ustrai Sa

EpidemiologyMolecular characterization

rdiaAsssidbe

ng snti

tation of genotyping data is complicated by the presence of multiple alleles that generate double peaks

ect nuirds. Its; apprrica ha2007)

via person-to person or animal-to-animal contact. Exposure to theacidic environment of the stomach provides the necessary stimulifor the excystation of the trophozoite from the cyst in the duode-num of the small intestine (Gardner and Hill, 2001). Trophozoitesundergo repeated mitotic division and are eventually triggered to

prises Giardia agilis in amphibians, Giardia ardeae and Giardia psit-taci in birds, Giardia microti and Giardia muris in rodents, andGiardia duodenalis in mammals. Another species, Giardia varani,has been described from a water monitor (Varanus salvator) (Uptonand Zien, 1997). This parasite lacks median bodies and had binu-cleated cysts but its identity has not been conrmed genetically.

Giardia duodenalis (syn. Giardia intestinalis, Giardia lamblia) isthe only species found in humans, although it is also found in manyother mammals including pets and livestock (Feng and Xiao, 2011).

Corresponding author. Tel.: +61 08 9360 2482; fax: +61 08 9310 4144.

International Journal for Parasitology 43 (2013) 943956

Contents lists available at

na

.e lE-mail address: Una.Ryan@murdoch.edu.au (U. Ryan).which is the infective stage. Infection is initiated either by con-sumption of contaminated food or water or by the faecal-oral route

Currently, six Giardia spp. are accepted by most researchers onthe basis of the morphology of trophozoites and/or cysts. This com-The life cycle of Giardia is direct and involves just two majorstages, the trophozoite, which is the replicative stage, and the cyst,

2. Taxonomy0020-7519/$36.00 2013 Published by Elsevier Ltd. on behalf of Australian Society for Parasitology Inc.http://dx.doi.org/10.1016/j.ijpara.2013.06.001of giardiasis are quite variable and range from the absence ofsymptoms to acute or chronic diarrhoea, dehydration, abdominalpain, nausea, vomiting and weight loss (Eckmann, 2003; Cacciand Ryan, 2008). Prevalences of giardiasis in humans are generallylower in developed countries with 0.47.5% reported for developedcountries and 830% for developing countries (Feng and Xiao,2011).

(Rendtorff, 1954). Most outbreaks of giardiasis have been linked tothe consumption of contaminated drinking water. In a recent re-view it was reported that of the 199 published outbreaks causedby protozoa during the period 20042010, 70 (35%) were causedby Giardia (Baldursson and Karanis, 2011).1. Introduction

Species of the genus Giardia inffrom mammals to amphibians and bmon intestinal parasites of humanpeople in Asia, Africa and Latin Ametions (WHO, 1996; Yason and Rivera,in sequencing les from PCR products, and by the potential exchange of genetic material among isolates,which may account for the non-concordance in the assignment of isolates to specic assemblages. There-fore, a better understanding of the genetics of this parasite is required to allow the design of more sen-sitive and variable subtyping tools, that in turn may help unravel the complex epidemiology of thisinfection.

2013 Published by Elsevier Ltd. on behalf of Australian Society for Parasitology Inc.

merous hosts, rangingis one of the most com-oximately 200 millionve symptomatic infec-. Clinical manifestations

form environmentally resistant cysts in response to the bile condi-tions of the small intestine, which are then shed in faecal material.Cysts are immediately infectious when excreted in faeces, areremarkably stable and can survive for weeks to months in the envi-ronment. As a result of this, environmental contamination can leadto the contamination of drinking water and food (Feng and Xiao,2011). In humans, the infective dose is approximately 10100 cystsGiardiaZoonosis

animal species, some of which may have zoonotic potential. Multi-locus typing data, however, has shownthat in most cases, animals do not share identical multi-locus types with humans. Furthermore, interpre-Invited Review

Zoonotic potential of Giardia

Una Ryan a,, Simone M. Cacci ba School of Veterinary and Life Sciences, Murdoch University, Murdoch, 6150 Western AbDepartment of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore d

a r t i c l e i n f o

Article history:Received 6 May 2013Received in revised form 12 June 2013Accepted 14 June 2013Available online 13 July 2013

Keywords:

a b s t r a c t

Giardia duodenalis (syn. Giaand mammals worldwide.acterization as there is congroups (assemblages) havemals, whereas the remainiveys of single loci have ide

International Jour

journal homepage: wwwlia, Australianit, Viale Regina Elena 299, Rome 00161, Italy

lamblia and Giardia intestinalis) is a common intestinal parasite of humansessing the zoonotic transmission of the infection requires molecular char-erable genetic variation within G. duodenalis. To date eight major geneticen identied, two of which (A and B) are found in both humans and ani-ix (C to H) are host-specic and do not infect humans. Sequence-based sur-ed a number of genetic variants (genotypes) within assemblages A and B inSciVerse ScienceDirect

l for Parasitology

sevier .com/locate / i jpara

A considerable amount of data has shown that G. duodenalis shouldbe considered as a species complex, whose members show littlevariation in their morphology, yet can be assigned to at least eightdistinct genetic groups or assemblages (A to H) based on protein orDNA polymorphisms (Andrews et al., 1989; Monis et al., 2003; Cac-ci and Ryan, 2008) (Table 1). The genetic distances that separateassemblages are very large and recent comparisons at the wholegenome level (Franzen et al., 2009; Jerlstrm-Hultqvist et al.,2010) have reinforced the notion that assemblages A, B and E(the three assemblages for which genome sequences are currentlyavailable) represent distinct species. It has been proposed to adoptthe names of G. duodenalis for assemblage A, Giardia enterica forassemblage B, Giardia canis for assemblages C and D, Giardia bovisfor assemblage E, Giardia cati for assemblage F and Giardia simondifor assemblage G (Monis et al., 2009; Thompson and Monis, 2004,2011; Thompson et al., 2008). However, due to the uncertainty re-lated to the identity of the parasites in their initial descriptions,these species need to be re-described and their names validated/conrmed with biological and molecular data in compliance withthe International Code of Zoological Nomenclature (ICZN) beforethey can be accepted as valid species. In addition, controversialpoints still remain, mostly concerning the names to be given toassemblage H and the fact that the two distinct assemblages (Cand D) that infect dogs will be grouped in a single species, G. canis,despite the rather large genetic variability observed at the genesinvestigated.

It is important to clarify the terminology used by differentresearchers to describe the genetic variants found within eachassemblage, as this has generated quite some confusion (Thomp-son and Monis, 2012). As stated above, extensive analysis of pro-tein and DNA polymorphisms has revealed that G. duodenalis is aspecies complex, and the major genetic groups are now knownas assemblages (and may correspond to distinct species). Furthergenetic variation within assemblages has also been reported anda number of sub-assemblages identied, which have been referredto as AI, AII, etc. (that is to say, using the Roman numeral as a suf-x; Monis et al., 2003). Isolates that belong to sub-assemblages aregenetically close, but not identical: i.e., sub-assemblages are clus-ters of closely related isolates. Confusion has mostly arisen fromthe terminology used to describe those closely related isolatesand from the fact that many of the isolates that have been usedto dene sub-assemblages by protein polymorphisms have notbeen studied at the DNA level (Monis et al., 1999). Indeed, in theabsence of a standardized terminology, isolates showing novelDNA sequences have been labelled as A3, A4, A5, etc. (e.g., Lalleet al., 2005a), or as S1, S2, S3, etc. (S for subtype; Sulaiman et al.,2003), or even as EI, EII, EIII, etc. (Zhang et al., 2012a). These geneticvariants should be described as genotypes and, when multiple lociare investigated, as multi-locus genotypes (MLGs). Since in the ori-ginal description of sub-assemblages the Roman numeral sufxwas used, this should not be used to describe genotypes (e.g.,one should not use A IV to describe a novel genotype, as in Zhang

Table 1The currently recognised assemblages of Giardia duodenalis, their host distribution and previously proposed taxonomy.

