1-s2.0-s0304401704004005-main

Review

The zoonotic significance and molecular

epidemiology of Giardia and giardiasis

R.C. Andrew Thompson*

WHO Collaborating Centre for the Molecular Epidemiology of Parasitic Infections and

Western Australian Biomedical Research Institute, Veterinary and Biomedical Sciences,

Murdoch University, South Street, Murdoch, WA 6150, Australia

Abstract

The taxonomy and molecular epidemiology of Giardia and Giardia infections are reviewed in the

context of zoonotic and waterborne transmission. Evidence to support the zoonotic transmission of

Giardia is very strong, but how frequent such transmission occurs and under what circumstances,

have yet to be determined. Zoonotic origin for waterborne outbreaks of Giardia infection appears to

be uncommon. Similarly, livestock are unlikely to be an important source of infection in humans. The

greatest risk of zoonotic transmission appears to be from companion animals such as dogs and cats,

although further studies are required in different endemic foci in order to determine the frequency of

such transmission.

# 2004 Elsevier B.V. All rights reserved.

Keywords: Giardia; Taxonomy; Molecular epidemiology; Zoonoses; Transmission; Waterborne

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

2. What is Giardia? Historical perspectives and evolutionary biology . . . . . . . . . . . . 17

3. Current taxonomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

4. Developmental biology and pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

www.elsevier.com/locate/vetpar

Veterinary Parasitology 126 (2004) 1535

* Tel.: +61 08 9360 2466; fax: +61 08 9310 4144.

E-mail address: [email protected].

0304-4017/$ see front matter # 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.vetpar.2004.09.008

5. Transmissionthe impact of molecular epidemiology . . . . . . . . . . . . . . . . . . . . . 21

5.1. Host specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

5.2. Cycles of transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

5.2.1. Humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

5.2.2. Cattle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

5.2.3. Dogs and cats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

5.2.4. Wildlife . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

5.3. Zoonotic and waterborne transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

6. Diagnosis and detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

7. Treatment and control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

8. Conclusions and looking to the future . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

1. Introduction

Although humans have undoubtedly suffered the consequences of Giardia infection for

thousands of years, we had to await the invention of the microscope before it was observed

for the first time, and another 200 years until it was properly described (Lambl, 1859).

Today, Giardia is one of the most widely studied organisms. Not only because of its

ubiquity as a parasite, but also because of its importance in evolutionary biology and

molecular genetics.

Giardia duodenalis has a global distribution causing an estimated 2.8 108 cases perannum (Lane and Lloyd, 2002), and is the most common intestinal parasite of humans in

developed countries. In Asia, Africa and Latin America, about 200 million people have

symptomatic giardiasis with some 500,000 new cases reported each year (WHO, 1996). It

is also a frequently encountered parasite of domestic animals, especially livestock, dogs

and cats, and numerous species of wild mammals and birds have been documented as hosts

of Giardia.

As a parasite, Giardia has a broad host range, although the adverse consequences of

Giardia infection and its pathogenic potential are best recognised in humans. Its simple life

cycle involving an environmentally resistant cyst, provides ample opportunities for the

parasite to be transmitted directly from one infected individual to another, or indirectly

through contamination of the environment or food. In this respect, water is an important

vehicle for the transmission of Giardia to people. Giardiasis is the most frequently

diagnosed waterborne disease and along with Cryptosporidium, is the major public health

concern of water utilities in both developed and developing nations (Levine et al., 1990;

Thurman et al., 1998; Hoque et al., 2002; Leclerc et al., 2002). However, although we

understand much about the waterborne transmission of Giardia, the public health

significance of infected non-human hosts as sources of water contamination remains an

unresolved issue. Indeed, the role of zoonotic transmission in the epidemiology of human

Giardia infections has yet to be resolved.

R.C.A. Thompson / Veterinary Parasitology 126 (2004) 153516

2. What is Giardia? Historical perspectives and evolutionary biology

The characteristic and distinctive morphological features of Giardia are well known and

were described initially in the latter part of the 1800s. The non-encysted, motile

trophozoite is bilaterally symmetrical, piriform to ellipsoidal in shape, 1215 mm 68 mm, with a convex dorsal surface and large unique adhesive or sucking disc on theventral surface. It is binucleate, with four pairs of flagella and a pair of distinctive median

bodies (Fig. 1). Giardia, along with other multi-flagellates are grouped in the Class

Zoomastigophorea and Order Diplomonadida.

The phylogenetic affinities of Giardia have been a matter of controversy for a number of

years. However, there is now broad consensus of Giardias primitive origins as an early

branching eukaryote lineage that diverged before mitochondrial acquisition (Simpson et

al., 2002). Giardia has thus become a key organism in attempts to understand the evolution

of eukaryotic cells. Giardia has a very simple intracellular organization, with no

mitochondria or peroxisomes but does have a primitive vesicular secretory system that has

been proposed as the archetype of the Golgi secretory apparatus in higher organisms (Marti

et al., 2003a,b).

3. Current taxonomy

More than 50 species of Giardia have been described, the majority between 1920 and

1930 (Kulda and Nohynkova, 1996; Thompson et al., 1990; Thompson, 2002). The small

number recognised today (Table 1) follows a comprehensive re-evaluation and

rationalization proposed by Filice (1952) based on the morphological similarity of the

described species and doubts over the validity of host specificity as a criterion for

taxonomic recognition.

