Giardia—From Genome to Proteome

CHAPTER

2 Giardia—From Genome to Proteome R.C. Andrew Thompson* and Paul Monis†

Contents

Abstract

2.1. Introduction 2.2. Current Status of Genome and Proteome Projects 2.3. What is Giardia?—Evolutionary Biology and Phylogeny 2.4. Taxonomy and Nomenclature 2.5. The Maintenance of Giardia in Nature 2.5.1. Life cycle and development 2.5.2. Hosts 2.5.3. Transmission 2.6. Interaction Between Cycles 2.6.1. Zoonotic transmission 2.7. Functional Significance of Genetic Variation 2.7.1. Developmental biology 2.7.2. Pathogenesis, variation in virulence and polyparasitism 2.8. Conclusions References

58 59 60 62 66 66 68 72 74 75 79 79 81 83 84

In this review, the current status of genomic and proteomic research on Giardia is examined in terms of evolutionary biology, phylogenetic relationships and taxonomy. The review also describes how characterising genetic variation in Giardia from numerous hosts and endemic areas has provided a better understanding of life cycle patterns, transmission and the epidemiology of Giardia infections in humans, domestic animals and wildlife. Some progress

* School of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, West Australia, Australia {

Australian Water Quality Centre, South Australian Water Corporation, Adelaide, South Australia, Australia

Advances in Parasitology, Volume 78 ISSN 0065-308X, DOI: 10.1016/B978-0-12-394303-3.00003-7

#

2012 Elsevier Ltd. All rights reserved.

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has been made in relating genomic information to the phenotype of Giardia, and as a consequence, new information has been obtained on aspects of developmental biology and the host– parasite relationship. However, deficiencies remain in our understanding of pathogenesis and host specificity, highlighting the limitations of currently available genomic datasets.

2.1. INTRODUCTION The taxonomy, life cycle patterns and zoonotic potential of Giardia (Fig. 2.1) infecting mammals and birds have been poorly understood and controversial for many years (Thompson and Monis, 2004). However, the development of molecular tools for characterising isolates of Giardia directly from faeces or environmental samples has helped to resolve the taxonomy of the most common forms of Giardia parasitising mammals, and we use the nomenclature proposed in this review (Table 2.1). In addition, major advances have been made in understanding the transmission and epidemiology of Giardia and giardiasis. The availability of full genome sequences for several species of Giardia now offers the potential to better understand host specificity and pathogenesis which will require not only a greater emphasis on bioinformatic analysis but also the application of proteomic technologies to Giardia in order to fully realise the value of the available genomic data. In this review, which seeks to ‘update’ our earlier review on genetic variation in Giardia (Thompson and Monis, 2004), we discuss how ‘marrying’ available genetic and phenotypic data in the context of improvements in proteomics will provide important insights into the host–parasite relationship.

FIGURE 2.1 Scanning electron micrograph of Giardia trophozoites showing ventral adhesive disc anteriorly and flagella in the trophozoite at top right (courtesy of Dr. Peta Clode, Centre for Microscopy, Characterisation and Analysis, University of Western Australia).

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TABLE 2.1 Species of Giardiaa Species

Assemblage

Host(s)

G. duodenalis

A

G. enterica

B

G. canis G. bovis G. cati G. simondi G. ?b G. ?b G. microti G. psittaci G. ardeae G. muris G. agilis

C/D E F G H ?

Humans and other primates, dogs, cats, livestock, rodents and other wild mammals Humans and other primates, dogs, cats and some species of wild mammals Dogs, other canids Cattle and other hoofed livestock Cats Rats Pinnipeds Marsupial (Quenda, bandicoot) Rodents Birds Birds Rodents Amphibians

Details in Monis et al. (2009) and Thompson and Monis (2011). a Designation based on original taxonomic description. b Novel lineages of Giardia distinct from the described species, likely new species but not yet formally described.

2.2. CURRENT STATUS OF GENOME AND PROTEOME PROJECTS Currently, there are genome sequences available from three isolates of Giardia: WB, GS and P15, representing Giardia duodenalis (syn. Giardia intestinalis; Giardia lamblia), Giardia enterica and Giardia bovis (Assemblages A, B and E—Table 2.1), respectively ( Jerlstrom-Hultqvist et al., 2010a). One of the limitations of the genome data is the quality of the GS and P15 assemblies in GC-rich regions. Different genome sequencing platforms have different strengths and weaknesses, including how well they deal with repeat regions or extremes in GC content, and so additional data from alternative sequencing approaches will likely be beneficial for improved data for these regions ( Jerlstrom-Hultqvist et al., 2010b). In addition, genome sequences have been obtained from Spironucleus salmonicida and Spironucleus barkhanus (Roxstrom-Lindquist et al., 2010), demonstrating large genome differences between the morphologically identical species. Comparison of the Giardia genomes has identified a core set of proteins common to the three assemblages studied, as well as

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major differences in particular gene families (such as variant-pecific surface antigens), which could underlie the biological differences (such as host range, disease type) between assemblages (Franzen et al., 2009; Jerlstrom-Hultqvist et al., 2010a,b). A useful tool for data mining the Giardia genome data is GiardiaDB (http://giardiadb.org/giardiadb/), which provides an integrated and searchable database that combines genome sequence data and chromosome maps with gene and protein expression data, including cell localisation of proteins (if known) (Aurrecoechea et al., 2009). The Giardia genome data have been used to further elucidate lipid metabolism (Yichoy et al., 2011), and data mining has provided insight into the evolution of Giardia and eukaryotic cells (discussed below). A combination of proteome and genome data has been used to identify unique basal body proteins (Lauwaet et al., 2011). Proteomic analysis has also been used to study metabolism in mitosomes ( Jedelsky et al., 2011). Transcriptomes and proteomics from different growth stages are starting to be generated and promise to provide further insight into processes such as excystation and encystation (Birkeland et al., 2010; Kim et al., 2009). Most recently, proteomic analysis of ventral disc extracts and comparison with the Giardia genome database has been used to identify novel proteins associated with the ventral disc and lateral crest (Hagen et al., 2011). The localisation of these proteins was confirmed by the expression of green fluorescent protein fusion constructs in trophozoites (Hagen et al., 2011).

2.3. WHAT IS GIARDIA?—EVOLUTIONARY BIOLOGY AND PHYLOGENY Giardia has long been considered to be a primitive, early diverging eukaryote due to the apparent lack of typical eukaryotic organelles such as peroxisomes and mitochondria and on the basis of phylogenetic analysis of conserved gene or protein sequences. The early branching of Giardia (and other diplomonads) was first suggested following the characterisation of ribosomal RNAs (Edlind and Chakraborty, 1987; Sogin et al., 1989; van Keulen et al., 1993) and later by analysis of conserved proteins such as the elongation factors (Hashimoto et al., 1994, 1995). However, on the basis of morphology and life history, Giardia had previously been suggested to be the most derived member of the order (Brugerolle, 1975). The status of Giardia as a ‘‘missing link’’ was further challenged by phylogenetic analysis of the Diplomonadida using morphological characters, which also suggested that Giardia was the most highly derived genus in the order (Siddall et al., 1992). Furthermore, the accuracy of the placement of Giardia and other diplomonads relative to other eukaryotes has been

