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Antimicrobial properties of Allium sativum (garlic)

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Abstract

Although garlic has been used for its medicinal properties for thousands of years, investigations into its mode of action are relatively recent. Garlic has a wide spectrum of actions; not only is it antibacterial, antiviral, antifungal and antiprotozoal, but it also has beneficial effects on the cardiovascular and immune systems. Resurgence in the use of natural herbal alternatives has brought the use of medicinal plants to the forefront of pharmacological investigations, and many new drugs are being discovered. This review aims to address the historical use of garlic and its sulfur chemistry, and to provide a basis for further research into its antimicrobial properties.
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Critical Reviews in Microbiology
ISSN: 1040-841X (Print) 1549-7828 (Online) Journal homepage: https://www.tandfonline.com/loi/imby20
Current Trends in Research into the Waterborne
Parasite Giardia
Samantha Lane & David Lloyd
To cite this article: Samantha Lane & David Lloyd (2002) Current Trends in Research
into the Waterborne Parasite Giardia, Critical Reviews in Microbiology, 28:2, 123-147, DOI:
10.1080/1040-840291046713
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Published online: 29 Sep 2008.
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Critical Reviews in Microbiology, 28(2):123–147 (2002)
Current Trends in Research into the
Waterborne Parasite
Giardia
Samantha Lane and David Lloyd
*
School of Biosciences, Microbiology (BIOSI 1), Main Building, Cardiff University, PO
Box 915, Cardiff CF10 3TL, Wales, U. K.
*Correspondence: Prof. David Lloyd, tel. +44-29 20874772, fax +44-29 20874305, E-mail lloydd@cf.ac.uk
ABSTRACT: The waterborne flagellated parasite Giardia intestinalis continues to be the most
frequent protozoan agent of intestinal disease world-wide, causing an estimated 2.8 × 108 cases
per annum. Severe symptoms of diarrhea and sickness can be persistent and even life threatening
in the immunocompromised, in infants, and in the aged, although self-limiting in the majority of
patients. Despite a growing awareness and intensified research many uncertainties remain,
especially with respect to the risk of potential zoonotic transmission. Water supplies can be
monitored for cysts using automated cytofluorimetric immunoassays, but this does not measure
infectivity. Filtration provides the best protection, because cysts are highly resistant to chlorine
and ozone. Other incompletely elucidated aspects include mechanisms of pathogenicity, host
reaction to infection, immunity and parasite control using vaccines or antigiardial compounds;
the 5-nitroimidazole metronidazole is the most effective of these. Molecular typing of various
isolates indicates that most animal parasites are not infective to humans, but those that are can
be genotypically classified as assemblage A or B. The phylogeny of the organism remains
uncertain, but there is a growing opinion that Giardia is not an ancient primitive eukaryote, but
that it is derived from a more complex mitochondria-containing protozoon.
KEY WORDS: protozoal parasite, dysentery, diarrhea, chemotherapy, pathogenicity.
I. INTRODUCTION
Giardia is a waterborne, flagellated, bi-
nucleated protozoan, discovered by Van
Leeuwenhoek in 1681.1 Belonging to the class
Zoomastigophorea and order Diplomonadida,2
it is a parasite of mammals and other animals,
including reptiles and birds. Giardia is the
most common source of intestinal infection in
the developed world,3 with an estimated 2.8 ×
108 infections per year in humans,4 and is a
serious cause of disease in developing coun-
tries. It causes the condition giardiasis, which
is characterized by diarrhea and malabsorp-
tion.5 Giardia has a characteristic morphol-
ogy, the vegetative trophozoites are about
15 µm in length, teardrop shaped (in Giardia
lamblia) with two nuclei of equal size at the
anterior end. They have an adhesive disc made
of microtubules, and two median bodies. There
are four pairs of flagella, one anterior pair,
two posterior pairs, and a caudal pair (Figures
1a and 1b). Trophozoites of Giardia swim
freely in the duodenum and ileum but also
attach to the intestinal epithelium.
The parasite has a two-stage life cycle
consisting of the trophozoite, and a 10- to
12-µm-long cyst, which is the infectious
form. In its encysted stage the organism is
highly resistant to chlorination and ozo-
nolysis and can remain viable for several
months, especially in cold water lakes,
124
FIGURE 1b. Ventral surface at magnification ×10,000. The ventral disc (D), its dense lateral
crest (L) and the ventrolateral flange (F) are all seen in the anterior of the cell. Midway, the
ventral groove (G) appears with the emerging ventral flagella and the anteriolateral flagella (A)
emerge at the periphery. Posterior, the posteriolateral flagella (P) emerge and the caudal (C)
flagella emerge from the tail. (Figure courtesy of Dr. Janine C. Harris and Mr. Michael Turner.)
FIGURE 1a. Dorsal surface of a two trophozoite and its structures at a magnification of x 7500.
(Figure courtesy of Dr. Janine C. Harris and Mr. Michael Turner.)
125
reservoirs, and streams. Infection occurs
when the cyst, usually transmitted by con-
taminated water, is ingested. Excystation
then occurs in the upper intestine. This
produces two trophozoites, which then
replicate asexually by binary fission.1 Gia-
rdiasis can often eventually be fought off
unaided by a healthy immune system, but
in individuals with compromised immu-
nity it is extremely dangerous and some-
times fatal.6 Giardia species names were
originally based on the host in which the
organism was found. However, new iden-
tification based on genetic analysis dis-
covered that some of those found in differ-
ent hosts were identical, and species are
now based on slight morphological differ-
ences. The teardrop-shaped Giardia
lamblia (synonyms Giardia intestinalis and
Giardia duodenalis) infects humans and
other mammals, whereas Giardia muris,
which is round in shape infects rodents.7
Reported cases of giardiasis are increas-
ing,8,9 and the possibility of zoonotic trans-
mission is becoming a major concern.3 If
it were possible for the protozoa to pass
from animals to humans, then cattle and
other livestock as well as domestic pets
would be a large reservoir of infection.
The elimination of the parasite from the
natural environment and supplies of po-
table water is not possible, and methods
for identification using cytological and im-
munological techniques, monitoring and
control by filtration are assiduously re-
searched. Together with the work on in-
fectivity and pathogenicity as well as che-
motherapy, these are the major topics of a
burgeoning literature. The continuing threat
to public health has led to continuing re-
search into Giardia. This review examines
the main areas of current research being
carried out into Giardia and highlights the
aspects of the subject that require further
investigation.
II. EVOLUTIONARY POSITION AND
SIGNIFICANCE
Giardia is of notable evolutionary im-
portance. It is amitochondriate and therefore
was considered one of the earliest diverging,
and thus most primitive, eukaryotic organ-
isms, splitting from the phylogenetic tree
before the development of mitochondria.
However, more recently there has been a
discovery of mitochondrial genes such as
chaperonin and valyl-tRNA synthetase in the
organism.10,11 Two explanations for the pres-
ence of these genes have been explored.7,12 It
could mean that Giardia spp. at one time
possessed mitochondria or an endosymbi-
otic progenitor and have since lost them. It is
also possible that Giardia were never en-
dowed with mitochondria but had endosym-
bionts of alpha-proteobacterial sources in the
past.7 Numerous analytic techniques have
shown that Giardia are primitive, and this
idea is generally accepted, although one phy-
logenetic analysis involving tubulin align-
ments in fact gave the lowest phylogenetic
position to Entamoeba histolytica.13 There-
fore, the phylogenetic position of Giardia is
currently a matter of considerable contro-
versy.
