19

Chapter I

Introduction / Literature review

1. Introduction

Cryptosporidium and Giardia are protozoa. They are single-celled organisms that

belong to the kingdom Protista. They have a low infection dose necessary to infect

humans, with possible as few as 10 organisms in some cases (USDA). They have

emerged in the last two decades as intriguing microbes with an enormous impact in

Animal (including wildlife species) and Human Health. Both can cause mild to severe

diarrhea. No specific therapy has proven to be effective, but immunocompetent individuals

generally recover within a week (USDA). However, immunocompromised individuals may

be unable to clear the parasites and, therefore, suffer chronic and debilitating illness. They

have been recognized as important pathogens in contaminated drinking water due to two

main reasons pointed out: 1) their resistance and biological viability under conventional

drinking water treatment conditions (chlorination and filtration); 2) the occurrence of

cryptosporidiosis and giardiasis outbreaks associated with the consumption of

contaminated water. This was the case in an outbreak in Milwaukee (Wisconsin, USA) in

1993, the largest waterborne disease outbreak reported all over the World. An estimated

400,000 people were reported ill (USDA).

The taxonomic and filogenetic relationships of Cryptosporidium and Giardia remain

poorly defined; thus, the understanding of their transmission dynamics has been limited. A

consensus has been adopted: with molecular techniques, the ability to observe extensive

genetic variation within Cryptosporidium and Giardia species is leading to a better

understanding of the taxonomy and zoonotic potential of these variants, and the

epidemiology of the diseases. Namely, genotyping of samples using molecular analysis at

informative loci is necessary to distinguish species and genotypes that are involved and

their zoonotic potential. Interestingly, the controversies around these concepts, the

complexity of the molecular analysis tools, all together, were additional challenge of

personal motivation. The present approach, my work as molecular biologist, integrates a

multidisciplinary and national program for the study of both protozoa in Portugal. Since

2004, this program was extended to Galicia in Spain. Molecular tools and strategy used in

this approach were further developed and refined in the ISS, Rome, Italy. Emphasis was

done in Real-Time PCR approaches to Giardia genotyping. Briefly, this is our contribution

for a better comprehension of transmission dynamics of both diseases in the Norwest of

the Iberia Peninsula.

20

2. The Protozoa Cryptosporidium and Giardia

In the last two decades a very rich amount of information and scientific evidences

has been produced in different domains of both parasites and diseases. This Literature

Review subsequently presented, does not pretend to be a whole revision in all domains.

Our effort was focused on the main aspects interesting for our work: genetic and

molecular biology of both protozoa, and their implications on transmission dynamics.

2.1. Historical aspects

Ernest Edward Tyzzer, in 1907, described life cycle stages of a protozoan parasite

that he frequently found in the feces and gastric glands of mice (Tyzer, 1907). Later, in

1910, he described, with remarkable detail, what he identified as “in form flask-shaped,

either spheroidal or ellipsoidal”. According to him “all forms (…) possess a relatively thin

membrane, an organ of attachment (…)” and identified that each oocyst contains four

sporozoites (Tyzer, 1910). He proposed Cryptosporidium muris as a new genus and

classified in the Family Asporoctstidae. In 1912, after observing parasite stages

developed only in the small intestine of mice and presenting oocysts smaller than those

described to C. muris, he reported a new species, C. parvum (Tyzer, 1912). A long period

without any significant changes passed. Suddenly, a new species, C. meleagridis, was

reported in turkeys (Slavin, 1955). In 1971, Panciera et al. have reported, by the first time

the association of the parasite with bovine diarrhea (Panciera et al., 1971). However, a

low interest on this parasite was evident until the first identification of human cases (Nime

et al., 1976). The authors have reported two cases of cryptosporidiosis in

immunocompromised individuals. The concept of cryptosporidiosis as a zoonotic disease

was born. In 1982, a report from Center for Diseases Control and Prevention – CDC

(Georgia, USA) described the clinical situation of 21 men in six cities with concurrent

cryptosporidiosis and AIDS (Goldfarb et al., 1982). The concept of cryptosporidiosis as a

human opportunistic disease was born. In the beginning of 1990s, molecular techniques

brought additional evidences on the identification of Cryptosporidium, as well as new

controversies regarding the organization of species and host specificity. After this, with the

occurrence of the greatest outbreak of cryptosporidiosis associated with drinking water in

the world, in Milwaukee, Wisconsin (MacKenzie et al., 1994), cryptosporidiosis was faced

as a water-borne disease.

Giardia duodenalis (syn. Giardia lamblia, Giardia intestinalis) was initially

described by van Leeuwenhoek in 1681, the Dutch tradesman that dedicated his life to

science and improved the microscope. He made this first observation of Giardia

duodenalis on the examination of his own diarrheal stools under the microscope. Two

21

hundred years later, in 1859, the organism was described in greater detail by Vilem

Lambl, a Czech physician that gives his name to the parasite, when observing the stools

of children with diarrhea. However, he believed that the protozoa were a commensal

microbe not responsible for the diarrhea (Lambl, 1859). Curiously, this concept remains

for a long time in the mind of many physicians, even on the twenty century. In 1888,

Blanchard suggested the name lamblia intestinalis to the parasite described by Vilem

Lambl (Blanchard, 1888). Later it was modified to G. duodenalis by Stiles in 1902.

