ArticlePDF AvailableLiterature Review

Antimicrobial properties of Allium sativum (garlic)

November 2001
Applied Microbiology and Biotechnology 57(3):282-6

DOI:10.1007/s002530100722

Source
PubMed

Authors:

Janine Harris

Cultech

Simon Cottrell

Public Health Wales

David Lloyd

Cardiff University

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

To link to this article: https://doi.org/10.1080/1040-840291046713

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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

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

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

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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Antifungal Activity of Plant Waste Extracts against Phytopathogenic Fungi: Allium sativum Peels Extract as a Promising Product Targeting the Fungal Plasma Membrane and Cell Wall

Article

Full-text available

Jan 2023

The harmful effect of synthetic fungicides on the environment and the development of resistance by fungi raises concerns about their security and future efficacy. In this work, we investigated plant by-products with the antifungal activity that could be safe alternatives to conventional fungicides. The in vitro antifungal potential of plant by-product extracts showed that garlic peel extract (GPE) was the most effective against several phytopathogenic fungi. Accordingly, in ex situ assays with apples, GPE significantly reduced the lesion size caused by subepidermal inoculation with Colletotrichum acutatum spores. In addition, Saccharomyces cerevisiae mutant strains affected in ergosterol synthesis showed higher resistance to GPE than the parental strain, indicating that the extract might target an intermediate of this pathway. Moreover, GPE affects the cell wall, given that bck1 and mkk1/mkk2 mutants were less able to cope with the stress because of the impairment of the remodeling mechanisms. Regarding the apoptosis-deficient mutant yca1, sensitivity was similar to that of the parental strain, suggesting that the extract does not induce apoptosis. A diverse group of sulfur compounds was identified by gas chromatography-mass spectrometry (GC/MS). Our findings contribute to the elucidation of the antifungal mechanism of GPE and highlight its potential as an alternative biofungicide in agriculture.

Synergy of Garlic Extract and Deep Eutectic Solvents as Promising Natural Antibiotics: Experimental and COSMO-RS

Article

Full-text available

Jan 2023
J MOL LIQ

The growing concern of antibiotic resistance calls for the identification of new and sustainable solutions. In this study, we employed the COSMO-RS model to select promising deep eutectic solvents (DESs) for investigating the antibacterial properties of Allium sativum L. (garlic) extract. Through computational analysis, we selected two hydrogen bond acceptors (HBAs) and three hydrogen bond donors (HBDs) and found that DESs containing choline chloride (ChCl) as an HBA were the most optimal for this research. Our experiments revealed that the chosen mixtures, which consisted of 50% garlic extract and 50% DES, displayed outstanding activity against 11 microbial strains including gram-positive and gram-negative bacteria as well as a fungal strain commonly found in hospital settings with inhibition zones ranging from 18 to 50 mm. Notably, the selected mixtures displayed a synergistic effect against B. subtilis, S. aureus, and L. monocytogenes. Furthermore, the minimum inhibitory and minimum bactericidal concentrations (MIC and MBC) recorded were between 0.0125 and 0.4 µl/ml, respectively. These findings confirm the potent antibacterial properties of Allium sativum L. extract and the enhancement of its effectiveness when combined with DESs. This study represents a significant step towards the development of natural and sustainable alternatives to combat antibiotic resistance.

Pharmacological properties and phytoconstituents of garlic (Allium sativum L.): A review

Article

Full-text available

Dec 2022

Medicinal plants have been used since ancient times for human healthcare in the form of traditional medicines, spices, and other food components. Throughout history, many different cultures have recognized the potential use of garlic for the prevention and treatment of different diseases. Garlic is a common bulb vegetable or spice that is used as an herb and to flavor food. The plant contains biologically active components that contribute to its pharmacological properties. The medicinal effects of garlic were known for 5,000 years. Garlic is recommended as a nutritive element in the treatment of various health problems and the prolongation of human life. The chemical constituents of garlic have also been investigated for the treatment of aging, Alzheimer’s disease, anti-fungal/bacterial/viral/ Protozoans, atherosclerosis, blood pressure, cancer, cardiovascular disease, diabetes, neuro/ nephroprotection, osteoporosis, stress, hyperlipidemia, and, wound healing and highly praised by several authors. Therefore, this paper is reviewed to inspire and impress young researchers about the medicinal values of garlic.

