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International Journal of Hygiene and Environmental Health 213 (2010) 321–333

Contents lists available at ScienceDirect

International Journal of Hygiene andEnvironmental Health

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Review

Giardia taxonomy, phylogeny and epidemiology: Facts and open questions

Judit Plutzera,∗, Jerry Ongerthb, Panagiotis Karanisc

a National Institute of Environmental Health, Department of Water Hygiene, Gyáli ut 2-6, Budapest H-1096, Hungaryb Civil Mining & Environmental Engineering, University of Wollongong, Wollongong, NSW 2522, Australiac Medical School, University of Cologne, Center of Anatomy, Institute II, Molecular and Medical Parasitology, Cologne, Germany

a r t i c l e i n f o

Article history:Received 24 February 2010Received in revised form 25 May 2010Accepted 2 June 2010

Keywords:GiardiaTaxonomyCultivationMolecular detectionEpidemiologyWater transmission

a b s t r a c t

Giardia duodenalis (synonymous Giardia lamblia and Giardia intestinalis) is a flagellated protozoan para-site that reproduces in the small intestine causing giardiasis. It is a cosmopolitan pathogen with a verywide host range, including domestic and wild animal species, as well as human beings. In this paper thecurrent knowledge about the taxonomy and phylogeny of G. duodenalis is summarized from the inter-national literature and data on the detection and epidemiology are also reviewed concentrating on thelast 20 years. Authors highlighted the current knowledge and some aspects on G. duodenalis in partic-ular, water transmission and in vitro cultivation. The review sheds light on the difficulties of the straindifferentiation and multilocus molecular analysis of Giardia strains especially when applied to watersamples containing low numbers of cysts and components complicating the problem of tracking sourcesof contamination. Genetic elements determining or conferring traits such as infectivity, pathogenicity,virulence, and immune interaction contributing to clearance are currently not well established, if at all.These should be useful and important topics for future research.

© 2010 Elsevier GmbH. All rights reserved.

Contents

Giardia taxonomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322Giardia species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322Giardia assemblages, subassemblages and genotypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323

Waterborne transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323Giardiasis outbreaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323Drinking water treatment process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324Post-treatment contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324Giardia presence and survival in the environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324The prevalence of Giardia in different water sources in Europe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325

Foodborne transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325Zoonotic risk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325The relevance of host factors in giardiasis transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326

Detection of Giardia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326Giardia detection in water samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326Giardia detection in stool samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327Giardia detection in food samples. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327

The importance of G. duodenalis cultivation in vivo and in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327General conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329

∗ Corresponding author. Tel.: +36 1 476 1200; fax: +36 1 476 1200.E-mail address: plujud@yahoo.com (J. Plutzer).

1438-4639/$ – see front matter © 2010 Elsevier GmbH. All rights reserved.doi:10.1016/j.ijheh.2010.06.005

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Giardia taxonomy

In the widely used 1980 classification the Protozoa is consid-ered as a subkingdom with seven phyla. The Sarcomastigophora(containing the Mastigophora and Sarcodina), Apicomplexa,Microspora, Myxozoa and Ciliophora contain important parasites.The most recent classification establishes the Protozoa as thebasal eukaryotic kingdom and recognizes 13 phyla. The flagellates,belonging to former Mastigophora are now distributed betweenfour phyla, Metamonada, Parabasalia, Percolozoa and Euglenozoa.These new insights have come not just from molecular sequencestudies but by integrating them with numerous other lines of evi-dence, genetic, structural and biochemical (Cavalier-Smith, 2003;Cox, 2002). Therefore, old systematic based on morphology, Giardiabelongs to Phylum Sarcomastigophora, Subphylum Mastigophora(=Flagellata), Class Zoomastigophorea, Order Diplomonadida andFamily Hexamitidae (Morrison et al., 2007). According to thenew systematic based on genetic, structural and biochemicaldata Giardia belongs to Phylum Metamonada, Subphylum Tri-chozoa, Superclass Eopharyngia, Class Trepomonadea, SubclassDiplozoa, Order Giardiida and Family Giardiidae (Cavalier-Smith,2003).

Giardia is a very unusual, seemingly ancient, eukaryotic sin-gle cell organism as it shares many characteristics with anaerobicprokaryotes. Giardia lacks common eukaryotic subcellular com-partments such as mitochondria, peroxisomes, and apparently alsoa traditional Golgi apparatus. During encystation, developmentallyregulated formation of large secretory compartments containingcyst-wall material occurs. Despite the lack of any morphologicalsimilarities, these encystation-specific vesicles (ESVs) show sev-eral biochemical characteristics of maturing Golgi cisternae (Martiet al., 2003). Molecular evidence implies that all known groups ofanaerobic, amitochondrial protists evolved by the loss of mitochon-drial genomes, cytochromes and oxidative phosphorylation andthe conversion of mitochondria into double-membraned organellessuch as mitosomes in Giardia (Cavalier-Smith, 2003; Dolezal et al.,2005). Based on the comparison of the small nucleolar RNAs iden-tified from Archaea and unicellular eukaryotes shows that Giardiaemerged somewhat later than Trypanosoma and Euglena during theearly evolution of Excavata eukaryotes (Luo et al., 2009). Further-more, the analysis of the cystatin superfamily (cysteine proteaseinhibitors that play key regulatory roles in protein degradationprocesses) provided strong evidence for the emergence of thissuperfamily in the ancestor of eukaryotes and the progenitor wasmost probably like the extant Giardia cystatin (Kordis and Turk,2009).

Giardia species

Six species of Giardia have been distinguished on the basisof light microscopic (shape of trophozoit and median body)and electro-microscopic (feature of ventrolateral flange, marginalgroove, ventral disc, flagellum) characteristics (Adam, 2001). Fivespecies are represented by isolates from amphibians (G. agilis),birds (G. ardeae, G. psittaci), mice (G. muris) and voles (G. microti)(Cacciò et al., 2005; McRoberts et al., 1996; Adam, 2001). Thesixth species includes Giardia strains isolated from a large rangeof other mammalian hosts, grouped by Filice (1952) into a sin-gle species because they share morphological features and inparticular, have similar median body structures. Filice (1952)named this species G. duodenalis. Later all species have beendefined using ribosomal RNA gene sequencing and supportedby sequencing of other genes. The Giardia species are shown inTable 1.

Table 1Giardia species, assemblages and the SSU rRNA GenBank accession numbers.