Subsequently, Kofoid and Christiansen proposed the names G. lamblia in 1915 (Kofoid

and Christiansen, 1915) and G. enterica in 1920 (Kofoid and Christiansen, 1920). It was

the beginning of a controversy about the number of species in the genus of Giardia. In

1952, Filice detailed the morphology of Giardia and proposed three species based on the

morphology of the median body: G. duodenalis, G. muris and G. agilis (Filice, 1952). The

electron microscopy has allowed the description of additional species, G. psittaci from

parakeets, G. ardeae from herons and G. microti from voles and muskrats (Erlandsen and

Bemrick, 1987; Erlandsen et al., 1990; Feely, 1988). These authors thought that the new

species belonged to G. intestinalis, described by Filice. The most significant waterborne

Giardia outbreak described to date occurred in Norway between October and December

2004, affecting more than 1500 cases. G. duodenalis assemblage B, described as closely

related to sub-genotype B3 has been described as the etiological agent (Robertson et al.,

2006). However, in Portugal, in Madeira Island, it was reported a high incidence of

diarrhea in a group of 1400 American tourists in October 1976. The diarrheal symptoms

lasted for longer than a week, and the drinking tap-water as well as the consumption of ice

creams and raw vegetables was implicated (Lopez et al., 1978). This report was highly

significant.

2.2. Taxonomy

Cryptosporidium taxonomy is organized as: Kingdom, Protista; Subkingdom,

Protozoa; Phylum, Apicomplexa; Class, Sporozoasida; Subclass, Coccidia; Order,

Eucoccidiorida.

A lack of consensus still exists in the taxonomy of Cryptosporidium. This is mainly

due to the fact that members of this protozoan genus in the phylum Apicomplexa were

thought to be closely related to the coccidian, but, despite strong morphological

similarities to the coccidian throughout the life cycle and the presence of mitochondrion-

specific genes, it has not been shown that C. parvum possesses a mitochondria-like

organelle as found in classical coccidia (Riordan et al., 1999; Tetley et al., 1998).

Moreover, molecular data suggest that Cryptosporidium may be more closely related to

22

Table 1. Species of Cryptosporidium and typical hosts. From Xiao and Fayer, 2008

gregarines, fact that is also supported by similar life cycle stages in both organisms

(Fayer, 2004; Hijjawi et al., 2002).

Giardia belong to the Kingdom, Protista; Subkingdom, Protozoa; Phylum,

Sarcomastigophora; Subplylum, Mastigophora; Class, Zoomastigophora; Order,

Diplomonadida; Family, Hexamitidae. Similar to Cryptosporidium taxonomy, the

classification based on molecular tools, have shown a great value in the understanding of

the pathogenesis, and the host range of Giardia isolates obtained from humans and from

a variety of other mammals. This molecular data shows a number of assemblages (similar

to genotypes) of G. duodenalis, although they are morphologically identical (Adam, 2001;

Thompson, 2004).

2.2.1. Species and Genotypes

The taxonomic status of Cryptosporidium and the naming of the species are

undergoing rapid change. The early classification of Cryptosporidium relied in the host

occurrence which, combined with the lack of morphological characters to differentiate

variants, created a huge debate on the taxonomy and specie organization. Furthermore, it

was not obvious to understand whether phenotypic differences were a consequence of

genetic differences or a result of host or environmental induced changes. In recent years,

molecular characterization of Cryptosporidium helped to clarify the confusion in

Cryptosporidium taxonomy that derived for the morphological data (Fayer, 2004; Xiao et

al., 2004). These tools also have the enormous advance of being applied in the direct

characterization of oocysts recovered from fecal or environmental samples. This

elucidated the real nature of variation in Cryptosporidium.

23

Species of Cryptosporidium have been named and are nowadays organized as is

in Table 1.

Filice, in 1952, made a comprehensive re-evaluation and rationalization of Giardia

species based on morphological similarities of the species and isolates, particularly the

morphology of the median body. He organized Giardia in three species: G. duodenalis, G.

muris and G. agilis (Adam, 2000, 2001). This was a major step forward in the taxonomy of

Giardia. Later, and as a consequence of the development of axenic culture of Giardia,

molecular characterization of Giardia begins by using zymodeme analysis (Adam, 2001).

As a result, the precedent taxonomic structure of genus Giardia was solidified and, more

important, considerable levels of genetic diversity within the G. duodenalis group were

observed. Data became even more solid with the development of pulsed-field gel

electrophoresis and Variant-specific proteins (VSP’s) analysis (Adam, 2001). Recently,

the application of PCR-based procedures facilitated and solidified largely the taxonomy

and organization of Giardia species and mainly G. duodenalis group (Monis et al., 1998;

Thompson, 2004). Using these procedures, a number of laboratories worldwide have

contributed to reach a consensus in Giardia species and genotypes organization (Monis et

al., 1999; Monis et al., 1998). Giardia genus currently comprises these six species,

distinguishable on the basis of the morphology and ultrastructure characteristics of their

trophozoites: Giardia agilis in amphibians, Giardia muris and Giardia microti in rodents,

Giardia duodenalis in mammals, Giardia ardeae and Giardia psittaci in birds (Table 2).

