerophilic lifestyle rather than diverging before the endosymbiosis of the mitochondrial ancestor [2,3]. Giardia is the most common enteric protozoan pathogen of humans, domestic animals and wildlife (Figure 2). Children, particularly those in developing countries and living in disadvantaged community settings, are most at risk from the clinical consequences of Giardia infection. In September 2004, Giardia was included in the ‘Neglected Diseases Initiative’ of the WHO [4]. However, despite its long history and ubiquity, our understanding of the pathogenesis of Giardia infections and its relationship with its host is limited, and we do not know why clinical disease occurs 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. ([email protected]).

variable expression of surface proteins might enable evasion of host immune responses and adaptation to different environments [3]. Giardia has a simple life cycle comprising rapidly multiplying non-invasive trophozoites on the mucosal surface of the small intestine and the production of environmentally resistant cysts that are passed in the faeces and can be transmitted directly or indirectly. Giardia has long been considered to reproduce asexually by simple binary fission, but there is increasing evidence from epidemiological and molecular genetic studies that Giardia is capable of sexual reproduction [4–6]. However, the frequency of recombination is not known, nor is its impact on the epidemiology of giardiasis and the extensive genetic diversity that characterizes the forms of Giardia that infect mammals. This genetic diversity undoubtedly has impacted upon the taxonomy of Giardia and contributed to many years of controversy and confusion. Box 1 summarizes changes in nomenclature and the taxonomic history of Giardia, Box 2 summarizes our current knowledge about sex in Giardia, and described species are listed in Table 1 and 2. Apart from those in the species listed in Table 1, there are no reliable morphological features that can be used to distinguish other species of Giardia or genotypes/assemblages that have been described. However, as a consequence of genetically characterizing isolates from many different hosts and being able to identify genotypic groupings (Table 2), a clearer picture of host specificity has been obtained (see below). 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 Refs [2,7]). Unfortunately, many of the early studies that have investigated phenotypic differences were conducted before the recognition of the current genetic groupings, or assemblages, and so it has been difficult to correlate phenotypic differences with particular assemblages. However, a recent study using comparative proteomics has found distinct differences in several proteins between Giardia isolates from assemblages A and B [8]. In a comprehensive evaluation of described species, Filice [9] recognised the inherent variability within

1471-4922/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.pt.2008.11.006 Available online 8 January 2009

93

Review

Figure 1. Trophozoite of Giardia duodenalis, with characteristic duplication of organelles, nuclei, median bodies and four pairs of flagella, and ventral disc.

Giardia affecting mammals, but without the tools available to discriminate reliably between variants, he created a ‘holding position’ by placing many described species under the Giardia duodenalis ‘umbrella’.

Trends in Parasitology Vol.25 No.2

Genetic basis for phenotypic variation Two main techniques have been used to characterize Giardia isolates: enzyme electrophoresis and DNA-based analyses. Early enzyme electrophoretic studies revealed extensive genetic heterogeneity within G. duodenalis (reviewed in Ref. [2]), prompting the proposition that it comprises cryptic species [10]. Mayrhofer et al. [11] demonstrated that all human-derived isolates belonged to one of two genetic assemblages, designated assemblage A and assemblage B. Assemblage B isolates seemed to be highly heterogeneous, with only two isolates exhibiting the same enzyme profiles, whereas assemblage A isolates displayed less variation and all clustered within one of two groups [11,12]. Enzyme electrophoretic studies also provided the first evidence that some assemblages of isolates seemed to be associated with particular host species (reviewed in Ref. [2]) and suggested sub-structuring within some assemblages (particularly assemblages A and E) [13]. Importantly, the levels of enzyme variation observed within the assemblages of G. duodenalis were similar to or greater than that observed among isolates of Giardia muris [13,14] (Figure 3). DNA analyses using a variety of fingerprinting techniques have confirmed the genetic polymorphism among isolates (reviewed in Ref. [2]), and DNA-based diagnostic assays have confirmed the widespread distribution of the assemblages, particularly A and B (see, for example, Refs [15–19]), as well as the host association of particular assemblages [19]. DNA sequencing and phylogenetic analyses of several genes have confirmed the enzyme electrophoresis groupings [20,21] and shown that the

Figure 2. Major cycles of transmission of Giardia duodenalis. Some assemblages/species are host specific and cycle between their respective hosts (blue), whereas others have low host specificity and are capable of infecting humans and other animals (red).