G. lamblia is considered a model organ-
ism for studying evolution because of its
basal position in the phylogenetic tree. By
comparing processes in Giardia with those
in organisms higher up the tree, it is thought
possible to see how these processes have
evolved, or if they have been conserved
through evolutionary time. Giardia may also
provide clues to the origins of eukaryotic
processes and structures. Using Giardia as
an evolutionary ‘tool’ has been exploited in
many areas of science. Studies on three novel
Giardia lamblia protein-disulfide isomerases,
which have a role in nascent protein folding
in the endoplasmic reticulum, have shown
that Giardia has unusual enzymes with only
126
one active site.14 However, these enzymes
have an active site sequence motif (CGHC)
that resembles those in eukaryotic protein
disulfide isomerases with multiple sites and
differs from those belonging to other groups
with only one active site. The three proteins
examined were fully able to rearrange disul-
fide bonds and one (giardial protein-disul-
fide isomerase-2) showed some oxidant and
reductant activities. This is the first time
protein-disulfide isomerases with only one
active site have been shown to play a com-
plex role in protein cross-linking. It is hoped
that this will provide information on the
evolution of other activities carried out by
the eukaryotic endoplasmic reticulum, in-
cluding protein disulfide formation and
isomerization.14
Giardia is sometimes thought of as a ‘link’
between prokaryotes and eukaryotes.15 It is
considered eukaryotic (e.g., its enolase has
characteristic insertions and deletions).16 Yet,
it has some characteristics that are not dem-
onstrated by, or are rare in, other eukaryotes,
such as anaerobic metabolism.17 It expresses
a proteobacterial-like Dna K HSP70 homo-
logue with an extremely divergent sequence
and with unique insertions.18 Although this is
most similar to proteobacterial and mitochon-
drial sequences, unlike cpn60 it could not be
assigned a specific phylogenetic relationship.
Despite this there now appears to be evidence
that Giardia is a ‘typical’ eukaryote in many
ways.19 The sequencing of Giardia histone
genes has been carried out.20 This could aid
the understanding of histone evolution and
also displays evidence for the eukaryotic na-
ture of Giardia. The genes coding for core
histones H2a, H2b, H3, and H4 have been
sequenced. A conserved, gene-specific ele-
ment (GRGCGCAGATTVGG) is reported to
sit upstream from the coding region in each
core histone gene. The authors describe how,
“the derived amino acid sequences of all four
histones were similar to their homologs in
other eukaryotes”.20 This study also reports
that “the G. lamblia proteins do not represent
an intermediate stage between archaeal and
eukaryotic histones”. Archeal-type prolyl and
alanyl t-RNA synthetases have been described
in Giardia.21 In addition to this, a similar
finding comes from sequences of Giardia
lamblia ribosomal proteins.19
Rat or yeast cells were used to represent a
‘typical’ eukaryote.19 The report explains that
Giardia has shorter small and large subunit
rRNAs than those of ‘typical’ eukaryotes, and
thus has smaller ribosomes. However, the au-
thors claim their results show the protein com-
ponents of the ribosome to be ‘typically’ eu-
karyotic. It concludes that many features of
diplomonads (e.g., structures, processes, genes,
and proteins) are typically eukaryotic; thus, the
posttranslational polyglycylation of alpha and
beta tubulin, which occurs widely in eukary-
otes, has also been found to occur in Giardia
lamblia.22 Although experiments with gamma
tubulin have shown Giardia to have unusual
mitotic spindle assembly, a role for gamma
tubulin is also ubiquitous among eukaryotic
evolution.23
III. THE
GIARDIA
GENOME
The Giardia genome project, started in
1998, intends to map almost the entire ge-
nome of G. lamblia and will help provide
answers to many questions about the organ-
ism and its origins. The project uses a shotgun
approach to sequence the WB strain of
G.lamblia. This strain is used as it is represen-
tative of all Giardia spp. and has no endo-
symbionts or double-stranded RNA viruses.24
Preliminary data from the project is displayed
and updated on the Giardia genome project
database website: www.mbl.edu/Giardia. The
structure of the Giardia genome is especially
interesting, early estimates have given way to
a reported genome complexity of 1.2 × 107 bp
of DNA,7 and a GC content of 46%.25 Giardia
possess two nuclei in the vegetative phase,
127
and research has been carried out to discover
whether both nuclei perform the same func-
tions. They are morphologically identical and
have both been shown to be transcriptionally
active. Investigations have looked into whether
the two are also genetically identical. Adam7
reports that current evidence suggests that
they are indistinguishable (although see Lloyd
and Wallis26). They contain equal copy num-
bers of rRNA genes and as most of these are
contained on a single chromosome (chromo-
some 1). It is probable that both nuclei con-
tain this chromosome. Why such a simple
organism has evolved with two nuclei carry-
ing out the same functions remains a mystery;
the Giardia genome project may also help to
answer this question.
A review of the genome7 discusses Gia-
rdia chromosomes. Originally using light
microscopy, four chromosomes were identi-
fied. This estimate was increased to five when
more sensitive confocal microscopy was
used. Evidence for the presence of five chro-
mosomes also comes from the use of pulsed-
field gel electrophoresis followed by bind-
ing of chromosome-specific probes. Five
stained bands were identified. A large band
previously thought to be one chromosome
was, in fact, two. The presence of some
weakly stained (minor) bands in other ex-
periments carried out could be due to the
presence of size variants of strongly stained
(major) chromosome bands. A subsequent
study, which used chromosome-specific
probes, has verified that some minor bands
were size variants.7 An investigation into
chromosome size variants has indicated the
genome ploidy of G. lamblia. There is evi-
dence that G. lamblia trophozoites are tetra-
ploid for at least some of their chromosomes.7
It has also been suggested that during the
vegetative cycle each nuclus alternates be-
tween diploid (2N) and tetraploid (4N), hence
the cell cycles between 4N and 8N.27
Further studies of the G. lamblia chro-
mosomes have demonstrated that recombi-
nation occurs only at the telomeres of the
chromosomes.28,29 Here there are repeated
sequences added by the telomerase enzyme.
The repeated sequences in Giardia are
TAGGG; this is similar to other eukaryotes
(which shows Giardia to be ‘typical’ in this
respect). The evolution of the catalytic sub-
unit of telomerase is interesting because its
sequence appears to be related to that of
“reverse transcriptases encoded by eukary-
otic retrotransposable elements”. In an effort
to discover the origins of the subunit, Giar-
dia has been studied.30 The study cites two
possible sources of the telomerase subunit; it
could have been present in the first eukary-
otes, or been derived from the replication
machinery of a retrotransposable element,
early in eukaryotic evolution. That the re-
port suggests the similarity of the Giardia
telomerase sequence compared with that of
other eukaryotes indicates that telomerase is
evolutionary ancient.
IV. ORGANELLES
As well as the absence of mitochondria,
Giardia differ from the rest of eukaryotes by
lacking a variety of organelles, these include
peroxisomes and nucleoli. Giardia tropho-
zoites also lack an early identifiable Golgi
complex, although during encystation one
can be seen. Recently, research has shown
the existence of a Golgi-like structure in
trophozoites.31 Using confocal laser scan-
ning microscopy on labeled cells, and trans-
mission electron microscopy on sections of
freeze-fractured cells, the presence of a mem-
brane cisterne was shown. The membranes
appeared densest in the area surrounding the
nuclei, thus resembling the Golgi apparatus
of other eukaryotic cells.