Although species of Giardia inhabit the intestinal tracts of virtually all classes of

vertebrates, G. duodenalis (sometimes referred to as Giardia intestinalis; Giardia

lamblia) is the only species found in humans and the majority of domestic and wild

mammals from which Giardia has been examined (see Thompson, 2002). It was to

this species that Filice (1952) allocated the majority of species previously described.

R.C.A. Thompson / Veterinary Parasitology 126 (2004) 1535 17

Fig. 1. Giemsa-stained trophozoite of Giardia duodenalis showing multiple flagella, nuclei and median bodies

(400). [Figure courtesy of Nicolette Binz.].

He recognised that his proposal was only a temporary solution in view of growing

evidence of phenotypic variability between isolates within the species G. duodenalis.

However, at the time, the methodology was not available to reliably discriminate

between these variants.

Filices taxonomic rationalism was a major step forward, as was the recognition by the

World health Organization (WHO) of the zoonotic potential of Giardia as a result of

epidemiological data and cross-infection experiments (WHO, 1979). The subsequent

development of axenic in vitro culture procedures that enabled laboratory amplification of

isolates of Giardia in axenic culture was also an important advance. It was now possible to

grow individual isolates of the parasite in the laboratory and produce sufficient numbers to

genetically characterise the isolates using procedures such as allozyme electrophoresis. As

a result, research over the last ten years has produced valuable information about the

genetic structure of Giardia populations and has demonstrated considerable levels of

genetic diversity within the G. duodenalis group.

Until recently it has been difficult to interpret the extensive genetic variability within G.

duodenalis in a taxonomic perspective. Although the ability to axenize and amplify isolates

of G. duodenalis in laboratory culture was a tremendous breakthrough in terms of

providing sufficient material for genetic characterisation, not all isolates of the parasite can

be established in axenic in vitro culture. This includes a significant proportion of human

isolates. Isolates from some species such as dogs are largely refractory to in vitro culture as

well. As a consequence it has not been possible, until recently, to genetically characterise

many isolates of G. duodenalis. Much of the available genetic data were therefore based on

a small pool of culture-selected isolates that could not be considered to be representative of

the extensive gene pool existing in nature. The inability to amplify all isolates obtained

from the field in in vitro culture has also been a major limitation to our understanding of the

epidemiology and transmission of Giardia.


Table 1

Recognised species in the genus Giardia (from Thompson, 2002)

Species Hosts Morphological characteristics Trophozoite dimensions:

length/width (mm)

G. duodenalis Wide range of domestic

and wild mammals

including humans

Pear-shaped trophozoites

with claw-shaped median bodies

1215/68

G. agilis Amphibians Long, narrow trophozoites with

club-shaped median bodies

2030/45

G. muris Rodents Rounded trophozoites with small

round median bodies

912/57

G. ardeae Birds Rounded trophozoites, with

prominent notch in ventral disc

and rudimentary caudal flagellum.

Median bodies round-oval to

claw-shaped

10/6.5

G. psittaci Birds Pear-shaped trophozoites, with no

ventro-lateral flange. Claw-shaped

median bodies

14/6

The recent application of PCR-based procedures has circumvented the need for in vitro

culture and amplification, with direct characterization of the parasite possible from faecal

and environmental samples, and has enabled the characterisation of previously inaccessible

genotypes (Van Keulen et al., 1998; Monis et al., 1998; Hopkins et al., 1997, 1999). Using

PCR-based procedures in conjunction with analysis of conserved genetic loci such as

rDNA and a variety of housekeeping genes including that coding for glutamate

dehydrogenase (GDH), elongation factor 1- alpha (ef1-a) and triose phosphate isomerase(tpi), combined with much larger data sets, it has been possible to elucidate the

fundamental genetic divisions within the G. duodenalis group (Table 2). A number of

laboratories in different countries have contributed to this work and a consensus has

emerged (Hopkins et al., 1997, 1999; Monis et al., 1996, 1998, 1999). This shows that G.

duodenalis is clearly not a uniform species but a species complex comprising a variety of

genetically and phenotypically distinct, yet morphologically similar genotypes which also

exhibit differences in host specificity (Thompson, 1998; Thompson et al., 1999, 2000;

Monis and Thompson, 2003). These genotypic groupings of G. duodenalis are also

geographically widely distributed and genetically conserved.

Giardia isolates recovered from humans fall into one of the two major genotypic

groupings or assemblages (Table 2). Distributed world-wide, these two assemblages

comprise several genotypes and are now widely referred to as Assemblages A and B.

Molecular analyses have shown that the genetic distance separating these two assemblages

exceeds that used to delineate other species of protozoa (Andrews et al., 1989; Mayrhofer

et al., 1995; Monis et al., 1996; Monis and Thompson, 2003). Although all the genetic

evidence suggests that separate species names for each of these assemblages may be

warranted, there is also significant genetic sub-structuring within each assemblage.

Assemblage A consists of isolates that can be grouped into two distinct clusters; AI consists

of a mixture of closely related animal and human isolates which are geographically

widespread, and most attention regarding the zoonotic potential of Giardia has focused on

this AI subgroup. In contrast, the second subgroup, AII consists entirely of human isolates.

Assemblage B comprises two subgroups, III and IV, of which the latter appears to be

human-specific.