94

Review

Trends in Parasitology

Vol.25 No.2

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 International 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

assemblages represent distinct evolutionary lineages. In some cases, the divergence among the lineages is of a similar order of magnitude to that separating the recognized species of Giardia [21]. Correlation of genotype with phenotype Correlations between particular genotypes and phenotypes were first reported in the early to mid-1990 s, with the observation that different culture conditions selected for a particular genotype from a mixture of genotypes. In particular, assemblage A isolates seem to have a selective advantage compared with assemblage B isolates under axenic in vitro culture conditions and vice versa for passage in suckling mice [22–24]. Infectivity and the development of clinical disease could also be related to genotype or interactions between genotype and environmental factors. A study by Geurden et al. [25], found the prevalence of assemblages 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 frequently detected (74% of cases) in calves with clinical disease compared to assemblage A (26% of cases). Conflicting results have been reported for the correlation between disease and genotype in humans. A recent survey conducted in Ethiopia found a significant correlation between symptomatic infection and the presence of assemblage B [26]. A similar correlation was reported by Homan and Mank [27], with assemblage B isolates associated with persistent diarrhoea, whereas assemblage A infections were associated with intermittent diarrhoea. However, in a case-control study in Bangladesh, Haque et al. [28] reported that, although assemblage B was the

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 morphological 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.

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

95

Review

Trends in Parasitology Vol.25 No.2

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

Hosts

Morphological characteristics

G. duodenalis

Pear-shaped trophozoites with claw-shaped median bodies

G. agilis G. muris G. ardeae

Wide range of domestic and wild mammals, including humans Amphibians Rodents Birds

G. psittaci

Birds

G. microti

Rodents

Long, narrow trophozoites with club-shaped median bodies Rounded trophozoites with small round median bodies Rounded trophozoites, with prominent notch in ventral disc and rudimentary caudal flagellum. Median bodies round-oval to claw shaped. Pear-shaped trophozoites, with no ventro-lateral flange. Claw-shaped median bodies. Trophozoites similar to G. duodenalis. Mature cysts contain fully differentiated trophozoites.

Trophozoite dimensions Length Width 12–15 mm 6–8 mm 20–30 mm 9–12 mm 10 mm

4–5 mm 5–7 mm 6.5 mm

14 mm

6 mm

12–15 mm

6–8 mm

Table 2. Genotypic groupings (assemblages) of Giardia duodenalis and speciesa Species (= assemblage) G. duodenalis (= assemblage A) G. enterica (= assemblage B) G. agilis G. muris G. psittaci G. ardeae G. microti G. canis (= assemblages C/D) G. cati (= assemblage F) G. bovis (= assemblage E) G. simondi (= assemblage G)

Host Humans and other primates, dogs, cats, livestock, rodents and other wild mammals Humans and other primates, dogs, some species of wild mammals Amphibians Rodents Birds Birds Rodents Dogs, other canids Cats Cattle and other hoofed livestock Rats

a

Designation based on original taxonomic descriptions.

for all three studies (62% [26], 57% [28] and 67% [29] symptomatic). The key difference was that 100% of detected assemblage B infections in Ref. [26] were associated with diarrhoea, compared with 16% of infections resulting in diarrhoea in Ref. [28] and 42% in Ref. [29]. One factor that was not considered was the degree of genetic variation within assemblage B, which could possibly account for the differences between the studies. It is also likely that the outcome of infection is a complex phenotype and that host factors will also affect the development of disease. Molecular epidemiology and host specificity The question of host specificity has dominated debate on the taxonomy of Giardia for nearly 100 years. Indeed, until Filice’s revision [9], the majority of species had been described principally on the basis of host occurrence. As well as taxonomy, a major driver in studies on host specificity has been the question of zoonotic potential. To this end, numerous cross-transmission experiments have been undertaken for both taxonomic and epidemiological reasons to determine whether G. duodenalis is strictly host specific and to elucidate whether humans might be susceptible to infection with isolates of G. duodenalis from other animals. The majority of experiments have involved trying to establish infection with human isolates of Giardia in a variety of animal species, and very few experiments have involved the attempted infection of humans with isolates from other animals (reviewed in Refs [2,30]). There has been great variability in results among different laboratories, and the accurate interpretation of data has been difficult, largely because of procedural factors (for example, differences in the number of cysts dosed and the 96