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questioned because of the observed large differences in GþC composition that can bias phylogenetic analysis (Leipe et al., 1993) and due to the possible effects of long branch attraction (Dacks et al., 2002). Lateral gene transfer has also complicated the elucidation of the evolutionary history of Giardia, with many genes involved in anaerobic metabolism in Giardia having been acquired from prokaryotes (Andersson et al., 2003; Nixon et al., 2002a). More recent molecular data support Siddall’s original proposal that Giardia are highly derived, rather than primitive organisms. It is likely that the divergence of the lineage giving rise to Giardia was subsequent to the acquisition of introns during eukaryote evolution, following the detection of a spliceosomal intron and eukaryote-specific spliceosomal peptides in Giardia (Nixon et al., 2002b). More recent genome sequence data have added further weight to these findings, identifying additional cis-spliced introns and also finding novel trans-spliced introns, where exons are dispersed throughout the genome and a single transcript is produced by trans-splicing (Kamikawa et al., 2011; Roy et al., 2011). Analyses of genomic data have also demonstrated the presence of numerous eukaryotic features, such as sequences encoding eukaryotic RNA processing machinery (Chen et al., 2011), Scaffold/Matrix attachment regions that are involved in chromatin attachment/DNA organisation in other eukaryotes (Padmaja et al., 2010), the presence of nucleoli ( JimenezGarcia et al., 2008), meiosis-specific genes (Ramesh et al., 2005) and pathways for RNA regulation such as RNA silencing (Ullu et al., 2004) and microRNAs (Zhang et al., 2009). The presence of mitochondrial remnants, called mitosomes, has been demonstrated in Giardia (Tovar et al., 2003) and other amitochondriate protists, as have genes encoding components of Golgi bodies (Dacks et al., 2003). The sequences involved in protein targeting for mitosomes have been shown to be conserved and recognised by the hydrogenosomes of Trichomonas and related to translocases in mitochondria (Dolezal et al., 2005). Phylogenetic analysis of the genes encoding type II DNA topoisomerases found that Giardia diverged after mitochondriate kinetoplastids and that amitochondriate protists were polyphyletic, adding further evidence that the Giardia lineage diverged after the acquisition of mitochondria by eukaryotes and that secondary loss or alternative evolution of organelles has occurred independently multiple times (He et al., 2005a,b). A further understanding of Giardia evolution has been gained from analysis of genome data. While the genome of Giardia is thought to be compact through the reduction or loss of many metabolic pathways, 40% of genes (predominantly variant-specific surface proteins) were found to be duplicates (Sun et al., 2010). Interestingly, phylogenetic analysis of the duplicated genes suggested that expansion of the variant-specific surface proteins coincided with the radiation of placental mammals (Sun et al., 2010).

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Comparison of predicted small nucleolar RNAs in Giardia demonstrated similarity with Dictyostelium, Plasmodium, fungi and metazoans, which were all different to those from Euglenozoa, suggesting that the lineage containing Giardia diverged later than Trypanosoma and Euglena (Luo et al., 2009). This observation is concordant with earlier analyses using multigene phylogenies, which separated the excavates into three major lineages: Diplomonads, Parabasalids and Carpediemonas; Trimastix and Oxymonads; Euglenozoa, Heterolobosea and Jakobids, with the lineage including Giardia and other Diplomonads closest to the fungi and animals (Simpson et al., 2006). However, a more recent study using phylogenomics suggests that long branch attraction may have affected previous attempts to elucidate the relationships of the excavate groups, and exclusion of the most rapidly evolving genes or species from analyses supports the monophyly of the Excavata (Hampl et al., 2009), leaving the placement of the excavates and the major lineages within it uncertain. The phylogeny of the Diplomonads has been further elucidated, with Octomitus being shown to be a sister taxon to Giardia (Keeling and Brugerolle, 2006) and with Spironucleus, Hexamita and Trepomonas being shown to be a separate lineage from Giardia/Octomitus (Kolisko et al., 2008). Interestingly, none of the Enteromonads were basal to the Diplomonads (as suggested by Brugerolle, 1975; Siddall et al., 1992) but were instead polyphyletic within the lineage including Spironucleus, suggesting either multiple origins of diplokarya or multiple instances of secondary loss of the duplicated nucleus (Kolisko et al., 2008). A genome comparison between Spironucleus and Giardia found evidence of lateral gene transfer from both prokaryotes and eukaryotes, distinct biases in mutations and polyadenylation signals and differences in codon usage (Andersson et al., 2007). Of more interest, large genomic differences were found between the morphologically indistinguishable species S. barkhanus and S. salmonicida, including differences in codon usage, the frequency of allelic sequence variation and genome size (Roxstrom-Lindquist et al., 2010). Interestingly, a similar observation has been made for the difference in allelic sequence variation observed between G. duodenalis and G. enterica (Assemblages A (WB) and B (GS)) (Roxstrom-Lindquist et al., 2010).

2.4. TAXONOMY AND NOMENCLATURE The taxonomic placement of Giardia has changed following revisions to higher order classifications. A key change has been the replacement of the Diplomonadida with two new orders, the Distomatida Klebs 1892 (Trepomonas, Hexamita, Spironucleus) and the Giardiida CavalierSmith 1996 (Giardia, Octomitus), which are both in the subclass Diplozoa Dangeard 1910 stat. nov. Cavalier-Smith 1996 (Cavalier-Smith, 2003). As a

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consequence, the family Hexamitidae is now in the order Distomatida, with no apparent families within the Giardiida (Brands, S.J. (comp.) 1989– present. Systema Naturae 2000. The Taxonomicon. Universal Taxonomic Services, Zwaag, The Netherlands. [http://taxonomicon.taxonomy.nl/]. Access date: 17-12-2011). The classification suggested by Cavalier-Smith (2003) still recognised the Enteromonadida, which has more recently been shown to be polyphyletic within the Distomatida and so should be reconsidered (Kolisko et al., 2008). At the higher level, there have been proposed changes to the kingdoms at the base of the eukaryotes, with Euglenozoa being removed from the infrakingdom Excavata, which still contains Metamonada (Cavalier-Smith, 2010). Following these revisions, Giardia belongs to the order Giardiida Cavalier-Smith 1996, subclass Diplozoa Dangeard, 1910, class Trepomonadea, superclass Eopharyngia, subphylum Trichozoa (Cavalier-Smith, 2003), phylum Metamonada Grasse´ 1952 stat. nov. et emend. Cavalier-Smith, 1981 (Cavalier Smith, 1993). Multigene phylogenetic analyses support previous suggestions that Giardia is the most highly derived member of the order (Brugerolle, 1975), but as described above, the molecular data do not support Brugerolle’s (1975) proposed evolution, which has Giardia and Octomitis being derived from Trepomonas, Hexamita and Spironucleus. The morphological descriptions and taxonomic history of Giardia have been reviewed extensively elsewhere (Monis et al., 2009; Thompson and Monis, 2004, 2011) and so will not be redescribed here. Despite critical reviews of the history of Giardia, taxonomic descriptions to clarify timelines and the validity of particular names (Monis et al., 2009; Thompson and Monis, 2004, 2011), there continues to be debate regarding nomenclature, with invalid species names such as G. lamblia still in use by some investigators. For this review, we will use the nomenclature proposed by Monis et al. (2009), which allocates previously described species to the currently recognised assemblages on the basis of apparent host preference (Table 2.1). This is consistent with the spirit of Filice’s (1952) original proposal, which was at pains to emphasise that the rationalisation of Giardia taxonomy was only a temporary solution in the absence of valid discriminatory criteria for recognising additional species. The nomenclature is also consistent with the relationships inferred by phylogenetic analysis of alloenzyme electrophoretic data (Monis et al., 2003), illustrated in Fig. 2.2, and of DNA sequence data from multiple independent loci (Monis et al., 1999). The sequence-based phylogenies suggest that G. duodenalis, Giardia cati and G. bovis were clustered together but could not resolve the branching order, with Giardia canis, G. enterica and Giardia simondi being external to these three species (Monis et al., 1999). The available genome data support that G. bovis and G. duodenalis are more similar to each other than either are to G. enterica ( Jerlstrom-Hultqvist et al., 2010b).

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G. muris (rodent)

G. ardeae (bird)

G. canis (dog)

G. simondi (rat) (Human)

(Various, bIII)

(Mamoset) (Siamang) (Dog)

G. enterica

(Human, BIV)

G. cati (Cat)

(Sheep)

(Cattle) G. bovis (Pig)

(Various, A?)