Further evidence to confirm the pres-
ence of an active Golgi complex comes from
studying another feature of G. lamblia tro-
phozoites. Trophozoites can “undergo anti-
128
genic variation as a result of alterations in
the cell surface protein composition”.32 These
changing, cysteine-rich surface proteins are
known as variant surface proteins (VSPs).
Giardia only expresses one particular VSP
at any one time, but they have the ability to
switch to another one. Nash et al.33 report
that this switching occurs at a frequency of
between 10–3 and 10–4. The VSPs all have
“highly conserved carboxy-terminal trans-
membrane domains and a short invariant
cytoplasmic tail”.32 It has been discovered
that one VSP (VSP4A1) in trophozoites has
a carbohydrate side chain attached to it. In
addition, palmitic acid is also attached to
VSP4A1. There is now evidence that
palmitoylation and glycosylation are com-
mon features of Giardia VSPs.32 Because in
higher eukaryotes similar modification to
plasma membrane proteins is carried out by
Golgi vesicles, this adds to the evidence that
an active Golgi complex is present in tro-
phozoites.
V. PHYSIOLOGY
Giardia spp. are very resistant to changes
in their local environment. During passage
through the gut they are subjected to fluctu-
ating concentrations of ions and pH levels.
Understanding how this resistance occurs
could help the development of new treat-
ments and inactivation techniques. For in-
stance, alcohol dehydrogenase actively plays
an essential role in maintaining the anaero-
bic redox balance of the organism.34 The
maintenance of a membrane potential across
the plasma membrane differs in higher and
lower eukaryotes. In higher eukaryotes the
ψm occurs due to the diffusion of inorganic
ions, such as K+ and Cl, down concentration
gradients previously set up by active trans-
porter molecules, whereas, in lower eukary-
otes (including other protozoa), the potential
depends on the action of H+ pumps.35 Flow
cytometric measurement indicates a mem-
brane potential of –134 mV for Giardia tro-
phozoites. This is similar to that of other
protozoa (for example, Leishmania major
has a potential of –113 mV), although these
values could vary depending on the tech-
niques used to estimate them. The value of –
134 mV is higher than that found in eukary-
otes that are more evolved; a human nerve
cell has ψm of –70 mV.36
Ions and chemical agents that act on the
ψm of Giardia were investigated.35 Of the
agents tested, either the H+ ionophore CCCP
(carbonylcyanide m-chlorophenylhydra-
zone) or the H+ pump inhibitor DCCD
(dicyclohexylcarbodiimide) almost halved
the ψm. This indicates the presence of H+
pumps (Table 1), as in other protozoa. The
ψm of Giardia trophozoites also has a depen-
dence on K+ concentration. Thus, ψm de-
creased as the extracellular K+ concentration
was raised, again as in higher eukaryotes.
Interestingly, in this experiment, despite all
the treatments, the Giardia always remained
viable, as the membrane potential was never
completely depolarized. This confirms that
Giardia can be very resistant to adverse en-
vironments.
Another study also showed how Giardia
cysts maintained their viability despite the
stress of experimental conditions; this inves-
tigated the triggers of encystation, high bile
concentration and alkaline pH.37 However,
Giardia cannot tolerate oxidative stress.
Thus, over a certain limit, the organism fails
to detoxify O2, and when this happens, H2O2
builds up. This can lead to a significant col-
lapse of the membrane potential (decreased
to –20 mV) and total loss of the organisms
ability to take up oxygen.38
The resistant nature of Giardia has been
investigated with respect to the role of potas-
sium in response to hypoosmotic stress by
G. Intestinalis.39 The trophozoites are very
efficient at maintaining cell volume despite
changing environmental conditions. Maroulis
129
et al.39 describe how when Giardia tropho-
zoites are subjected to hypo-osmotic swell-
ing, rapid regulatory volume decrease (RVD)
occurs. Fifty percent of this decrease can be
accounted for by an efflux from the highly
concentrated intracellular pool of alanine
(~50 mM) and other amino acids through an
activated channel. Giardia differs from
higher eukaryotes in using alanine, because
the osmolyte taurine is more usual.39 This
also has an evolutionary significance because
mechanisms in cell volume regulation are
evolutionarily ancient.
VI. PATHOGENESIS
A fuller understanding of the mecha-
nisms involved in pathogenesis could help
the discovery of better treatments and vacci-
nation methods. The trophozoite has a ven-
tral disc made up of various microtubules,
which allows it to attach to intestinal epithe-
lium; alterations to the microvilli in the brush
border of the jejunum lead to malabsorption
in the small intestine.40 There is also a sig-
nificant reduction in the activity of maltase
and sucrase,41 and effects on intestinal ultra-
structure are still being investigated. Giar-
dia may cause rearrangements to F-actin and
α-actinin in the monolayers of the duode-
num, causing transepithelial electrical resis-
tance to decrease.42 This effect was investi-
gated by exposing monolayers to sonicated
trophozoites and live G. lamblia cultures.
Localized condensation of F-actin and loss
of perijunctional α-actinin was observed.
These epithelial rearrangements were re-
ported to be “due at least in part to trophozo-
ite products”. Diffuse shortening of microvilli
was also reported; this causes inhibition of
enzymes produced by the microvilli that are
vital for nutrient transport.3 This report also
suggests that toxins produced by trophozoi-
tes act on the mucosa; this is supported by
evidence for Giardia cells excreting prod-
ucts such as metalloproteases that harm epi-
thelial cells.43
Proteolytic enzymes detected in media
collected from in vitro cultures have been
examined. Gelatin-substrate polyacrylamide
gel electrophoresis showed six bands exhibit-
ing proteolytic activity, two of these were
inhibited by iodoacetic acid, suggesting they
TABLE 1
The Effect of Ions and Inhibitors on ψm of
Giardia intestinalis
trophozoites (control value –134 ± 3mV)
Adapted from Biagini
et al
.35
130
could be cysteine proteases. Another band
was inhibited by the metal chelating agent
EDTA, indicating that metalloproteases could
also be present.43 Another study explains how
the secretion of toxins may be dependent on
the prevailing environmental conditions within
the intestine, for example, the bacteria that
are present.3 The influence of the duodenal
microbiota is also seen as significant in an-
other study.44 This study compared the effect
of giardiasis on conventional and germ-free
mice. The germ-free mice were less affected
by Giardia duodenalis infection, suggesting
that the flora already in the intestine may have
a stimulatory effect on giardial pathogenesis.