Giardia species Assemblages GenBank accessionnumber (18S rRNA)

G. duodenalis(intestinalis, lamblia)(Leeuwenhoek, 1681a;Lamb, 1859a)

Assemblage A (Polish)(Homan, 1992;Mayrhofer, 1995;Karanis and Ey, 1998)

AF199446 (A2)AB159796 (A1)X52949 (A1)M54878 (A1)

Assemblage B (Belgian)(Homan, 1992;Mayrhofer, 1995)

U09491 (B)AF199447 (B)AF113898 (BIV)AF113897 (BIII)

Assemblages C and D(Meloni andThompson, 1987;Monis et al., 1998)

AF113899AF199443 (C)AF113900AF199449 (D)

Assemblage E (Ey et al.,1997)

AF199448

Assemblage F (Monis etal., 1999)

AF113901

Assemblage G (Moniset al., 1999)

AF199450AF113896

G. agilis (Kunstler,1882, 1883a)

– Not available

G. muris (Grassi, 1879a) – X65063AF113895

G. psittaci (Erlandsenand Bemrick, 1987)

– AF473853

G. ardeae (Noller,1920a; Erlandsen etal., 1990)

– Z17210U20351

G. microti (Feely, 1988) – AF006676AF006677AF473852

a See references McRoberts et al. (1996) and Adam (2001).

Giardia assemblages, subassemblages and genotypes

G. duodenalis genotypes are named after substantial sequencedifferences found in the genes such as glutamate dehydroge-nase/gdh, triosephosphate isomerase/tpi, and �-giardin/bg genes.This naming is done after phylogenetic analysis has eliminatedthe possibility that the differences are because of heterogene-ity between copies of the gene or intragenotypic variations. Theclosely related genotypes were then grouped into assemblagesand subassemblages. Recently the genotypic division within Gia-rdia species seems certainly much more complex than previouslybelieved since the given isolate could not be grouped into thesame (sub)assemblage based on the genotyping data of differ-ent loci, therefore the rule of the determination of subassemblagehas changed. The subassemblage of a given isolate is determinedbased on three different loci, not based on one gene. The usage ofgenotype, assemblage, subassemblage and subgenotype in severalpapers in the international literature is not consistent. We suggestthe usage of genotype, assemblage and subassemblage and avoidusing the subgenotype, because subgenotype means “under” thecategory genotype so it is not relevant to use “above” the genotypecategory.

Human-derived Giardia were earlier assigned to a separatespecies (G. lamblia) and the major lineages have been defined byanalysis of human-derived Giardia isolates: Mayrhofer (1995) des-ignated assemblages A and B, which include all the human isolatesand they correspond, respectively, to groups I plus II and III plus IV ofAndrews et al. (1998), Karanis and Ey (1998), to Polish and Belgiangenotypes of Homan (1992) and to group 1 plus 2 and group 3 ofNash and Keister (1985). The animal derived G. duodenalis exhibita similar genetic spectrum, although some isolates appear to besimilar or identical to particular human-derived genotypes, othersrepresent unique genotypes that seem likely to be host specific.All these findings bring into focus the question whether giardiasis

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is a zoonosis involving different G. lamblia/duodenalis isolates andwhether animals contribute significantly to the disease in humans(see section Zoonotic risk). Besides assemblages A and B a numberof other distinct assemblages (C–G) have also been identified withinthe G. duodenalis morphological group, and they all appear each tobe associated with a single species of mammalian host (Hunter andThompson, 2005; Cacciò et al., 2005). The non-human, host-specificassemblages found in dogs, cats, rats, voles and muskrats, are quitedistinct from assemblages A and B. In contrast, the assemblagesidentified in hoofed livestock and cats appear to be closely relatedto isolates in the major assemblages (Thompson, 2000). However,few characteristics of biological or epidemiological significance canconsistently distinguish between species-specific isolates. Differ-ences that may be observed include metabolism, biochemistry, invitro growth rates, infectivity, duration and nature of infection, drugsensitivity, pH preference, and susceptibility to infection with adsRNA Giardia virus. The apparent host specificity of genotypes tohosts including dogs, cats, livestock and rats appear most consistent(Monis et al., 2009; Cacciò et al., 2005).

The database of the Giardia genome project (McArthur et al.,2000) provides an online resource for G. duodenalis (WB strain,clone C6) genome sequence information. Franzén et al. (2009)found that the partial genomes of G. duodenalis WB (assemblageA) and GS (assemblage B) strains show 77% nucleotide and 78%amino-acid identity in protein coding regions. Comparative analy-sis identified 28 unique GS and 3 unique WB protein coding genes,and the variable surface protein (VSP) repertoires of the two iso-lates are completely different. The promoters of several enzymesinvolved in the synthesis of the cyst-wall lack binding sites forencystation-specific transcription factors in GS. Several syntony-breaks were detected and verified. The tetraploid GS genome showshigher levels of overall allelic sequence polymorphism (0.5% ver-sus <0.01% in WB). The genomic differences between WB and GSmay explain some of the observed biological and clinical differ-ences between the two isolates, and it suggests that assemblagesA and B Giardia can be two different species. Furthermore, anotherstudy using comparative proteomics has found distinct differencesin several proteins between Giardia isolates from assemblages Aand B. An assemblage A-specific protein of human infective G.duodenalis; alpha 2 giardin has been identified. Alpha 2 giardin isknown to be a structural protein and it is associated with the cau-dal flagella and the plasma membrane although its exact function isunknown. Although several proteins unique to assemblage B werealso observed, it was impossible to identify these proteins due to alack of genomic data available for assemblage B isolates. Together,these proteins represent distinct phenotypic differences betweenthe human infective assemblages of G. duodenalis and support theneed to revise the taxonomy of this parasite (Steuart et al., 2008).

The supposedly separate species could be: G. canis (=assem-blage C/D), G. simondi (=assemblage G), G. cati (=assemblage F), G.bovis (=assemblage E), G. enterica (=assemblage B) and G. duode-nalis (=assemblage A) (Monis et al., 2009). However, it is still underdebate, whether presently available data are sufficient for soundspecies differentiation. The compact data on the Giardia assem-blages are shown in Table 1.