use of isolates that have not been characterized genetically) and the unknown contribution of host and/or parasite factors to the results. Suffice to say, such crossinfection experiments have contributed little to elucidating taxonomic issues, although they have questioned the notion of host-adapted species as a tenable criterion for species recognition. It has been the ability to apply PCR-based tools directly to faecal or environmental samples, without a reliance on subsequent laboratory amplification, that has helped to address the question of host specificity between isolates of Giardia [27,31,32]. Such molecular epidemiological studies have demonstrated that there are four main cycles of transmission in which host-specific and zoonotic assemblages of Giardia can be maintained in nature (Figure 2). Thus, assemblages A and B can be maintained by direct transmission between humans (e.g. between infants in a day-care centre), assemblage E between livestock (e.g. dairy cattle in the enclosed environment of a barn), assemblage C/D between dogs (e.g. puppies in a breeding kennel) and novel wildlife genotypes between various wildlife species. However, assemblage A and, to a lesser extent, assemblage B, can infect all host populations shown in Figure 2. For example, several studies have shown that zoonotic genotypes of Giardia can occur frequently in individual pet dogs living in urban areas (reviewed in Ref. [33]), highlighting their potential role as reservoirs of human infection. However, although such studies on the occurrence of the different assemblages of Giardia in different host species serve to emphasize the potential public health risk from domestic dogs, cats and livestock, and the potential for wildlife to act as reservoirs of human infection, data on the frequency of zoonotic Giardia trans-

Review

Trends in Parasitology

Vol.25 No.2

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].

mission is lacking [31,33]. Such information can be obtained from molecular epidemiological studies that genotype parasite isolates of the parasites from susceptible hosts in localized endemic foci of transmission or as a result of longitudinal surveillance and genotyping of positive cases. Recent research in localized endemic foci of transmission has provided evidence in support of the role of dogs in cycles of zoonotic Giardia transmission involving humans and domestic dogs from communities in tea-growing areas of Assam in India, and in temple communities in

Bangkok, Thailand [34,35]. In both these studies, some dogs and their owners sharing the same living area were shown to harbour isolates of G. duodenalis from the same assemblage. Phylogenetic relationships The phylogenetic position of the genus Giardia has been studied since the late 1980 s, examining the position of Giardia both within the ‘tree of life’ (see, for example, Refs [36,37]) and within the Diplomonadida [38]. Interestingly, 97

Review

Trends in Parasitology Vol.25 No.2

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].

an early phylogenetic analysis using morphological characters from members of the Diplomonadida, including Giardia, was the first to propose that Giardia is not a primitive protozoan and does not hold a pivotal position in the evolution of eukaryotes [39]. This has been supported by molecular data, which show: (i) that Giardia is from a lineage of early diverging eukaryotes but, like many other protistan parasites, it is highly evolved; and (ii) that the absence of organelles and biochemical pathways is due to secondary loss [40]. A detailed phylogenetic analysis of G. duodenalis was not conducted until the late 1990 s, when Monis et al. [15] used four loci (fragments of the genes encoding glutamate dehydrogenase, triose phosphate isomerase, elongation factor 1 a and small-subunit rRNA) to examine the phylogenetic relationships of the major genotypes comprising G. duodenalis. This study provided comprehensive evidence that the assemblages of isolates identified by enzyme electrophoretic analysis (reviewed in Ref. [2]) represent distinct evolutionary lineages. Furthermore, there was general agreement between the relationships inferred from the enzyme electrophoresis data and the DNA sequence data (Figures 3 and 4). Neighbour-joining analysis of enzyme electrophoretic data from a larger set of isolates from diverse hosts provided evidence of further sub-structuring within the recognized assemblages, some of which seemed to indicate host specificity or host restriction [13]. The cluster of assemblage A isolates from non98