Assemblage Host distribution Proposed species name

A Humans and other primates, livestock, dogs, cats and some species of wild mammals Giardia duodenalisB Humans and other primates, dogs, cats and some species of wild mammals Giardia entericaC Dogs and other canids Giardia canisD Dogs and other canids

AII-

sem

944 U. Ryan, S.M. Cacci / International Journal for Parasitology 43 (2013) 943956E Hoofed livestockF CatsG RatsH Marine mammals (pinnipeds)

Assemblage(e.g., A)

Sub-assemblage(e.g., AI)

Sub-assemblage(e.g., AII)

Sub-as

(e.g., A

Fig. 1. Sub-assemblages and genotypes within Giardia duodenalis aGiardia bovisGiardia catiGiardia simondi?

Genotypes of AI

Genotypes of AII

AI-1AI-2

AII-12

AII-3

AII-4

AII-5

blage

Genotypes of AIIIAIII-1

AI-3AI-4III)

ssemblage A as dened by using a multi-locus typing scheme.

et al., 2012a). A possible terminology was introduced by Cacciet al. (2008), where MLGs were dened as AI-1, AI-2, AII-1, AII-2etc. In such a way, isolates are rst assigned to the sub-assemblagelevel (AI or AII, etc.) and then further identied by an Arabicnumeral sufx (Fig. 1). Admittedly, this nomenclature is of use inhumans, but may be less applicable to animals, due to the greatvariability found in studies carried out on various animal species(Lebbad et al., 2010; Gmez-Muoz et al., 2012). To avoid furtherconfusion, in this review we will refer to genotypes without men-tioning the names that were assigned in the original publications,and will simply indicate to which sub-assemblages these geno-types belong.

3. Molecular tools for genetic characterization of Giardia

The introduction of molecular techniques, in particular thosebased on the in vitro amplication of nucleic acids (i.e., PCR and re-lated methodologies), has revolutionised the study of the epidemi-

studies. In addition, the GC richness of the 18S locus in particularrequires special PCR buffers for GC rich templates to improve thesensitivity of the detection.

Some researchers have considered that Giardia has a clonal pop-ulation structure and that the use of a single marker with high ge-netic heterogeneity can provide a resolution as high as multilocussequence typing (MLST; Sulaiman et al., 2003). In more recentstudies, however, the use of a MLST scheme has been shown torepresent a more informative approach for genotyping this para-site (Cacci et al., 2008; Lebbad et al., 2010).

Current MLST schemes are predominantly based on housekeep-ing genes and the variation found in these markers seems sufcientfor genotyping. Highly variable molecular markers such as micro-satellites and other repeated DNA sequences will be useful forsub-typing but it appears that these sequences are rarely repre-sented in the genome.

ion

bilit

embembemb

embembembembembembembembemb

embemb

U. Ryan, S.M. Cacci / International Journal for Parasitology 43 (2013) 943956 945Glutamate dehydrogensase Housekeeping enzyme G. duodenalis assTriose phosphate

isomeraseHousekeeping enzyme G. duodenalis ass

Beta-giardin Structural protein G. duodenalis assElongation factor 1-a Involved in translation G. duodenalis assFerredoxin Mediates electron transfer G. duodenalis assHistone H2B Nucleosomal protein G. duodenalis assHistone H4 Nucleosomal protein G. duodenalis assActin Structural protein G. duodenalis assa-tubulin Structural protein G. duodenalis assChaperonin 60 Heat shock protein G. duodenalis assOpen reading frame C4 Hypothetical heat shock

proteinG. duodenalis ass

18S rDNA Small subunit of the ribosome G. duodenalis assIntergenic ribosomal

spacerNon-coding ribosomal G. duodenalis assology of giardiasis. Molecular tools are thought to provide highersensitivity and specicity compared with both microscopic orimmunological assays, and offer the possibility to identify Giardiaisolates at the level of species, assemblage, sub-assemblage andgenotype (Cacci and Ryan, 2008).

The rst PCR assays targeted fragments of well-conservedeukaryotic genes, sometimes using degenerated primers (18SrRNA, glutamate dehydrogenase (gdh), elongation factor 1-alfa(el1-a, triose-phosphate isomerase (tpi); Monis et al., 1999), orgenes uniquely associated with the parasite (-giardin; Lalleet al., 2005a,b). In more recent studies, a number of other PCR as-says have been developed and tested for their applicability fordetection and typing of G. duodenalis; a list of the currently avail-able markers is given in Table 2.

It is important to note that these markers differ widely in termsof genetic variability (Wielinga and Thompson, 2007); indeedsome, such as the 18S rRNA and the el1-a, are strongly conservedand can be used to identify G. duodenalis assemblages, but are oflittle use for studies where genetic variation within assemblagesneeds to be determined. Nevertheless, due to the multicopy natureof the 18S rRNA, the PCR that targets this locus has a high sensitiv-ity and is often used for detection of Giardia from different matrices(e.g., human and animal faeces, water samples). However it shouldbe noted that many 18S PCR assays target very small fragmentswith a heavy reliance on a few polymorphic sites which may bepartially responsible for the discordant ndings among some

Table 2List of the genetic loci used for genotyping, their function and availability of informat

Genetic marker Function Sequences availa

Mlh1 Involved in DNA repair G. duodenalis assITS1, ITS2 and 5.8S rDNA Ribosomal G. duodenalis assembRibosomal protein L7a Ribosomal G. duodenalis assembfrom different Giardia spp. or Giardia duodenalis assemblages.

y Reference

lage A and B Lasek-Nesselquist et al. (2009)lages, Giardia muris, Giardia ardeae Monis et al. (1999)lages, Giardia. microti, G. muris, G. ardeae Sulaiman et al. (2003)

lages, G. muris Cacci et al. (2002)lages, G. muris, G. ardeae Monis et al. (1999)lage A and B Lasek-Nesselquist et al. (2009)lage A and B Lasek-Nesselquist et al. (2009)lage A and B Lasek-Nesselquist et al. (2009)lage A and B, G. ardeae Drouin et al. (1995)lage A and B Kim et al. (2009)lage A and B Lee et al. (2009)lage A and B Yong et al. (2002)

lages, G. muris, G. microti, G. ardeae, Giardia agilis Monis et al. (1999)lage A and B Lee et al. (2006)4. Molecular epidemiology of giardiasis in humans

Humans are infected by two G. duodenalis assemblages, namelyassemblages A and B (Mayrhofer et al., 1995). Molecular analysis ofmore than 2,800 samples (Table 3) indicates that assemblage B(58%) has a higher prevalence than assemblage A (37%) world-wide. This proportion does not change when data from eitherdeveloped or developing countries are analysed. However, theprevalence of mixed infections is higher (5.2%) in developing coun-tries than in developed ones (3.2%). It should be noted that preva-lences of mixed infection are likely to be grossly underestimated,as conventional PCR assays are biassed towards detection of themost abundant parasite population (see section 4.1).