The infectious dose of Giardia is thought
to be very small.45 An explanation of why
this is the case stems from the life cycle of
the organism. Experiments that looked at the
genome ploidy of Giardia lamblia have
shown that a single cyst divides to form
multiple cells.27 Thus, a low number of in-
gested cysts can quickly multiply to infec-
tious levels. The phenomenon of VSP switch-
ing in Giardia is also interesting from the
point of view of pathogenesis, as a reason
for its occurrence could be to evade the im-
mune system of the host.46 Although there is
“little evidence that [VSP switching] is re-
sponsible for chronicity of infections”, it is
involved in pathogenesis.3 VSPs are selected
against in immunocompetent hosts and in
immunosuppressed hosts, some are selected
for and others are selected against.46
Host defense has also been reported as
having a large bearing on the duration of
infection, and how severe are the symptoms.3
A suggested role for T cells is in the control
of acute Giardia infections.47 Nitric oxide
(NO) produced by human epithelial cells
may act as a host defense against the proto-
zoa.48 Several NO donors were shown to
inhibit giardial growth, but the cells remained
viable, so NO may be cytostatic. NO donors
also inhibited giardial differentiation. It was
shown that Giardia actively respond to this
host defense mechanism. In models of hu-
man intestinal epithelium, G. lamblia inhib-
ited host NO production by increased con-
sumption of arginine, the substrate used by
NO synthase in the formation of NO.48
VII. HUMAN VACCINATION
A better understanding of the methods
by which Giardia spp. cause infection, and
especially of its antigenicity, will help to
highlight new vaccine targets. Vaccines are
important not only in preventing clinical
symptoms of infection, but also to reduce
cyst shedding from infected animals, as this
would help to reduce transmission of the
disease and lower any zoonotic risk. Indica-
tions that a vaccination may be an effective
option in the control of Giardia have been
described.3 These include the fact that
immunocompromised individuals have much
more severe and chronic symptoms than those
with healthy immune systems and that people
living in endemic areas often show some
innate resistance to the disease.
Humoral immunity is important in elimi-
nating the disease. Good candidate molecules
for vaccine development would be those
antigens that are highly immunogenic
(e.g., cytosolic and cytoskeletal antigens).
Giardia toxins and toxoids have also been
suggested as vaccine components that could
protect the host from infection.3 Subunit
vaccines have not proven as effective as
whole trophozoite preparations. Cytosolic
proteins may be the best candidates of all, as
they are surface expressed and might have
toxin activity. Interestingly, “animals which
have been immunised by whole cell prepara-
tions, are able to recognize common anti-
gens from a wide variety of Giardia iso-
lates”.3 This is an ideal situation for vaccine
development, as a preparation using a given
strain would protect the host from many
strains.
131
VIII. TREATMENT
Many studies have been carried out to
assess the efficacy of currently available
Giardia treatments for animals and humans.
The most widely used animal treatments
currently are benzimidazole carbamates such
as albendazole and mebendazole.49 However,
“there have been relatively few agents used
in therapy”50: alternative treatment regimes
and drug resistance patterns exhibited by
Giardia are major topics. There is again an
emphasis on reducing cyst shedding by live-
stock to the environment; as livestock infec-
tions contaminate water supplies, this could
be of zoonotic concern.
The economic impact of infected cattle
for the farmer must also be considered. An
investigation was carried out to study the ef-
fects of fenbendazole treatment on dairy
calves.51 This study found no benefit of the
treatment regarding dairy production, as calves
from both groups had the same mean body
weight, gained weight at the same speed, and
showed no difference with respect to feed
intake. Despite this, fenbendazole treatment
did reduce the number of calves shedding
cysts, and decreased the duration of episodes
of diarrhea compared with those of a placebo
group. This has implications regarding the
reduction of cyst transmission to the environ-
ment. Nevertheless, reinfection of the ani-
mals occurred within 2 weeks, causing the
number of animals demonstrating fecal cyst
shedding to “return to placebo levels”.51
The occurrence of reinfection shows
fenbendazole treatment to be of only limited
use in reducing transmission. O’Handley et
al.51 suggest that “continuous low dose treat-
ment” could prevent reinfection of calves.
This, however, could eventually lead to the
development of resistance in Giardia, al-
though the report claims that Giardia resis-
tance to benzimidazoles has not yet been
documented. An important result of know-
ing that reinfection occurs at high levels
draws attention to the need for strict hygiene
controls on farms. Despite this, the reinfec-
tion in this study occurred despite the daily
cleaning of pens and the use of disinfectant.
This may be due to the highly resistant na-
ture of Giardia cysts. Environmental rein-
fection was also demonstrated in a study on
the efficacy of oxfendazole for kennelled
dogs suffering from giardiasis.52 In kennels
with poor hygiene, dogs began to excrete
cysts again after the treatment, whereas in
kennels that where disinfected no recurrence
of cyst egestion occurred.
A further study looking at the effects of
fenbendazole treatment in dairy calves, ex-
amined its effect on intestinal function.53 In
this study, the intestinal cells of calves treated
with 5 mg/kg fenbendazole once daily for
3 days were compared with those treated
with a saline placebo. The segments for
examination were taken from the duodenum,
the proximal, and distal jejunum and the
ileum. No trophozoites were found in any of
the intestinal sections taken from the group
treated with fenbendazole, yet all sections of
the placebo group contained numerous tro-
phozoites. Villus height and crypt depth in
the segments showed no significant differ-
ence found between the measurements taken
from the two groups; perhaps the time be-
tween treatment and examination was inad-
equate to reveal any changes.
As the jejunum plays an important role in
nutrient digestion and absorption, it was ex-
amined more closely for the effects of
giardiasis. The jejunal microvilli of the
fenbendazole group had a significantly higher
surface area than the placebo group. The drug-
treated group also had significantly higher
brush border maltase activity, and noticeably
increased lactase activity, although this was
not significantly higher than in the saline
group. This shows that fenbendazole caused a
recovery in the normal absorptive function of
the small intestine ultrastructure, and the di-
gestive disaccharidase enzymes produced
132
there, the activity of which is adversely ef-
fected by giardiasis. Interestingly, the number
of lymphocytes within the epithelium was
significantly reduced in the duodenum and
jejunum segments of the fenbendazole group
compared with those of the saline group. An
increase in intraepithelial lymphocytes is also
associated with the infection of Giardia (Table 2).
This is the first time the effects of fenbendazole
treatment have been examined in cattle in
vivo. They relate their work to an earlier study,
stating that the improvements seen in the drug-
treated cattle examined explain the positive
effects observed on fenbendazole treatment,
such as shorter episodes of diarrhea.51 The
results of this later study, however, offer no
clue as to why it was found that fenbendazole
treatment did not improve dairy production in
the earlier study. Fenbendazole is undoubt-
edly of clinical benefit to cattle, but further
work needs to be carried out to investigate
whether it is also of economic benefit with
regard to production, because this now seems
to be unclear.
Resistance to metronidazole has been
documented in Giardia;54,55 conversion of
the compound into its toxic form, a nitro
radical anion, occurs by a coupled reaction
involving the decarboxylation of pyruvate
by the enzyme PFOR (pyruvate ferredoxin
oxidoreductase) and the reduction of ferre-
doxin. Ferredoxin is responsible for reduc-
ing metronidazole; it is able to do this be-
cause it has a low redox potential. Ellis et
al.54 were able to show that in some resistant
stocks NADPH oxidase is elevated, thereby
increasing competition for electrons away
from the metronidazole electron accepting
mechanism. Liu et al.55 suggest another
mechanism whereby decreased PFOR activ-
ity and decreased ferredoxin levels reduce
the ability of Giardia to convert metronida-
zole into the nitro radical anion. Survival “in
concentrations of the drug as high as 100 µM
can be demonstrated in these resistant or-
ganisms.
Due to the burgeoning threat of metron-
idazole resistance, scientists are actively seek-
ing new compounds with anti-giardial activ-
ity; several unusual alternatives have been
investigated recently. Colloidal bismuth
subcitrate (CBS) has antitrophozoite activity.