Natural products-based: Synthesis and antifungal activity evaluation of novel L-pyroglutamic acid analogues

Article

Full-text available

Dec 2022

Botanical pesticides are one of the sources of third-generation pesticides, which have received much attention at home and abroad in recent years due to their degradable and pollution-free advantages in nature. This article explored a concise approach toward synthesizing a series of novel L-pyroglutamic acid analogues from L-hydroxyproline. Furthermore, bioassay studies of these sulfonyl ester derivatives against Pyricularia oryzae, Fusarium graminearum, Alternaria brassicae, Valsa mali, and Alternaria alternariae showed moderate antifungal activity. For instance, C08a and C08l provide potential lead agents for controlling Fusarium graminearum because of their inhibitory activity.

INFLUENCE OF GARLIC EXTRACT AND PACKAGING METHODS ON THE QUALITY PARAMETERS OF FISH FILLETS STORED AT LOW TEMPERATURES

Article

Full-text available

Nov 2022

Fish meat has very high water content, a very low content of connective tissue but has high digestibility. Raw fish is a very perishable food because it has all substances necessary for the development of microorganisms. Fish meat lipids are also very sensitive because they oxidize very quickly during storage. The aim of our research was to examine the influence of garlic extract and packaging methods on the total volatile basic nitrogen (TVB-N), thiobarbituric acid content and pH value in fish fillets during storage at low temperatures. 48 fillets of rainbow trout (Oncorhynchus mykiss) were studied. The fish fillets were randomly assigned to four batches of 12 samples each (C-control and experimental batches E1, E2 and E3) and marinated in a solution with garlic extract in proportion of 0‰ (C), 3‰ (E1), 6‰ (E2) and 9‰ for 12 hours. For each batch, six fillets were individually vacuum packed and six fillets were individually packed in plastic boxes. The samples were stored in the refrigerator at a temperature of 0...+4°C for six days. The studied parameters were determined after marinating, on the 1st, 3rd and 6th day of storage.The raw data obtained was processed by biostatistical methods using the One-Way Anova test, including the Tukey HSD test of the SPPS Statistics application. In the storage period, the amount of TBA increased progressively at each stage of the examination (30 min., 1, 3 and 6 days, respectively), and the amounts in the control samples were significantly higher (p ≤ 0.05) than those in the E1, E2 and E3 samples. Also, the lowest values after 6 days of refrigeration were recorded in the marinated samples and vaccum packed. The lower values recorded in marinated samples were influnced by the antioxidant effect of the garlic extract used. Regarding the TBA amount in fish and fish products, the admissibility limit recommended for the consumer is 2.0 mg MDA/kg, and in our case the values have been much lower.

USE OF DIETARY GARLIC (Allium sativum L.) AND VIETNAMESE BALM (Elsholtzia ciliata) EXTRACT FOR PREVENTION OF BACILLARY NECROSIS IN PANGASIUS (BNP) IN STRIPED CATFISH (Pangasianodon hypophthalmus)

Article

Full-text available

Jan 2022

Garlic (Allium sativum) and Vietnamese balm (Elsholtzia ciliata) are well-known herbs that have been widely used in Vietnamese traditional medicines. However, studies on the effects of these plants in improving the immune system of fish have received less attention. This study aimed to investigate the effect of ethanolic leave extracts from garlic and Vietnamese balm leaves on the growth and immune response of striped catfish (Pangasianodon hypophthalmus) for the prevention of Bacillary Necrosis of Pangasius. Bacillary Necrosis of Pangasius (BNP) caused by Edwardsiella ictaluri is the most serious disease occurring in farmed striped catfish in Vietnam. The experiment was in a completely randomized design with five triplicated treatments including 0% (control); 2% and 4% of garlic and 2% and 4% of Vietnamese balm leaves extract. Fish were soaked with E. ictaluri. Samples were collected on the days 7 th and 14 th of the growth experiment and days 3 rd , 5 th and 7 th of the challenge test. Fish mortalities and immune response among challenge tests were also recorded. The results suggest that plant extracts possibly modulate the striped catfish's immune response in a time and dose dependent manner. Specifically, diets enriched with extracts of A. sativum at 4% have great potential than Vietnamese balm extraction for improving striped catfish health by enhancing the immune system. After challenging with E. ictaluri, the accumulative mortality of fish in garlic and Vietnamese balm leaves treatments was lower than that of the control. The mortality of fish fed with 4% garlic extract was the lowest. These results indicated that supplementation of 4% garlic extract significantly improved the resistance to the BNP infection in striped catfish and has the ability to add fish feed in reality.. Use of dietary garlic (Allium sativum L.) and Vietnamese balm (Elsholtzia ciliata) extract for prevention of Bacillary Necrosis in Pangasius (BNP) in striped catfish (Pangasianodon hypophthalmus). Academia Journal of Biology, 44(4): 65-76. https://doi.