Previous analysis of genetic variability within assemblages hasshown that isolates of assemblage A can be divided into foursub-assemblages (AI, AII, AIII and AIV) by protein polymorphismsof 23 loci (Monis et al., 2003), and the host distribution indicatedthat human isolates belonged to AI and AII, while animal isolatesbelonged to AI, AIII and AIV. Similarly, sub-assemblages BI, BII, BIIIand BIV were described in assemblage B and, as was the case forassemblage A, human isolates appeared to form two clusters (BIIIand BIV), whereas animal isolates (monkeys and a dog) belongedto sub-assemblages BI and BII (Monis et al., 2003). However, theBIII and BIV sub-assemblages identied by allozyme electrophore-sis are not supported by DNA sequence analysis (Feng and Xiao,2011).lages, G. muris, G. microti, G. ardeae Cacci et al. (2010)lage A and B Lasek-Nesselquist et al. (2009)

Table 3Giardia duodenalis prevalences and assemblages in humans in different regions of the world.

Location Total No. ofsamples

Positive samples(%)

No. of samplesgenotyped

AssemblageA

AssemblageB

Mixed infections References

EuropeAlbania 125 17.6 22 10 12 Feng and Xiao (2011)Belgium 373 4.0 72 18 54 Feng and Xiao (2011)France 25 9 16 Feng and Xiao (2011)Germany 202 1.5 3 3 Feng and Xiao (2011)Italy 120 65 39 16 (A + B) Giangaspero et al.

(2007)Netherlands 98 34 64 Feng and Xiao (2011)Netherlands 892 2.0 18 9 9 Feng and Xiao (2011)Norway 21 21 Feng and Xiao (2011)Norway 63 3 60 Feng and Xiao (2011)Poland 232 1.3 3 2 1 Feng and Xiao (2011)Portugal 190 3.7 7 7 Feng and Xiao (2011)Portugal 25 25 Feng and Xiao (2011)Spain 327 3.0 7 2 4 1 (A + B) Cardona et al. (2011)Spain 108 43 61 4 (A + B) Feng and Xiao (2011)Sweden 207 73 128 6 (A + B) Lebbad et al. (2011)United

Kingdom199 48 145 6 (A + B) Feng and Xiao (2011)

UnitedKingdom

33 9 21 3 (A + B) Feng and Xiao (2011)

1031 360 635 36

North AmericaCanada 658 0.5 3 3 Budu-Amoako et al.

(2012b)Canada 52 28.9 15 3 9 3 (A + B) Feng and Xiao (2011)Canada 6 6 Feng and Xiao (2011)United States 14 14 Feng and Xiao (2011)United States 2 2 Feng and Xiao (2011)

40 26 11 3

OceaniaAustralia 12 11 1 (A + B) Feng and Xiao (2011)Australia 353 7.6 23 7 16 Feng and Xiao (2011)Australia 124 31 93 Feng and Xiao (2011)New Zealand 66 7.6 5 1 4 Feng and Xiao (2011)New Zealand 30 23 7 Feng and Xiao (2011)

194 62 131 1

Central and Southern AmericaArgentina 43 40 3 Feng and Xiao (2011)Brazil 245 51.8 30 30 Santos et al. (2012)Brazil 366 23.8 62 62 Feng and Xiao (2011)Brazil 37 29 8 Feng and Xiao (2011)Cuba 20 9 11 Feng and Xiao (2011)Guatemala 645 5.4 20 7 12 1 (A + B) Velasquez et al. (2011)Mexico 19 19 Feng and Xiao (2011)Mexico 9 9 Feng and Xiao (2011)Mexico 12 12 Feng and Xiao (2011)Nicaragua 119 25 94 Feng and Xiao (2011)Peru 1531 20.4 167 66 81 20 (A + B) Feng and Xiao (2011)Peru 845 23.8 16 10 6 Feng and Xiao (2011)Peru 25 6 19 Feng and Xiao (2011)

579 294 264 21

AfricaEgypt 15 1 13 1 (A + C) Soliman et al. (2011)Egypt 52 34.6 18 1 14 1 (B + E), 2 (E) Feng and Xiao (2011)Ethiopia 59 31 13 8 (A + B), 7 (A + F) Feng and Xiao (2011)Guinea Bissau 50 56 25 3 22 Ferreira et al. (2012)Ivory Coast 307 19.8 61 25 36 Berrilli et al. (2012)Nigeria 157 3.2 5 5 Maikai et al. (2012)Sahrawi 120 34.2 32 12 18 2 (A + B) Feng and Xiao (2011)Uganda 427 20.1 34 5 25 4 (A + B) Ankarklev et al. (2012)Uganda 62 5.0 3 3 Feng and Xiao (2011)

252 86 141 25

AsiaBangladesh 2534 12.7 267 20 231 16 (A + B) Feng and Xiao (2011)China 8 4 4 Feng and Xiao (2011)China 18 12 6 Feng and Xiao (2011)India 51 27.4 14 6 8 Khan et al. (2011)India 16 5 8 3 (A + B) Feng and Xiao (2011)India 19 6 9 4 (A + B) Feng and Xiao (2011)Japan 3 2 1 Feng and Xiao (2011)

946 U. Ryan, S.M. Cacci / International Journal for Parasitology 43 (2013) 943956

dent mutations and therefore should carry different alleles. The

AsA

5 Feng and Xiao (2011)172358525

53194319

rna4.1. Mixed infections and recombination

It is clear that the molecular characterization of G. duodenalis iscomplicated by our incomplete understanding of the genetics ofthis organism. Many studies have been based on the analysis of asingle locus, particularly the 18S rDNA locus, but it was onlythrough the application of MLST schemes that researchers havebeen confronted with the lack of concordance between loci, result-ing in the amplication or lack of amplication of particular locifrom a given isolate, or in the detection of different assemblages(or species) when isolates were typed at more than one locus(Sprong et al., 2009). Of note, earlier studies using allozymes (An-drews et al., 1989) already demonstrated that some isolates (e.g.,BAH12) had multiple banding patterns consistent with those ex-pected from a diploid heterzygote. The non-concordance in typingresults has been observed in both human and animal (particularlydogs but also cattle) isolates of G. duodenalis (Sprong et al., 2009),and can be formally explained by two distinct mechanisms: (i) thepresence of genetically different cysts in a faecal sample coupledwith a preferential amplication of one assemblage at a particularlocus and of another assemblage at another locus (i.e., a true mixedinfection followed by PCR bias); or (ii) the existence of recombi-nant isolates in which genetic exchanges have occurred at thegene(s) targeted by the PCR assay(s). How common natural infec-tion with genetically different isolates are is only partially knownbut the use of assemblage-specic PCR assays, which are nowavailable as both conventional PCR (Geurden et al., 2009; Vanni

Table 3 (continued)

Location Total No. ofsamples

Positive samples(%)

No. of samplesgenotyped

Laos 5Malaysia 321 23.7 42Nepal 1096 4.1 35Saudi Arabia 1500 6.5 40South Korea 5Taiwan 209 3.8 8Thailand 6967 0.9 61Thailand 204 20.3 35

Thailand 531 6.2 12Thailand 189 5.8 10Turkey 44Yemen 503 17.6 65

707

Numbers in bold are the totals for each region.