Following the great advances on the Giardia molecular typing, the main

observation is that G. duodenalis is not a uniform species but a species complex

comprising a variety of genetically and phenotypically (yet morphologically similar)

genotypes (Monis and Thompson, 2003; Thompson, 2004). So, based on the genetic

structure of the species G. duodenalis, this can be assigned to at least seven genetically

Table 2. Species of Giardia and typical hosts. From Adam, 2001

24

distinct assemblages: A to G (Caccio et al., 2005; Hunter and Thompson, 2005; Monis

and Thompson, 2003). These genotypes are represented in Table 3.

Also, based on the genetic differences among the G. duodenalis complex, some

authors propose to classify these assemblages into six different species (Caccio et al.,

2005; Thompson and Monis, 2004). In this way, for assemblage A it was proposed the

name G. duodenalis, for assemblage B, G. enterica, for assemblage C, G. canis, for

assemblage E, G. bovis, for assemblage F, G. cati and for assemblage G, G. simondi

(Caccio et al., 2005; Thompson and Monis, 2004).

2.2.2. Host-specificity

Studies on Cryptosporidium isolates obtained from cattle, sheep, pigs, cats, dogs,

kangaroos, squirrels and other with mammals, have shown that most species are infected

with a restricted host-adapted Cryptosporidium species or genotypes (Xiao and Fayer,

2008; Xiao et al., 2004) (Table 1). The existence of host-adapted Cryptosporidium species

or genotypes indicates that cross transmission of Cryptosporidium among different groups

of animals is usually limited.

Cryptosporidium parvum has received a major attention concerning cross-species

transmission. First, C. parvum was thought to infect all animals. The use of molecular

tools proved its absence in infecting wild mammals. Nowadays, it is generally accepted

that it infects primarily ruminants and humans. Even in cattle, only calves aged less than 2

m-o, are frequently infected with C. parvum (Mendonca et al., 2007). In older dairy calves,

the majority of infections are caused by C. bovis and Cryptosporidium deer-like genotype.

In cattle, C. andersoni is the most prevalent parasite. Oocysts of C. parvum are not

commonly detected in sheep faeces (Castro-Hermida et al., 2007) (see chapters’ ahead).

Natural C. parvum infections have been found occasionally in other mammals such as

mice, raccoon dogs and dogs, although companion animals are most often infected with

host-specific Cryptosporidium spp. (Xiao and Fayer, 2008). In this way, dogs are almost

exclusively infected with Cryptosporidium canis and cats with Cryptosporidium felis. The

Table 3. Genotypes of Giardia duodenalis group. From Thompson, 2004

25

role of dogs and cats in the transmission of human cryptosporidiosis appears quite limited.

In fact, C. canis and C. felis infections are infrequently reported in humans, despite their

close and widespread contact. Further information regarding the typical hosts from the

remaining Cryptosporidium species can be found in Table 1.

Species of the genus Giardia, as referred to before, were defined according to the

hosts they infected. As it can be observed in Table 2, species of Giardia are very host-

specific. On the contrary, G. duodenalis shows an ability to infect a large range of hosts.

As previously stated, G. duodenalis is not a uniform species but a species complex.

These species named assemblages (genotypes) are morphologically similar but exhibit

differences in host specificity (Monis and Thompson, 2003; Thompson et al., 2000).

Evidences produced among laboratories all over the world have permitted to reach a

consensus regarding the genotypic grouping of G. duodenalis. Their zoonotic potential

was recognized. Giardia duodenalis is therefore organized in assemblages from A to G

(Table 3). Isolates recovered from humans fall into the assemblage A or B. Their

prevalence is geographically dependent. Remarkable, the molecular analysis taken into

these two assemblages shows a genetic distance that exceeds that one used to delineate

other species of protozoa (Mayrhofer et al., 1995; Monis and Thompson, 2003).

Furthermore, there is also a genetic sub-structure within each one of these assemblages;

a) assemblage A isolates can be grouped into two distinct sub-genotypes: A-I sub-group,

that received a particular attention in the molecular epidemiology of Giardia regarding its

zoonotic potential, because they have been found in animal and human isolates, and A-II

sub-group, that is human-restricted, although it has been found occasionally in bovine

fecal samples; b) assemblage B is similarly divided into two sub-groups: B-III and B-IV, in

which the latter appears to be human-specific. The remaining G. duodenalis assemblages

are quite uniform and confined to specific hosts. This fact supports their assignment as

separated species: assemblage C and D is recovered from cats, dogs, coyotes and

wolves; assemblage E is recovered in cattle, sheep, goats, pigs, water buffaloes and

muflons; assemblage F is recovered in cats; assemblage G is recovered in rats (Adam,

2001; Caccio and Ryan, 2008; Caccio et al., 2005; Hunter and Thompson, 2005;

Mayrhofer et al., 1995; Monis et al., 1999; Monis and Thompson, 2003; Thompson, 2004;

Thompson et al., 2000; Thompson and Monis, 2004).

2.3. Life cycle

The life cycle of Cryptosporidium is more complex than the one of Giardia.