G. duodenalis

(Various, AI)

(Guinea pig) (Cat) (Alpaca) (Cat) (Cat) (Dog) (Human)

(Human, AI) 0.1 Roger’s distance

FIGURE 2.2 Diagrammatic representation of the phylogenetic relationships of Giardia species and the possible infection cycles that occur within these species. The phylogeny was inferred by Neighbor Joining analysis of Roger’s Distances (modified from Monis et al. 2003). Host origin for species or lineages is indicated in parentheses.

Acceptance of suggested changes to Giardia nomenclature may have been hampered through the application of imprecise terminology, for example, the use of the term ‘genotype’ to describe a group of genetically diverse organisms such as the Giardia isolates comprising Assemblage B. A ‘genotype’ reflects the genetic constitution of an organism and can be broadly interpreted as a group or class of organisms having the same

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genetic constitution. The first detailed genetic studies of Giardia identified a number of genotypes, some of which were shown by clustering analysis to form distinct groups (Andrews et al., 1989; Meloni et al., 1988). More detailed analysis of human isolates of Giardia demonstrated quite a large degree of genetic diversity, but that all the human isolates formed two broad clusters and that there was some genetic substructuring within these clusters (Mayrhofer et al., 1995). This study gave rise to the concept of genetic assemblages of Giardia, with the two assemblages (A and B) each comprising clusters of related genotypes that were separated from each other by relatively large genetic distances, of the same order of magnitude as those separating G. duodenalis from G. muris (Ey et al., 1997; Mayrhofer et al., 1995). From this, it is evident that it is inappropriate to refer to the assemblages as genotypes and that to do so provides a false impression of homogeneity within these groups, de-emphasizing the magnitude of the differences between them, which are sufficient for them to be recognised as distinct species. Recent genome sequence data lend support to the distinct species status of the Giardia assemblages ( Jerlstrom-Hultqvist et al., 2010a). Sequence data are now available from representative isolates of G. duodenalis (Assemblage A) (Morrison et al., 2007; Svard et al., 2003), G. enterica (Assemblage B) (Franzen et al., 2009) and G. bovis (Assemblage E) ( Jerlstrom-Hultqvist et al., 2010b). Comparison of the genomes at the amino acid level ( Jerlstrom-Hultqvist et al., 2010a) supports earlier phylogenetic analyses that suggested that G. duodenalis and G. bovis were more closely related to each other relative to G. enterica (Monis et al., 1999). The GþC content of G. enterica and G. bovis was slightly lower than that of G. duodenalis, but the difference was ascribed to imperfect sequence assemblies of the two newer genome sequences ( Jerlstrom-Hultqvist et al., 2010b). The three species/ assemblages shared a core set of genes representing 91% of their genomes, but there were still large differences, particularly in Giardia-specific gene families and chromosomal rearrangements ( Jerlstrom-Hultqvist et al., 2010a). Comparison of the genomes of G. duodenalis and G. enterica found a higher frequency of allelic sequence heterozygosity in G. enterica, but additional data from other isolates of these two species are required to determine if this is a characteristic of the species/assemblages or the isolates (WB and GS, respectively) used to obtain the genome sequences (Franzen et al., 2009). Perhaps the most important indication of the species status of the assemblages has been demonstrated by a genome-based comparison of the divergence between the assemblages with divergence between species within other protozoan genera. The divergence between G. duodenalis and G. enterica (Assemblages A and B) and between G. enterica and G. bovis (Assemblages B and E) are similar to those separating Theileria parva from Theileria annulata, whereas the divergence between G. duodenalis and G. bovis (Assemblages A and E), while lower, are similar to those

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separating Leishmania major and Leishmania infantum ( Jerlstrom-Hultqvist et al., 2010b). Previous genetic studies of Giardia isolates have focussed on isolates from humans and animals in close contact with humans, especially pets and livestock. More recent studies have examined Giardia isolated from wildlife, identifying new assemblages or genotypes. A novel species, based on cyst morphology and phylogenetic analysis of SSU rRNA, and elongation factor 1 alpha, was isolated from a small marsupial in Australia (Adams et al., 2004). A survey of marine vertebrates identified a new lineage, named Assemblage H, which was isolated from grey seal and a single gull sample (Lasek-Nesselquist et al., 2010). A novel genotype belonging to Assemblage A has been isolated from a deer (Lalle et al., 2007), but it is not clear if this is similar to AIII reported from wild-hoofed mammals and a rabbit (Lebbad et al., 2010) or the novel non-AI, non-AII genotypes from animal hosts identified by alloenzyme analysis (Monis et al., 2003). Comparison between studies is made complicated by differences in terminology, for example, A1, A2, A3, etc., have been used to arbitrarily describe genotypes or subtypes within Assemblage A (EligioGarcia et al., 2005; Lalle et al., 2005) but without reference to other studies or earlier descriptions of genetic groups, such as AI and AII, which represent clusters of genotypes (Read et al., 2004).

2.5. THE MAINTENANCE OF GIARDIA IN NATURE 2.5.1. Life cycle and development The cyst is the most important transmissible stage in the life cycle of Giardia. Most authorities consider cysts to be immediately infective upon being passed in the faeces, but there is evidence that some cysts undergo a maturation period of up to 7 days before becoming infective (Caccio and Sprong, 2011; Grant and Woo, 1978; Thompson, 2011). Between 10 and 25 cysts are considered the minimum dose to initiate an infection (Caccio and Sprong, 2011; De Carneri et al., 1977; Rendtorff, 1954). Trophozoites are also capable of initiating an infection following ingestion, as demonstrated experimentally in mice (Thompson, 1999). However, trophozoites will only have transitory survival in the environment and initiate infection in circumstances where they are expelled in large numbers during diarrhoetic episodes leading to contamination of individuals or surfaces, for example, in day care centres, breeding kennels and dairies. Cysts can also initiate infections in similar situations and may lead to longer term contamination of surfaces and the external bodies of animals, as well as contaminating the environment which may lead to the establishment of foci supporting a sustained high frequency of

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transmission by the faecal–oral route, for example, community situations, kennels, dairies, etc. (see below). Following ingestion, excystation takes place shortly after cysts leave the stomach. The low pH of the stomach environment appears to be the major factor which initiates the excystation process (Bingham and Meyer, 1979; Boucher and Gillin, 1990; Lauwaet and Gillin, 2009). Excystation leads to rapid colonisation of the duodenum and jejunum, where the excysted trophozoites attach to the intestinal mucosa and multiply rapidly. Attachment is mediated by Giardia’s unique attachment organelle, the ventral adhesive disc (Fig. 2.1), and is an essential feature of the relationship between Giardia and its host and a prerequisite to sustained infection (Thompson, 2011). 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 (Cooper et al., 2007; Lasek-Nesselquist et al., 2009; Meloni et al., 1995; Siripattanapipong et al., 2011; and reviewed in Monis et al., 2009) and genes involved in meiosis have been identified in the Giardia genome (Ramesh et al., 2005). However, the frequency of recombination is not known, nor its impact on the epidemiology of giardiasis and the extensive genetic diversity that characterises the forms of Giardia that infect mammals. The evolutionary advantage of genetic exchange to Giardia would be the capacity to respond to adversity, for example, selection pressures imposed by regular exposure to antigiardial drugs or competition with cohabiting ‘strains’ in circumstances where the likelihood of mixed infections is common (Hopkins et al., 1999; Monis et al., 2009). As such, it may be a relatively rare event and further population genetic studies are required in foci of infection where the frequency of infection is high (Monis et al., 1999; Thompson and Monis, 2011). The fact that available data indicate that the genetic assemblages¼species (Table 2.1) of Giardia are conserved in terms of geographic location and host occurrence suggests that any recombination is not reflected at the species level (Monis et al., 2009). The incubation, or pre-patent period, before cysts appear in the faeces, may be short in both humans and other animals, commencing as early as 3 days postinfection but can range up to 3 weeks depending upon host species (Flanagan, 1992; Hopkins and Juranek, 1991; Rendtorff, 1954; Thompson et al., 2008). The duration of infection may vary from a few days to several months. During the course of infection, trophozoites will encyst in the posterior small intestine. The trigger for trophozoites to encyst in vivo is not completely understood but appears to be initiated by the presence of bile salts and cholesterol (Lauwaet and Gillin, 2009; Sener et al., 2009). There have been no comparative studies of encystation in different species of Giardia, with transcriptional analysis of encysting