U. Ryan, S.M. Cacci / International Jouet al., 2012) or real-time PCR (Almeida et al., 2010), has revealedthat mixed infections are not uncommon and that the prevalenceis higher in infected individuals living in developing countries(e.g., Gelanew et al., 2007; Cooper et al., 2010) (Table 3).

With regard to genetic exchanges, initial observations sup-ported the possibility of recombination between isolates of assem-blages A and B (Teodorovic et al., 2007), but subsequentcomparison at the whole genome level of the WB and GS strains(Franzen et al., 2009) could not conrm these data. However,recombination between isolates of sub-assemblage AII, genotypeA2, was demonstrated in human isolates from an endemic country(Cooper et al., 2007, 2010) and, similarly, evidence from severalstudies suggest that recombination occurs among assemblage Bisolates (Lasek-Nesselquist et al., 2009; Siripattanapipong et al.,2011). The Giardia genome contains many (21 of 29) of the genesrequired for meiosis (Ramesh et al., 2005), albeit their exact roleis unknown, as they can be involved in other functions such asDNA repair. Thus, whether a true meiotic process occurs in G. duo-denalis remains to be established. Based on experimental evidenceof plasmid transfer between nuclei in the cyst, and fusion betweencyst nuclei, it has been proposed that somatic homologousnding of a very low level of ASH in the whole WB genome (assem-blage A) obtained from a population of trophozoites was thereforea surprise (Morrison et al., 2007), and indicates that alleles withinnuclei as well as those from opposite nuclei are nearly identical,suggesting the presence of homogenisation mechanisms. However,when the genome of the GS strain of assemblage B was studied bypyrosequencing (Franzen et al., 2009), a much higher level of ASHwas found (0.5% versus

rnaof genetic loci from clinical samples and that provide sufcient ge-netic discrimination to infer origins or sources of transmission forGiardia are yet to be developed. When using molecular analysis toidentify assemblages in animal hosts, the classication of geneticheterogeneity within an assemblage should be approached withcaution and should be completed based on MLST data, wherenon-concordance between loci is a serious issue. In addition, com-parison of assemblage-specic primers versus PCR followed bysequencing has revealed a high prevalence of mixed infectionsand therefore the use of assemblage-specic primers in additionto MLST data is advocated in molecular epidemiological surveys,as mixed infections are likely to be underestimated (Leveckeet al., 2007; Geurden et al., 2009). Mixed infections are likely tobe one of the main reasons why multiple genetic markers can iden-tify different assemblages in the same sample. The data presentedin the following sections and tables should be viewed with theselimitations in mind and more emphasis is placed on those obtainedby a MLST approach.

5.1. Giardia in farm animals

Most cattle, sheep and pigs are infected with the host-specicassemblage E (livestock genotype) (Barigye et al., 2008; Thompsonet al., 2008; Xiao and Fayer, 2008; Feng and Xiao, 2011; Fayer et al.,2012; Muhid et al., 2012). Although assemblage E generally pre-dominates in cattle, assemblage A is increasingly being detected,which indicates that assemblage A is probably more widespreadin the bovine population than initially assumed (Table 4). Forexample, a recent multicentre trial, which examined Giardia in cat-tle in Germany, UK, France and Italy, identied an overall preva-lence of 45.4% (942/2072) for Giardia, with a high overallprevalence of assemblage A (43%) (Geurden et al., 2012). The prev-alence of assemblage A ranged from 61% for France to 41% for Ger-many, 29% for the UK and 28% for Italy. Importantly, 32% of thesamples had a mixed assemblage A and E infection, indicating thatprevalence is more correctly estimated using assemblage-specicassays (Geurden et al., 2012).

Few studies have typed assemblage A isolates in cattle but sub-assemblage AI is the most common (Table 4). In one study con-ducted in Europe, among 113 samples tested, 62% belonged tosub-assemblage AI, 39 belonged to sub-assemblage AII, and fourbelonged to sub-assemblage AIII (Sprong et al., 2009). Zoonotictransmission between cattle and humans has been proposed innumerous studies (e.g. Uehlinger et al., 2006; Khan et al., 2011).In the most recent study in India, genotype A1 was identied atthe bg locus in cattle and in dairy farm workers from the samefarms (Khan et al., 2011). As cattle are more commonly infectedwith genotypes in sub-assemblage AI (mostly genotype A1),whereas humans are more commonly infected with genotypes insub-assemblage AII (Xiao and Fayer, 2008), the nding of genotypeA1 in the farms workers was used to support the suggestion thatthe infection in the farm workers was acquired zoonotically (Khanet al., 2011). In addition to direct animal to human contact, trans-mission of zoonotic assemblages from cattle is also likely to occurthrough the contamination of surface water used for the produc-tion of drinking water (Feng and Xiao, 2011; Budu-Amoako et al.,2012a).

Assemblage B is not commonly found in farm animals (Feng andXiao, 2011) (Table 4). One of the few exceptions to this is in NewZealand, where assemblage B (in addition to assemblage A) wascommonly identied in cattle, whereas assemblage E was not de-tected at all (Hunt et al., 2000; Learmonth et al., 2003; Winkworthet al., 2008), indicating that the signicance of New Zealands dairy

948 U. Ryan, S.M. Cacci / International Jouherd as a potential reservoir of zoonotic Giardia is probably greaterthan that of dairy cattle in other countries. However a more recentstudy identied both assemblages E and A in cattle isolates fromNew Zealand with a higher prevalence of assemblage E (Abeywar-dena et al., 2012). Another recent study in cattle in China identiedan overall prevalence of 5.2% for Giardia by microscopy, of which43.8% (7/16) were genotypes in assemblage B as determined bytyping at the tpi locus (Liu et al., 2012).

An age-associated difference in the distribution of assemblagesA and E has been reported in cattle. Some studies have reportedthat assemblage A was predominantly identied in pre-weanedcalves (

omp

rnaTable 4Giardia duodenalis assemblages A and B and sub-assemblages identied in farm and c

U. Ryan, S.M. Cacci / International Jouet al., 2010). Most studies have identied the host-specic assem-blages C and D in dogs (Mark-Carew et al., in press; Liang et al.,2012; Ballweber et al., 2010; Upjohn et al., 2010; Feng and Xiao,2011; Beck et al., 2012). However assemblage A and occasionally

Geographic location Host Assemblage Loci tested

USA and Peru AlpacasandLlamas

A bg, gdh, 18SrRNA

Australia Cats B gdhBrazil Cats AI gdhEurope (Germany, Italy, Poland,

Spain, Sweden)Cats AI, AII, AIII, B [gdh, bg

(RFLP)], bg,gdh, tpi

Japan Cats AI gdh, tpiUSA Cats AI and B gdh, tpiCanada Cats A and B 18S rRNAArgentina Dogs B tpi RFLPAustralia Dogs A and B gdhCanada Dogs A, AI and B gdh, tpi 18S

rRNAChina Dogs AI gdhEurope (Belgium, Germany, Italy,

Sweden, Poland, TheNetherlands)