Cryptosporidium has a heteroxenic life cycle, completely developed inside the host. The

life cycle of Cryptosporidium comprises an asexual stage and sexual stage;

26

Figure 1. Life cycle of

Cryptosporidium parvum or

Cryptosporidium hominis . From

CDC (www.cdc.gov).

Cryptosporidium has a spore phase named oocyst, which represents the infectious stage

of Cryptosporidium and is the resistant form found in the environmental; the oocyst is

extraordinary resistant to common disinfectants of water, such as chloride, and the water

represents the primary transmission route.

The infection begins with the ingestion of oocysts through contaminated water or

food, or by fecal-oral contact. Inhalation is also reported as a possible way why infection

occurs. After ingestion, the excystation of oocyst is induced by the acidic nature of

stomachic lumen and the presence of enzymes. However, this event occurs in the small

intestine favored by the presence of the neutral pH, bile salts and fatty acids. Each oocyst

contains four sporozoites. They are released and try immediately to infect epithelial cells

of the gastrointestinal tract. Infections in the epithelium of the respiratory tract have been

described. Inside the epithelial cells, the parasite differentiates into a trophozoite and

undergoes asexual multiplication by multiple fission, a process known as Schizogony or

Merogony. According to the process, the trophozoites develop into Type 1 meronts that

contain 8 daughter cells; these daughter cells are Type 1 merozoites, which get released

by the meronts. As can be seen in Figure 1, the merozoites can cause autoinfection by

attaching to epithelial cells, or evolve to Type II meronts, which contain 4 Type II

merozoites; a sexual cycle initiates at this point

by the release of merozoites that attach to

epithelial cells and become either macrogamonts

(female) and microgamonts (male). This sexual

multiplication is known as Gametogony. The

sexual cycle becomes complete upon the

fertilization of the macrogamonts by the

microgametes released from the microgamonts,

forming the zygote. This biological form evolves

into oocysts of two types: a) oocysts with thin

wall, which can reinfect the host by rupturing and

releasing sporozoites that starts the process

again (autoinfection process); b) oocysts with

thick wall, which are excreted into the

environment. At the end of this endogenous

cycle, sporulated oocysts are formed which, once

shed in the environment with feces, are ready to

infect a new suitable host. The prepatent period,

which means the time between the ingestion of

27

Figure 3. The morphology of

apicomplexan parasites. From

(Morrissette and Sibley, 2002)

infecting oocysts and the excretion of a new generation of oocysts, varies with the host

and species of Cryptosporidium, but usually it ranges from 4 to 22 days. The patent

period, which means the duration of oocyst excretion, ranges from 1 to 20 days. As

previously stated, transmission of Cryptosporidium may occur through contact with

contaminated water and food. In fact, many

reported outbreaks occurred in water parks,

pools and day care centers. Compatible

species of parasite and host are required

for infection to occur: the zoonotic and

anthroponotic transmission of C. parvum

and anthroponotic transmission of C.

hominis occur through exposure to infected

animals or exposure to water contaminated

by feces of infected animals.

The life cycle of Giardia is

monoxenic, and comprises two stages: the

cyst and the trophozoite (Figure 2). The

cysts are the resistant form and are

approximately 7 to 10 µm length and oval in

shape; cysts can be found in feces and are

released into the environment where they

can survive and remain viable for several

months in cool or moist conditions. They are

responsible for disease transmission, and

they are able to survive under the standard

concentrations of chlorine used in water

treatment plants. The infection occurs after

ingestion of cysts in contaminated water,

food, or by the fecal-oral route in the absence

of hygienic conditions. The excystation of

cysts is induced by the acidic nature of

stomachic lumen and the presence of

enzymes. However, this event occurs in the

Figure 2. Life cycle of Giardia duodenalis.

From CDC (www.cdc.gov).

28

Figure 4. Mucosal surface

of the small intestine of a

gerbil infested with Giardia

sp. protozoa. The intestinal

epithelial surface is almost

entirely obscured by the

attached Giardia

trophozoites. Public Health

Image Library (PHIL) #

11632 www.cdc.gov

Figure 5. Trophozoite coronal section. A

coronal view of a trophozoite demonstrates

the nuclei (N), endoplasmic reticulum (ER),

flagella (F), and vacuoles (V). A mechanical

suction is formed when the ventral disk

(VD) attaches to an intestinal or glass

surface. Components of the ventral disk

include the bare area (BA), lateral crest

(LC), and ventrolateral flange (VLF). From

Adam, 2001

small intestine favored by the presence of the neutral pH, and bile salts and fatty acids.

Each cyst contains two trophozoites. Trophozoites have two distinct nuclei, four pairs of

flagellae, are 12 to 15 µm length. They multiply by asexual reproduction, longitudinal

binary fission, and colonize the lumen of the proximal small bowel, attaching to the

mucosa of the bowel using a ventral sucking disk. Trophozoites are responsible for the

clinical disease in the host. They are able to move toward

the colon. Here, an event named encystation occurs: the

trophozoite retreat into the cyst stage. Cysts are excreted

in the feces and became immediately infectious, making

possible the transmission from person-to-person

(http://www.dpd.cdc.gov/dpdx/HTML/Giardiasis .htm).

The prepatent period varies with the host and species of

Giardia, with a median value of 14 days.