Interestingly, some of the genotypic groupings, or assemblages, are genetically quite

uniform and appear to be confined to specific animal hosts. Giardia genotypes exhibiting a

limited host range include those recovered from cats, dogs, rats, voles/muskrats and hoofed

animals (Table 2). Unlike the uncertainty regarding the taxonomic status of genotypic


Table 2

Genotype and host range of isolates within the Giardia duodenalis morphological group

Genotype/Assemblage Host range

Zoonotic/A Humans, livestock, cats, dogs, beavers, guinea pig, slow loris

Zoonotic/B Humans, slow loris, chinchillas, dogs, beavers, rats, siamang

Dog/C, D Dog

Livestock/E Cattle, sheep, pigs

Cat/F Cat

Rat/G Domestic Rats

Muskrats/Vole Wild rodents

assemblages A and B, there is probably sufficient data supporting the restricted host

range of these genotypes to warrant species designation. Consequently, consideration

could be given to reinstating previously invalidated species such as G. canis and G. bovis

(Thompson, 2002). However, the situation is likely to be much more complicated than that

summarised in Table 2, as illustrated by the discovery of novel genotypes, such as those

occurring in Australian marsupials (Adams and Thompson, 2002).

4. Developmental biology and pathogenesis

Giardia is not invasive and lives and multiplies by asexual multiplication on the

lumenal surface of the small intestine of its vertebrate host. The pathogenesis of Giardia

is not clearly understood and symptoms which include acute or chronic diarrhoea,

dehydration, abdominal pain and weight loss are highly variable (Thompson et al.,

1993), and may not be evident in a significant proportion of infected individuals

(Rodriguez-Hernandez et al., 1996). However, recent research is starting to provide

information on the diversity of processes that are triggered by an infection with Giardia,

which all contribute to a complex pathophysiological process (Buret et al., 2002a). One

of the fundamental changes is altered epithelial permeability that appears to result from a

direct cytopathic effect induced by products of the parasite (Buret et al., 2002b).

Peripheral membrane proteins, and in particular the tight junction-associated protein

zonula occludin-1 (ZO-1), which play an important role in the regulation of epithelial

permeability are disrupted. The resultant increased epithelial permeability leads to an

inflammatory response and both digestive and absorptive changes, that correlate with

brush border injury and disaccharidase deficiencies (Scott et al., 2002). Giardia also

induces apoptosis that correlates with the loss of epithelial barrier function via the

disruption of tight junctional ZO-1 and the subsequent increase in permeability (Chin et

al., 2002). Interestingly, apoptosis and the severity of disease is determined by strain-

dependent virulence factors of the parasite, as well as by the developmental, nutritional

and immunological status of the host (Chin et al., 2002; Scott et al., 2002). It should also

be noted that increased intestinal permeability may result in the uptake of lumenal

antigens. This may exacerbate the occurrence of allergic disorders, a complication often

reported in humans infected with Giardia (Scott et al., 2002) and a factor that may

confuse the aetiology of nutritional disorders.

It is not surprising that such a multifactorial pathophysiological process that is both

parasite and host dependent is so variable in its expression in terms of symptomatology,

clinical consequences and severity. In this respect, the contribution of host factors has

not been given much attention. It seems likely that the pathophysiological changes

described above may occur in the majority of hosts infected with Giardia but that

the consequences of such changes may vary depending upon nutritional factors and

immune status, as well as concurrent enteric infections with other parasites.

For example, the chronicity of Giardia infections in disadvantaged children whose

nutrition may be suboptimal and who suffer infections with other gastrointestinal

parasites such as Hymenolepis or hookworm, is recognised as an important contributor

to poor growth (Thompson, 2000; Sackey et al., 2003). Similarly, in young animals


and birds that are nutritionally compromised or exposed to stress through overcrowding

or low temperatures, Giardia may be an additional factor that culminates in the

expression of severe disease. For example, this is likely to be the cause of mortalities in

nestling ibis (McRoberts et al., 1996). The risk factors for clinical giardiasis may be

closer to being resolved, and they clearly involve host and environmental factors, as well

as the strain of the parasite. The precise nature of these factors requires further

characterisation, as do the complex interactions in the host that result in the expression of

giardiasis.

5. Transmissionthe impact of molecular epidemiology

Based on what is known about the prevalence of Giardia in different animal species,

including humans, and our current understanding of the major genetic groupings in G.

duodenalis, there are four major cycles of transmission that maintain the parasite in

mammalian hosts (Fig. 2). However, we need to consider how these cycles may interact,

and try to determine the frequency of transmission of zoonotic genotypes.

5.1. Host specificity

The phenotypic trait that has provided most controversy and disagreement in the context

of species recognition in Giardia has been that of host specificity. Indeed, until Filices

revision (Filice, 1952), the majority of species had been described principally on the basis

of host occurrence (see above). However, the question of host specificity has not only had

an important influence on Giardia taxonomy but also on the ongoing debate as to whether

giardiasis is a zoonosis. Although, the results of experimental cross-infection experiments

questioned the notion of host adapted species as a tenable criterion for species recognition,

it is now clear that some species may be host specific and others are capable of infecting a

broad range of host species.