Wielinga and Thompson (2007) sorted and aligned in total 405G. duodenalis sequences to examine the substitutions within andbetween the assemblages. It was found that all of the genes couldreproducibly group isolates into their assemblages and A-I and A-IIsubassemblages were robust and identifiable at all loci. However, inone sample from surface water in Hungary a G. duodenalis-uniquesequence was identified based on the gdh (glutamate dehydroge-nase) sequence (Plutzer et al., 2008). According to the phylogenetictree this sequence could not be grouped to any assemblages andclustered close to the assemblage A. According to the SSU rDNAproduct, this sequence was identical to G. duodenalis assemblage A

(Plutzer et al., 2008). Further unique sequences have been reportedbased on gdh sequence in harbour seal by Gaydos et al. (2008) andin a Quenda (Australian southern brown bandicoot) based on SSUrDNA and elongation factor 1 alfa (EF1A) genes by Adams et al.(2004). Recent studies, based on multilocus approach have shownthat a number of isolates, of both human and animal origin, can-not be unequivocally assigned at the assemblage level (Gelanew etal., 2007; Cacciò et al., 2008; Robertson et al., 2006). Assignmentof assemblage B sometimes has been problematic, given that oneof four markers supported an assignment to assemblage A. Multi-locus sequence and phylogenetic analysis supported the existenceof A-I, A-II and a new A-III subassemblages and that the levels ofgenetic diversity in assemblage B have been greater than thosefound in assemblage A, with many of the subassemblages rep-resenting single isolates from specific hosts. On the other hand,variable levels of intra-isolate sequence heterogeneity have beenreported in assemblage B isolates. But, no comparable evidenceof intra-isolate sequence heterogeneity for assemblage A isolateswas observed (Cacciò et al., 2008). Another study also emphasizes,that multilocus genotyping is a highly discriminatory tool in thedetermination of zoonotic sub-groups within assemblage A, butless valuable for subtyping assemblage B due to the high frequencyof double peaks in the sequence chromatograms (Lebbad et al.,2009). Sequencing of Giardia isolates from non-human primatesrevealed the presence of new polymorphisms for both assemblagesand at the three loci (tpi, gdh, bg) examined. The majority of theassemblage B isolates could not be grouped into recently describedsubassemblages, particularly at the tpi gene. Isolates could onlybe allocated to a specific group when polymorphisms of the threeloci were combined (Levecke et al., 2009). Mixtures of genotypesin individual isolates were repeatedly observed, which makes themultilocus genotyping (MLG) more difficult and complex (Spronget al., 2009; Kosuwin et al., 2010; Geurden et al., 2009).

Epidemiology

Giardia cysts are transmitted by the faecal–oral route, eitherdirect or indirect. Potential mechanisms of transmission include:person to person, animal to animal, zoonotic (animal to human,human to animal), waterborne from humans or animals throughdrinking water or recreational contact such as in swimming andfoodborne from contamination of water used in food preparationand manufacture or from food handlers (Karanis et al., 2007; Porteret al., 1990; Shields et al., 2008; Takizawa et al., 2009).

Waterborne transmission

Giardiasis outbreaksMore than 100 waterborne giardiasis outbreaks have been

reported worldwide from the beginning of the previous centurytill 2004 (systematically reviewed by Karanis et al., 2007). In thelast 2 years interactive water fountain associated giardiasis out-break in Florida (USA), and drinking water associated outbreaksin New Hampshire (USA) and in Nokia (Finland) were reported(Daly et al., 2010; Rimhanen-Finne et al., 2010; Eisenstein et al.,2008). The largest drinking water related outbreak described todate occurred in Norway, 2004, affecting around 1500 people. Thisoutbreak was caused by G. duodenalis assemblage B, but genotyp-ing of patient samples was complex and gave conflicting results.Genotyping of Giardia cysts found in contaminated water was notpossible (Robertson et al., 2006).

Deficiencies in the drinking water treatment process are amongthe most frequently cited reasons for giardiasis outbreaks, includ-ing insufficient barriers, inadequate or poorly operated treatment(Karanis et al., 2007). The risk of contracting giardiasis increases

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with the time of exposure to pathogen contaminants (Aström etal., 2007). Particularly in areas where ambient Giardia concen-trations are likely to be high, this emphasizes the importanceof a well-conceived and well-implemented monitoring programthrough which Giardia concentrations are measured. The feasibilityof this monitoring program is discussed in the section General con-clusions. A coincidence of source and treatment system causativeevents resulting in outbreaks has been reported, however, distri-bution system causative events occurred less frequently. Livestockand rainfall in a catchment with inadequate filtration of sourcewater also contributed to outbreaks (Risebro et al., 2007). Some90% of protozoan outbreaks have been reported as due to filtra-tion deficiencies. By contrast, for bacterial, viral gastroenteritis andmixed pathogen outbreaks, 75% of treatment events were disin-fection deficiencies (Risebro et al., 2007). Mohammed Mahdy et al.(2008) highlighted the importance of water and food transmissionroutes of Giardia infections.

Drinking water treatment processTypically, surface water can become contaminated through the

discharge of untreated and treated human sewage and/or fromurban or rural land drainage containing animal faces. The relativesignificance of these sources will differ between watersheds. Largerivers and lakes often receive both agricultural run-off and treatedand untreated domestic wastewater (Hunter and Thompson, 2005).The cysts of Giardia are relatively small with a size of 8–12 �mand they are relatively efficiently removed during soil passagein bank filtration compared to bacteria or viruses (Plutzer et al.,2007). Characteristics of Giardia cyst removal by filtration processesincluding slow sand or rapid sand filtration and diatomaceousearth filtration are well established (Nieminski and Ongerth, 1995;Ongerth, 1990; Tanner and Ongerth, 1990). Filtration processesare important barriers for the removal of cysts in water treat-ment. The first consideration of the water industry was focused oncysts removal by filtration process and especially upgrading filterdesign and operations to optimise cyst removal. Full scale conven-tional treatment with coagulation, flock removal and rapid granularfiltration or slow sand filtration removes 1.5–2.5 logs (Nieminskiand Ongerth, 1995). Pressure driven membrane processes (micro-filtration, ultrafiltration, nanofiltration and reverse osmosis) areplaying an increasingly important role in drinking water produc-tion in the United States and in Europe. These processes are beingemployed in water treatment for multiple purposes including con-trol of disinfection by-products, pathogen removal, clarification,and removal of inorganic and synthetic organic chemicals. Differentmicrofiltration and ultrafiltration membranes have been shown toprovide >4–6 log removals of Giardia cysts (Jacangelo et al., 1995;Betancourt and Rose, 2004). A recent study confirmed, that theGranular Activated Carbon (GAC) adsorption filtration has certaincapacity to eliminate Giardia cysts (1.3–2.7 log) (Hijnen et al., 2010).The log credits are originating from the United States, where thedata from different studies are combined using mathematical orstatistical approaches and are an approximation for removal by welldesigned, maintained and operated treatment process (Medemaet al., 2006). Assavasilavasukul et al. (2008) showed that Giardiacyst removal by conventional treatment was dependent on initialpathogen concentrations, with lower pathogen removals observedwhen lower initial pathogen spike doses were used. In addition,higher raw water turbidity appeared to result in a higher logremoval of the cysts (Assavasilavasukul et al., 2008).