human mammalian hosts identified in this study to be external to the known AI and AII groups might be equivalent to the novel assemblage A subtype described from deer [41,42] in which (in both cases) the novel genotypes are external to the clustering of AI and AII. The host restriction exhibited by some of the assemblages has been supported further by phylogenetic analyses or molecular typing (e.g. assemblage E and livestock [43,44], assemblage F and cats [45], assemblages C/D and dogs [45], and assemblages A and B and humans [45– 47]). The phylogenetic relationship of the assemblages does not reflect that of their mammalian hosts (for example, dogs and cats are more closely related to each other than to artiodactylids, and all three are more closely related to each other than to other mammalian lineages such as rodents and primates, but such a pattern is not apparent for the assemblages). This indicates host switching and/or host adaptation rather than co-evolution as the basis for host specificity. Case for a revised taxonomy There are two broad reasons for revising the taxonomy. First, the taxonomy needs to recognize and reflect the biological and evolutionary differences within G duodenalis, particularly host specificity. Poulin and Keeney [48] emphasized the now-routine use of DNA sequences to identify and discriminate morphologically similar species and also emphasized that in many cases, we have pre-

Review viously underestimated the levels of host specificity shown by parasites in nature. We would argue that early workers on Giardia recognised such host specificity, as reflected in the largely host-related nomenclature they proposed, and that subsequent molecular studies have validated their proposals. Second, a formal nomenclature is essential for effective communication at all levels. Furthermore, as Bowman [49] suggests, the taxonomy can affect the way policy is made. Recognising the different G. duodenalis assemblages as distinct species can affect policy and ways of thinking in terms of zoonotic potential and human health threats. The fact that the genetic characteristics of the assemblages are maintained in sympatry in endemic areas where the cycles of transmission might overlap (Figure 2) reinforces the argument that the assemblages represent separate species. Nomenclature In 1952, Filice was at pains to emphasise that his rationalization of the species taxonomy of Giardia was only a temporary solution in the absence of valid discriminatory criteria other than morphology. We now have appropriate discriminatory tools, and molecular characterization of Giardia isolated from different host species has revealed the 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, respectively) or have a limited host range (e.g. assemblage E for hoofed livestock, particularly cattle). There is, thus, ample justification to reconsider the taxonomic status previously afforded to Giardia described in dogs, cats, rats and cattle as separate species, namely Giardia canis [50] Giardia cati (Deschiens, 1925, in Ref. [51]) Giardia simondi [52] and Giardia bovis (Fantham, 1921, in Ref. [51]) and, thus, give appropriate recognition to these original taxonomic descriptions. The genetic distance separating assemblages A and B is at the same level as that separating the other proposed species (see above), strongly suggesting that separate species names for each of these assemblages is warranted. This case is further strengthened considering the differences in in vitro and in vivo growth rates [22,23] and possible differences in clinical disease outcomes [27,53] (and see above). The most appropriate name requires further consideration, but Giardia enterica [54] might be a logical choice in view of its previous use to describe a form of Giardia in humans subsequent to Lambl’s description of Giardia in humans that was eventually named G. duodenalis (see Box 1). Table 2 summarizes the eleven species that we think should be recognized in the genus Giardia at the present time. The choice of species names reflects those afforded originally by the authors who proposed them. Although the descriptions provided varied in their detail, it is of little consequence given the lack of any useful morphological features to discriminate between variants of the G. duodenalis morphological group (reviewed in Ref. [2]). We hope that subsequent discussion of the arguments and evidence presented here will result in consensus and a new nomenclature for the assemblages of G. duodenalis.

Trends in Parasitology

Vol.25 No.2

Concluding remarks It has been more than 50 years since Filice’s landmark paper on the taxonomy of Giardia, but it is only within the past decade that appropriate tools have been developed to address outstanding questions on the taxonomic and epidemiological significance of variation in the G. duodenalis morphological group. However, this is not the end of the story. Increasing recognition of genetic subgroupings within assemblages and species will be a focus of future research, and it is likely that some of the underlying substructure within assemblages A and B will account for the apparently conflicting reports of different assemblages with different clinical outcomes. Genetic studies and sequencing of the Giardia genome have laid an important foundation for understanding this parasite. However, the complexity of any biological system, including Giardia, lies at the protein level and genomics alone cannot be used to understand these complexities. The phenotypic differences referred to above underline the need to obtain information from the entire proteome of Giardia to identify proteins associated with different phenotypic characteristics, particularly those associated with particular disease traits, and host infectivity. Acknowledgements We thank Mark Preston from Murdoch Design for the production of Figures 1 and 2.