Biotransformation of root and tuber crops by lactic acid bacteria into value-added bio-commodities

Chapter

Jan 2022

Effect of Achillea millefolium and Moringa oleifera supplementation on Productive, Immunological and Physiological Responses of Weaning V-line Rabbits

Article

Dec 2022

Medicinal Plants as Control for Prevalent and Infectious Diseases

Chapter

Jan 2023

The greatest loss to the world’s economy has resulted from the multidrug resistance (MDR) strains and other prevalent infectious diseases like leishmania, diarrheal diseases, malaria, tuberculosis, parasitic infections, pneumonia, and trypanosomiasis. Antibiotics have played the core role of wonder drugs in treating a variety of diseases. However, the irregular, unsuitable, and irrational uses of antibiotics have led to the emergence of antibiotic resistance. This has paved the way to an increased usage of medicinal plants as currently, many nutraceuticals and pharmaceuticals are plant derived. This chapter describes the huge repositories of secondary metabolites present in medicinal plants that may provide novel antibiotics to tackle all the infectious disease-causing pathogens and other prevalent diseases and reclaim the currently used antibiotics. The wide spectrum of phytochemicals in medicinal plants is to be explored as potential therapeutics for the sustainable use of plant resources. This chapter focuses on the diverse efficacy and effects of plant-derived multiple compounds for the development of bioactive therapeutics to identify novel antibiotics in controlling many infectious pathogens and finding a cure for the most common prevalent diseases. More research into unexplored plants is the need of the hour for global health benefits to identify novel antibiotics.KeywordsBio-moleculesAntimicrobialsAnti-dengueAnti-diarrhealBiofilmsLeishmaniasis

Evaluation of phytochemicals from Indian traditional medicinal plants as antihiv-1 Drugs

Article

Dec 2022

According to a global estimate, the growing infection by Human Immunodeficiency Virus type 1 (HIV-1) causing development of a dreaded disease i.e., Acquired Immuno-Deficiency Syndrome (AIDS) has been a major health risk resulting into loss of lives of about 25 million people in the last 25 years. The information available on basic biological processes in the HIV life-cycle suggest that it destroys a subpopulation of T-lymphocytes containing CD4 receptor and CXCR4 or CCR5 as a coreceptor on their surface. These molecules on cell surface are required by the virus for docking and internalization. Since the availability of a suitable vaccine against this virus is currently far from reach and the presently available synthetic antiHIV therapeutics exhibit severe toxicity and induce emergence of drug resistant mutations, it was imperative to explore plant-based principles targeting specific steps in the viral life-cycle. Moreover, the advancement in separation, purification and chemical characterization technologies for identification of natural compounds has offered promises for the usage of medicinal plants in modern drug discovery. With a few newly discovered natural products demonstrating anti-HIV potential in comparison to a vast number of previously known natural products exhibiting activity against HIV, a multiplex approach is required for exploring cost effective, safe, and efficient plant based anti-HIV drugs with very small LD50 value exhibiting efficacy at nanomolar / picomolar range. This article illustrates an updated account of information on the antiHIV-1 efficacy of some bioactive molecules isolated from different medicinal plants. Furthermore, the article highlights some chemical compounds isolated from weeds showing activity against functions of viral reverse transcriptase (HIV-1RT), protease and integrase enzymes.

Genotyping of Giardia lamblia Isolates from Humans in China and Korea Using Ribosomal DNA Sequences

Article

Full-text available

Aug 2000

Genetic characterization of a total of 15 Giardia lamblia isolates, 8 from Anhui Province, China (all from purified cysts) and 7 from Seoul, Korea (2 from axenic cultures and 5 from purified cysts), was performed by polymerase chain reaction amplification and sequencing of a 295-bp region near the 5' end of the small subunit ribosomal DNA (eukaryotic 16S rDNA). Phylogenetic analyses were subsequently conducted using sequence data obtained in this study, as well as sequences published from other Giardia isolates. The maximum parsi- mony method revealed that G. lamblia isolates from humans in China and Korea are divided into 2 major lineages, assemblages A and B. All 7 Korean isolates were grouped into assemblage A, whereas 4 Chinese isolates were grouped into assemblage A and 4 into assemblage B. Two Giardia microti isolates and 2 dog-derived Giardia isolates also grouped into assemblage B, whereas Giardia ardeae and Giardia muris were unique.