TABLE 2
Mean Number of Intraepithelial Lymphocytes, Microvillous Surface Area, and Intestinal
Disaccharidase Activity in
Giardia duodenalis
-Infected Calves, 7 Days Following
Treatment with Fenbendazole or Saline
Adapted from O’Handley
et al
.53
µ
133
CBS was found to rapidly disrupt the attach-
ment of trophozoites to the surfaces of culture
vials possibly by “binding to cytoskeletal
components”.56 Another group tested Allium
sativum (whole garlic); components were
found to be inhibitory toward Giardia, in-
cluding allyl alcohol and allyl mercaptan.57
Whole garlic and allyl alcohol were found to
“collapse the transmembrane electrochemical
membrane potential of the organism”, as well
as causing morphological changes and vacu-
ole formation. There have also been studies
on various isoflavones isolated from the bark
of the tulipwood Dalbergia frutescans, their
activity was tested on G. intestinalis.58 The
isoflavone formononetin was found an active
antigiardial agent; this has an IC50 value of
30 ng/ml; compared with that of metronida-
zole (100 ng/ml). Khan et al.58 suggest that
this compound could be used “as a probe for
a new mechanistic target”. Arrieta et al.59
have described another equally potent natural
product, asarinin.
IX. DETECTION OF GIARDIA AND
RECOVERY FROM WATER
The efficacies of detection and inactiva-
tion methods for Giardia are very important,
especially in drinking water, which is the
main route of transmission to humans. These
procedures are especially important given
that Giardia cysts have high resistance to
chlorine and other disinfectants,60 and even
trophozoites are “able to survive for long
periods in cold water”.61 There are several
current methods of detection. Oda et al.62
describe a size selective continuous flow fil-
tration method for detecting Giardia cysts in
finished water. Pairs of macro and micro
polycarbonate-track-etch membrane filters
with various pore sizes were tested for their
efficacy. It was found that a pair of mem-
branes with 10 µm and 3 µm pore sizes are
very efficient in the recovery of Giardia
cysts. This method is also quick, easy, and
cheap when compared with some previously
used techniques.62
Concentration methods are also used for
detecting Giardia in water, two of these are
the cartridge filtration method and the mem-
brane filtration method. They are evaluated
in a study by Hsu et al.63 The membrane
filtration method was seen to have a higher
recovery rate and more sensitive detection
limit. However, this technique is only prac-
ticable with low turbidity water, whereas the
cartridge method can be used for large sample
volumes of highly turbid water. This study
also makes the important point that many
methods are too expensive to be used in less
developed countries; the cost of equipment
should be considered when designing new
systems.
A method that is capable of showing up
viable cysts separately has also been re-
ported.64 Fluorescein diacetate (FDA) was
used to stain viable cysts, while nonviable
cysts remained unstained. Tetramethyl red
labelled anti-Giardia monoclonal antibodies
(TMR) were then used as a counterstain.
Viewed under epifluorescence microscopy,
viable cysts appear green with a red wall,
while those that are nonviable appear only
red. This allows simultaneous detection and
viability confirmation in a simple, quick
method. The study claims that the technique
can be used for environmental and drinking
water samples. Viable and nonviable cysts
of Giardia duodenalis can be differentiated
by electrorotation. Electrorotation is capable
of detecting morphological and physico-
chemical changes, it is easy, rapid, and can
be applied to drinking water and foodstuffs.65
Techniques that can distinguish live from
dead cysts were originally PCR based, tar-
geting the giardin gene,66 the more recent
techniques have built on this innovation and
they are now more widely applicable.
Studies have analyzed methods used to
detect Giardia cysts in sewage influent and
134
effluent, in sewage works in Scotland.67
Methods of sewage analysis and the effi-
ciency of sewage treatment works (STW) at
removing cysts were addressed. Yarn wound
filter and cartridge filtration methods of
sample collection for analysis were investi-
gated. Filtration through a yarn filter was
found impractical for use with influent as it
was easily clogged by debris; however, this
might be useful when used on effluent. The
cartridge filtration method was reported to
have low recovery efficiency over a wide
range (5.2 to 20%).67 This method was dis-
cussed above and was also found to have
only limited detection limits when applied to
water, although it could be useful for use on
large volume samples of highly turbid wa-
ter.63
The efficiency of treatment plants at re-
moving Giardia cysts was also investigated.
Considerable variation has been reported
between STW.67 It is reported that although
primary settlement can be successful, there
is a wide range of efficiency (63 to 90%),
and secondary treatments are of more im-
portance. Removal can be estimated at 60 to
90% in STW that have both primary and
secondary processes.67 Although high num-
bers of cysts are removed by routine treat-
ment, numbers of them may still be present
in sewage effluent.
Due to the drawbacks of many of the
techniques discussed, molecular methods for
the detection of Giardia have been investi-
gated. A new method for detecting Giardia
in stool samples that uses PCR has been
developed.68 This targets the intergenic spacer
region (IGS) of the multicopy rRNA gene.
The test is very sensitive and can detect 10
cells in100 µl of stool; Thus, it is more sen-
sitive and specific than more conventional
methods of diagnosis such as ELISA (en-
zyme linked immunosorbant assay).68 An-
other study reports that when using enzyme
immunoassay to detect Giardia in stool
samples, one sample is not sufficient to give
a reliable result, and at least two are needed.69
Typing Giardia isolates by molecular tech-
niques is another area being investigated.70
PCR is again used on the intergenic rDNA
spacer region in biotyping. A large bonus of
this method is that in vitro culturing is not
required, so it is a simpler and faster tech-
nique. Because the first use of PCR methods
for the unambiguous identification of Giar-
dia cysts and trophozoites in natural
samples,71 it has proven a very powerful
approach to a difficult problem and one that
has produced a burgeoning literature of its
own.
X. INACTIVATION AND
MONITORING OF CYSTS IN WATER
New and improved techniques to inacti-
vate Giardia have been tested; these meth-
ods are again important due to the resistant
nature of the cysts. Currently, filtration is
really the only method that guarantees cyst
removal. Electroporation could be a proce-
dure that increases the permeability of cysts
to chlorine.60 This study demonstrated that
by exposing Giardia muris cysts to short
bursts of electrical energy, they require a
lower chemical dose of free chlorine to inac-
tivate them. This is thought to work by dis-
rupting the protective cyst wall and increas-
ing its permeability to external chemicals.
Although electroporation applied alone did
not affect the cysts, it did show a statistically
significant improvement in inactivation when
combined with exposure to chlorine.60
The efficacy of chlorination with respect
to cyst viability can be determined by using
bacterial spores as indicators. This is useful,
because the direct detection of cysts can be
expensive and unreliable, as monitoring the
presence of protozoa themselves in finished
water is a regulatory requirement.72 Aerobic
bacteria are present at all stages of water
treatment. They are also resistant to disin-
135
fectants, and their methods of detection are
much simpler than those used for Giardia
directly (although as discussed these meth-
ods are improving). The experiments carried
out showed spores to be “a valuable indica-
tor of disinfecting efficiency”.72
Aerobically produced bacterial spores
can also be used to determine the efficacy of
cyst inactivation by ozone.73 This report also
highlights the problems of current methods
used to monitor cyst contamination in drink-
ing water; it mentions techniques such as
particle counting, microscopic particle analy-
sis, and turbidity. The authors point out the
shortcomings of these methods when used in
routine analysis, that is, they can be expen-
sive, suffer from interference, and those that
measure physical parameters do not indicate
viability. The report suggests that “bacterial
spores should be considered as a promising
indicator of protozoan cyst inactivation in
full scale treatment plants”. Compared with
techniques currently in use, this is simple,
cheap, and reliable and does not require
highly skilled workers.73
Due to the resistance of Giardia to dis-
infectants, other methods of inactivation have
been explored. These include the above-
mentioned ozone, and UV radiation also has
potential. Facile et al.73 state that “because
of its high efficiency, ozone is one of the
best oxidants to use to chemically inactivate
protozoan cysts”. The report goes on to as-
sess the use of UV radiation, saying that it is
“still too early to draw conclusions on their
[UV Technologies] performance in full scale
operations”. However, the use of ultraviolet
radiation to inactivate Giardia muris cysts
has been shown to be successful.74 Experi-
ments carried out on filtered drinking water,
where samples were exposed to radiation
from a medium-pressure UV lamp for vary-
ing periods, assessed the degree of inactiva-
tion using a mouse infection model. It was
found that low UV doses could inactivate
99% of Giardia cysts. Although the increase
in inactivation with rising UV dose reached
a plateau over 8 mJ/cm2, and there may be
some cysts that are more resistant to the
radiation than others, the results are positive.