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trophozoites restricted to the WB strain (Morf et al., 2010). Comparison of different in vitro encystation protocols identified upregulation of a core set of genes, as well as upregulation (and downregulation) of genes specific to each protocol (Morf et al., 2010). Comparison of the encystation-associated genes determined that they all possessed a motif for binding the Giardia Myb transcription factor (Morf et al., 2010). Further studies are required to determine what similarities or differences occur between different species of Giardia and whether differences in regulation could account for differences in virulence. The first appearance of any symptoms usually coincides with the onset of cyst excretion. Cyst excretion is characteristically intermittent in both humans and other animal species, and cysts are resistant, surviving for at least 2 months in suitable temperature and moisture conditions (Meyer and Jarroll, 1980; Thompson, 2011). Encystation must be considered a major virulence factor as differentiation into a form that can survive in the environment and infect a new host is vital for transmission and disease progression.

2.5.2. Hosts Numerous vertebrate species have been shown to harbour Giardia infections in nature. To some extent, the current taxonomy reflects the host range of Giardia (Table 2.1). The majority of species of Giardia appear to have a relatively restricted host range. However, the two most common species found in mammals, G. duodenalis and G. enterica, have a low host specificity and are considered to have zoonotic potential. As a consequence, most data on the distribution and prevalence of Giardia in vertebrates have come from studies on mammalian hosts, principally domestic animals. From these studies, three life cycle patterns have been well defined that maintain Giardia in domestic hosts, including humans (Monis et al., 2009). Interaction between these cycles occurs in terms of transmission between hosts (Fig. 2.3). In addition, Giardia cycles of transmission have been identified in numerous species of wildlife, but it is not clear how the parasite is maintained in nature in wildlife populations, nor the impact of domestic cycles on the perpetuation of Giardia infections in wildlife (Fig. 2.3).

2.5.2.1. Humans Giardia is today well recognised as one of the most prevalent intestinal infections of humans in both temperate and tropical areas, with prevalence rates varying between 2% and 7% in Europe, the United States, Canada and Australia, to over 40% in developing areas where living conditions are poor, nutritional levels are often inadequate and concurrent infections are common (reviewed in Feng and Xiao, 2011; Thompson,

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Dog/cat cycle

Frequency of transmission?

Direct (occasional waterborne)

Frequency of transmission?

Human cycle

Livestock cycle Direct (occasional waterborne)

Frequency of transmission?

Direct (occasional waterborne)

f yo ? nc ue ssion q Fre smi n tra

l

na sio ca e) c o n t ( rbor ec Dir wate

Wildlife cycle(s)

FIGURE 2.3 Major cycles of transmission of Giardia in mammalian hosts. Some assemblages/species are host specific and cycle between their respective hosts, whereas others have low host specificity and are capable of infecting humans and other animals (modified from Monis et al., 2009).

2009, 2011). In developed countries, infections with Giardia are most common in children, especially in day care centres, residents of institutions and travellers. A rising incidence in such settings has led to the designation of giardiasis as a re-emerging infectious disease in the developed world (Eckmann, 2003; Savioli et al., 2006; Thompson, 2000, 2004; Thompson and Monis, 2004). The emerging issue of Giardia infections in developing regions of the world and the impact on children was a major factor in the inclusion of Giardia in WHO’s ‘Neglected Diseases Initiative’ (Savioli et al., 2006). In developing countries, particularly in Asia, Africa and Latin America, about 200million people have symptomatic giardiasis with some 500,000

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new cases reported each year (Savioli et al., 2006; Thompson, 2009; WHO, 1996). Children living in communities in developing countries and among disadvantaged groups living in isolated communities such as indigenous Australians are most commonly infected (Al-Mekhlafi et al., 2005; Savioli et al., 2006; Thompson, 2000, 2009; Thompson and Smith, 2011; Thompson et al., 2001). These children are most at risk from the chronic consequences of Giardia infection, as well as the repeated exposure to potentially toxic drugs in some endemic regions (Thompson et al., 2001). In developed countries, epidemiological investigations have demonstrated that travel, swimming in surface water, contact with young children and institutional confinement are important risk factors associated with Giardia infection (Abe and Teramoto, 2012; Hunter and Thompson, 2005; Kettlewell et al., 1998; Stuart et al., 2003; Thompson, 2009). There is also evidence that contact with farm and companion animals are also risk factors for infection ( Jagai et al., 2010; Robertson et al., 2010; Warburton et al., 1994). Infection varies inversely with socio-economic status and is high in regions where water supplies are poor or non-existent and sanitation and personal hygiene standards are inadequate (Alvarado and Va´squez, 2006; Balcioglu et al., 2007; Hesham et al., 2005; Hunter and Thompson, 2005; Savioli et al., 2006; Thompson, 2011). Living in community settings with other animals has also been shown to heighten the risk of infection with Giardia (Inpankaew et al., 2007; Marangi et al., 2010; Salb et al., 2008; Traub et al., 2003, 2004). Risk factors identified as important in facilitating emergence of Giardia infection include high environmental faecal contamination, lack of potable water, inadequate education and housing, overcrowding and high population density and animal reservoirs of infection (reviewed in Thompson, 2011).

2.5.2.2. Dogs and cats Giardia is a common parasite of dogs and cats globally and is the most frequently diagnosed enteric parasite of dogs and cats in developed countries (Ballweber et al., 2010; Scaramozzino et al., 2009; Thompson et al., 2008; Tangtrongsup et al., 2010). Prevalence rates vary (Ballweber et al., 2010; Feng and Xiao, 2011) and are influenced by the sampling strategies and diagnostic methods used (Epe et al., 2010). Surveys of a variety of canine and feline populations reveal prevalences of between 10% in well-cared-for dogs, 36–50% in puppies and kittens, and up to 100% in breeding establishments and kennels where the frequency of transmission will be higher (Hahn et al., 1988; Kirkpatrick, 1988; and reviewed in Ballweber et al., 2010; Feng and Xiao, 2011; Thompson et al., 2008). A recent study in the United States found that dog park-attending dogs were more likely to be positive for Giardia than non-dog park-attending dogs (Wang et al., 2011).

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Infection with Giardia will result from direct transmission between animals or from the environment. Dogs and cats may harbour hostadapted (G canis, Giardia felis) or zoonotic species of Giardia which can cycle between dogs or cats (Covacin et al., 2011; Thompson et al., 2008; Upjohn et al., 2010). Host-adapted species, such as G. canis in dogs, is likely to predominate in breeding establishments and pet shops (Itoh et al., 2011).