Effect of ajoene, a compound derived from Allium sativum, on phytopathogenic and epiphytic micro-organisms

Article

Full-text available

Jan 1993
Z PFLANZENK PFLANZEN

Ajoene, a compound derived from Allium sativum and produced by chemical synthesis, was investigated in vitro and in vivo for its activity against 20 phytopathogenic and epiphytic fungi and bacteria. The main interest was focused on the fungi which belonged to the group of obligate biotrophic parasites (powdery mildews), other leaf pathogens, soil-borne pathogens, vascular wilt fungi, and yeasts.The minimum inhibitory concentration, determined by in vitro tests, ranged from 2 to 200mg/l depending on the organism, method, and nutrient medium used. The phytopathogenic fungi Cladosporium fulvum and Verticillium dahliae and the phytopathogenic bacterium Erwinia amylovora were the most sensitive species. In greenhouse experiments, the inhibiting action of ajoene against Cladosporium fulvum could be confirmed with tomato plants after protective treatment. However, only powdery mildew of tomatoes (Oidium lycopersicum) and roses (Sphaerotheca pannosa var. rosae could be inhibited completely.

Death of Salmonella typhimurium and Escherichia coli in the Presence of Freshly Reconstituted Dehydrated Garlic and Onion

Article

Jun 1969
Appl Microbiol

The kinetics of the decline of populations of Salmonella typhimurium inoculated into freshly reconstituted dehydrated onion and garlic powders was studied. Measurable bactericidal activity was observed for onion and garlic concentrations of 1 and 5% (w/v), respectively, with maximal death rates occurring for concentrations of 5 and 10%. At these concentrations, the decimal reduction times were 1.1 and 1.2 hr, respectively, for resting cell cultures and 1.8 and 2.1 hr, respectively, for growing cultures. Of the major volatile aliphatic disulfide compounds of onions, n-propyl allyl and di-n-propyl, at concentrations of 0.1%, showed a comparable activity against resting cells but only a bacteriostatic effect toward actively growing cultures, which overcame this effect in 2 to 6 hr. At comparable concentrations, growing cultures of Escherichia coli were as susceptible to garlic, but apparently more resistant to onion, than were those of S. typhimurium.

Evaluation of Molecular Techniques to Biotype Giardia duodenalis Collected during an Outbreak

Article

Feb 2000

Twenty-seven Giardia duodenalis cyst-positive specimens (human, animal, or drinking water) were obtained from a waterborne outbreak in a community in British Columbia, western Canada. Parasite isolates were characterized using molecular techniques at 4 different steps of organism retrieval. None of the drinking water samples (n = 20) infected gerbils and none was successfully amplified using polymerase chain reaction (PCR). We were able to genotype 4 of 7 (human and animal) isolates by amplification of DNA from original specimens at the triosephosphate isomerase (tpi) gene locus using PCR followed by restriction fragment length polymorphism (RFLP) analysis. Five of the original specimens inoculated into Mongolian gerbils (Meriones unguiculatus) were infective and genotyped at the tpi locus using parasite material collected from the gerbil (cysts and trophozoites). Pulsed field gel electrophoresis (PFGE) was used to biotype trophozoites collected from the gerbils as well as trophozoites from the 4 isolates that adapted to culture. Four of these 5 isolates displayed the same (designated outbreak) biotype at all parasite retrieval steps with all molecular techniques including the originally amplified isolates. PCR-RFLP identified an additional biotype group. The 4 isolates that adapted to in vitro culture were also characterized by isoenzyme electrophoresis (IE). Bio-type groups identified in these axenized isolates were all the same with each molecular technique (PCR-RFLP, PFGE, IE) tested. Results of this study demonstrate a need for more sensitive molecular methods to detect and characterize Giardia in original host and environmental samples. Results are also consistent with evidence of biotype changes that occur during the presently used process of isolate retrieval.