References 1 Meyer, E.A. (1994) Giardia as an organism. In Giardia: From Molecules to Disease (Thompson, R.C.A., Reynoldson, J.A. and Lymbery, A.J., eds), pp. 3–15, CAB International 2 Thompson, R.C.A. and Monis, P.T. (2004) Variation in Giardia: implications for taxonomy and epidemiology. Adv. Parasitol. 58, 69– 137 3 Morrison, H.G. et al. (2007) Genomic minimialism in the early diverging intestinal parasite Giardia lamblia. Science 317, 1921–1926 4 Savioli, L. et al. (2006) Giardia and Cryptosporidium join the ‘Neglected Diseases Initiative’. Trends Parasitol. 22, 203–208 5 Cooper, M.A. et al. (2007) Population genetics provides evidence for recombination in Giardia. Curr. Biol. 17, 1984–1988 6 Poxleitner, M.K. et al. (2008) Evidence for karyogamy and exchange of genetic material in the binucleate intestinal parasite Giardia intestinalis. Science 319, 1530–1533 7 Caccio`, S.M. et al. (2005) Unravelling Cryptosporidium and Giardia epidemiology. Trends Parasitol. 21, 430–437 8 Steuart, R.F.L. et al. (2008) Alpha 2 Giardin is an assemblage A specific protein of human infective Giardia duodenalis. Parasitology 135, 1621– 1627 9 Filice, F.P. (1952) Studies on the cytology and life history of a Giardia from the laboratory rat. Univ. Calif. Publ. Zool. 57, 53–146 10 Andrews, R.H. et al. (1989) Giardia intestinalis: electrophoretic evidence for a species complex. Int. J. Parasitol. 19, 183–190 11 Mayrhofer, G. et al. (1995) Division of Giardia isolates from humans into two genetically distinct assemblages by electrophoretic analysis of enzymes encoded at 27 loci and comparison with Giardia muris. Parasitology 111, 11–17 12 Meloni, B.P. et al. (1995) Genetic characterization of isolates of Giardia duodenalis by enzyme electrophoresis: implications for reproductive biology, population structure, taxonomy, and epidemiology. J. Parasitol. 81, 368–383 13 Monis, P.T. et al. (1998) Novel lineages of Giardia intestinalis identified by genetic analysis of organisms isolated from dogs in Australia. Parasitology 116, 7–19 14 Monis, P.T. et al. (2003) Genetic diversity within the morphological species Giardia intestinalis and its relationship to host origin. Infect. Genet. Evol. 3, 29–38 15 Monis, P.T. et al. (1999) Molecular systematics of the parasitic protozoan Giardia intestinalis. Mol. Biol. Evol. 16, 1135–1144 99