This type of inactivation therefore has great
potential.74
XI. PREVALENCE OF GIARDIASIS
IN CATTLE
The prevalence of giardiasis in cattle from
different geographical locations has been
examined for patterns or distinct differences
between outbreaks. Researchers have looked
for a correlation between cases and farming
practices.6,75,76 Studies such as these also test
available detection protocols, because
samples need to be efficiently collected.
Calves very frequently suffer from giardia-
sis. Incidences have been reported from
across the globe and some areas have cases
of infection reaching 100%.77 A study car-
ried out in calves in two areas of New Zealand
also looked at strain differentiation of the G.
intestinalis involved.75 Calves become natu-
rally infected by Giardia in the first 8 weeks
of life, and providing they are healthy they
rid themselves of the infection.
Seven hundred and fifteen specimens
were collected during the spring calving sea-
sons of 1998 and 1999 from calves less than
8 weeks of age.75 This study concentrated on
the Manawatu and Waikato regions of North
Island in New Zealand. The specimens were
examined using a commercial immunofluo-
rescence antibody kit. Giardia cysts were
detected in 40.6% of the calves tested. This
was similar on all farms despite them being
separated geographically and having differ-
ent housing, feeding, and water management
practices on each farm. The time of infection
seemed to be significant. Infection is reported
to have generally occurred after the calves
were first transferred from their pens to pad-
docks. Animals that were infected but re-
136
mained in their pens throughout the study
were infected immediately after birth from
an unknown source. No relationship was
observed between Giardia cysts and the
consistency of fecal specimens, even though
other studies have found there to be a rela-
tionship here.6 The typing of isolates was
carried out using PCR amplification primers
that targeted a section of rDNA. Two se-
quences were found, but these did not corre-
late with a particular geographic region, sea-
son, or farming practice.75
A similar study was carried out on dairy
calves from Western Australia and Western
Canada.76 In this study, immunofluorescence
microscopy was used to examine specimens
from calves 2 to 10 weeks of age. The num-
ber of calves found positive for giardiasis
and the number of cysts shed by these calves
did not differ significantly between Austra-
lia and Canada. Prevalence was 58% and
57%, respectively. This is slightly higher
than that in the previous study in New
Zealand (40.6%).75 The calves screened were
housed in various ways, but no difference
was found between those in individual pens,
shared pens, or pastures. The genotypic char-
acterization done in this study showed that
most isolates were of the previously known
livestock type. The study looked at the pos-
sibility of this type being transferred to hu-
mans, but it reports that isolates with the
genetic sequences found in this study have
not been found in humans and may be host
specific. However, some (17%) of the iso-
lates from both countries were found to be of
the most common human genotype (assem-
blage A). This shows that in a minority of
cases, calves can become infected by a geno-
type that is infectious to humans. This is
very interesting from a zoonotic standpoint.
The authors76 also make the point that in-
fected calves shed from 105 to 106 cysts/g of
feces, and therefore even a small number of
calves carrying assemblage A could pose a
large threat to humans.
A third study carried out in different coun-
ties of southeast New York also investigated
the prevalence of Giardia in dairy herds.6
This time a link with the age of cattle was
looked for as well as any correlations be-
tween infection and the season. The popula-
tions of the farms were divided into three
groups; 0 to 6 months, 6 to 24 months, and
older than 24 months. The seasons of collec-
tion were defined as winter (January – March);
spring (April June); summer (July Sep-
tember); autumn (October – December). A
majority (70%) of the farms tested contained
Giardia infections. This makes Giardia much
more prevalent than Cryptosporidium parvum
and C. andersoni. Other protozoa examined
in the study that were found to be present at
13 and 18% of the farms, respectively.6 There
was a significant correlation between the age
of the animals and the risk of Giardia infec-
tion (Table 3).
This study also found that animals suf-
fering from diarrhea were two times more
likely to be infected with Giardia spp. than
animals that did not have diarrhoea. This is
in contrast to the previously discussed study
performed in New Zealand where no rela-
tionship was found between the consistency
of the fecal specimen and the presence of
Giardia.75 There was no relationship found
relating to the season and the infection risk.6
Another such study was carried out on
cattle in three Maryland farms. This looked
at post-weaned and adult cattle.77 Little is
known about the infections of Giardia in
mature animals. The experiments here were
carried out on healthy, asymptomatic cattle.
The chosen farms were a dairy research fa-
cility, commercial dairy, and a beef cattle
breeding and research facility. Giardia in-
fections were found at various levels. No
Giardia infections were found at the dairy
research facility. The commercial dairy had
a prevalence of 10.5% in cows and 17.4% in
heifers. The highest level of infection was
found at the third facility; this was 37.3%.
137
The report highlights the reasons why preva-
lence data vary considerably between stud-
ies. It cites that different detection proce-
dures are a main cause, because some
techniques are more sensitive than others.
The experiments carried out in this investi-
gation used cesium chloride density gradient
centrifugation and immunofluorescence mi-
croscopy, instead of the more commonly used
sucrose density gradients and bright field
and phase contrast microscopy. Tests car-
ried out by the authors77 showed that these
innovations improved the accuracy of the
methods. The study also explains that other
reports have suggested that older animals
are rarely infected by Giardia. Much more is
known about the infection in calves and there
is a risk of complacency when it comes to
adult animals. A danger with this “low-level,
asymptomatic” giardiasis is that it may never
be picked up or treated. Meanwhile, cysts
are continually being transmitted to the en-
vironment. Hence, the cattle may well act as
a reservoir for human infection.
XII. THE RISKS OF WASTEWATER
IRRIGATION
Giardiasis is a large problem in less-
developed countries. Here wastewater is of-
ten reused in traditional farming practices to
irrigate land. This clearly raises questions
about the effect on public health. Several
studies have been carried out to assess the
risk in such practices. In Marrakech, the
potential contamination to food crops has
been assessed.8 This detailed study assessed
three types of water; raw wastewater, treated
wastewater, and fresh water, during field
trials on coriander, carrot, mint, and radish.
Several crops were also collected and tested
in the laboratory. These were turnip, mar-
row, squash, potato, pepper, and eggplant.