Dogs A, AI, AII and B bg, gdh, tpi

India Dogs AI, AII, B (varioussub-assemblages)

tpi

Japan Dogs AI gdhMexico Dogs AI, AII bg, gdhThailand Dogs A and B 18S rRNAUSA Dogs AI, AII and B gdh, tpi, 18S

rRNAAustralia Cattle A gdh, 18S

rRNABrazil Cattle AI gdhCanada Cattle A and B bg, 18S rRNA

China AI and B tpiEurope (Belgium, Denmark,

France, Germany, Italy,Portugal, Spain, UK)

Cattle andpigs

AI, AII, AIII, B bg, gdh, 18SrRNA

India Cattle AI bgJapan Cattle AI gdhNew Zealand Cattle A and B bg, tpiTaiwan Cattle A bgUSA Cattle AI, AII and B tpi, bg, 18S

rRNAItaly Chickens

and ducksA, B, mixed Aand B

bg, 18S rRNA

Sweden Guineapigs

B bg, gdh, tpi

Australia and USA Horses AI, AII, B (varioussub-assemblages)

tpi

Columbia Horses bg, gdh, tpi,18S rRNA

Italy HorsesAustralia Pigs A 18S rRNACanada Pigs BEurope Pigs A, AI and B bg, gdh, tpi,

18S rRNAChina, USA Rabbits B (various sub-

assemblages)tpi

Japan Ferrets A1 gdh, bgAustralia Sheep A gdh, tpiChina Sheep and

goatsAI, AIV and B tpi

Europe (Belgium, Italy, Norway,Spain, Sweden, UK)

Sheep,goats andpigs

AI, AII, B (varioussub-assemblages)

bg, gdh, tpi,18S rRNA

USA Sheep A bg, gdh, tpianion animals.

l for Parasitology 43 (2013) 943956 949assemblage B have been identied (Table 4). As with most animals,sub-assemblage AI is the most commonly detected, with one Euro-pean study reporting sub-assemblage AI in 73% of 120 isolates andsub-assemblage AII in the remainder (27%) (Sprong et al., 2009).

References

Gmez-Couso et al. (2012) and Scorza et al. (2012)

Read et al. (2004)Souza et al. (2007)van Keulen et al. (2002), Papini et al. (2007), Caccio et al. (2008), Sprong et al.(2009), Lebbad et al. (2010), Jaros et al. (2011), Dado et al. (2012) and Sotiriadouet al. (2013)Suzuki et al. (2011)Vasilopulos et al. (2007), Oates et al. (2012) and Scorza et al. (2012)McDowall et al. (2011)Minvielle et al. (2008)Read et al. (2004) and Palmer et al. (2008)Himsworth et al. (2010) and McDowall et al. (2011)

Li et al. (2012)Lalle et al. (2005a), Zygner et al. (2006), Leonhard et al. (2007), Claerebout et al.(2009), Overgaauw et al. (2009), Lebbad et al. (2010), Marangi et al. (2010),Upjohn et al. (2010), Berrilli et al. (2012) and Sotiriadou et al. (2013)Traub et al. (2004)

Itagaki et al. (2005)Ponce-Macotela et al. (2002), Lalle et al. (2005b) and Eligio-Garca et al. (2008)Inpankaew et al. (2007) and Traub et al. (2009)Covacin et al. (2011), McDowall et al. (2011) and Scorza et al. (2012)

Ng et al. (2011)

Souza et al. (2007) and Dixon et al. (2011Uehlinger et al. (2011), Budu-Amoako et al. (2012a, 2012b, 2012c) and Paz e Silvaet al. (2012)Liu et al. (2012)van Keulen et al. (2002), Lalle et al. (2005a), Langkjaer et al. (2007), Mendonaet al. (2007), Geurden et al. (2008a), Sprong et al. (2009), Castro-Hermida et al.(2011), Geurden et al. (2012) and Minetti et al. (2013)Khan et al. (2011)Itagaki et al. (2005)Learmonth et al. (2003), Winkworth et al. (2008) and Abeywardena et al. (2012)Hsu et al. (2007)Trout et al. (2004, 2005, 2006, 2007), Coklin et al. (2007), Feng et al. (2008), Santinet al. (2009, 2012) and Mark-Carew et al. (2012)Berrilli et al. (2012)

Lebbad et al. (2010)

Traub et al. (2005) and Scorza et al. (2012)

Traversa et al. (2012)

Santn et al. (2013)Armson et al. (2009)Farzan et al. (2011)Langkjaer et al. (2007), Caccio et al. (2008) and Sprong et al. (2009)

Sulaiman et al. (2003), Lebbad et al. (2010) and Zhang et al. (2012b)

Abe et al. (2005, 2010)Ryan et al. (2005), Yang et al. (2009), Nolan et al. (2010), Sweeny et al. (2011)Zhang et al. (2012a)

Giangaspero et al. (2005), Aloisio et al. (2006), Castro-Hermida et al. (2007),Giangaspero et al. (2007), Geurden et al. (2008b), Gmez-Muoz et al. (2009),Sprong et al. (2009), Lebbad et al. (2010), Robertson et al. (2010), Castro-Hermidaet al. (2011), Gmez-Muoz et al. (2012) and Minetti et al. (2013)Santn et al. (2007), Scorza et al. (2012)

rnaGenotypes of sub-assemblage AII have also been reported in dogsamples in studies from Mexico, India and Belgium (Ponce-Maco-tela et al., 2002; Traub et al., 2004; Claerebout et al., 2009).

It has been suggested that two transmission cycles exist indomestic urban environments, with the transmission of dog-spe-cic assemblages among dogs and the possible transmission ofassemblage A between pets and humans. The transmission ofdog-specic genotypes may be favoured by intensive contactamong large numbers of dogs living together and may outcompetethe transmission of other assemblages. In household dogs, the fre-quency of dog-to-dog transmission may be lower and conse-quently infections with potentially zoonotic assemblages in dogsare likely to persist (Thompson and Monis, 2004; Claereboutet al., 2009). However, other studies on household dogs identiedmainly assemblages C and D (Itagaki et al., 2005; Palmer et al.,2008; Overgaauw et al., 2009) and kennel dogs were shown to beinfected predominantly with assemblage A (Itagaki et al., 2005;Leonhard et al., 2007). A more recent study revealed a variety ofGiardia assemblages in faeces from household dogs, with 28% and41% having assemblages A and B, respectively, and 15% and 16%having host-specic assemblages C and D, respectively (Covacinet al., 2011).

Several studies which suggested potential zoonotic transmis-sion between dogs and humans have been conducted. A cross-sec-tional study in tea-growing communities of Assam, India,identied sub-assemblages AI and AII as well as B sub-assemblagesin dogs and people (Traub et al., 2004). In a study conducted in 20temples in Thailand, stool samples were collected from 229 dogsand 204 people, and Giardia was genotyped in 13 dogs and threehuman samples using the 18S rRNA locus. All specimens from hu-mans had either assemblages A or B, as did samples from eightdogs, whereas the ve remaining dogs had assemblages C or D(Inpankaew et al., 2007). In a second report from Thai temples,18S rRNA-based genotyping identied assemblage A in 33 dogsand 26 people, and assemblage B in nine dogs and one person(Traub et al., 2009). In Argentina, out of 60 people and two dogs,one dog had assemblage B, whereas people had either assemblageA (sub-assemblage AII; n = 3) or B (n = 40) (Minvielle et al., 2008).A study in southern Italy, which screened faecal samples from chil-dren and dogs living in a small Rom community, identied sub-assemblage AI at the bg locus in all of the dog (n = 9) and human(n = 6) samples typed (Marangi et al., 2010). In contrast, in Aborig-inal communities in Australia, where Giardia infection is highly en-demic for both the human and dog populations, it was found thatassemblages C and D predominated in infected dogs (Hopkinset al., 1997). Some of these studies were conducted using onlyone locus and some relied on restriction fragment length polymor-phism (RFLP) analysis rather than sequencing. More sensitive sub-typing tools are therefore required to conrm zoonotictransmission.