2.4. Ultrastucture of the trophozoite of Giardia and

sporozoite of Cryptosporidium

Cryptosporidium as an Apicomplexa parasite,

shares a variety of morphological traits common of this

phylum (Figure 3). These protists share the organization

of unique

organelles

in a region

called the

apical

complex. These organelles include the

rhoptires, the micronemes, the spical polar

ring and the conoid, which are directly

involved in the host cell invasion and

interaction. Rhoptries, micronemes and

dense granules are unique secretary

organelles that contain products required

for motility, invasion, adhesion and

invasion of host cells and the

establishment of the parsasitophorous

vacuole (Carruthers et al., 1999;

Carruthers and Sibley, 1997; Morrissette

29

and Sibley, 2002; Scholtyseck and Mehlhorn, 1970). The conoid is a small cone-shaped

structure composed of a spiral of unidentified filaments that is thought to play a

mechanical role in invasion and can be protruded from or retracted into the apical polar

ring (Nichols and Chiappino, 1987; Scholtyseck and Mehlhorn, 1970). The apical polar

ring serves as microtubule-organizing centers (MTOC’s) in Apicomplexa. Spindle pole

plaques and centrioles/basal bodies are other MTOC’s. Parallel to this, the Apicomplexa

have other unique structural features, such the apicoplast: an essential chloroplast-like

organelle (Kohler et al., 1997; McFadden and Waller, 1997; Morrissette and Sibley, 2002;

Wilson and Williamson, 1997). The parasites are bounded by the pellicle, a composite

structure consisting of the plasma membrane and the closely apposed inner membrane

complex (IMC) (Morrissette and Sibley, 2002). The pellicle is intimately associated with a

number of cytoskeleton elements, including actin, myosin, microtubules and a network of

intermediate filament-like proteins. The endoplasmic reticulum surrounds the nucleus, and

the Golgi body is immediately above it. The apicoplast is immediately adjacent to the

Golgi body (Figure 3).

Morphological studies have shown that G. duodenalis presents one of the simplest

structural organizations found in a eukaryotic cell, in which structures such as

peroxisomes, mitochondria and a well-elaborated Golgi complex are not present. Authors

have postulated that Giardia can be derived from an aerobic mitochondria-containing

flagellate (de Souza et al., 2004; Lloyd et al., 2002; Lujan et al., 1995; Lujan et al., 1997).

Trophozoites form G. duodenalis have a characteristic pear shaped body, around

12-15 µm long and 5-9 µm wide (Figure 4). The cytoskeleton includes a median body, four

pairs of flagella (anterior, posterior, caudal and ventral), and a ventral disk (Figure 5).

Trophozoites have two nuclei without nucleoli which are located anteriorly and are

symmetric with respect to the long axis (Figure 5). Lysosomal vacuoles, ribosome and

glycogen granules are found in the cytoplasm. Stacked membranes suggestive of Golgi

complexes have been demonstrated (Adam, 2001; Lanfredi-Rangel et al., 1999; Soltys et

al., 1996). Scanning electron microscopy of the ventral side of the trophozoite reveals the

adhesive disk used for the protozoan attachment to substrates, and the four pair of

flagella. These flagella are responsible for the motility, trophozoite dislocation and

attachment (Campanati et al., 2002; de Souza et al., 2004; Ghosh et al., 2001; Holberton,

1973, 1974; Owen, 1980).

30

Figure 6. The ultrastructural morphology of a

Giardia protozoan’s ventral adhesive disk on

the left, and the circular lesion on the right,

which can be left on the intestinal mucosal

surface, as a result of the tight adhesion of this

disk to the intestine’s microvillous border.

Public Health Image Library (PHIL) # 11644

(www.cdc.gov)

2.5. Pathophysiology and clinical features

Giardia is an entero-pathogen, non-cell-invasive which causes giardiasis. The

most prominent clinical signs of the disease are abdominal pain, nausea, followed by

severe watery diarrhea, dehydration, malabsorption (particularly lipids and lipid soluble

vitamins) and weight loss. Chronic courses are characterized by recurrent brief or

persistent episodes of diarrhoea. The

resolution of the infection may, in

some cases, occur after few weeks

but may also evolve to a chronic

state. The symptoms of the infection

are variable, depending on the

immunological status of the infected

individuals, as well as other non-

immunological factors, involving the

host-parasite interaction. However,

the pathophysiology associated with

these symptoms are still incompletely

understood (Eckmann and Gillin,

2001; Muller and von Allmen, 2005).

The intestinal colonization by the

parasite seems to cause villous

flattening or atrophy and microvillus

shortening (Figure 4 and 6).

Therefore, during the infection, a combination of malabsorption and hyper secretion of

electrolytes seems to be responsible for fluid accumulation in the intestinal lumens, which

leads to diarrhoea (Buret, 2008; Scott et al., 2000; Scott et al., 2004; Williamson et al.,

2000). For reasons that remain obscure, symptoms can be present in the absence of any

significant morphological injury to the intestinal mucosa, and infections may remain

asymptomatic or become chronic (Buret, 2008; Gascon, 2006; Roxstrom-Lindquist et al.,

2005). The chronicity of the infection maight be linked to the phenomenon of antigenic

variation, namely surface proteins implicated on evasion mechanisms to host immunity

(Buret, 2008; Muller and von Allmen, 2005; Roxstrom-Lindquist et al., 2006).