Of these vegetable crops, Giardia cysts were
only found on potatoes. Twenty-five percent
of the potato crop was contaminated with an
average of 5.1 cysts/kg. The field trials in-
volving raw wastewater showed high levels
of Giardia cysts (Table 4). None of the ex-
periments using treated wastewater or fresh
water were found to have any Giardia cysts,
demonstrating the importance of water treat-
ment.
The report tries to explain the varying
concentrations of the contamination found.
It suggests that the frequencies of cysts found
depend on the structure of crop foliage. It is
suggested that the foliage of coriander pro-
vides a large surface area for contact with
the sewage, and this type of foliage acts as a
shelter for the cysts. The study also included
the detection of the eggs of the helminth
Ascaris lumbricoides. These were found in
TABLE 3
Prevalence (%) of
Giardia duodenalis
Cysts Found in Samples from Dairy Calves
of Different Ages in Southeastern New York
Adapted from Wade
et al
.6
138
all four vegetable crops examined in the field
trials. They were also found in high concen-
trations in ground surface crops such as
squash and marrow. The explanation given
for this is that these vegetables are in direct
contact with the raw sewage as well as the
contaminated soil. If this is the case, then
why were these vegetables found free of
Giardia cysts? A reason for this could be
that the size of the cysts prevents them from
remaining on these vegetables. Giardia cysts
(12 µm diameter) could be washed deeper
into the soil than Ascaris eggs (65 µm), and
not remain in contact with the surface.
The study also shows the persistence of
the cysts in the environment following irri-
gation; the average cyst number decreased
with time and all cysts had disappeared
3 days after the last irrigation. The authors
explain that this time could vary depending
on the environmental conditions, desicca-
tion due to sunlight, and high temperatures
may be deleterious. However, Giardia cysts
are notoriously resistant to changing envi-
ronmental conditions, and further studies
examining their persistence during different
seasons or times of day are urgently needed.
The fact that wastewater, from waste stabi-
lization (sedimentation) ponds, did not trans-
mit any cysts shows that this is an effective
method of breaking the fecal-oral route of
the giardiasis.
A study has been carried out in Marrakech
to test the efficacy of sedimentation ponds in
the removal of protozoan cysts, including those
of Giardia.78 This study tested the cyst con-
tent of water at the inlet and outlet of pilot
waste sedimentation ponds. Although there
were high concentrations of cysts in the waste-
water entering at the inlet, no cysts were de-
tected at the outlet. By analyzing sludge from
the bottom of the ponds, it was shown that the
cysts had sunk to the sediment. Therefore,
ponds of this nature are a simple and efficient
procedure for removing pathogenic cysts;
however, high concentrations of cysts then
build up in the sludge at the bottom of the
ponds, where they may persist for some
months as potentially infective agents.78
The practice of wastewater irrigation has
also been assessed in agricultural villages in
Mexico.79 Three exposed populations were
examined. These were individuals exposed
to untreated wastewater, a group using efflu-
ent from reservoirs, and those from rain-fed
agricultural villages. Untreated wastewater
samples were tested for the presence of Gia-
rdia, and it was found that some had as
TABLE 4
Contamination Levels of
Giardia
Cysts Found during Field Tests on Various
Crop Plants Irrigated with Raw Wastewater in Marrakech.
Adapted from Amahmid
et al
.8
139
many as 300 Giardia cysts/liter. The water
from reservoirs, which had been subjected
to hydraulic retention lasting 3 to 7 months,
was of an improved quality, with only five
or less Giardia cysts/liter. Despite this, those
individuals exposed to untreated wastewater
were not found to be more at risk than con-
trols, neither were those using water from
the reservoirs. The study also showed there
was no significant risk from agricultural prac-
tices, although it does say that people buying
vegetables at the city market had higher rates
of infection than those buying from the local
village shop.
Another study describes how the con-
centrations of cysts in small water systems
in Taiwan increase proportionately with the
population using the water systems.80 These
studies show that the consumption of con-
taminated crops can be unsafe. However,
these are practices that have been carried out
for decades, and they are convenient in many
poorer parts of the world. From 1970 to
1994 the volume of wastewater in Morocco
increased from 129 to 470 million cubic
metres per year,8 using it for irrigation is
seen as simple and economic. These studies
also show the improvement that simple wa-
ter treatment methods, such as sedimenta-
tion ponds, can make to water quality.
XIII. ZOONOTIC TRANSMISSION
The zoonotic potential of Giardia has
been suspected for a long time. Giardiasis
earned the nickname ‘beaver fever’ after an
outbreak in the Banff National Park in Canada
some 30 years ago. The outbreak was attrib-
uted to wild beavers contaminating the wa-
ter supply used by campers. Campers are
still encouraged to follow safety guidelines
when drinking water from certain areas. The
zoonotic problem of Giardia is indicated by
the need for the new system of nomencla-
ture. Initially, species were divided accord-
ing to the host from which they were first
isolated, but with improved microscopy it
was seen that some Giardia isolates found
on different hosts were morphologically iden-
tical.7
Giardia duodenalis is capable of infect-
ing a range of mammals (Table 5), but to
what extent this occurs remains unclear.76
There is a large amount of interest in this
currently, with the focus being on livestock
and domestic pets as the most concerning
sources of human infection. It has been sug-
gested that the large numbers of VSP genes
in Giardia could allow it to infect different
hosts, and antigenic variation may be a
mechanism for the protozoon to expand its
host range.46 Research discussed earlier
showed that isolates of Giardia duodenalis
isolated from cattle were genetically identi-
cal to an assemblage that infects humans.76
Cattle are a considerable threat as a reser-
voir, as large numbers of cysts are shed by
herds, and can easily enter watercourses
through farm and field run-off. The resulting
contamination of drinking water, which
would be a major problem in places with
inadequate water treatment, especially be-
cause Giardia is relatively resistant to chlo-
rination. Giardia cysts are also small enough
to enter groundwater systems,81 thus the prob-
lem could be even more widespread, and
harder to combat.
Domestic pets are also a source of
zoonotic concern, because many of them are
owned by young children. A report has stated
that incidences of Giardia in cats and dogs
in Australia are increasing, possibly only as
a result of better diagnostic methods.9 How-
ever, as the increase in Giardia cases is ac-
companied by a decrease in other disease-
causing parasites (e.g., Toxocara and
Ancylostoma) that are susceptible to the
antihelminth treatments currently being used.
Giardia may be filling the niche left by these
declining parasites because it is more resis-
tant to the treatments currently used.9 Inter-
140
estingly, this review also cites different de-
tection techniques as a reason for discrepant
results, also a view expressed in an earlier
discussed work.77
The Robertson study9 addresses simple
ways to prevent pet-human transmission
(e.g., washing hands after handling pets or
emptying litter trays and supervising tod-
dlers while playing with pets). This study
claims that the person- to-person transmis-
sion of giardiasis is “more important than
zoonotic transmission”, but does accept that
dogs and cats carry strains of Giardia that
could infect humans. A main theme in this
report is that immunocompromised individu-
als are more at risk of contracting the disease
than people with healthy immune systems
and therefore should take extra care. The
authors also highlight the importance of edu-
cation in preventing giardiasis. A study re-
viewed in the Robertson report carried out
by Bugg et al.82 showed that none of the pet
shop owners interviewed were aware of the
zoonotic potential of Giardia. Robertson et
al.9 identify vets and other knowledgeable
medical professionals as important sources
of educational information for the public.