Animal infectivity experiments have shown that cysts are sen-sitive to UV light and that it is probably an effective disinfectionmeasure for human strains of G. duodenalis (Karanis et al., 1992)comfirmed later also by others (Belosevic et al., 2001; Campbelland Wallis, 2002; Linden et al., 2002; Mofidi et al., 2002). Li et al.(2008) suggested that some G. duodenalis trophozoites may sur-

vive or are reactivated following exposure to UV at fluences up to10 mJ/cm2. Evidence of survival or reactivation at UV fluences of 20and 40 mJ/cm2 was ambiguous and statistically inconclusive, whileat 100 mJ/cm2 there was no evidence of survival or reactivation.These findings have implications for criteria used by the drinkingwater and wastewater treatment industries to ensure safe inacti-vation of G. duodenalis cysts by UV disinfection processes. All thesefindings support the use of UV disinfection in water for Giardia cystscontrol.

Disinfection with chlorine and chloramines has always beenan important barrier for waterborne pathogens, but they wereless effective against Giardia cysts (Khalifa et al., 2001). Chlorinedioxide treatment may result in inactivation, but the required prod-uct of concentration and contact time is still high especially atlow temperatures (Medema et al., 2006; Winiecka-Krusnell andLinder, 1998). Ozone is the most potent chemical against protozoa,although the effectiveness of ozone also reduces at lower temper-atures and the Ct (contact time) values required are high (Bukhariet al., 2000; Khalifa et al., 2001; Haas and Kaymak, 2003; Finch etal., 1993). Chemical disinfectants cannot be dosed at too high con-centrations, because toxic by-products are formed by the reactionwith compounds in the water, such as trihalomethanes by chlo-rine, nitrite by monochloramine, chlorite by chlorine dioxide andbromate by ozone (von Gunten, 2003).

Exposure of protozoa to multiple disinfectants has been shownto be more effective than expected from data on single disinfec-tants acting alone. The multiple stresses that protozoa encounterin the environment and during treatment might limit their infec-tivity including Giardia cysts (Medema et al., 2006). How the cystsoccur in water (suspended or attached to particles) is relevant forwater treatment (sedimentation, filtration) and cysts readily attachto particles in sewage effluent, which influences sedimentation ofthem in surface water (Medema et al., 1998).

Post-treatment contaminationPost-treatment contamination is another reported cause of

waterborne giardiasis (reviewed by Karanis et al., 2007). Whenwater in the distribution system or in storage reservoirs becomescontaminated any disinfectant residual that may be present is notsufficient to inactivate Giardia and to prevent the ingestion ofinfectious cysts. Post-treatment contamination may occur throughinfiltration of contaminants in the distribution system throughleaks (during surges), in open distribution reservoirs or otheropen connections or through improper or inadequate disinfec-tion following construction or repair. Cross-connections and backsiphonage may draw water from faecal contaminated sources intothe network. Storage tanks used in houses or multi story build-ing (i.e. in stand pipe systems) may also become contaminatedby animals (Neringer et al., 1987; Robertson et al., 2009). Biofilmsaccumulated in transmission and reticulation (distribution) pip-ing may contribute as a potential secondary source of drinkingwater contamination by Giardia cysts by serving as an accumulationmedium during low flow periods (Helmi et al., 2008).

Giardia presence and survival in the environmentLarge numbers of cattle and sheep around water supplies and

their excretion of high numbers of Giardia cysts can make infectedanimals important sources of water contamination. G. duodenalisassemblages A and B carriage have been reported for five live-stock species (goat, sheep, cattle, pig, and horse) from diversegeographic localities (Spain, Belgium, Denmark, Potrugal, Brazil,Australia, and Canada) (Castro-Hermida et al., 2007; Geurden etal., 2008; Langkjaer et al., 2007; Mendonca et al., 2007; Souza et al.,2007; Traub et al., 2005; Uehlinger et al., 2006). Thus, it is essentialfor the management of animal farming in watersheds to ensure thatthe deposition of faces in or along water sources is controlled to the

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J. Plutzer et al. / International Journal of Hygiene and Environmental Health 213 (2010) 321–333 325

extent practical to minimize the waterborne transmission. Someepidemics in North America and Spain have been linked circum-stantially to contamination of water by cysts excreted from animalssuch as muskrats, beaver, nutria and wild otter. The reported preva-lence of Giardia in individual species can reach 33% in beaver, 73.3%in nutria, 75.2% in muskrat, and 6.8% in wild otter (Dunlap and Thies,2002; Fayer et al., 2006; Heitman et al., 2002; Karanis et al., 1996a;Méndez-Hermida et al., 2007). The contribution of aquatic birdsto environmental contamination by G. duodenalis cysts has beenalso reported (carriage rate 5–49%) (Majewska et al., 2009; Plutzerand Tomor, 2009). Unlike coccidian parasites and helminths, Giar-dia does not require a period of maturation of cysts after sheddingin faeces (Svärd et al., 2003). Upon excretion, they are immediatelyable to infect a new host.

Protozoan cysts including Giardia can survive for months in sur-face water and in soil. Based on studies by Robertson and Gjerde(2004) in Norwegian terrestrial environment it has been postu-lated that shear forces generated during freeze–thaw cycles havedisintegrated the cysts of Giardia exposed to the Norwegian win-ter and retrospective laboratory studies supported this theory.These results suggested that Giardia cysts do not persist in terres-trial environment over winter, and when detected, will have beenexcreted since the previous winter. However, in several countries(e.g. Mediterranean countries) the winter temperatures remainabove zero.

The infectivity of at least some isolates of Giardia is high,although evidence suggests considerable variation among isolates(Fantham et al., 1916; Homan and Mank, 2001; Karanis and Ey,1998; Thompson, 2004). Nevertheless, no consistent evidence ofthe genetic basis for infectivity, virulence, host specificity, andrelated disease factors has been reported (Robertson et al., 2010).