Experimental cross transmission studies have often been questionable because of

uncertainty about the Giardia-free status of experimental animals and the common use

of high doses of cysts that are unlikely to represent a natural infection. (Thompson et al.,

1990; Monis and Thompson, 2003). This has been further emphasised by recent results

which have raised the possibility that infected hosts may not produce cysts in sufficient

quantities to be detected by microscopy (McGlade et al., 2003; Traub et al., 2004). Cross

transmission studies have also used uncharacterised isolates, limiting their usefulness in

determining the host specificity of the different genotypes. For example, an avian isolate

of G. duodenalis exhibiting aggressive pathogenesis has been shown experimentally to

establish infections in domestic kittens and lambs (McDonnell et al., 2003). However,

the isolate has yet to be characterised genetically with respect to the known genetic

groupings (Table 2) thus limiting the significance of the study. An interpretation of the

results of cross transmission studies is also difficult when some report successful cross

infection between species while others are unsuccessful. Such results may reflect the

genetic diversity of the isolates being used, differences in the viability of the cysts,

variation in the immune status of experimental hosts, or insensitive detection techniques.


From a review of the results of cross-infection experiments (Thompson et al., 1990;

Monis and Thompson, 2003), probably the most valuable results from epidemiological

and zoonotic perspectives are that: it has been possible to infect dogs with G. duodenalis

from Assemblage A, group I; and that beavers are susceptible to infection with isolates

of human origin (see Monis and Thompson, 2003).


Fig. 2. Major transmission cycles of Giardia duodenalis.

5.2. Cycles of transmission

5.2.1. Humans

Human to human transmission of Giardia can occur indirectly through the accidental

ingestion of cysts in contaminated water or food, or directly in environments where

hygiene levels may be compromised, such as day care centres or disadvantaged community

settings. In environments where the frequency of transmission is high and/or conditions are

conducive to direct person-to-person transfer, such as localised endemic communities or

institutional settings such as day care centres, it would be expected that competitive

interactions might result in the predominance of particular genotypes. However, this does

not appear to be the case. A study that examined Giardia from humans and dogs in the same

community found that all of the human isolates were either Assemblage A or B and that all

of the dog isolates, with a single exception, were Assemblage C or D (dog-specific

assemblages) (Hopkins et al., 1997). The exception was a dog that had a mixed infection of

Assemblage B and C, which suggests that perhaps the dog was infected from a human

source. Similarly, a recent molecular epidemiological investigation on the aetiology of

giardiasis in gorillas in a remote area in Uganda found that they were infected with

Assemblage A, and that human rangers were the likely source of the infections as a result of

indiscriminate defaecation (Graczyk et al., 2002).

A recent UK study that examined 35 human clinical samples found that 64% were

Assemblage B, 27% were Assemblage A genetic group II and the remainder were a

mixture of Assemblage B and Assemblage A genetic group II (Amar et al., 2002).

Similarly, an institutional survey in Australia found that infections with Assemblage B

were more prevalent (70%) than Assemblage A (30%) (Read et al., 2001). The Assemblage

B genotype was also found to be responsible for an outbreak in a nursery in the UK where

21 of 24 (88%) cases were infected with this genotype (Amar et al., 2002). In tea growing

communities in Assam, India, the proportion of Assemblage B and A infections in 18

infected people was 61% and 39%, respectively (Traub et al., 2004). Apart from these

studies, no large-scale surveys of Giardia infections in humans have isolated and

genetically characterised the isolates of Giardia detected and so it is not possible to

determine the distribution and prevalence of human-infective genotypes. Such surveys will

provide valuable data since there is evidence that Assemblages A and B genotypes differ in

virulence. A longitudinal study in day-care centres in Perth, Western Australia, found that

children infected with isolates of Giardia belonging to Assemblage A were 26 times more

likely to have diarrhoea than children infected with Assemblage B isolates (Read et al.,

2001). Thus children infected with isolates of Giardia from Assemblage B will not be

excluded from such day-care centres since exclusion is dependent on the occurrence of

diarrhoea. This would explain why infections with Assemblage B are more common in

such environments. Children with such infections are likely not to be treated which also

raises questions about the long term consequences of such chronic infections if they persist

and there is no self cure. This is thought to be significant in situations where infected

children are disadvantaged in terms of nutrition and exposure to concurrent enteric

infections with other parasites such as Hymenolepis and Ancylostoma. This is the situation

in isolated Aboriginal communities in northern Australia, where Giardia infections are

recognised as contributing to nutritional disorders and poor growth. In such communities,


infections with isolates of Giardia from Assemblage B are more common than those

with Assemblage A (Meloni et al., 1995; Thompson and Meloni, 1993; Hopkins et al.,

1999).

5.2.2. Cattle

Attention has focussed on the widespread and exceptionally high levels of infection of

Giardia in young livestock, particularly calves (Thompson, 2000; Olson et al., 2004).

Giardia has been found in both beef and dairy cattle throughout the world, and longitudinal

studies have consistently demonstrated prevalence rates of 100% (OHandley, 2002;

OHandley et al., 1999; Ralston et al., 2003; Xiao and Herd, 1994). The infection pattern of

Giardia is similar between beef and dairy cattle (OHandley et al., 1999; Ralston et al.,

2003) with cysts appearing in the faeces at approximately 4 weeks of age. Calves have the

highest excretion intensities of 105106 cysts per gram of faeces between 4 and 12 weeks of

age (OHandley et al., 1999; Ralston et al., 2003). A periparturient rise in cyst excretion has

also been demonstrated (Ralston et al., 2003). Transmission occurs among infected calves

as well as chronically infected adults, but the frequency of transmission is particularly high

amongst dairy calves (Xiao and Herd, 1994; OHandley et al., 1999, 2000).

Giardia infections in cattle are clinically important and may be of economic

significance by impairing performance (OHandley et al., 2001; Olson et al., 2004).