2.5.2.3. Livestock In livestock, Giardia infections have been reported in cattle, both dairy and beef, sheep, goats, horses, pigs and cervids (Dixon et al., 2011; Farzan et al., 2011; Feng and Xiao, 2011; O’Handley and Olson, 2006). Although all ruminants are likely to be exposed to Giardia shortly after birth, infections are most common towards the end of the neonatal period and in calves can be as high as 100% (O’Handley and Olson, 2006; Olson et al., 2004). Direct contact between young livestock appears to be the most likely source of transmission (Becher et al., 2004; Dixon et al., 2011; O’Handley et al., 1999; St Jean et al., 1987; Wade et al., 2000; Xiao et al., 1993). Grouping behaviour of calves in pens or paddocks provides ample opportunities for the transmission of Giardia. As with dogs and cats, livestock may harbour host-adapted (G. bovis) or zoonotic species of Giardia, although G. bovis tends to be more prevalent in cattle (Dixon et al., 2011; Khan et al., 2011). However, G. duodenalis is most common in young animals (Mark-Carew et al., 2011), and in a recent survey of pigs in Ontario, Canada, G. enterica was the most common species found (Farzan et al., 2011). The role of zoonotic transmission is discussed below, but the introduction of zoonotic species of Giardia by humans into environments where cattle are housed may result in infections in cattle which can then be transmitted between cattle.

2.5.2.4. Wildlife Although numerous species of wild mammals have been reported to be infected with Giardia, both in the wild and captivity, the majority of infections are with zoonotic species (Levecke et al., 2011; Martinez-Diaz et al., 2011; Siembieda et al., 2011; Soares et al., 2011; and reviewed in Thompson et al., 2010a). These are considered to have been introduced into wildlife habitats and once established would appear to be maintained by direct contact or via the environment even in terrestrial and aquatic environments presumed to be pristine, for example, muskoxen in the Arctic and beavers in pristine mountain streams (Thompson et al., 2010a). Distinct species and genotypes of Giardia have been recovered from amphibia, reptiles, rodents, bandicoots and birds (Adams et al., 2004;

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McRoberts et al., 1996; Monis and Thompson, 2003). Although the ecology of infections with these host-restricted species of Giardia is not well understood, it is presumed that infections cycle directly between hosts and/or the environment. However, there is limited information on the prevalence of infections in nature. A recent study in Australia found that infections in bandicoots were not common raising questions about how the parasite is maintained in nature (Thompson et al., 2010b).

2.5.3. Transmission 2.5.3.1. Faecal–oral transmission In humans, transmission of Giardia is principally by faecal–oral contamination, which is reflected by higher levels of infection where levels of hygiene and sanitation are compromised, particularly in tropical and subtropical environments (Alvarado and Va´squez, 2006; Balcioglu et al., 2007; Savioli et al., 2006). As such, direct person-to-person transmission is considered to be more important than waterborne, foodborne or zoonotic transmission (Hesham et al., 2005; Hunter and Thompson, 2005; Pawlowski et al., 1987; Schantz, 1991; Thompson, 2004; Thompson and Smith, 2011). Other environmental factors which will exacerbate the frequency of faecal–oral transmission include day care centres where conditions conducive to faecal–oral contamination are common and high prevalence rates of Giardia infection have often been observed (Thompson, 2000, 2011). Indirect transmission, where infection results through the mechanical transmission of cysts on, for example, flies (Szostakowska et al., 2004) or other animals such as dogs or livestock, poses a significant threat particularly in the developing world (Thompson and Smith, 2011). In domestic animals, Giardia infections are most common in situations where the levels of environmental contamination with cysts are high, such as breeding establishments, kennels, catteries, dog parks, pet shops, dairies, cattle sheds and in the case of dogs communities with free roaming dogs (Itoh et al., 2011; Thompson, 2011; Wang et al., 2011). In addition, direct transmission from the contaminated coats of animals in breeding and weaning areas will be common.

2.5.3.2. Waterborne transmission Giardiasis is a frequently diagnosed waterborne disease in developed countries (Karanis et al., 2007; Levine et al., 1990; Robertson and Lim, 2011; Smith et al., 2007; Thompson, 2004). The consumption of drinking water other than metropolitan mains, or other filtered supplies, represents a significant risk for giardiasis (Robertson and Lim, 2011). The majority of waterborne giardiasis outbreaks in humans have occurred in unfiltered surface or groundwater systems impacted by surface run off or

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sewage discharges ( Jakubowski and Craun, 2002; O’Reilly et al., 2007; Robertson and Lim, 2011) or systems that have been poorly maintained (Daly et al., 2010). Irrigation waters used for food crops that are traditionally consumed raw may also represent a high risk as a source of Giardia (Thurston-Enriquez et al., 2002). Environmental contamination of such water systems and supplies may result from human, agricultural and wildlife sources (Heitman et al., 2002). Waterborne transmission is also a well-documented cause of Giardia infection in travellers who usually contract infection from drinking local tap water (Hunter and Thompson, 2005). In the developed world, waterborne transmission is usually the result of contamination with Giardia of human origin or a process failure by water utilities, industry or in swimming pools (Dale et al., 2010; Shields et al., 2008; Stuart et al., 2003). Such contamination may impact negatively on ecosystem health leading to infections in aquatic wildlife which may then establish reservoirs of human infection. The role of the beaver as a ‘spill back’ reservoir of Giardia in North America is the best known example (Thompson et al., 2009a). Recent studies have also demonstrated that filter-feeding molluscs and freshwater fish are useful indicators of the presence of waterborne pathogens, including Giardia of human origin (Lucy et al., 2008; Miller et al., 2005; Nappier et al., 2010; Thompson and Smith, 2011). In the developing world, there is a much greater reliance on lakes, streams and other natural surface water sources for drinking, food preparation, washing clothes and personal hygiene exacerbating the chances of waterborne infection (Hunter and Thompson, 2005; Thompson and Smith, 2011). Areas which are prone to flooding face an increased risk of waterborne infection particularly where basic sewerage systems are used and containment likely to be compromised (Thompson and Smith, 2011). Kutz et al. (2009a) also emphasised that climate change has been proposed to cause increased frequency and magnitude of flooding enhancing transmission of waterborne pathogens such as zoonotic species of Giardia, in and between terrestrial and marine systems.

2.5.3.3. Foodborne transmission Giardia is one of the several enteric protozoa that is known to be readily transmitted on food (Robertson and Lim, 2011; Thompson, 2011), and in some parts of the world, foodborne transmission may be enhanced through the use of human waste as fertiliser and inadequate pasteurisation techniques (Thompson and Smith, 2011). However, most foodborne transmission is considered to be associated with infected food handlers and poor hygiene, usually at a local level rather than as the source of outbreaks of Giardia infection (Barnard and Jackson, 1984; Mintz et al., 1993; Petersen et al., 1988; Robertson and Lim, 2011; Smith et al., 2007). Overall, it has been estimated that the number of cases of foodborne

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transmission range from 13 to 76 million globally (Thompson and Smith, 2011). Foods associated with cyst contamination have included canned salmon, salads, sandwiches, raw vegetables and ice (Robertson and Lim, 2011; Smith et al., 2007). The reason there are fewer reported outbreaks of Giardia infection involving contaminated foods is likely due to the lack of appropriate tools, or their application, in the past, the sporadic nature of such outbreaks, lack of awareness and under reporting (Robertson and Lim, 2011; Thompson and Smith, 2011).