By implication, where transmission by water is of interest andwhere the exposed population is large, even very small concentra-tions of virulent and infectious cysts may contribute a detectableoutbreak. For example in a community of 10,000, assuming percapita water consumption of 2 L/day, and if a single cyst is capa-ble of causing a case of giardiasis, then if the cyst concentrationwere 1 cyst/200 L, 1% of the population (100 people) would becomeinfected.

The prevalence of Giardia in different water sources in EuropeThe number of European countries is around 45, of which 20

countries published scientific research on the prevalence of Giar-dia in humans and in water samples. These data are summarizedin Table 3. Based on these publications the prevalence of giardia-sis in humans is reported between 1% and 17.6% in Europe (de Witet al., 2001; Almeida et al., 2006; Karanis and Ey, 1998; Hörmanet al., 2004; Bajer, 2008; Davies et al., 2009; Berrilli et al., 2006;Spinelli et al., 2006; Geurden et al., 2009). Giardia spp. cysts havebeen detected in different European water sources in 15 countriesand Giardia spp. cysts occurred sometimes in huge numbers in rawwater samples (above 10 cysts/L) (Lobo et al., 2009; Plutzer et al.,2007; Dolejs et al., 2000; Mons et al., 2009; Karanis et al., 2006;Castro-Hermida et al., 2008a; Karanis et al., 1998). Several authorsdiscussed the importance of urban (treated and untreated) sewageeffluents in contamination of surface waters in Europe (Aströmet al., 2009; Bertrand and Schwartzbrod, 2007; Castro-Hermidaet al., 2008b; Ottoson et al., 2006; Plutzer et al., 2008; Touron etal., 2007). The removal of Giardia cysts during sewage treatment is0.18–2.6 log (Ottoson et al., 2006). Only one paper reported data onthe concentration of Giardia cysts in backwash water from drinkingwater treatment plants in Germany (Karanis et al., 1996c). Becauseusually backwash water is returned to the raw water it may beresponsible for an increased risk to feed the parasites into the drink-ing water. Ten countries reported data on the presence of Giardiacysts in recreational water or swimming pools sometimes with high

cyst numbers (10–722 cysts/L) (Schets et al., 2004, 2008; Wicki etal., 2009; Karanis et al., 2002, 2005; Plutzer et al., 2008; Coupe et al.,2006; Karanis et al., 2006; Bajer, 2008; Briancesco and Bonadonna,2005).

Foodborne transmission

The contamination of fruit, vegetables and shellfish with Giar-dia cysts is an important public health consideration, because theseproducts are frequently consumed raw without thermal process-ing (Blasi et al., 2008; Pozio, 2008). Through filter feeding, oysters(Crassostrea spp.) have been shown to accumulate Giardia cysts(Schets et al., 2007; Graczyk et al., 1998, 2006) and also several otherclam species (Macoma spp., Corbicula fluminea, Dreissena polymor-pha, Mytilus galloprovincialis, and Anadonta piscinalis) (Graczyk etal., 1999a,b, 2003; Gómez-Couso et al., 2005; Hänninen et al., 2005).These data suggest that consumption of raw bivalve molluscs maylead to cases of gastro-intestinal illness.

Cook et al. (2007) found one Giardia cyst/50 g on examining a“natural food” sample of organic watercress, spinach, and rocketsalad as a surface contaminant. Dill, lettuce, mung bean sprouts,radish sprouts and strawberries have also been found contaminatedby Giardia cysts (mean 3 cysts per 100 g) as reported by Robertsonand Gjerde (2001a).

Poor personal hygiene of food handlers causing giardiasis out-break have been reported several times (Mintz et al., 1993; Porteret al., 1990; Smith et al., 2007).

In the 27 Member States of the European Union, zoonotic para-sites transmitted by food are circulating with different prevalenceaccording to the country, the environmental conditions, the humanbehaviour, and the socio-economic level (Pozio, 2008).

Zoonotic risk

Infectivity studies using Giardia cysts isolated from both humansand animals have shown that zoonotic transmission is possible(Monis and Thompson, 2003). The zoonotic potential of G. duode-nalis has been discussed by various authors (van Keulen et al., 2002;Traub et al., 2004; Lalle et al., 2005; Savioli et al., 2006) but its realclinical significance is not clear. The major zoonotic risk should befrom those genotypes of Giardia in assemblage A and to a lesserextent, genotypes in assemblage B (Thompson, 2000). However,when genotypes are defined using a multilocus sequence typingshame (see section Giardia assemblages, subassemblages and geno-types) only assemblage A and not assemblage B appeared to havea zoonotic potential (Lebbad et al., 2009; Sprong et al., 2009).

Humans, dogs, cats, domestic livestock (cattle, sheep, pig, horse,goat) and certain species of wildlife (Table 2) were described asnatural hosts of G. duodenalis assemblage A (Armson et al., 2009;Geurden et al., 2008; Langkjaer et al., 2007; Leonhard et al., 2007;Mendonca et al., 2007; Robertson et al., 2007; Souza et al., 2007;Traub et al., 2005; Uehlinger et al., 2006; Yang et al., 2009).

Humans, dogs, guinea pig, rabbit, domestic livestocks (cattle,sheep, horses) and some species of wildlife (Table 2) have beendescribed as natural hosts of G. duodenalis assemblage B (Aloisio etal., 2006; Castro-Hermida et al., 2007; Coklin et al., 2007; Itagaki etal., 2005; Mendonca et al., 2007; Minvielle et al., 2008; Traub et al.,2005; Lebbad et al., 2009). G. duodenalis assemblages A and B havebeen detected in marine animals too, such as dolphins, porpoises,seals (ringed seal), common eiders, and thresher shark. The possi-bility of human infection from these animals is very low, howeverthey can contaminate water that is used by humans for recre-ation activities (Lasek-Nesselquist et al., 2008; Dixon et al., 2008;Yang et al., 2010). Furthermore, Giardia cysts have been detected in7.3% of flies and 5–49% in the faecal droppings of wild birds (bothassemblages A and B), indicating the potential role of such vectors

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326 J. Plutzer et al. / International Journal of Hygiene and Environmental Health 213 (2010) 321–333

Table 2The reported natural wild hosts of Giardia duodenalis assemblages A and B.