Recent studies have shown that G. duodenalis may induce enterocytic apoptosis,

in an assemblage dependent manner. The resulting disruption of tight junctional integrity

could be inhibited with the apical administration of epidermal growth factor (Buret et al.,

2002; Chin et al., 2002). Also, the Giardia-induced apoptosis of enterocyte cells was

confirmed in human patients with chronic giardiasis. Apparently, this fact involves

31

Figure 7. Electron microscopic evaluation of

HT29.74 cells infected with C. parvum. A) oocyst

after 1 hour in culture in the process of

excysting, which release four sporozoites (three

are visible). B) sporozoites after 6 hours in

culture in the process of infecting HT29.74 cell.

C) mature schizont 24 hours later. The schizont

is intracellular, yet extracytoplasmic. A dense

band and feeder layer which interface between

the parasite and host cell are present. Eighty

fully developed merozoites are visible. D) mature

schizont at 24 hours has rupted, releasing the

merozoites. From Flanigan et al., 1991

caspase-3 activation and other apoptotic pathways (Panaro et al., 2007; Troeger et al.,

2007). In this way, it was recently verified in chronic giardiasis clinical cases a down

regulation in the intestinal barrier function (Troeger et al., 2007). The increased epithelial

permeability allows luminal antigens to activate host immune-dependent pathological

pathways (Buret, 2008; Buret et al., 2002). In a more detailed form, the disruption of

cellular F-actin and tight junctional ZO-1 seems to be modulated by myosin-light-chain

kinase. Also, Giardia disrupts

enterocytes cells α-actinin, a

component of the actomyosin ring

that regulates paracellular flow

across intestinal epithelia (Buret,

2008; Chin et al., 2002; Teoh et al.,

2000).

In Cryptosporidium

infections, parasites invade cells

(Figure 7). Primarily this organism

infects the microvillous border of

the intestinal epithelium, and to

lesser extent extra intestinal

epithelia, causing acute

gastrointestinal disturbs (Fayer,

2004). The duration of infection and

the ultimate outcome of intestinal

cryptosporidiosis greatly depend on

the immune status of the patient. In

fact, immunologically healthy

patients usually recover

spontaneously in a week. The

clinical signs can range from

asymptomatic to acute, severe and

persistent diarrhea and their

potential for Cryptosporidium

transmission can persist for weeks

after symptoms cease (Deng et al.,

2004; Fayer, 2004; Hunter and

Thompson, 2005). Diarrhea is usually watery with mucus, and without blood or

32

leukocytes. Often stomach pains or cramps and low fever, as well as nausea, vomiting,

malabsorption and dehydration may occur, leading to anorexia and weight loss.

In immunocompromised patients, diarrhea caused by the infection of the

gastrointestinal tract becomes progressively worse and may be a major factor contributing

to death (Colford et al., 1996; Deng et al., 2004). These patients have a higher risk for

Cryptosporidium infection (Hunter and Nichols, 2002). Unusual complications of

cryptosporidiosis are directly related to low CD4 cells count (< 500 cells/mL). Other

tissues were reported to be infected in association with other disturbs: gastric colonization

by the parasite, pneumatosis cystoides intestinalis (caused by gas-containing cysts in

intestinal wall), esophagus infection, disturbs in the biliary tract (cholangitis), pancreatitis

and disturbs in the respiratory tract (Deng et al., 2004; Hunter and Nichols, 2002). In

cryptosporidiosis, progress has been made in understanding the machinery of the parasite

invasion and histopathological changes in the infected tissues (Deng et al., 2004; Tzipori

and Griffiths, 1998). On the other hand, little is known about the mechanisms involved in

the uptake of nutrients or how protozoan proteins traffic to the infect cell cytoplasm to

control host cell processes.

Cryptosporidium resides in the apical surface of intestinal epithelial cells and elicits

a strong cell mediated response (Riggs, 2002). The infection initiates by the ingestion of

the oocysts that undergo excystation releasing of the sporozoites. These cells attach to

host epithelial cells by their anterior pole, followed by invagination of the host cell

membrane (Aji et al., 1991; Deng et al., 2004; Lumb et al., 1988). With the process of

invagination, the surface of the sporozoites is completely surrounded, forming the

parasitophorus vacuole at intracellular space with extracytoplasmatic location. Here,

sporozoites undergo further development (Figure 7) (Aji et al., 1991; Deng et al., 2004;

Flanigan et al., 1991). The attachment can be affected by several factors, such as pH,

status of host cell differentiation and may be inhibited by the use of polyclonal and

monoclonal antibodies reacting Cryptosporidium surface molecules (Elliot et al., 1997; Joe

et al., 1998; Langer and Riggs, 1996; Riggs, 2002).

Receptor/ligand interactions between C. parvum and the surface of host epithelial

cells have been investigated. Recent studies suggested that several C. parvum (glycol)-

proteins of sporozoites are involved in attachment and invasion of host epithelial cells,

namely, GP900 (Barnes et al., 1998; Bonnin et al., 2001), galactose-N-

acetylgalactosamine (Gal/GalNAc)-specific lectin (Chen and LaRusso, 2000; Joe et al.,

1998), gp15/40 (Cevallos et al., 2000a; Cevallos et al., 2000b; Strong et al., 2000),

thrombospondin-related anonymous proteins (TRAP’s) (Spano et al., 1998b), CP47

(Nesterenko et al., 1999) and CSL glycoprotein (Langer and Riggs, 1999).