Cats are infected with assemblages A and F, with the cat-specicassemblage F being found more frequently (Ballweber et al., 2010;Feng and Xiao, 2011) (Table 4). Recent studies have identiedgenotypes of sub-assemblage AII and AIII in cats on the basis ofMLST (Caccio et al., 2008; Sprong et al., 2009; Lebbad et al.,2010; Suzuki et al., 2011). Few studies have identied assemblageB (Read et al., 2004; Palmer et al., 2008; Sprong et al., 2009; Jaroset al., 2011; McDowall et al., 2011), or assemblage D (Read et al.,2004; Jaros et al., 2011) in cats.

Little is known about G. duodenalis infection in horses and thereis no denitive evidence of the role played by infected horses as apotential source of infection for people (Feng and Xiao, 2011). Anearly study reported that horses harbour genotypes within sub-

950 U. Ryan, S.M. Cacci / International Jouassemblages AI, AII and B (Traub et al., 2005). A later study in Italyidentied only assemblage E in horses by typing at the 18S rRNAlocus (Veronesi et al., 2010), whereas assemblages A, B and E wereidentied using both 18S rRNA and bg loci in another study in Italy(Traversa et al., 2012). The most recent study tested 195 horses inColombia and, based on MLST at four loci, identied assemblage Ain two horses, and assemblage B (genotypes B1 and B2) in 32horses (Santn et al., in press).

In other companion animals, various genotypes of assemblage Bhave been identied in rabbits (Sulaiman et al., 2003; Lebbad et al.,2010; Zhang et al., 2012b) and assemblage A has been identied inferrets (Abe et al., 2005, 2010).

5.3. Giardia in sh and marine mammals

Very little is known about the prevalence and genetic diversityof species of Giardia in marine environments and the role that mar-ine animals play in transmission of these parasites to humans.Giardia spp. have been reported in sea lions, seals and whales (Ta-ble 5) (Olson et al., 1997; Measures and Olson, 1999; Deng et al.,2000; Hughes-Hanks et al., 2005). Giardia duodenalis assemblagesA and B have been reported in the faeces of dolphins, porpoises,seals, a thresher shark and a Mako shark (Table 5) (Bogomolniet al., 2008; Dixon et al., 2008; Lasek-Nesselquist et al., 2008,2010; Appelbee et al., 2010). Assemblage D and assemblageC- and D-like sequences have been isolated from harbour seals(Gaydos et al., 2008).

The non-zoonotic assemblage H was recently described fromthe faeces of grey seals (Halichoerus grypus) and from one gull sam-ple (Larus argentatus) from a northern location on Cape Cod, USA(Lasek-Nesselquist et al., 2010). Phylogenetic analysis revealedthat assemblage H is genetically distinct and shares 80% similar-ity with other G. duodenalis assemblages. The discovery of a previ-ously uncharacterised lineage of G. duodenalis suggests that thisparasite has more genetic diversity and perhaps a larger host rangethan previously believed (Lasek-Nesselquist et al., 2010).

There have been a number of reports of potential transmissionof Giardia from sh to humans, one of which involved an intestinalinfection in Thai labourers in Taipei resulting from the consump-tion of uncooked freshwater sh (Cheng and Shieh, 2000) andthe consumption of raw sh in Laos (Soukhathammavong et al.,2008). Infections have also been linked to home-canned salmonin the US (Osterholm et al., 1981).

Little is known, however, about the prevalence and assemblagesof Giardia in sh, but preliminary evidence suggests that sh maybe a potential reservoir for zoonotic assemblages of G. duodenalis(Yang et al., 2010; Ghoneim et al., 2012). For example, in onestudy predominantly assemblages A (AII) and B (various sub-assemblages) were detected by PCR at various loci in 3.8% (27/709) of sh studied, including eight species of cultured ngerlings(n = 227), ve species of wild marine sh (n = 255), and eight spe-cies of wild freshwater sh (n = 227) (Yang et al., 2010). The high-est prevalence (8.4%) was in cultured ngerlings of various species.Examination of tissue sections demonstrated trophozoites in 10samples and it was reported that several isolates had large num-bers of parasites (>100 Giardia per eld of view) (Yang et al., 2010).

In a more recent study, 92 faecal samples from both culturedand wild caught sh (Tilapia nilotica and Mugil cephalus) from dif-ferent governorates in Egypt were screened using an ELISA (Gho-neim et al., 2012). The overall prevalence of G. duodenalis was3.3% (3/92), and 2.9% (2/68) and 4.2% (2/44) for T. nilotica andM. cephalus, respectively. Typing of the positive samples usingassemblage A- and B-specic tpi primers identied assemblage Ain all samples (Ghoneim et al., 2012). The prevalence of assemblageA in wild sh was 4.5%, while that of the farmed sh was 2.1%(Ghoneim et al., 2012).

l for Parasitology 43 (2013) 943956Another study investigated the possibility of establishingassemblage A (and D) infections in common laboratory zebrash(Danio rerio) (Tysnes et al., 2012). However, although a single

mm

sem

AII,semicrotB

AII,sem

, AII

and

, AII

and

0 an

rnatrophozoite was observed in one sh 3 days post-exposure, anestablished, propagative infection could not be demonstratedusing direct microscopy and immunouorescent antibody tests(Tysnes et al., 2012).

Giardia duodenalis cysts can enter the marine environmentthrough agricultural runoff and sewage efuents, and the occur-rence of Giardia increases in water with decreasing temperaturesand increasing turbidity of the water (Appelbee et al., 2005; Mag-ana-Ordorica et al., 2010). One study suggested that the microcrus-tacean, Artemia franciscana, could serve as a disseminating vehiclefor Giardia (and Cryptosporidium) in aquatic environments, as un-

Table 5Giardia duodenalis assemblages and sub-assemblages identied in sh and marine ma

Fish/marine mammal species As

FishFingerlings:Barramundi (Lates calcarifer), Black bream (Acanthopagrus

butcheri), Mulloway (Argyrosomus japonicus), Snapper (Pagrus auratus)A,asm

Freshwater Fish:Western minnow (Galaxias occidentalis), Nile tilapia (Tilapianilotica)

A,

Marine sh:Thresher shark (Alopias vulpinus), Mako shark (Isurus paucus),Flathead Sea mullet (Mugil cephalus)

A,as

Marine mammalsPorpoises (Phocoena phocoena), Dolphins (Delphinus delphis and Grampus

griseus)AI

Harbour seals (Phoca vitulina) CRinged seals (Phoca hispida) BHarp seal (Phoca groenlandica) ADolphin (Delphinus delphis), Grey seal (Halichoerus grypus), Pacic harbour

seals (Phoca vitulina richardsi), Atlantic harbour seal (Phoca vitulinavitulina)

AI

a Analysed using a combination of some or all of the following loci: 18S, tpi, gdhb Analysed using tpi assemblage A- and B-specic primers.c Analysed at the 18S locus.d Analysed at the tpi locus.e Analysed using a combination of some or all of the following loci: gdh, mlh, tpi5

U. Ryan, S.M. Cacci / International Jouder experimental conditions it was capable of ingesting andspreading the xed (oo)cysts of Cryptosporidium and Giardia (Mn-dez-Hermida et al., 2006).