Review 16 Andrews, R.H. et al. (1998) Comparison of the levels of intra-specific genetic variation within Giardia muris and Giardia intestinalis. Int. J. Parasitol. 28, 1179–1185 17 Amar, C.F. et al. (2002) Sensitive PCR-restriction fragment length polymorphism assay for detection and genotyping of Giardia duodenalis in human feces. J. Clin. Microbiol. 40, 446–452 18 Caccio, S.M. et al. (2002) Sequence analysis of the beta-giardin gene and development of a polymerase chain reaction-restriction fragment length polymorphism assay to genotype Giardia duodenalis cysts from human faecal samples. Int. J. Parasitol. 32, 1023–1030 19 Homan, W.L. et al. (1998) Characterization of Giardia duodenalis by polymerase-chain-reaction fingerprinting. Parasitol. Res. 84, 707–714 20 Monis, P.T. et al. (1996) Molecular genetic analysis of Giardia intestinalis isolates at the glutamate dehydrogenase locus. Parasitology 112, 1–12 21 Read, C.M. et al. (2004) Discrimination of all genotypes of Giardia duodenalis at the glutamate dehydrogenase locus using PCR-RFLP. Infect. Genet. Evol. 4, 125–130 22 Andrews, R.H. et al. (1992) Selection of specific genotypes of Giardia intestinalis by growth in vitro and in vivo. Parasitology 105, 375–386 23 Binz, N. et al. (1992) Comparative studies on the growth dynamics of two genetically distinct isolates of Giardia duodenalis in vitro. Int. J. Parasitol. 22, 195–202 24 Thompson, R.C. and Lymbery, A.J. (1996) Genetic variability in parasites and host-parasite interactions. Parasitology 112, S7–S22 25 Geurden, T. et al. (2008) Mixed Giardia duodenalis assemblage A and E infections in calves. Int. J. Parasitol. 38, 259–264 26 Gelanew, T. et al. (2007) Molecular characterization of human isolates of Giardia duodenalis from Ethiopia. Acta Trop. 102, 92–99 27 Homan, W.L. and Mank, T.G. (2001) Human giardiasis: genotype linked differences in clinical symptomatology. Int. J. Parasitol. 31, 822–826 28 Haque, R. et al. (2005) Giardia assemblage A infection and diarrhea in Bangladesh. J. Infect. Dis. 192, 2171–2173 29 Sahagun, J. et al. (2008) Correlation between the presence of symptoms and the Giardia duodenalis genotype. Eur. J. Clin. Microbiol. Infect. Dis. 27, 81–83 30 Monis, P.T. and Thompson, R.C.A. (2003) Cryptosporidium and Giardia-zoonoses: fact or fiction? Infect. Genet. Evol. 3, 233–244 31 Hunter, P.R. and Thompson, R.C.A. (2005) The zoonotic transmission of Giardia and Cryptosporidium. Int. J. Parasitol. 35, 1181–1190 32 Thompson, R.C.A. (2004) The zoonotic significance and molecular epidemiology of Giardia and giardiasis. Vet. Parasitol. 126, 15–35 33 Leonhard, S. et al. (2007) The molecular characterisation of Giardia from dogs in southern Germany. Vet. Parasitol. 150, 33–38 34 Traub, R.J. et al. (2004) Epidemiological and molecular evidence supports the zoonotic transmission of Giardia among humans and dogs living in the same community. Parasitology 128, 253–262 35 Inpankaew, T. et al. (2007) Canine parasitic zoonoses and temple communities in Thailand. Southeast Asian J. Trop. Med. Public Health 38, 247–255 36 Sogin, M.L. et al. (1989) Phylogenetic meaning of the kingdom concept: an unusual ribosomal RNA from Giardia lamblia. Science 243, 75–77 37 Hashimoto, T. et al. (1994) Protein phylogeny gives a robust estimation for early divergences of eukaryotes: phylogenetic place of a mitochondria-lacking protozoan, Giardia lamblia. Mol. Biol. Evol. 11, 65–71 38 van Keulen, H. et al. (1993) Unique phylogenetic position of Diplomonadida based on the complete small subunit ribosomal RNA sequence of Giardia ardeae, G. muris, G. duodenalis and Hexamita sp. FASEB J. 7, 223–231 39 Siddall, M.E. et al. (1992) Phylogenetic analysis of the Diplomonadida (Wenyon 1926) Brugerolle, 1975: evidence for heterochrony in protozoa