33

Figure 8. The most important cycles of

transmission for maintaining Giardia and

Cryptosporidium. Besides direct

transmission, water and food may also

play a role in transmission. The frequency

of interaction between cycles is not

known. From Hunter and Thompson, 2005

Intestinal mucosa epithelial cells are critical for initiation of the mucosal immune

response to different enteric pathogens. Adherence and invasion by the obligate

intracellular parasite, usually induces cytoskeleton rearrangement within the host cells as

a prelude to membrane penetration and cytoplasmatic intrusion (Theriot, 1995). In

contrast to other Apicomplexa, C. parvum sporozoites do not actively penetrate the host

cell membranes (by an actin polymerization process). So, successive intermediate stags

develop within the extracytoplasmatic space in the parasitophorous vacuole. Parasite

attachment to host plasma membrane is a primary event in the initial hosp-parasite

interaction and a prerequisite for the pathophysiological consequences (Elliott et al.,

2001). By the action of the microfilaments of the parasite, the host cell cytoskeleton is

modified originating a unique structure at the host-parasite interface (Elliott et al., 2001). In

the host, a rapid onset of phospholipid and protein kinase activities is observed after

sporozoites attachment. Also, host cytoskeleton actin and actin-binding protein villin are

focused and aggregated in the parasitophorous vacuole (Chen and LaRusso, 2000).

Cryptosporidium parvum also seems to induce apoptosis in host cells (Certad et

al., 2007; Mele et al., 2004). Nuclear condensation and increasing apoptotic cell number

was observed in in vitro HCT-8 cell cultures (Chen and LaRusso, 2000; Ojcius et al.,

1999). The role of caspases and other apoptotic signals were investigated. It has been

suggested that C. parvum has developed

strategies to limit apoptosis in order to

facilitate its growth and maturation in the

early period after epithelial cell infection

(McCole et al., 2000). Namely, infection

with C. parvum is associated with the

recruitment of leukocytes to the lamina

propria of the mucosa and with the

regulation of the expression of pro-

inflammatory cytokines and several

immune modulators. This suggests that

intestinal epithelial cells play an important

role in initiating the mucosal immune

response to C. parvum infection (Deng et

al., 2004; Riggs, 2002).

34

2.6. Epidemiology of Giardia and Cryptosporidium

Giardia and Cryptosporidium share common characteristics that influence greatly

the epidemiology of their infections. They are maintained in a variety of transmission

cycles, independently, not requiring interaction between them (Figure 8). Giardia can be

maintained in independent cycles involving wildlife or domestic animals, and similarly,

Cryptosporidium can be maintained in cycles involving livestock, especially cattle. As

observed in Figure 8, the circumstances under which such cycles may interact and where

zoonotic transfer occurs are not completely understood (Hunter and Thompson, 2005).

Cysts and oocysts are the stage transmitted from an infected host to a susceptible host by

the fecal-oral route. Several common transmission routes exist, and include a) person-to-

person through direct or indirect contact, where sexual activities may potentiate

transmission, b) from animal-to-animal, c) animal-to-human, d) water-borne through

drinking water and recreational water, and, e) food-borne (Caccio and Ryan, 2008; Caccio

et al., 2005; Fayer et al., 2000; Hunter and Thompson, 2005). The infective dose of both

parasites, in human infections, was calculated taking into account statistical data and

experimental infection studies: the ID50 varies regarding the isolates, ranging from 9 to

1042 oocysts for Cryptosporidium and 1 to 10 cysts for Giardia (Adam, 2001; Fayer et al.,

2000; Okhuysen et al., 1999). These features markedly influence the epidemiology of

these infections: a) the infective dose is low for both parasites; b) cysts and oocysts are

immediately infectious when excreted in faeces, and possess several transmission routes;

c) cysts and oocysts are very stable and can survive for weeks to months in the

environment; d) water and food may became contaminated due to the environmental

dispersal. The transmission of these infections, either direct or indirect, is favored by

several factors such as high population densities and close contact with infected hosts or

contaminated water or food. These factors are dependent on the infecting species, either

in zoonotic and anthroponotic transmissions. Recent studies suggested separated risks

for C. hominis (such as travel abroad and contact with infected diarrheic individuals) and

C. parvum (contact with cattle) (Caccio et al., 2005; Hunter et al., 2004). In sporadic

cryptosporidiosis, risk factors include the age of patients (children under five years of

age), travelling, contact with infected individuals and contact with farm animals (Caccio et

al., 2005). Furthermore, swimming in public swimming pools or recreational areas

represents a risk of infection, as suggested by Australian and US studies (Robertson et

al., 2002; Roy et al., 2004). Curiously, authors have postulated that, although

Cryptosporidium is transmitted through contaminated food, a small number of parasites in

these samples may not induce infection with clinical symptoms but a protective immunity

(Meinhardt et al., 1996).