Identification and Partial Characterization of Excretory/Secretory Products with Proteolytic Activity in Giardia intestinalis

Article

Aug 2000

This report examines the presence of proteolytic activity detected in media collected from in vitro cultures of Giardia intestinalis, and the partial characterization by gelatin-substrate polyacrylamide gel electrophoresis and inhibition studies. Gelatin-substrate polyacrylamide gel electrophoresis revealed 6 bands with proteolytic activity, with estimated molecular weights of 36, 59, 63, 72, 103, and 175 kDa. These bands were not present in the control medium. On the other hand, G. intestinalis trophozoite lysates showed proteolytic bands at 16, 20, 66, 82, 108, and 120 kDa, thus indicating that intracellular proteases could be different from the excretory/secretory (E/S) products. Based on inhibition studies, 2 bands of 59 and 63 kDa were inhibited by iodoacetic acid, indicating the presence of cysteine proteases. Partial inhibition of a band of 36 kDa was found with EDTA, a metal-chelating agent, suggesting the possible presence of metalloproteases. The presence of aspartic and serine proteases were not detected under the assay conditions used. As G. intestinalis E/S may be involved in differentiation mechanisms of the parasite and also be responsible for the mucosal alterations that occur in giardiasis, the characterization of these proteases may facilitate their evaluation as targets in the therapy of the disease.

Encystation of Giardia lamblia by high bile and alkaline pH and its ultrastructural changes during encystation

Article

Jun 2000

Giardia lamblia, a human pathogen causing outbreaks of diarrhea, recently became a focus of great concerns in the fields of both medical and environmental microbiology. To develop the experimental tools to study giardiasis, encystation, one of the major processes in its life cycle, was reconstituted by inducing an axenic culture of a flagellated form of G. lamblia into a cyst form under high concentration of bile and alkaline pH condition. The successful induction was confirmed by Northern analysis of resulting increased expression of the CWP1 gene encoding the cyst wall protein 1. An examination of the encystation process with SEM (scanning electron microscopy) and TEM (transmission electron microscopy) revealed that the trophozoite, a flagellate with a bilateral symmetry, was transformed to a cyst form with an oval-shape and defined filamentous wall. The encystation was found to cause a disappearance of the flagella and an invagination of the adhesive disc. An extensive formation of rER (rough endoplasmic reticulum) was observed after 24 h of induction, indicating an active synthesis and export of proteins during this process. The vital staining of the in vitro- induced cysts showed that most cysts maintained their viability.

Antiviral Constituents from Allium sativum

Article

Feb 1989

Occurrence and removal of protozoan cysts and helminth eggs in waste stabilization ponds in Marrakech

Article

Nov 2000
WATER SCI TECHNOL

The present study aimed to determine the occurrence and removal of protozoan cysts and helminth eggs from municipal wastewater by pilot waste stabilisation ponds in the arid climate of Marrakech (Morocco). Protozoan cysts detected in raw wastewater at the entrance of the system were identified as Entamoeba histolytica, Giardia sp. and Entamoeba coli at a mean concentration of 1.5x104 cysts/l. Helminth eggs identified were Ascaris, Trichuris, Hymenolepis, Enterobius and Taenia with an average of 7.6 eggs/l. The effluent at the system outlet did not contain detectable cysts or eggs. Sludge samples collected in the pond basins resulted in detection of protozoan cysts and helminth eggs. Sedimentation therefore seems to be the main factor for cyst and egg elimination during wastewater treatment by waste stabilisation ponds. This phenomenon leads however to parasite accumulation in the sediment.

Giardia: A Missing Link between Prokaryotes and Eukaryotes

Article

Jan 1991

Size selective continuous flow filtration method for detection of Cryptosporidium and Giardia

Article

Dec 2000
WATER RES

We describe a size selective continuous flow filtration method for detecting Cryptosporidium oocyst and Giardia cyst in finished water. Twenty liters of finished water spiked with Cryptosporidium oocysts (110) and Giardia cysts (60) were filtered through macro (5.0–10.0 μm) and micro (3.0 μm) pore-sized polycarbonate-track-etch (PCTE) membrane filters housed into two separate 47 mm diameter filter holders joined with a stainless steel nipple. The macro-pore-sized filters removed larger unwanted particles while allowing the Cryptosporidium oocysts and Giardia cysts to pass through, these being subsequently captured on the micro-pore-sized filter. The eluted oocysts and cysts were transferred onto the well-slide, stained by IFA and examined microscopically. The mean recovery efficiencies obtained for Cryptosporidium oocysts using the filter pairs of macro 5 μm and micro 3 μm, macro 8 μm and micro 3 μm, and macro 10 μm and micro 3 μm were 67.3%, 77.2–85.0% and 76.9–82.2%, respectively. Whereas the recovery rates for Giardia cysts using the same filter pairs were 0, 55 and 77.1%. The filter pair having the pore size of macro 10 μm and micro 3 μm was found to be most efficient in the simultaneous detection of Cryptosporidium and Giardia in the finished water. This method is simple, easy, rapid and economical.

Antimicrobial properties of Allium sativum (garlic)

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