Giardia has been implicated as an aetiological agent alone and in combination with other

enteric pathogens in calf diarrhoea (Xiao and Herd, 1994; Olson et al., 1995; OHandley et

al., 1999; Huetink et al., 2001). These studies demonstrated that concurrent infections with

Giardia and Cryptosporidium were common in calves and the primary cause of diarrhoea

in calves less than 30 days of age, whereas Giardia alone was associated with diarrhoea in

older calves. The impact of chronic giardiasis in calves on performance may be reflected in

a reduced rate of weight gain, impaired feed efficiency and decreased carcass weight as

demonstrated in experimentally infected lambs (Olson et al., 1995). However, this has yet

to be demonstrated, and in a preliminary study giardiasis was not found to affect average

daily gain, feed intake or feed efficiency in feedlot calves, but there may have been an

insufficient number of animals to demonstrate a production effect (Ralston et al., 2003).

Recent studies have demonstrated that calves in dairy and beef herds may harbour one

of two genotypes of G. duodenalis. Although the livestock genotype (Assemblage E) of

Giardia appears to occur most frequently in cattle, studies in Canada and Australia have

shown that a small proportion of cattle in a herd,

low sensitivity of conventional detection methods, the fact that the parasite may be present

at subclinical levels and the intermittent nature of cyst excretion (McGlade et al., 2003).

Although Giardia is common in dogs and cats, it is rarely associated with clinical

disease in these animals. However, if clinical giardiasis is reported, it is usually associated

with kennel or cattery situations, where the effects of over crowding may cause stress and

exacerbate the effects of an infection (Robertson et al., 2000). Treatment of Giardia-

infected dogs and cats is usually recommended whether or not they are clinically ill,

because of the perceived potential for zoonotic transmission. The recent development of

vaccines for the treatment and prevention of Giardia infections in dogs and cats and their

apparent ability to reduce the duration of shedding of cysts may provide an alternative to

drugs for reducing carrier rates in pets and subsequent environmental contamination

(Olson et al., 2000).

Molecular epidemiological studies have shown that dogs may be infected with their

own, host-adapted genotype of Giardia, as well as with zoonotic genotypes (see below).

5.2.4. Wildlife

Although wildlife are susceptible to infection with zoonotic genotypes of G. duodenalis,

the limited evidence collected under natural, pristine conditions suggests that wildlife

harbour their own genotypes/species of Giardia.

For example, genotypic characterisation of Giardia from native marsupials in Australia

has shown that they are infected with a novel, genetically distinct genotype of Giardia

(Adams and Thompson, 2002). Thus the finding of high prevalence rates of G. duodenalis

in a wide range of native animals in Tasmania may not necessarily constitute a risk to

public health as recently proposed (Kettlewell et al., 1998). Distinct genotypes have also

been recovered from microtine rodents and the majority of birds where genotypic

characterisation of the isolates has been undertaken (McRoberts et al., 1996; Thompson,

2002; Monis and Thompson, 2003). However, a recent study in Australia comparing the

disease status of populations of house mice on subantarctic Macquarie Island and

Boullanger Island which has an arid Mediterranean climate, found that the mice harboured

several different genotypes of Giardia (Moro et al., 2003). Zoonotic genotypes were found

in mice on both islands, but on Macquarie Island, mice were also infected with novel

genotypes of G. duodenalis. The source of infection in these mice has still to be

determined.

Animals such as beaver, nutria and deer are also commonly infected with Giardia in

North America with prevalence rates often over 50% (Dixon et al., 2002; Dunlap and

Thies, 2002; Heitman et al., 2002; Rickard et al., 1999) but there is surprisingly little

information on what genotype of Giardia they carry. Recent studies have, however,

confirmed that beavers and white-tailed deer in the wild can harbour infections with

zoonotic genotypes of G. duodenalis (Appelbee et al., 2002; Trout et al., 2003).

5.3. Zoonotic and waterborne transmission

Molecular data have shown that livestock, pets and wildlife may harbour zoonotic

genotypes of G. duodenalis, as well as genotypes that appear to be host specific

(Thompson, 2002). However, although the WHO has considered Giardia to have zoonotic


potential for over twenty years (WHO, 1979), either through direct faecal-oral or

waterborne routes of transmission, direct evidence has been lacking (Thompson, 1998).

The consumption of drinking water other than metropolitan mains, or other filtered

supplies represents a significant risk for giardiasis (Hoque et al., 2002; Jakubowski and

Graun, 2002). The majority of waterborne giardiasis outbreaks in humans have occurred in

unfiltered surface or groundwater systems impacted by surface run off or sewage

discharges (Jakubowski and Graun, 2002). Irrigation waters used for food crops that are

traditionally consumed raw may also represent a high risk as a source of Giardia (Thurston-

Enriquez et al., 2002). Environmental contamination of such water systems and supplies

may result from human, agricultural and wildlife sources (Heitman et al., 2002). These

latter authors undertook a two-year investigation to determine the significance of each

source with regard to the presence of Giardia in the environment and found sewage effluent

to have the highest prevalence of Giardia, although the concentration of cysts was minimal

compared with that detected in cattle faeces. Cow-calf sources contained the highest

concentration of Giardia. Although the overall prevalence of Giardia was lower in wildlife

than in the other two sources tested, the prevalence in aquatic mammals such as beaver and

muskrat was quite high, as for similar studies (see above). An interpretation of these results

in the context of the source(s) of human infections with Giardia should be made in

conjunction with data on the genotypes of Giardia in these animals.