2.6. INTERACTION BETWEEN CYCLES Although we may have a growing understanding of how Giardia is maintained in cycles involving domestic animals and wildlife, the question of how these cycles may interact, and the host range of the various genotypes of Giardia involved, is largely unresolved. This is particularly important with respect to zoonotic and waterborne transmission. The molecular characterisation of Giardia isolates from different species of mammalian hosts throughout the world has confirmed the existence of host-specific species and two species with broad host ranges which are zoonotic (Table 2.1). This revised taxonomy largely reflects the species nomenclature reported by early workers in the field (Monis et al., 2009; Thompson and Monis, 2011) and helps to better understand host specificity in terms of the epidemiology of Giardia infections. The two zoonotic species of Giardia are geographically widespread, and as more isolates are genotyped, some patterns are emerging on host occurrence. Overall in humans, the distribution of G. duodenalis and G. enterica is similar in both developed and developing countries, with G. enterica more common (58%) in developing than developed countries (55%) compared to G. duodenalis (37% vs. 40%), but mixed infections are more common in developing countries (8% vs. 2%) (data from Feng and Xiao, 2011). In dogs, recent studies have shown that it is not possible to extrapolate from one geographical region to another in terms of the species/assemblage composition of Giardia infections in dogs (Ballweber et al., 2010; Covacin et al., 2011). In Europe, studies had suggested that Assemblage B has a predominantly human distribution (Sprong et al., 2009), but a recent study in the United States found a higher frequency of infections in dogs with G. enterica than G. duodenalis, which has not been reported elsewhere (Covacin et al., 2011). This suggests that in North America at least, we cannot assume that G. duodenalis is the most common of the zoonotic species found in non-human hosts (Covacin et al., 2011). Indeed, in wildlife, G. enterica often predominates (e.g. Johnston et al., 2010), whereas in cattle, G. duodenalis is most often reported (Sprong et al., 2009). However, there is an extensive genetic substructuring within

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G. enterica, and it is possible that some subgroups are more commonly associated with zoonotic infections than others. In humans, there is some evidence of geographic substructuring (Siripattanapipong et al., 2011; Wielinga et al., 2011), and G. enterica may be more common in isolated and/or community settings where the frequency of transmission is high (Thompson, 2000). Under such circumstances, the parasite is likely to be exposed to greater selection pressure in terms of exposure to antigiardial drugs and competitive interactions which might explain why evidence of recombination in Giardia is mostly confined to G. enterica isolates (LasekNesselquist et al., 2009; Siripattanapipong et al., 2011; Thompson and Monis, 2011).

2.6.1. Zoonotic transmission The application of molecular tools for ‘typing’ isolates of Giardia in faecal samples from human and non-human mammalian hosts in different parts of the world has produced a wealth of information on the distribution of host-specific and zoonotic species of Giardia. From these studies, there is clear evidence that cysts of zoonotic Giardia do contaminate the environment in areas where the potential for zoonotic transmission exists. The epidemiological value of these studies varies with the number of loci used for genotyping and the number of host species sampled. However, in most cases, it is possible to extrapolate that a risk of zoonotic transmission exists, but evidence of how frequently it occurs requires focal studies in defined endemic areas where transmission dynamics and host range are known.

2.6.1.1. Dogs and cats The significance of Giardia infection in domestic dogs and cats is considered to be primarily a public health issue, and the clinical impact on dogs and cats is generally believed to be minimal (Thompson et al., 2008). However, Giardia may be associated with gastrointestinal disorders in dogs (Barutzki et al., 2007; Epe et al., 2010), and more studies are required to determine whether there are differences in clinical impact between infections with zoonotic Giardia species and G. canis in dogs, and similarly, whether mixed infections of Giardia species may be clinically more apparent in dogs. The zoonotic potential of Giardia infections in dogs and cats was proposed long before genotyping data was available, but cross-infection experiments proved difficult to interpret (Thompson and Monis, 2004; Thompson et al., 1990). Apart from limitations in experimental design, variability in results will have been affected by the differences in host specificity of the Giardia isolates used which has now been confirmed from molecular epidemiological data (Monis et al., 2009).

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A number of studies have been undertaken in which domestic dogs, and to a lesser extent, cats, living in urban areas of developed countries have been sampled. In the majority of studies, both host-specific, G. canis/ G. felis, and zoonotic species, G. duodenalis and G. enterica, and subgenotypes have been identified, albeit in varying proportions (Ballweber et al., 2010; Covacin et al., 2011; Feng and Xiao, 2011; Leonhard et al., 2007; Suzuki et al., 2011; Volotao et al., 2011). Mixed infections of G. canis and G. duodenalis or G. enterica have also been reported. As discussed above, the distribution of zoonotic species varies; for example, in Europe, G. duodenalis has been reported more commonly than G. enterica which a recent study found to be the dominant zoonotic species in dogs in the United States (Covacin et al., 2011). From an epidemiological perspective, interpretation of the results of these studies demonstrate that a potential environmental reservoir of Giardia infection exists in urban areas but without concurrent data from owners or known handlers, information on the frequency of zoonotic transmission is lacking. However, Bugg et al. (1999) found that dogs from multi-dog households were more commonly infected with Giardia than dogs in single-dog households, emphasising the potential ease with which Giardia can be spread to in-contact animals and therefore presumably humans (Bugg et al., 1999). In contrast, a few studies have been undertaken in defined endemic foci in which both humans and dogs have been sampled and isolates of Giardia characterised genetically. Results from these molecular epidemiological studies have provided more definitive support for zoonotic transmission but have also highlighted the importance of understanding the transmission dynamics of Giardia infections. The first multilocus molecular epidemiological studies to address the issue of zoonotic transmission were undertaken by Traub et al. (2004) in tea growing communities in Assam, north-east India, where Giardia occurs in both humans (up to 21%, depending upon age) and dogs (20%). Traub and her colleagues found that all infected dogs harboured zoonotic species of Giardia: G. duodenalis and G. enterica, with some mixed infections. These studies by Traub et al. (2004) provided the first direct evidence of zoonotic transmission between dogs and humans, by finding the same genotype of Giardia in people and dogs, not only in the same village but also in the same household. Evidence for zoonotic transmission was supported by strong epidemiological data showing a highly significant association between the prevalence of Giardia in humans and the presence of a Giardia positive dog in the same household. A similar situation was found in Temple communities in Bangkok (Inpankaew et al., 2007) and northern Canadian indigenous communities (Salb et al., 2008). Both studies demonstrated zoonotic species of Giardia infecting dogs and their owners sharing the same living area. In contrast, a molecular epidemiological investigation in remote indigenous communities in

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northern Western Australia, which represent highly endemic foci of Giardia transmission with high rates of infection in children and dogs, often greater than 50% (Meloni et al., 1993; Thompson, 2000), found that all but one dog (1/12 dogs) were infected with G. canis (Hopkins et al., 1997). This result was interpreted as evidence of competitive exclusion, since the frequency of Giardia transmission is so high in these communities, with dogs equally likely to be exposed regularly to infection with G. canis and zoonotic species, mostly G. enterica. Such competitive interactions are likely to ensure that the host-adapted genotypes predominate in respective host species, as with G. bovis in dairy cattle (Hopkins et al., 1997, 1999; Thompson and Lymbery, 1996; Thompson and Monis, 2004, 2011; Thompson et al., 1996). Such an interpretation is supported by a recent study undertaken in a desert community in Peru where 16% of dogs and 20% of humans were infected with Giardia with all dogs apart from one infected with G. canis (Cooper et al., 2010). One dog had a mixed infection with G. canis and G. enterica. In domestic, urban environments, and in the communities in Assam, Bangkok, northern Canada and Peru, the frequency of dog-to-dog transmission will be less frequent, and thus infections acquired with zoonotic species in dogs are likely to persist. It should be emphasised, however, that the fact that dogs have contact with young children passing Giardia cysts, as well as discarded nappies/ diapers, means that dogs are likely to act as mechanical transmitters of zoonotic Giardia since their coats are likely to be contaminated with cysts. Although competitive interactions between different species of Giardia have been proposed to explain the predominance of single species infections in both dogs and cattle (see below), this may reflect the consequences of mixed infections in endemic foci where the frequency of transmission is very high. In other situations where transmission is sporadic, mixed infections may coexist and have been increasingly reported from multilocus studies in several countries in humans, dogs and cattle (e.g. Covacin et al., 2011; Dixon et al., 2011; Hussein et al., 2009; Sprong et al., 2009). The reason why mixed Giardia infections are more common in domestic dogs in urban areas of developed countries is not clear. Perhaps it is a reflection of the lower frequency of transmission and/or dietary differences between well-cared-for dogs living in more affluent environments and those on a poorer plane of nutrition which do not provide an intestinal environment supportive of mixed infections.