100

Trends in Parasitology Vol.25 No.2

40 41 42

43

44 45

46 47

48 49 50 51

52 53

54 55 56 57 58 59 60 61 62 63

64 65

66

and against Giardia lamblia as a ‘‘missing link’’. J. Protozool. 39, 361– 367 Dacks, J.B. et al. (2008) Implications of the new eukaryotic systematics for parasitologists. Parasitol. Int. 57, 97–104 Lalle, M. et al. (2007) A novel Giardia duodenalis assemblage A subtype in fallow deer. J. Parasitol. 93, 426–428 van der Giessen, J.W. et al. (2006) Genotyping of Giardia in Dutch patients and animals: a phylogenetic analysis of human and animal isolates. Int. J. Parasitol. 36, 849–858 Sedinova, J. et al. (2003) Use of random amplified polymorphic DNA (RAPD) analysis for the identification of Giardia intestinalis subtypes and phylogenetic tree construction. J. Eukaryot. Microbiol. 50, 198–203 Trout, J.M. et al. (2006) Prevalence and genotypes of Giardia duodenalis in 1-2 year old dairy cattle. Vet. Parasitol. 140, 217–222 Souza, S.L. et al. (2007) Molecular identification of Giardia duodenalis isolates from humans, dogs, cats and cattle from the state of Sao Paulo, Brazil, by sequence analysis of fragments of glutamate dehydrogenase (gdh) coding gene. Vet. Parasitol. 149, 258–264 Lebbad, M. et al. (2008) Dominance of Giardia assemblage B in Leon. Nicaragua. Acta Trop. 106, 44–53 Yason, J.A. and Rivera, W.L. (2007) Genotyping of Giardia duodenalis isolates among residents of slum area in Manila, Philippines. Parasitol. Res. 101, 681–687 Poulin, R. and Keeney, D.B. (2008) Host specificity under molecular and experimental scrutiny. Trends Parasitol. 24, 24–28 Bowman, D. (2005) What’s in a name. Trends Parasitol. 21, 267–269 Hegner, R.W. (1922) A comparative study of the Giardia living in man, rabbit and dog. Am. J. Hyg. 2, 442–454 Kulda, J. and Nohy´nkova´, E. (1978) Flagellates of the human intestine and of intestines of other species. In Parasitic Protozoa (Kreier, P.P., ed.), pp. 83–89, Academic Press Lavier, G. (1924) Deux especes de Giardia du rat d’egout parisien (Epimys norvegicus). Ann. Parasitol. Hum. Comp. 2, 161–168 Kofoid, C.A. (1920) A critical review of the nomenclature of human intestinal flagellates, Cercomonas, Chilomastix, Trichomonas, Tetratrichomonas and Giardia. Univ. Calif. Publ. Zool. 20, 145–168 Read, C. et al. (2002) Correlation between genotypes of Giardia duodenalis and diarrhoea. Int. J. Parasitol. 32, 229–231 Kunstler, J. (1882) Sur cinq protozoaires parasites nouveaus. C. R. Seances Soc. Biol. Fil. 95, 347–349 Blanchard, R. (1888) Remarques sur le megastome intestinal. Bull. Soc. Zool. de France 30, 18–19 Lambl, W. (1859) Mikroskopische untersuchungen der darm-excrete. Vierteljahtsschrift fur die Praktisch Heikunde (Prag) 61, 1–58 Alexeieff, A. (1914) Notes protistologiques. Zool. Anz. 44, 193–213 Kofoid, C.A. and Christiansen, E.B. (1915) On binary and multiple fission in Giardia muris (Grassi). Univ. Calif. Publ. Zool. 16, 30–54 Kofoid, C.A. and Christiansen, E.B. (1915) On Giardia microti sp. nov., from the meadow mouse.. Univ. Calif. Publ. Zool. 16, 23–29 Dobell, C. (1940) Vilem Lambl (1824-1895) – a portrait and a biographical note. Parasitology 32, 122–125 Diesing, C.M. (1850). Systema Helminthium. Sumptibus Academiae Caesareae Scientiarum. Vindobonae. Gerald’s Sohn, Vienna. Davaine, C. (1875) Monadiens. In Dictionnaires encyclopedique des sciences medicales. (Ser. 2, Vol. 9) (Asselin, P. and Masson, G., eds). Place de l’Ecole-de-Medecine, Paris Meyer, E.A. (1985) The epidemiology of giardiasis. Parasitol. Today 1, 101–105 Ramesh, M.A. et al. (2005) A phylogenomic inventory of meiotic genes; evidence for sex in Giardia and an early eukaryotic origin of meiosis. Curr. Biol. 15, 185–191 Hopkins, R.M. et al. (1999) PCR-based DNA fingerprinting of Giardia duodenalis isolates using the intergenic rDNA spacer. Parasitol. 118, 531–539