As mentioned above, the greatest zoonotic risk is from genotypes of Giardia in

Assemblage A, particularly those in the AI subgroup, and to a lesser extent genotypes in

Assemblage B. In contrast, the animal-specific genotypes appear to be host-adapted,

restricted to livestock, dogs and rodents (Table 2). There is no epidemiological evidence

to suggest that they occur frequently in the human population and thus their zoonotic risk

appears minimal. This would certainly appear to be the case in cattle. Although there is

clearly a definite potential for microbial contamination of ground and surface waters

from livestock operations (Donham, 2000), studies suggest that the public health risk

from cattle may be minimal, at least in North America and Australia where genotyping

has been undertaken and has shown that the livestock genotype appears to predominate

in cattle (OHandley et al., 2000; Hoar et al., 2001). Cattle are susceptible to infection

with zoonotic genotypes of Giardia and it has been shown that calves infected with

Giardia commonly shed from 105 to 106 cysts per gram of faeces (Xiao, 1994;

OHandley et al., 1999). Thus, even a few calves infected with genotypes in Assemblage

A could pose a significant public health risk directly to handlers or indirectly as an

important reservoir for human waterborne outbreaks of giardiasis. This is of potential

public health significance and may put producers, and other members of the community,

at risk. However, longitudinal studies in Australia suggest that zoonotic genotypes may

only be present transiently in cattle under conditions where the frequency of

transmission with the livestock genotype (Assemblage E) is high and competition is thus

likely to occur. In contrast, a recent molecular epidemiological study in Uganda where

humans appear to have introduced Giardia into a remote national park are also thought to

have been the source of Giardia in a small number of cohabiting dairy cattle (Graczyk et

al., 2002).

The occurrence of Giardia in wildlife, particularly of isolates that are morphologically

identical to G. duodenalis, has been the single most important factor incriminating Giardia


as a zoonotic agent. It is therefore surprising that there is so little evidence to support the

role of wildlife as a source of disease in humans, since this has dominated debate on the

zoonotic transmission of Giardia, and in particular when water is the vehicle for such

transmission. Indeed, it was the association between infected animals such as beavers and

waterborne outbreaks in people that led the WHO (1979) to classify Giardia as a zoonotic

parasite. It is therefore surprising that so little information is available on the genotypes of

Giardia affecting wildlife, as well as in people infected with Giardia as a result of a

waterborne outbreak.

Although wildlife, particularly aquatic mammals, are commonly infected with Giardia

there is little evidence to implicate such infections as the original contaminating source in

water borne outbreaks. It would appear that such animals are more likely to have become

infected from water contaminated with faecal material of human, or less likely, domestic

animal origin. They will thus serve to amplify the numbers of the originally contaminating

isolate (Bemrick and Erlandsen, 1988; Monzingo and Hibler, 1987; Thompson, 1998;

Thompson et al., 1990).

Some studies (e.g. Isaac-Renton et al., 1993) have genetically characterised isolates

associated with waterborne outbreaks, but the typing schemes used did not allow

correlation with the currently recognised assemblages. The one study that did genotype

Giardia of beaver origin, confirmed previous suggestions that the source of Giardia

infection in beavers was likely to be of human origin (Dixon et al., 2002; Monzingo and

Hibler, 1987; Rickard et al., 1999). In this study, 12 of 113 (10.6%) beaver faecal samples

from 6 of 14 different riverbank sites in southern Alberta, Canada, were positive for

Giardia, and all those genotyped using 16S-rRNA gene belonged to the zoonotic genotype,

Assemblage A (Appelbee et al., 2002).

At the present time, it is not known whether zoonotic transmission impacts significantly

on the aetiology of waterborne outbreaks of giardiasis. To date, Giardia of human origin

appears to be the main source of water contamination and as such may impact negatively

on ecosystem health leading to infections in aquatic wildlife. Recent studies have

demonstrated that filter-feeding molluscs are useful indicators of the presence of

waterborne pathogens. Genotypic characterisation was recently utilised in a study that

isolated Giardia cysts from clams in an estuary in North America (Graczyk et al., 1999).

All isolates were identified as belonging to genotype Assemblage A, highlighting

contamination with faeces of mammalian origin, most probably human, that contained G.

duodenalis cysts of public health importance. Such filter-feeding molluscan shellfish can

concentrate waterborne pathogens and thus in combination with appropriate genotyping

procedures can serve as biological indicators of contamination with Giardia cysts and can

thus be used for sanitary assessment of water quality.

The possibility of other animals acting as sources of waterborne contamination with

Giardia of zoonotic significance seems to be minimal. Dogs and cats are susceptible to

infection with zoonotic genotypes of Giardia but the chances of a contamination event

from a dog or cat leading to a waterborne outbreak in humans would seem unlikely.

However, in the case of the Sydney, Australia 1998 water crisis, dead dogs found near a

water supply (McClellan, 1998), were incriminated as a possible source of contamination

with Giardia, but no evidence of infection in the dogs, nor whether any isolates recovered

were genotyped, was provided.


Although the clinical significance of Giardia in dogs and cats appears to be minimal,

there has been much speculation about the public health significance of such infections in

pets. In domestic, urban environments of Australia, genotypes from Assemblage A and the

dog genotype, Assemblage D, are both equally common in dogs (Thompson et al., 1999).