As well as domesticated animals, wild
animals are still thought to be the source of
some outbreaks of the disease. This concern
is likely to gain support; recently published
reports into Giardia detected in species of
animals hitherto not considered hosts. Thus
Giardia spp. have been found for the first
time in the Netherlands white stork (Ciconia
ciconia),83 the Californian sea lion (Zalophus
Californianus),84 and also in pinnipeds from
the eastern coast of Canada.85 The tropho-
zoites found in a fecal sample of a 6-week-
old asymptomatic stork chick resembled
Giardia ardeae, which had been isolated
previously from some species of wading birds
of the order Ciconiiformes. The parasites
from the chick did not serologically react
with anti-Giardia intestinalis monoclonal
antibodies, showing they were a separate
TABLE 5
The Host Range of
G. duodenalis
Isolates
Adapted from Thompson2
141
genotype, and therefore not of zoonotic con-
cern.83 However, fecal samples from the
Californian sea lion contained cysts con-
firmed by morphology, immunology, and
PCR to be Giardia duodenalis, showing that
these animals could act as a reservoir for
environmental transmission and potentially
for human infection. Further research is
needed in this area, and the increased use of
molecular techniques to biotype Giardia from
diverse sources will provide vital informa-
tion in the future.
XIV. CONCLUSIONS AND FUTURE
WORK
Giardia intestinalis studies not only pro-
vide excitement and fascination to a new
generation of research workers and challenge
basic biological ideas, but also present a dif-
ficult and acute biomedical problem. There
are clearly very varied areas of research cur-
rently being carried out into Giardia. The
evolutionary significance of the organism is
one of the most interesting aspects, and a
source of continuing debate. Although it is
generally accepted that it is an evolutionary
ancient and thus primitive organism, there
remains doubt as to whether Giardia is the
deepest branch of the eukaryotic tree or a
‘link’ between eukaryotes and prokaryotes.20
There is also speculation over the function
of the two nuclei; although they are thought
to be identical,7 this is not accepted by ev-
eryone and questions are still being asked,
“is one nucleus transcriptionally silent for
VSPs?”.86 Despite these controversies, some
researchers have employed Giardia as a use-
ful tool for gaining information, especially
in the examination of evolutionary processes.
The work carried out into structures such as
protein disulfide isomerases and telomerase
catalytic subunits,14,30 coupled with investi-
gations into the physiology of Giardia have
resulted in increased knowledge of the or-
ganism itself. In addition to this, the investi-
gations have also provided novel insights
into the evolution of eukaryotic processes,
for example, those involved in the elabora-
tion of the mechanisms of the maintenance
of plasma membrane potential.35 This study
had to adapt previously used flow cytometric
techniques to be suitable for use on Giardia.
Developing tools in this way may prove use-
ful for the study of other protozoa. Many
still unanswered questions about the rather
atypical organellar structures in Giardia
should be solved alongside the completion
and analysis of the Giardia genome project.
Little was known until recently about the
mechanisms by which the parasite causes gia-
rdiasis. Although there are still many aspects
that are poorly understood, we are now more
familiar with the interactions of cysts and
trophozoites with the host within the small
intestines in humans and cattle.41,53 There is
now evidence that trophozoites excrete prod-
ucts, which damage the intestinal epithelium.
The identification of metalloproteases and the
involvement of the jejunal microbiota in the
severity of infection are also very interest-
ing.43,44 An increased understanding of these
processes has led to the recognition of new
targets for the prevention and treatment of the
disease. A vaccine is now available in North
America that prevents clinical signs of giar-
diasis in humans.3 There have been searches
for alternative methods of treatment, for ex-
ample, whole garlic,57 and these approaches
may have potential for future development
and will prove useful if resistance to currently
used antigiardial agents becomes widespread.
Scientists have tried to discover the ex-
tent to which giardiasis has affected cattle.6,75
The effects of fenbendazole treatment on
cattle has been examined in detail.51,53 This
has shown that the compound can reduce
symptoms and cyst shedding, but the prob-
lem of rapid reinfection remains unsolved.
This problem arises in cattle treated with
fenbendazole despite the use of disinfectant,51
142
but did not occur in disinfected dog kennels
after treatment with oxfendazole.52 Further
comparable studies are needed to explore
the extent of reinfection and methods of pre-
venting it. Also for the future are investiga-
tions into the economic impact of giardiasis
infections of the farmer. One study showed
that treatment did not improve dairy produc-
tion.51 More detailed research is required to
clarify these issues. Asymptomatic giardia-
sis may have important implications in the
mechanisms by which the disease is trans-
mitted.
The need for improved techniques in the
detection of Giardia cysts in water and stool
samples has been highlighted repeatedly in
the literature. A lack of consistency between
methods was reported in several papers.72,77
A study of the recovery of cysts using a
membrane filter dissolution method reports
an incredibly large range of recovery from
3.2% to 90.3%.87 An explanation of varied
results obtained from one study was that
“detection procedures…differ significantly
from laboratory to laboratory”.77 Therefore,
there is a call for “more sensitive molecular
methods to detect and characterise Giar-
dia”.88 Recently developed PCR methods will
lead to more reproducible techniques. If an
efficient, uniform detection procedure comes
into routine use, studies into the prevalence
of giardiasis from different geographical lo-
cations could be fairly compared. The com-
parison of similar studies could give impor-
tant information into patterns of giardiasis
outbreaks.
The resistant nature of Giardia is well
documented: the organism “synthesizes and
deposits large amounts of extracellular ma-
trix into an extracellular cyst wall”.89 This
means that very sensitive methods of detec-
tion are needed to monitor levels of Giardia,
especially in treated water, because cysts
can evade chlorination and the only guaran-
teed removal method is by filtration. Meth-
ods that are quick, cheap, reliable, and are
not labor intensive are required. There are
some successful new techniques that have
improved inactivation and thus the removal
of cysts. Electroporation could prove very
useful in aiding the inactivating effects of
chlorine on the parasite.60 UV light and ozone
also have the potential for routine use in
water treatment plants.73,74 There is a remark
made in the literature about the expense of
some techniques putting them out of reach
of developing countries.80 It is in these coun-
tries where the severity of giardiasis is exac-
erbated by a lack of nutrition and poor sani-
tation. It is these conditions that make it
more likely that giardiasis will be fatal.
Simple and inexpensive methods of control-
ling transmission of the disease are therefore
much needed in these areas, and the suitabil-
ity of new methods for adoption in develop-
ing countries therefore should be considered
during their development.
As well as molecular detection methods,
procedures to type Giardia isolates by PCR
have now been developed.70,71,90 These will
prove useful in monitoring the risks of
zoonoses; sources of outbreaks will be more
easily traced.91 PCR-based fingerprinting is
carried out without the need for in vitro cul-
ture. This makes the method simpler and
faster.90 Although the same genotypes have
been found in different hosts, this does not
automatically mean that transmission is oc-
curring between them. One review explains
that by looking in greater detail at the trans-
mission between hosts living in the same
area, more evidence for zoonosis may be
found.91 It is important to establish who
(if anyone) is most at risk of contracting the
disease and from which animals. Although it
is clear that observing zoonotic transmission
directly is not easy, with improved genetic
fingerprinting perhaps in the future a form of
genetic labeling will be employed.
Sensitive molecular methods are being
employed to revolutionize the study of Gia-
rdia. This together with the ongoing Giardia
143
genome project puts researchers in an excel-
lent position in the near future to unravel the
remaining mysteries connected with this
unique parasite.
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