2.6.1.2. Livestock Livestock infected with Giardia, particularly cattle, has long been considered to represent a public health risk as a source of waterborne outbreaks of giardiasis in humans. This is because livestock is known to be susceptible to infection with zoonotic species of Giardia as well as G. bovis, and thus the potential for livestock operations to contaminate ground and surface waters

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and considering the large numbers of cysts shed by infected cattle (Donham, 2000). It has been shown that calves infected with Giardia commonly shed from 105 to 106 cysts per gram of faeces (O’Handley et al., 1999; Xiao, 1994). However, of the 132 documented waterborne outbreaks (Robertson and Lim, 2011), there is no evidence incriminating infected cattle in any outbreak (Hunter and Thompson, 2005; Olson et al., 2004; Thompson, 2004). Although it would seem likely that runoff and flooding would result in contamination events, molecular epidemiological data suggest cattle operations are a minimal risk as a source of environmental contamination with zoonotic Giardia. Although Giardia is common in both dairy and beef cattle, it is principally dairy cattle that harbour zoonotic species, usually G. duodenalis and less commonly G. enterica (Dixon et al., 2011; Feng and Xiao, 2011), but only as transitory infections in young animals less than 3 months of age. Older animals only seem to support infections with G. bovis which may also be related to competitive exclusion operating in older animals (Thompson and Monis, 2011). Longitudinal studies in Australia and the United States (Becher et al., 2004; Mark-Carew et al., 2011) suggest that zoonotic genotypes may only be present transiently in cattle under conditions where the frequency of transmission with the livestock species, G. bovis (Assemblage E), is high and competition is thus likely to occur (Becher et al., 2004; Thompson, 2004; Thompson and Monis, 2004, 2011). A recent survey of pigs on 10 farms in Ontario, Canada, found that over 50% of pigs were infected on all farms and that 92.1% of isolates were G. enterica, the remainder being G. bovis (Farzan et al., 2011). These authors considered that there was potential for zoonotic transmission via cyst-contaminated water. Animal handlers are at risk from contracting Giardia from dairy cattle as recently demonstrated in a molecular epidemiological study in India (Khan et al., 2011). However, reverse zoonotic transmission should be considered as the possible source of zoonotic Giardia infections in cattle, particularly in dairy cattle because of more frequent contact with handlers (Dixon et al., 2011). A molecular epidemiological study in Uganda where humans appear to have introduced Giardia into a remote national park are thought to have been the source of Giardia in a small number of cohabiting dairy cattle (Graczyk et al., 2002).

2.6.1.3. Wildlife The occurrence of Giardia in wildlife has been the single most important factor incriminating Giardia as a zoonotic agent. As such, it was the association between infected animals such as beavers and waterborne outbreaks in people that led the WHO (1979) to classify Giardia as a zoonotic parasite. It is therefore surprising that there is so little evidence to support the role of wildlife as a source of disease in humans, since this has dominated debate on the zoonotic transmission of Giardia and, in particular, when water is the vehicle for such transmission (Welch, 2000).

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Indeed, there is increasing evidence to suggest that Giardia infections in wildlife result from environmental contamination from domestic sources, that is, reverse zoonotic events. Although wildlife, particularly aquatic mammals, is commonly infected with Giardia, there is little evidence to implicate such infections as the original contaminating source in waterborne outbreaks (Appelbee et al., 2005; Thompson, 2004). It would appear that such animals are more likely to have become infected from water contaminated with faecal material of human, or less likely, domestic animal origin (Thompson, 2011). Wildife may thus serve to amplify the numbers of the originally contaminating isolate (Bemrick and Erlandsen, 1988; Kutz et al., 2009b; Monzingo and Hibler, 1987; Thompson, 2004, 2011; Thompson et al., 1990; Thompson et al., 2009a), and depending upon the nature of the particular ecosystem, a zoonotic reservoir may be established, as was the case with beavers in North America. The few studies that have genotyped Giardia of beaver origin, in both Canada and the United States, have confirmed previous suggestions that the source of Giardia infection in beavers was likely to be of human origin (Appelbee et al., 2002, 2005; Sulaiman et al., 2003). The latter authors also examined Giardia from eight muskrats from the same region and only three were infected with the expected Giardia microti, and the remaining five muskrats were infected with zoonotic Giardia, G. enterica. Several more recent reports have also shown that ‘reverse zoonotic transmission’ is an important factor that must be considered in understanding the epidemiology of Giardia infections in wildlife. Humans are considered to be the source of infection in non-human primates and painted dogs in Africa, marsupials in Australia, coyotes in North America, muskoxen in the Canadian Arctic, house mice on remote subarctic islands and marine mammals in various parts of the world (Appelbee et al., 2010; Ash et al., 2010; Dixon et al., 2008; Graczyk et al., 2002; Johnston et al., 2010; Kutz et al., 2008; Moro et al., 2003; Teichroeb et al., 2009; Thompson et al., 2009b, 2010b). These reports raise important issues for conservation because we do not understand the impact Giardia may have on what are possibly naı¨ve hosts. They may have been exposed to the parasite relatively recently, as a consequence of habitat disturbance and human encroachment, impairing health and fitness ( Johnston et al., 2010; Thompson et al., 2010a).

2.7. FUNCTIONAL SIGNIFICANCE OF GENETIC VARIATION 2.7.1. Developmental biology Widespread differences have been reported between isolates of Giardia (representing G. duodenalis and G. enterica) in a variety of areas, including biochemistry, growth rates (in vitro and in vivo), DNA content, drug

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sensitivity, site and duration of infection, pH preference, virulence and susceptibility to infection with a dsRNA virus (Binz, 1996; Binz et al., 1992; Farbey et al., 1995; Hall et al., 1992; Monis et al., 1996; Reynoldson, 2002; Thompson et al., 1996). Genome comparative analyses are in their relative infancy, but they are already revealing some interesting findings. There is significant variation between the genomes of G. duodenalis, G. bovis and G. enterica in terms of gene content and polymorphism, chromosome structure and gene families encoding surface antigens and kinases ( Jerlstrom-Hultqvist et al., 2010b). G. duodenalis and G. bovis can both be readily cultivated axenically (Ey et al., 1997), while G. enterica is more difficult to establish in vitro, growing slower than G. duodenalis in vitro and appearing to grow better than G. duodenalis in suckling mice (Andrews et al., 1992). At a gross level, G. duodenalis and G. bovis are overall more similar to each across their genomes than either are to G. enterica ( Jerlstrom-Hultqvist et al., 2010b). A comparison of promoter regions for major cyst wall proteins has found conserved promoters present in both WB and GS isolates of G. duodenalis and G. enterica, respectively, suggesting regulation of these proteins is similar in both isolates (Franzen et al., 2009). A similar promoter sequence has also been found in front of a key regulatory enzyme in WB and the P15 isolate of G. bovis, but the GS sequence lacks the same promoter (Franzen et al., 2009; Jerlstrom-Hultqvist et al., 2010b). This variation has been suggested to cause a difference in the regulation of cyst wall sugar synthesis in GS and may be the cause of the poor encystation observed in vitro of GS (Franzen et al., 2009). The metabolic gene content for GS and WB is the same (Franzen et al., 2009). Comparison of VSP, NEK kinases and high cysteine membrane proteins found some that were conserved between WB and GS and some that were highly divergent (Franzen et al., 2009). The genomic organisation of the VSP genes has only been analysed in detail for WB, finding that many genes occur in clusters and that recombination has occurred between different VSP clusters (Adam et al., 2010). The regulation of Giardia VSPs is likely to be different to that of other protozoan parasites, with the WB VSPs predominantly occurring at internal chromosome locations, whereas subtelomeric location of surface antigen genes is more common (and in some cases, required for expression) in trypanosomes or Plasmodium (Adam et al., 2010). The P15 genome appears to be poorer in VSPs compared to WB, although this could be due to incomplete sequencing of those regions ( JerlstromHultqvist et al., 2010b). The differences in some of these gene families may explain the differences in host range ( Jerlstrom-Hultqvist et al., 2010b). The genome organisation is different between the three species (Jerlstrom-Hultqvist et al., 2010b), but the biological significance of this is not clear.