5.4. Giardia in wild mammals

Giardia duodenalis has been identied in both captive and wildanimals (Table 6) and the detection of potentially zoonotic sub-assemblages in captive animals may be due to reverse zoonosisor spill-back transmission from humans to animals due to theclose contact that captive animals have with their care-takers.

Early epidemiological studies which linked giardiasis in camp-ers in Canada with drinking stream water contaminated withGiardia cysts from beavers led the World Health organisation(WHO) to list Giardia as a zoonosis (Thompson, 2004). Molecularcharacterization has conrmed the identication of assemblagesA and B in beavers (Appelbee et al., 2002; Sulaiman et al., 2003;Fayer et al., 2006) (Table 6), yet convincing evidence of zoonotictransmission from beavers to humans was never obtained.

Rodents are most commonly infected with assemblage G (Fengand Xiao, 2011) but zoonotic assemblages have been identied inchinchillas, muskrats, Patagonian maras and Prevosts Squirrel(Sulaiman et al., 2003; Beck et al., 2011a; Levecke et al., 2011;Veronesi et al., 2012).

Both assemblages A and B are commonly found in other wildmammals indicating that these assemblages have a very wide hostrange (Table 6). Wild ungulates are mostly infected with genotypesof sub-assemblages AI and AIII. Sub-assemblage AIII appears to bespecically associated with wild ungulates (Robertson et al., 2007;Caccio et al., 2008), but has been identied in a cat (Lebbad et al.,2010) and a few cattle isolates (Sprong et al., 2009). It has not beenreported in humans to date.

Giardia is commonly found in the stools of wild and captivenon-human primates (NHPs) (Ghandour et al., 1995; Hope et al.,2004; Levecke et al., 2007; Salzer et al., 2007) and is a signicantcause of diarrhoea and failure to thrive, particularly in young ani-mals (Hamlen and Lawrence, 1994; Kalishman et al., 1996). Poten-tially zoonotic genotypes have been identied in a range of NHPsincluding lemurs, monkeys, chimpanzees, gibbons and gorrillas(Graczyk et al., 2002; Itagaki et al., 2005; Levecke et al., 2007; Cac-

als.

blage Reference

B (various sub-blages), E(and Giardiai)

Yang et al. (2010)a

Ghonim et al., 2012)b, Yang et al. (2010)a

B (various sub-blages)

Lasek-Nesselquist et al. (2008)e, Lasek-Nesselquistet al. (2010)a, Yang et al. (2010)a, Ghoneim et al.(2012)b

and B Lasek-Nesselquist et al. (2008)e

D Gaydos et al. (2008)c

Dixon et al. (2008)d

Appelbee et al. (2010)c

, B (and H) Lasek-Nesselquist et al. (2010)a

bg.

d tpi30.

l for Parasitology 43 (2013) 943956 951cio et al., 2008; Volotao et al., 2008; Lebbad et al., 2010; Beck et al.,2011a; Martnez-Daz et al., 2011; Ye et al., 2012). To date onlyassemblages A and B have been identied in NHPs with assem-blage B predominating. A recent study in China identied geno-types A2 and B1 in rhesus monkeys from a public park in Chinawhere the monkeys have close contact with humans (Ye et al.,2012). The dominant B1 genotype found in the park was previouslyidentied in humans in China (Wang et al., 2011).

Little is known of the prevalence and genotypes of Giardiainfecting marsupials, despite marsupials being one of the dominantmammalian groups within watersheds in Australia (Power et al.,2005). It is therefore important to investigate the occurrence ofGiardia in marsupials to determine whether infections pose a po-tential threat to public health, so that appropriate catchment man-agement strategies may be implemented. Giardia cysts have beenidentied in faecal samples from Tasmanian devils (Sarcophilusharrisii), southern brown bandicoots (Isoodon obesulus), long-nosedbandicoots (Perameles spp.), northern brushtail possums (Trichosu-rus vulpecula arnhemensis), kangaroos (Macropus spp.) and opos-sums (Didelphis albiventris) (Mackerras, 1958; Bettiol et al., 1997;Milstein and Goldsmid, 1997; Zanette et al., 2008). However, nomolecular studies were conducted on these isolates.

A novel host-adapted quenda genotype has been identied insouthern brown bandicoots (also known as quendas) (I. obesulus)in Western Australia (Adams et al., 2004; Thompson et al., 2010)and assemblage A was identied from several western grey kanga-roos (Macropus fuliginosus) (McCarthy et al., 2008), also in WesternAustralia. A recent study identied a low prevalence (4.8% 17/351) in marsupials and mainly non-zoonotic assemblages were

ge

Beaver (Castor canadensis) A and B

B

B

rnaBirds:Common eider (Somateria mollissima), Herring gulls (Larusargentatus), Sulphur-crested Cockatoo (Cacatua galerita)a

AI, AII and

Collared peccary (Pecari tajacu)a A1

Coatimundi (Nasua narica)a A

Hoofed animals:Fallow Deer (Dama dama), Red deer (Cervus elaphus),Roe deer (Capreolus capreolus), White-tailed Deer (Odocoileusvirginianus), Muskoxen (Ovibos moschatus), Scimitar-horned oryx(Oryx dammah)a, Water buffalo (Bubalus bubalis)a, Wild boar (Susscrofa)

A, AI, AIII

Non-human Primates (NHPs)a:Red ruffed lemur (Varecia rubra), A, AI, AII,Table 6Giardia duodenalis assemblages A and B and sub-assemblages identied in wildlife.

Wildlife species Assembla

Anteater (Tanamuda sp.) B

952 U. Ryan, S.M. Cacci / International Jouidentied, including assemblage C and E (in addition to the quendagenotype) (Thompson et al., 2010). In contrast, another study re-ported a somewhat higher prevalence (8.7% 39/445) from bothcaptive and wild marsupials, and identied only assemblages Aand B (Thompson et al., 2008). In the latter study, assemblage Bsubtypes were identied in captive marsupials from wildlife parks,which had direct and indirect contact between marsupials and hu-mans, but was not identied in wild marsupials. One of the assem-blage B gdh sequences was identical to a gdh sequence obtainedfrom a human-derived isolate from Western Australia. This sug-gests that human interaction may have been a source of infectionin marsupials (Thompson et al., 2008).