Giardia duodenalisassemblage

Natural host Reference

Assemblage A Fallow deer Lalle et al. (2007) andCacciò et al. (2008)

White tailed deer Trout et al. (2003)Reindeer Robertson et al. (2007)Coyote Thompson et al. (2009)Fox, Norwegian wildred fox

Hamnes et al. (2007)and McCarthy et al.(2008)

Australian house mice Moro et al. (2003)Wild moose Robertson et al. (2007)Muskoxen Kutz et al. (2008)Southern brownhowler monkey

Volotão et al. (2008)

Black howler monkey Vitazkova and Wade(2006)

Alpaca Trout et al. (2008)Water buffalo Cacciò et al. (2007)Wild boar Cacciò et al. (2008)Western grey kangaroo Thompson et al. (2008)

and McCarthy et al.(2008)

Common brushtailpossum

Thompson et al. (2008)

Mountain brushtailpossum

Thompson et al. (2008)

Swamp wallaby Thompson et al. (2008)Koala Thompson et al. (2008)Domestic ferret Abe et al. (2005, 2010)Red deer Bajer (2008)Roe deer van der Giessen et al.

(2006)Marmoset van der Giessen et al.

(2006)Common planigale Thompson et al. (2010)Quenda Thompson et al. (2010)

Assemblage B Beaver Fayer et al. (2006)Mandrill Cacciò et al. (2008)Macaque Cacciò et al. (2008) and

Itagaki et al. (2005)Chimp Cacciò et al. (2008)Black howler monkey Vitazkova and Wade

(2006)Coyote Trout et al. (2006)Western grey kangaroo Thompson et al. (2008)Norwegian wild red fox Hamnes et al. (2007)Chinchilla van der Giessen et al.

(2006)

in transmission of intestinal parasites (Szostakowska et al., 2004;Kuhn et al., 2002; Medema, 1999; Plutzer and Tomor, 2009).

Foronda et al. (2008) detected also G. duodenalis assemblageE in human stool samples in Egypt based on the tpi gene. How-ever, further analyses of a second locus are required to confirm thisresult.

The relevance of host factors in giardiasis transmission

The spectrum of clinical manifestations in human giardiasisis broad ranging from brief, mild, and transient intestinal com-plaints, that resolve completely to a more characteristic complexof symptoms consisting of an acute onset of diarrhoea, abdomi-nal cramps, bloating and flatulence often accompanied by nauseaand weight loss lasting for up to 7 weeks. In some cases giar-diasis has been episodic with symptomatic periods recurring forseveral years. A typical feature of Giardia infections in any host isintermittency of cyst shedding. In undernourished hosts and in chil-dren, the infection can become chronic with profound diarrhoea,weight loss, disturbance of absorption and growth (Farthing, 1996;Lebwohl et al., 2003). Furthermore, people may be infected with-

out any relevant symptoms, and it has even been suggested thatsome people benefit from their carrier state, e.g. healthy day carechildren with asymptomatic G. duodenalis infection showed no dis-advantage and perhaps even an advantage in nutritional status andfreedom from other illnesses (Ish-Horowicz et al., 1989). It is notfully understood why some individuals develop clinical giardiasiswhile others remain asymptomatic, however host factors such asimmune status, nutritional status, age, concurrent enteric infec-tions and environmental factors as well as differences in virulenceand pathogenicity of G. duodenalis strains are probably responsiblefor the severity of infection (Thompson, 2004).

Activities with increased risk for Giardia infection include travel-ers (endemic areas), children in child care settings, close contacts ofinfected persons, persons who ingest contaminated drinking water,persons who swallow contaminated recreational water, personstaking part in outdoor activities who consume unfiltered, untreatedwater or who fail to practice good hygienic behaviours and personswho have contact with infected animals (Stuart et al., 2003).

Detection of Giardia

Giardia detection in water samples

Measuring the concentration of Giardia cysts in water samplestypically requires three steps: (1) sampling and particle collec-tion; (2) particle segregation, and (3) cyst detection. The samplevolume required to avoid non-zero results must be planned withprior knowledge of conditions at the sampling location and char-acteristics of the analytical procedure. Conceptually, the presenceof Giardia cysts has been shown to be continuous rather than inter-mittent (Hansen and Ongerth, 1991) and the concentration of cystsin the water is affected by the run-off from agricultural land andsewage discharges causing concentration peaks. For example dur-ing the River Danube monitoring in 2004–2005, the highest Giardialevels have been detected in January to April in both 2004 and 2005with a second peak in November 2005. During these times a recordhigh of 1020 Giardia cysts/100 L was found with mean numbersranging between 260 and 550 cysts/100 L. The lowest Giardia levelswere found to be in July and August each year with mean numbersranging between 16 and 67 cysts/100 L (Plutzer et al., 2007).

The distinction is subtile but critical for planning catchmentmanagement and water quality monitoring. It also emphasizes theimportance of processing a sufficient water volume to permit find-ing at least one cyst. The recovery efficiency of the total procedurecan be measured by processing a seeded sample with known num-bers of cysts.

Forming the initial particle concentration in water includ-ing chemical flocculation, separating has been accomplished byvarious means membrane or cartridge filtration, followed by cen-trifugation. Other filtration options (Hemoflow and Hollow-fiberultrafilters, adsorption–elution (VIRADEL) technique using 1MDSelectropositive filters) have been described demonstrating thatrecoveries of cysts from water samples vary significantly, depend-ing on both procedure and water quality (Ferguson et al., 2004;Hill et al., 2009). The most practical method depends on the qual-ity of water being processed and experience of the laboratory inwhich the work is performed. Particle segregation to select the frac-tion of initial pellet containing Giardia has been accomplished byseveral means including gradient centrifugation and immunomag-netic separation (IMS). The imposition of strict analytical procedureto meet regulatory requirements in the USA has restricted mostanalyses since 2001 to use of IMS (USEPA, 2005).