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2.7.2. Pathogenesis, variation in virulence and polyparasitism A variety of symptoms are associated with Giardia infections. With the genomic information now available, it should be possible to correlate this with clinical expression and identify factors associated with virulence. However, this remains a major challenge, given the variables that need to be considered. Most information on the pathogenesis of Giardia infections has been obtained from studies in rodent models and in vitro culture, which have shown that Giardia damages brush border microvilli, thus limiting intestinal barrier function resulting in malabsorption and maldigestion (reviewed in Cotton et al., 2011; Humen et al., 2011; Shukla and Sidhu, 2011). Although such observed changes help in understanding how diarrhoeal disease may occur in Giardia infections, it is not clear how the pathophysiological changes described in rodent models can be extrapolated to humans and other vertebrate hosts, since infection may not result in overt clinical symptoms. Symptoms are also influenced by species/ breed of host, species/assemblage of Giardia, age, immune competence, frequency of infection, nutrition and concurrent infections. In humans, acute and chronic giardiasis present as two very different diseases. In the former, acute episodes of diarrhoea are most commonly associated with infection, whereas chronic giardiasis is not characterised by diarrhoea but is associated with failure to thrive and is often exacerbated by poor nutrition and polyparasitism (Thompson, 2008; Thompson and Smith, 2011). Furthermore, there is emerging evidence that Giardia infections may induce post-infectious gastrointestinal symptoms including irritable bowel syndrome (Hanevik et al., 2009; Kampitak, 2010; Wensaas et al., 2010). Unfortunately, the impact on health of concurrent/coinfections (polyparasitism) has not been adequately taken into account. Giardia commonly occurs with other genera of intestinal parasites, particularly in the developing countries (Thompson and Smith, 2011), and this will influence the clinical impact of Giardia infections. This makes it difficult to determine the contribution of each cohabiting pathogen to the clinical consequences of such mixed infections. For example, the chronicity of Giardia infections in disadvantaged children whose nutrition may be suboptimal and who suffer infections with other gastrointestinal parasites such as Entamoeba, Blastocystis, Hymenolepis and/or hookworm is recognised as an important contributor to poor growth (Sackey et al., 2003; Thompson, 2000; reviewed in Thompson and Smith, 2011). However, the situation is further complicated by the fact that mixed infections with G. duodenalis and G. enterica are also common. A number of studies have provided data suggesting that acute and chronic giardiasis may be associated with different species/assemblages of Giardia (Gelanew et al., 2007; Haque et al., 2005; Homan and Mank, 2001;

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Molina et al., 2011; Read et al., 2002; Sahagun et al., 2008). Based on available data, it had been proposed that G. duodenalis may be more commonly associated with acute giardiasis and G. enterica with chronic infections (Thompson and Monis, 2011). In contrast, some recent reports found that diarrhoea was more common in individuals infected with G. enterica (Al-Mohammed, 2011; Mahdy et al., 2008, 2009; Pelayo et al., 2008). However, these reports were from developing and/or rural regions and are difficult to interpret since Giardia was one of the several other cohabiting enteric parasites, and in such cases of polyparasitism, it is very difficult to conclude that non-specific symptoms such as diarrhoea are only due to Giardia. The clinical impact of enteric protozoan infections is greatest in the developing world where inadequate sanitation, poor hygiene and proximity to zoonotic reservoirs, particularly companion animals and livestock, are greatest. In such circumstances, it is not surprising that infections with more than one species of enteric protozoan and helminth are common, and in fact, single infections are rare (Thompson and Smith, 2011). Interpretation of the results is also complicated by differences in study design and sampling strategy. From what has been reported in the literature, there is evidence that infections with G. enterica in humans are more common in rural areas, particularly in developing countries, and community situations, where the frequency of transmission is high (Boontanom et al., 2011; Mahdy et al., 2009; Molina et al., 2011; Yason and Rivera, 2007). This would suggest that G. enterica is better adapted to such situations which are characterised by prolonged infections/regular reinfections where acute diarrhoeal episodes are not in the best interests of the parasite, allowing better survival in mixed infections. The lack of overt symptoms such as diarrhoea would explain why infections with G. enterica are more common in such environments (Molina et al., 2011). Children with such infections are likely not to be treated, which also raises questions about the long-term consequences of such chronic infections if they persist and there is no ‘self cure’. This is thought to be significant in situations where infected children are disadvantaged in terms of nutrition and exposure to concurrent enteric infections. A number of mechanisms have been proposed to explain how Giardia attaches to intestinal epithelial cells, but most evidence indicates that the ventral disc plays the major role in attachment and that the cytoskeletal elements of the disc are the major mediators in this process (Palm and Sva¨rd, 2009). This is indicated by the fact that microtubule inhibitors, including known b-tubulin antagonists, have been shown to inhibit adherence in vitro (Edlind et al., 1990; Magne et al., 1991; Meloni et al., 1990). It is therefore interesting that a prominent cytoskeletal protein of the ventral adhesive disc, alpha 2 giardin, which is present in G. duodenalis (Assemblage A) isolates is absent in G. enterica (Assemblage B) isolates which may explain the differences emerging in the clinical consequences of infection with these two species (Steuart et al., 2008).

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It is not known whether the complexity of symptomatology that is seen in humans is seen in other hosts infected with the same species/ assemblage of Giardia. For example, it is not known whether there is any difference in the clinical outcome in dogs infected with the zoonotic species or G. canis.

2.8. CONCLUSIONS The data from Giardia genome sequences (and other related protozoans) have already improved our understanding of the evolution of Giardia and eukaryotes in general and have identified some unique strategies that Giardia has developed during its evolution, such as split introns. The genome data are also improving our understanding of the metabolism and cellular processes within Giardia. Comparison of the available Giardia genomes supports the species status of the currently recognised assemblages, suggesting genome-wide differences equivalent to those separating species in other genera such as Theileria and Leishmania. The differences that have been identified so far might also explain observed phenotypic differences, such as differences in encystation caused by differences in the regulation of key enzymes. These are relatively early days in the comparative genomics of the different lineages of Giardia, and more work is required to further compare the regulation of cellular processes and to determine if there are differences that correlate with variation in characters such as host range. Importantly, more genome sequences are required, both from the different species and from multiple isolates within the same assemblage/species, so that we can determine the levels of intra- and interspecific differences, and if key differences in chromosome arrangements or gene family repertoires are conserved within species. Considering the level of genetic diversity within G. enterica, it will be particularly important to compare the intraspecific variation since this may underlie differences in host infectivity/disease outcome among different isolates of G. enterica. The cost of genome sequencing is continually decreasing, so the challenges to come will be more in the collection of type material for sequencing, with the largest challenge to conduct the necessary bioinformatic analysis to make best use of the large amount of data that can now be readily generated. There has been a progression in the development of molecular tools for the identification of Giardia in recent years (Smith and Mank, 2011), but the challenge for the future is the development of diagnostic assays that will support clinical management and treatment decisions. For example, an ELISA-based assay for use with dogs and cats that will provide not only sensitive detection of Giardia but also information on species will support the need for treatment in terms of public health significance and

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possibly clinical prognosis. We already have a good stable of drugs with antigiardial efficacy (Lalle, 2010), but there are limitations due to toxicity, specificity, dosage, palatability and, possibly, resistance. Mining the genome and proteome of Giardia will allow the development of new classes of compounds with improved specificity, thus avoiding any impact on normal gut microflora as well as improved compliance in terms of palatability and dosage.

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