It is therefore considered that there are probably two cycles of transmission taking place in

domestic urban environments with the possibility of zoonotic transmission of Assemblage

A genotypes between pets and their owners. This was highlighted in the study by Bugg et

al. (1999) which found that dogs from multi-dog households were more commonly

infected with Giardia than dogs in single-dog households, emphasising the potential ease

with which Giardia can be spread to in-contact animals and therefore presumably humans

(Bugg et al., 1999). In contrast, a recent survey of domestic dogs in Japan found all isolates

to belong to the dog-specific genotype, Assemblage D (Abe et al., 2003).

From the point of view of direct zoonotic transmission, the finding that similar

genotypes are dispersed in different hosts is not by itself conclusive evidence that zoonotic

transmission is taking place. A better assessment for this can only come from studies which

examine the dynamics of Giardia transmission between hosts living in the same

geographical area or localised endemic focus. Molecular epidemiological studies in

localised endemic foci where the frequency of transmission of zoonotic and non-zoonotic

genotypes is high, such as in Aboriginal communities in Australia, have shown that the dog

genotype predominates in infected dogs (Hopkins et al., 1997). In contrast, in remote tea

growing communities in Assam northeast India, where Giardia occurs in both humans and

their dogs, 20% of dogs were found to be infected with Giardia, but they were all infected

with zoonotic genotypes, mostly from Assemblage A. This difference may reflect a closer

association between individual dogs and their owners in the tea growing communities, and

the frequency with which dogs are able to eat human faeces in these communities (Traub et

al., 2002). In Aboriginal communities in Australia, such behaviour by dogs is less common

and the dogs tend to stay together in packs. In environments where the infection pressure is

less, such as domestic households in urban settings, dogs are just as likely to harbour

zoonotic genotypes of Giardia from Assemblage A as they are their own dog genotype

(Assemblage D).

Presumably, in environments where the frequency of transmission of different

genotypes is high, as in Aboriginal communities in Australia, competitive interactions are

likely to ensure that the host adapted genotypes predominate in respective host species, as

with the livestock genotype in dairy cattle (see above). Under such conditions where

Giardia infections are common in humans and dogs, dogs would be equally likely to be

exposed regularly to infection with both dog and zoonotic genotypes of G. duodenalis

(Hopkins et al., 1997; Thompson, 2002). Australian Aboriginal communities represent

highly endemic foci of Giardia transmission with high rates of infection in children and

dogs, often greater than 50% (Meloni et al., 1993; Thompson, 2000). Under these

circumstances, experimental evidence suggests that the dog-adapted genotype will, by

competitive exclusion, out-compete other genotypes preventing their colonisation in the

dog intestine (Thompson et al., 1996). In domestic, urban environments, and in the tea

growing communities in Assam, India, the frequency of dog to dog transmission will be

less frequent and thus infections acquired with Assemblage A genotypes in dogs are likely

to persist.


Although within-host competition between genotypes may preclude the establishment

of infections with zoonotic genotypes in dogs living in environments where the frequency

of transmission is very high, the role of dogs as mechanical vectors of zoonotic Giardia

genotypes in such situations must be considered. Under such conditions, dogs are very

likely to be contaminated on their coats and muzzle with cysts of Giardia which could be

readily transferred to children.

The study in Assam, India by Traub et al. (2004), has provided the first direct evidence

of zoonotic transmission between dogs and humans, by finding the same genotype of

Giardia in people and dogs, not only in the same village, but also in the same household.

Giardia isolates were characterised at three different loci; the SSU-rDNA, elongation

factor 1-alpha (ef1-a) and triose phosphate isomerase (tpi) gene. Evidence for zoonotictransmission was supported by strong epidemiological data showing a highly significant

association between the prevalence of Giardia in humans and the presence of a Giardia

positive dog in the same household. A major finding of this study was the importance of

using multiple loci when inferring genotypes to Giardia in epidemiological investigations

(Traub et al., 2004).

6. Diagnosis and detection

Diagnosis of Giardia by traditional microscopic methods following the application of

faecal concentration techniques, especially zinc sulphate flotation and centrifugation

(Zajac et al., 2002), remain a reliable indicator of infection. However, the detection of G.

duodenalis by microscopy or faecal ELISA is of limited epidemiological value, especially

in terms of the source of infection. The development of direct immunofluorescence

microscopy has generally improved the sensitivity of detecting and quantitating faecal

Giardia cysts and may allow for more accurate determination of prevalence rates and cyst

excretion intensities compared to conventional microscopy (OHandley, 2002). However,

it is still not possible to discriminate between morphologically identical, or similar,

organisms that are genetically different using immunofluorescene. Such genotypic

identification is not possible using microscopy alone, and as with any epidemiological

investigation, there is a need for sensitive and specific diagnostic procedures for detecting

the aetiological agents of infectious disease. With Giardia, molecular techniques,

particularly PCR-based procedures have greater sensitivity and specificity than

conventional diagnostics that are reliant on microscopy and/or immunodiagnosis

(McGlade et al., 2003). Molecular techniques have been developed that can provide

information on the genotype or species of Giardia present by combining PCR with

restriction fragment length polymorphism (RFLP) analysis, without having to resort to

costly and time-consuming sequencing (Groth and Wetherall, 2000; Amar et al., 2002;

Caccio et al., 2002). In addition to their excellent sensitivity and specificity, such

procedures are quick and amenable to large sample throughput (Morgan, 2000). Their cost

is declining but there is a need for better technology transfer so that such diagnostic

procedures can be applied directly in the laboratories of developing countries.