For the final detection part of analysis immunofluorescenceassay (IFA or IFT) has become a standard test in recent yearsfor the detection of G. duodenalis cysts in environmental sam-

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J. Plutzer et al. / International Journal of Hygiene and Environmental Health 213 (2010) 321–333 327

ples (USEPA, 2005). By specifically targeting the cysts it providesmore reliable results in a shorter time than conventional meth-ods (Ongerth, 1989; Dixon et al., 1997). The available standardssuch as US EPA Method 1623 and ISO 15553 (Europe) specify amethod that is applicable for the detection and enumeration ofGiardia cysts in water (USEPA, 2005; Anonymous, 2006). It is appli-cable for the examination of surface and ground waters, treatedwaters, mineral waters, swimming pool and recreational waters.These methods do not allow identification to species level, the hostspecies of origin or the determination of viability or infectivity ofany Giardia cyst, which may be present. However, the application ofDAPI [2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloridestaining] and DIC (Differential Interference Contrast) microscopygive some indication about the internal structure and thereforeviability of the cysts detected. Molecular techniques, particularlyPCR-based procedures have higher specificity than microscopy andthey have been developed to provide information on the geno-types or species of Giardia, combining PCR with RFLP analysis,without having to resort to costly and time-consuming sequenc-ing (Appelbee et al., 2003; Amar et al., 2004; Read et al., 2004).Plutzer et al. (2008) were able to genotype cysts (using PCR tar-geting SSU rRNA gene and sequencing) in 69% of the sewage IFTpositive water samples, 36% of the raw water, and 8% of the surfacewater IFT positive samples. Similar results were published by Loboet al. (2009) in Portugal (13.4% of the Giardia IFT positive water sam-ples were genotyped using PCR targeting the �-giardin gene andsequencing). Using real-time PCR methods (TaqMan probes basedon �-giardin gene), the detection of Giardia cysts was 100% in IFTpositive wastewater samples and a methodology for dealing withqPCR inhibitors involving the use of Chelex 100 and PVP 360 wasdeveloped (Guy et al., 2003; Bertrand and Schwartzbrod, 2007).These findings emphasize, that extraction of high quality DNA isa key step in PCR detection. A major disadvantage of PCR is itsstrong inhibition by various substances commonly present in watersamples. Environmental samples rich in humic acids (organic richsoils, composts, decaying litter, etc.) and polysaccharides (biofilms,cyanobacteria, microbial mats, etc.) can contribute to poor qualityDNA extracts. This has been particularly important for the molec-ular identification of Giardia in raw and highly polluted surfacewaters (Jiang et al., 2005). Loop mediated isothermal amplifica-tion (LAMP) is an emerging technology (Karanis and Ongerth, 2009)and is rapidly becoming recognized as a useful diagnostic toolin the field of parasitology. LAMP relies on auto cycling stranddisplacement DNA synthesis by Bst polymerase and it has beendemonstrated for Giardia by Plutzer and Karanis (2009). LAMP assaywas able to detect picograms of G. duodenalis assemblages A and BDNA in the investigated samples and is proving to be a powerful andflexible tool to enhance the molecular detection process. The majoradvantages of the LAMP reaction are that it is highly selective, vir-tually free from false positives, does not require a thermocycler andis not affected by the presence of inhibitors (Karanis and Ongerth,2009).

Giardia detection in stool samples

Since about 1990, the preferred method of analysing stoolsamples for suspected giardiasis is ELISA kits for free stool anti-gen (Stibbs et al., 1988). In clinical or laboratory applicationswhere the objective is to find, identify, and isolate Giardia cysts,microscopy following immunofluorescence assay (IFA or IFT) iscommonly employed. IFT microscopy has been previously eval-uated for Giardia cysts and directly compared to phase contrastmicroscopy (Karanis et al., 1996b). Species identification was pos-sible only by phase contrast microscopy but the results suggestedthat immunofluorescence microscopy was superior for the detec-tion of Giardia cysts in animal faeces. Rimhanen-Finne et al. (2007)

tested IFT and ELISA in canine faeces and the Giardia ELISA cor-related well with IFT. Depending on the objective concentrationsimple techniques such as zinc sulphate flotation, formol-ethyl sed-imentation and centrifugation may be applied (Zajac et al., 2002).

Giardia detection in food samples

The methods used for the detection of Giardia in/on food is basedon four basic steps: extraction of cysts from the foodstuffs, con-centration of the extract by centrifugation and separation of thecysts from food materials by IMS, immunofluorescence staining ofthe cysts to allow their visualization, and identification of cysts bymicroscopy (Cook et al., 2007; Graczyk et al., 1999a,b; Schets et al.,2007; Robertson and Gjerde, 2001a). Currently there are no inter-national guidelines for determining cyst numbers contaminationin or on foodstaffs.

The importance of G. duodenalis cultivation in vivo and invitro

The cultivation of Giardia strains dates back to more than fourdecades (Karapetyan, 1962). Meyer and Pope (1965) grew Giardiafrom the rabbit and chinchilla by modifying Karapetayan’s originalmethod and in the year 1970, Bingham and Meyer (1979) discov-ered dependency of excystment by pH and established the routinecultivation system in vitro. The next major advance in cultivationfollowed the reports of Belosevic et al. (1983) and Faubert et al.(1983) on development of the Mongolian gerbil as an animal modelfor the in vivo Giardia production. Completion of the life cycle suc-ceeded in vitro by the group of Francis Gillin and the roles of bile,lactic acid, and pH in the completion of the life cycle in vitro has beenemphasized (Gillin et al., 1989). Recently the earlier finding wasconfirmed, that cholesterol and bile salts are relevant modulatorsof encystation (Argüello-Garciá et al., 2009). Ability to complete thelife cycle of Giardia in both directions (Boucher and Gillin, 1990)is fortuitous, permitting a broad scope of research targeting manyaspects of Giardia and giardiasis and made also a major contributionto research on the control of Giardia in water (e.g. the possibility ofpropagation of Giardia cysts isolated from water sources). However,generalizations should be proposed with caution as, for example,intraspecific variation of the in vitro encystation of G. duodenalis iso-lates from humans and animals has been observed (Karanis, 1994).In that work mass production of G. duodenalis cysts in vitro wassuccessful but only for few strains, specifically, human isolates ofG. duodenalis assemblages A-II and B-IV and another isolate from ared howler (Alouatta seniculus) of the Cologne Zoo (Karanis and Ey,1998). Measurement of in vitro growth kinetics for 12 of the Giardiaisolates revealed 3 phenotypes (‘rapid’, ‘medium-rate’ and ‘slow’growers) characterized by generation times of 9–11 h (5 isolates),12–15 h (5 isolates) and ≥18–20 h (2 isolates), respectively. Clonedsublines exhibited growth rates similar to those of the parent iso-lates. The in vitro growth rates correlated strongly with genotype,group I or group II (assemblage A) genotypes accounting for all ofthe ‘rapid’ and ‘medium-rate’ cultures and both assemblage B iso-lates being ‘slow growers’. Lyophilized bovine bile obtained fromlocal meat producers induced higher levels of encystation thanporcine or bovine bile available from the commercial suppliers, butwas dependent on pH (Karanis and Ey, 1998). In the encystationprocess of the motile, flagellated, binucleate trophozoite gradu-ally transforms into a form that can survive in the environmentand infect a new host and involves differentiation and antigenicvariation (Nash, 2002; Bittencourt-Silvestre et al., 2010). However,generalizing to all Giardia based on data developed in the labora-tory using few poorly characterized (if at all) isolates is problematicin light of the physiological and genetic diversity of Giardia as cited