6. Perspectives

The use of molecular epidemiological tools, and particularlysub-typing tools, has greatly changed our understanding of zoo-notic transmission of Giardia spp. Genetic typing supports the zoo-notic potential of Giardia cysts shed by animals. Sub-assemblagesAI and AII are found in both humans and animals but sub-assem-

Mayotte lemur (Eulemur fulvus mayottensis), Ring-tailed lemur(Lemur catta), Black-capped squirrel monkey (Saimiri boliviensis),Black-headed spider monkey (Ateles fusciceps), Barbary macaques(Macaca sylvanus), Common chimpanzee (Pan troglodytes), Cotton-top tamarin (Saguinus oedipus), Japanese macaque (Macacafuscata); Javan lutung (Trachypithecus auratus), Mantledguereza (Colobus guereza), Mandrill (Mandrillus sphinx), Pygmymarmoset (Cebuella pygmaea), Rhesus monkeys (Macaca mulatta),Southern Brown Howler Monkeys (Alouatta clamitans), Mountaingorilla (Gorilla beringei beringei), Vervet monkey (Cercopithecus sp.),White-Handed Gibbon (Hylobates lar)

Marsupials:Common planigale (Planigale maculata), Quenda (Isoodonobesulus), Quokka (Setonix brachyurus), Red kangaroo (Macropusrufus), Swamp wallaby (Wallabia bicolor), Tammar wallaby(Macropus eugenii), Western grey kangaroo (Macropus fuliginosus),Yellow-footed rock-wallaby (Petrogale xanthopus)

A, AI, B

Moose (Alces alces) and reindeer (Rangifer tarandus) A and AIRodents:Chinchillas (Chincilla lanigera), Muskrat (Ondatra zibethicus),

Patagonian mara (Dolichotis patagonum)a, Prevosts Squirrel(Callosciurus prevosti)a

A, B

Rock hyrax (Procavia capensis)a B

Sun bear (Ursidae Helarctos malayanus)a B

Wild felids:Cheetah (Acinonyx jubatus)a, Lynx (Lynx lynx)a, Snowleopard (Uncia uncial)a

AI

Wild canids: African painted dog (Lycaon pictus), Coyote (Canislatrans), Fox (Vulpes vulpes) Wolf (Canis lupus)a, Maned wolf(Chrysocyon brachyurus)a, Jackals (Canis aureus)

AI, AII, B(various sub-assemblages)

a Captive mammals.Loci tested References

bg Solarczyk (2009)tpi, bg Appelbee et al. (2002), Sulaiman et al. (2003) and Fayer et al.

(2006)gdh, mlh,tpi5andtpi3

Lasek-Nesselquist et al. (2008) and Nolan et al. (2011)

bg, tpi, ITS1rRNA, 18SrRNA

Beck et al. (2011a)

bg, tpi, ITS1rRNA, 18SrRNA

Beck et al. (2011a)

tpi, bg Trout et al. (2003), Lalle et al. (2007), Caccio et al. (2007, 2008),Solarczyk (2009), Lebbad et al. (2010), Beck et al. (2011b) andSolarczyk et al. (2012)

bg, gdh, tpi Graczyk et al. (2002), Itagaki et al. (2005), Levecke et al. (2007,

l for Parasitology 43 (2013) 943956blage AI is preferentially found in livestock and pets whereassub-assemblage AII is predominantly found in humans. Sub-assemblage AIII is almost exclusively found in wild ungulatesand is likely to be non-zoonotic. The host distribution of assem-blage B is predominantly human and NHPs and to a much lesserextent wildlife and dogs.

There is currently no consensus on the genotyping scheme to beadopted for the characterisation of G. duodenalis isolates, as differ-ent groups use different loci, with some still using analysis of the18S rRNA locus alone, which has generated the most discordantdata among studies. To allow the comparison of data among stud-ies, hosts and regions, we recommend a MLST approach includingassemblage-specic detection. As a minimum, the gdh, tpi and bgloci should be used. Sequence analysis of short fragments of the18S rRNA locus should be avoided.

More variable loci are required to conrm zoonotic transmis-sion, however sub-typing tools that can provide sufcient geneticdiscrimination to infer origins or sources of transmission for Giar-dia are currently lacking and need to be developed. The increasinguse of whole genome analysis using next generation sequencingtechnologies will undoubtedly assist in the identication of more

2009), Caccio et al. (2008), Kutz et al. (2008), Volotao et al.(2008), Lebbad et al. (2010), Beck et al. (2011a), Martnez-Dazet al. (2011) and Ye et al. (2012)

bg, 18SrRNA

McCarthy et al. (2008) and Thompson et al. (2008, 2010)

gdh, bg Robertson et al. (2007) and Lebbad et al. (2010)bg, tpi, ITS1rRNA

Sulaiman et al. (2003), Beck et al. (2011a), Levecke et al. (2011)and Veronesi et al. (2012)

bg, tpi, ITS1rRNA

Beck et al. (2011a)

bg, tpi, ITS1rRNA

Beck et al. (2011a)

bg, tpi, ITS1rRNA

Beck et al. (2011a)

bg, tpi, bg,18S rRNAITS1 rRNA

Hamnes et al. (2007), Thompson et al. (2009), Ash et al. (2010),Beck et al. (2011a,b) and Oates et al. (2012)

epidemiology of Giardia duodenalis in an endangered carnivore-the African

rnapainted dog. Vet. Parasitol. 174, 206212.Baldursson, S., Karanis, P., 2011. Waterborne transmission of protozoan parasites:

review of worldwide outbreaks an update 20042010. Water Res. 45, 66036614.

Ballweber, L.R., Xiao, L., Bowman, D.D., Kahn, G., Cama, V.A., 2010. Giardiasis in dogsand cats: update on epidemiology and public health signicance. TrendsParasitol. 26, 180189.

Barigye, R., Dyer, N.W., Newell, T.K., Khaitsa, M.L., Trout, J.M., Santin, M., Fayer, R.,2008. Molecular and immunohistochemical detection of assemblage E, Giardiaduodenalis in scouring North Dakota calves. Vet. Parasitol. 157, 196202.

Beck, R., Sprong, H., Bata, I., Lucinger, S., Pozio, E., Cacci, S.M., 2011a. Prevalenceand molecular typing of Giardia spp. in captive mammals at the zoo of Zagreb.Croatia Vet. Parasitol. 175, 4046.

Beck, R., Sprong, H., Lucinger, S., Pozio, E., Cacci, S.M., 2011b. A large survey ofCroatian wild mammals for Giardia duodenalis reveals a low prevalence andlimited zoonotic potential. Vector Borne Zoonotic Dis. 11, 10491055.

Beck, R., Sprong, H., Pozio, E., Cacci, S.M., 2012. Genotyping Giardia duodenalisisolates from dogs: lessons from a multilocus sequence typing study. Vectorvariable loci. Those tools will also allow us to determine the extentto which reverse zoonosis or spill-back transmission from hu-mans to animals occurs, which will be important for conservationmanagement.

A better understanding of the transmission of Giardia betweenanimals and humans also requires a thorough understanding ofthe population genetics of G. duodenalis, particularly populationsub-structure, geographic and host segregations. As advances aremade in whole genome sequencing of Giardia isolates and moresensitive sub-typing tools are systematically used in well-designedepidemiological investigations in both endemic and epidemic set-tings, we will undoubtedly gain a more comprehensive under-standing of the host-specicity and transmission cycles of Giardia.

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Abe, N., Tanoue, T., Noguchi, E., Ohta, G., Sakai, H., 2010. Molecular characterizationof Giardia duodenalis isolates from domestic ferrets. Parasitol. Res. 106, 733736.

Abeywardena, H., Jex, A.R., Nolan, M.J., Haydon, S.R., Stevens, M.A., McAnulty, R.W.,Gasser, R.B., 2012. Genetic characterisation of Cryptosporidium and Giardia fromdairy calves: discovery of species/genotypes consistent with those found inhumans. Infect. Genet. Evol. 12, 19841993.

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