One of the major advantages of such PCR-based procedures is the ease of interpretation

which usually involves the visualization of a small number of bands on a gel. However, the


very high sensitivity of most PCR-based procedures can also present problems of

interpretation. For example, a recent survey of parasites of domestic cats using microscopy

found that 5% were infected with Giardia whereas PCR revealed that 80% were positive, a

level supported by the detection of faecal antigen (McGlade et al., 2003). Similarly, a

survey of dogs in India found a 3% prevalence using microscopy, compared to 20% with

PCR (Traub et al., 2004). Such results could not be accounted for by the intermittent nature

of cyst shedding by Giardia (McGlade et al., 2003) and raise questions concerning both the

clinical and epidemiological significance of such presumably low-level infections with

Giardia that result in minimal excretion of infective stages.

A greater awareness of parasite contamination of the environment and its impact on

health has precipitated the development of better detection methods for waterborne

pathogens such as Giardia (Slifko et al., 2000). Filtration, flocculation, flow cytometry,

immunomagnetisable separation and immunofluorescence with monoclonal antibodies

together constitute the currently acceptable methodology for detecting Giardia in drinking

water (Slifko et al., 2000). These approaches are also used for testing raw and treated

waters, although PCR-based procedures are increasingly being used to complement

immunofluorescence and add a measure of quality control. In addition, molecular

techniques can provide genotypic characterisation of the parasites isolated from water, thus

providing valuable data for determining the source of contamination.

7. Treatment and control

Nitroimidazoles and benzimidazoles are effective antigiardial drugs for treating

infections in humans. The superior palatability and efficacy of benzimidazoles such as

albendazole, offer a useful alternative to the nitroimidazoles, particularly where mass

chemotherapy is required, compliance is an issue, or where treatment failures have

occurred following nitroimidazole treatment. In dogs and cats, benzimidazoles such as

fenbendazole/febantel are proving very valuable alternatives to nitroimidazoles (Barr et al.,

1998; Zajac et al., 1998).

In livestock, benzimidazoles, such as fenbendazole and albendazole, have been shown

to be effective in the elimination of Giardia from both housed and range calves (Xiao et al.,

1996; OHandley et al., 2001; Garossino et al., 2001). Treating calves with fenbendazole

was also able to improve the mucosal microvillus structure and function within seven days

of initiating treatment (OHandley et al., 2001).

Although chemotherapy may be highly effective in eliminating infection, re-infection

frequently occurs if the sources of environmental contamination are not eliminated and the

frequency of transmission is high. This applies to both human and animal infections,

particularly in localised endemic foci such as communities and institutions where hygiene

is inadequate or easily compromised, dairy farms, kennels and catteries (Reynoldson et al.,

1998; Thompson, 1998; OHandley et al., 2001).

Infections with Giardia stimulate humoral immunity that results in self-limiting

infection in many animal species (Olson et al., 2000). Unfortunately, it may take several

months for the host to produce protective antibodies that can eliminate the parasite. Calves

have been shown not to mount an effective humoral immune response against Giardia even


after 100 days of infection (OHandley et al., 2003). Lactating cows produce colostrum and

milk with anti-giardial activity and the consumption of antibodies in colostrum may afford

protection to young calves against infection, since Giardia is usually observed in calves

greater than 34 weeks of age. Dogs and cats have been protected following vaccination

with a commercially available trophozoite extract vaccine (Olson et al., 2000). Such a

prophylactic approach may have potential application in beef and dairy cattle but has not

yet been evaluated experimentally in livestock.

8. Conclusions and looking to the future

The genetic characterisation of Giardia from different hosts and geographical areas has

now provided a wealth of data to support a revised taxonomy of Giardia affecting

mammalian hosts. As a result of such studies, the zoonotic potential of Giardia is no longer

in doubt, but there is limited data on the frequency of zoonotic transmission. What data

there is available suggests that animals are unlikely in most cases to be the original

contaminating source of Giardia in waterborne outbreaks, although aquatic wildlife are

capable of amplifying zoonotic genotypes of Giardia that may contaminate water.

However, there is still very little information available on the genotypes of Giardia

naturally occurring in wildlife, both aquatic and terrestrial. In contrast, a number of studies

have genotyped Giardia in domestic animals, particularly livestock and companion

animals, and have found that they may be infected with zoonotic or species-specific

genotypes. The frequency of infection with zoonotic genotypes appears to be much less in

livestock than in companion animals, particularly domestic dogs. However, the role of dogs

in the zoonotic transmission of Giardia is clearly dependent upon the nature of the

interaction between dogs and their owners and further studies are required in different

endemic foci. Understanding the transmission dynamics of Giardia in endemic areas where

the frequency of transmission is high will require the application of more discriminatory

genotyping tools than those currently available. The search for such tools is an essential

pre-requisite in the development of genetic markers for important phenotypic traits of

Giardia, particularly drug sensitivity and virulence, both of which have been shown to vary

between isolates of the parasite.

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The zoonotic significance and molecular epidemiology of Giardia and giardiasisIntroductionWhat is Giardia? Historical perspectives and evolutionary biologyCurrent taxonomyDevelopmental biology and pathogenesisTransmission-the impact of molecular epidemiologyHost specificityCycles of transmissionHumansCattleDogs and catsWildlife

Zoonotic and waterborne transmission

Diagnosis and detectionTreatment and controlConclusions and looking to the futureReferences

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giardia infections

giardia duodenalis

hostsof giardia

waterborne transmission

cycles of transmission

zoonotic origin

thecontext of zoonotic

frequency ofsuch transmission