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328 J. Plutzer et al. / International Journal of Hygiene and Environmental Health 213 (2010) 321–333

Tab

le3

The

Gia

rdia

pre

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dat

are

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ted

for

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ds

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ets

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.(20

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58.6

%/5

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ge:

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.(20

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16.8

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mar

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2004

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81%

2.97

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2004

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2004

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above. Results of cell culture viability assays have revealed limi-tations in their use for quantitative and other purposes, becauseGiardia is an extracellular parasite. Cell lines are not suitable to cul-ture Giardia in vitro. This has been found in several internationalstudies and corresponds to our own observations. Compared to anin vivo model, an in vitro system is less expensive and more advan-tageous for screening anti-giardial strategies and in assessing theefficacy of drugs. Moreover, the in vitro axenic system providesaccess to stage-specific parasite antigens and such a system hasbeen achieved for practical application. All in all, the cultivationof Giardia in vitro axenic system is very useful to apply for mul-tiple giardiasis research questions, but limitations are the type ofthe isolate, whether possible to culture it or not and also the firstestablishment of the isolate in culture needs expertise.

General conclusions

As it has been mentioned above, research is needed to beinitiated in pan-European level and worldwide for beneficialresearch outcomes to reduce the concentration of cysts in theraw/recreational/irrigation water sources (Lonigro et al., 2006).Protection of the water from contamination and “multiple bar-rier concepts” comprise the only effective approach to treatmentstrategies at present. Essential components combine effective pol-lution control and catchment management, and efficient watertreatment. Optimal conventional water treatment includes welldesigned and operated processes: coagulation–flocculation, sedi-mentation and/or flotation followed by granular media rapid ratefiltration and high-performance disinfection by chemical or physi-cal processes. Long-term objectives are to develop a cheap, simpleand sensitive technique for monitoring water supplies for thedetection of Giardia from environmental samples as it has beenalready suggested and reported by the LAMP detections applica-tions of Giardia (Karanis and Ongerth, 2009; Plutzer and Karanis,2009) and it has been already applied in combination with a newfiltration technique for the detection of giardial DNA in water sup-plies (Plutzer et al., 2010). The objectives would be to provide earlywarning system to water industries to permit the possibility for fastresponse in a risk situation in order to prevent outbreaks throughdrinking water in the human population. Priority should be givento those water supplies or water sources with a high risk of con-tamination. Giardiasis has been recently included as “NeglectedDiseases” by the WHO but as it is indicated in Table 3, it is notreported by many countries of the world leaving major gaps inunderstanding of its importance. Water shortage is an increas-ingly urgent public health problem facing many countries. Ongoingdemographic changes can affect both water quality and demandthrough increased agricultural, industrial and domestic use, whileunplanned urbanization leads to over-exploitation of local waterresources and threatens the quality of local water bodies throughrelease of inadequately treated wastewater. Effective wastewatertreatment is a critical link to safeguard public health against Giar-dia infections. Special emphasis will be needed to cope with thelikely effects of climate changes on the emergence of giardiasistransmitted through the aquatic environment and food.

Increasing knowledge of the Giardia genome will improveour understanding of Giardia contamination sources, transmissionroutes and features contributing to human illness. Yet no unifiedand widely accepted typing scheme is available. Working towardsuch tools would contribute significantly to enable identification ofindividual Giardia isolates according to their disease risk potential.Genetic elements determining or conferring traits such as infectiv-ity, pathogenicity, virulence, and immune interaction contributingto clearance are currently not well established, if at all. These shouldbe useful and important topics for future research.

It is clear that Giardia is an unusual organism biologically andof scientific and public health interest. Ability to cultivate Giardiain vitro and to complete its life cycle independent of an infectedhost enables pursuit of biological details that are important and notfully understood yet. This also permits reproducible investigationsof properties of Giardia cysts that are essential to effective watertreatment and environmental control.

Because the host origin of cysts in water cannot usually be iden-tified with certainty by either field or laboratory investigationsand because the zoonotic potential of giardiasis remain an unre-solved issue, health authorities are often forced to adopt policiesand water management practices that consider any Giardia cystsas potentially infectious for humans. There are a lot of indicationsthat animals and humans serve as contaminants of water supplies,but the frequency of zoonotic, anthroponotic, anthropozoonotic,or zooantroponotic transfer of Giardia infections and the ques-tion who infects whom it remains an unclear issue. Identifying theenvironmental sources of contamination and routes of infection,particularly for human giardiasis, requires an in depth understand-ing of which subassemblages or genotypes are host adapted (and donot infect humans) and which are transmissible to humans. Accord-ing to the molecular investigations performed in the last 10 yearsthis could not be determined, however multilocus identificationin the future may help to resolve this problem. Source trackingwould also be possible if it could be confirmed that the same strainsisolated from water could be isolated from infected people whoconsumed this water. For this purpose also multilocus analysis isnecessary, however it is problematic in case of water samples con-taining few numbers of cysts for molecular investigations.

The emergence and spread of giardiasis in human populationsis an increasing problem around the world. While developed coun-tries have been more successful in part to control waterbornegiardiasis, water quality problems are still prevalent. Safe drinkingwater supplies are critical for protecting public health depending onthe availability of a safe drinking water. The status of Giardia threatsto drinking waters and aquatic ecosystems is not known in mostcountries and future investigations are needed to shed a light onthe current situation of water supply contamination along with andin combination with molecular studies directed to understandingthe significance of local Giardia genotypes. Surveillance and scien-tific research as indicated above required to better understandingand the control of transmission of this public health related oldpathogen.

Acknowledgements

This work was supported by funds from the European Commis-sion for the HEALTHY WATER project (FOOD-CT-2006-036306) andby Institutional Funding of Cologne University in Germany. Theauthors are solely responsible for the content of this publication.It does not represent the opinion of the European Commission. TheEuropean Commission is not responsible for any use that might bemade of data appearing therein.

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