35

Table 4. Prevalence of five common Cryptosporidium species in humans. From Xiao et al., 2004

Similar studies regarding Giardia transmission and sporadic giardiasis performed

by authors in UK, reveled as main risks the swallowing of water while swimming, drinking

treated tap water, contact with fresh water and easting lettuce (Figure 8) (Stuart et al.,

2003). The introduction of molecular tools analysis on the epidemiological field can

produce useful information allowing to a better understanding about the origin of

contamination, the genetic characterization of involved species/genotype/assemblage and

their zoonotic potential: a new field in the modern molecular epidemiology.

2.6.1. Cryptosporidiosis and giardiosis in humans

Cryptosporidiosis has been reported worldwide, in more than 90 countries over 6

continents. It was reported in individuals aged from 3 d-o to 95 y-o, although young

children appear to be more susceptible to infection. The firsts human cases of

cryptosporidiosis were reported in 1976, as referred before. Immunocompromised

individuals represent a serious group risk. The consequences of cryptosporidiosis as an

opportunistic infection are well known (Fayer, 2004; Fayer et al., 2000). Consecutive

observations showed that children in day-care centers represented another important

group at risk.

In 1986, the Center for Diseases Control (CDC) has reported an important

observation regarding the impact of cryptosporidiosis on Human Health. According to

CDC, 3.6% out of 19817 AIDS clinical cases had cryptosporidiosis, among

Cryptosporidium-infected patients the fatality rate was 61% (Fayer, 2004).

Several epidemiological studies, regarding the evaluation on geographic

distribution of cryptosporidiosis, based on the detection of oocysts in fecal samples and

seroprevalence: European countries (0.1-14.1%); North America countries (0.3-4.3%);

African countries (2.6-21.3%); Central and South American countries (3.2-31.5%); Asian

countries (1.3-13.1%). Probably these results are consequence of sanitation conditions,

quality of food and water. Obviously developing countries populations have a higher risk

36

Table 5. Species and assemblages of

Giardia. From Xiao and Fayer, 2008

of Cryptosporidium-infection, particularly children, undernourished individuals and a range

of immunocompromised individuals such as AIDS patients, transplant recipients, patients

receiving chemotherapy for cancer, institutionalized patients and patients with

immunosuppressive infectious diseases) (Fayer, 2004; Fayer et al., 2000).

Until now, different molecular studies show that C. parvum and C. hominis were

the major species responsible for human cryptosporidiosis. C. meleagridis, C. felis and C.

canis, traditionally associated with animals, were found in AIDS patients (Table 4) (Xiao

and Fayer, 2008; Xiao et al., 2004). Also, C. suis and C. muris are, to a less extent,

reported in human infections (Xiao and Fayer, 2008; Xiao et al., 2004).

Geographic and disease burdens differences were reported to C. parvum and C.

hominis (Xiao and Ryan, 2004). In the UK, early studies showed a higher prevalence of C.

parvum over C. hominis. In opposition, a more recent surveys have showed the reverse

situation: 50.3% out of 13112 cases of cryptosporidiosis were associated with C. hominis

and 45.6% with C. parvum (Nichols et al., 2006). Similar studies made in other European

countries showed the same trend: no correlation in prevalence rates of C. parvum and C.

hominis (Llorente et al., 2007; Wielinga et al., 2008). In general, C. hominis is more

prevalent C. parvum in the USA, Canada, Australia, Japan and developing countries

where molecular tools have been used to identify specimens (Xiao and Fayer, 2008).

The nomenclature for Giardia is confusing and, although the modern genetic

analysis tools helped in this organization,

there is still lack of clarity (Xiao and Fayer,

2008). The species Giardia agilis, Giardia

ardeae, Giardia muris, Giardia microti and

Giardia psittaci have not been found to

infect humans, but animals; G. duodenalis

is the only species infecting humans,

particularly the established assemblages A

and B (Table 3 and 5) (Caccio et al., 2005).

Both assemblages A and B are also able to

infect animals, which imply that the

zoonotic transmission plays an important role in the epidemiology of human giardiasis

(Caccio and Ryan, 2008; Xiao and Fayer, 2008). The prevalence of each assemblage

varies considerably from country to country with assemblage B appearing to be more

common overall (Table 7). However, the number of molecular epidemiological studies

concerning giardiasis in humans is smaller and, until now, do not evidence clear

geographic or socioeconomic differences in the distribution of assemblage A and B, or,

37

Table 6. Cryptosporidium spp. and genotypes that infects humans and other hosts. From Xiao and Fayer, 2008.

Table 7. Prevalence of Giardia duodenalis assemblage A and B in humans. From

(Caccio and Ryan, 2008)

moreover, clearly indicate the role of zoonotic infections in human giardiasis (Caccio and

Ryan, 2008; Caccio et al., 2005; Xiao and Fayer, 2008).

2.6.2. Cryptosporidiosis and giardiosis in animals

As referred to before, most

Cryptosporidium and Giardia species and

genotypes are host-adapted in nature,

having a narrow spectrum of natural hosts.

This indicates that the majority of species

probably do not have high infectivity to

humans, since one species or genotype,

usually infects only a particular specie or a

group of related animals (Fayer et al.,

2000). However, there are exceptions and

some species of Cryptosporidium and

Giardia have been recognized as having

zoonotic potential. Several studies about

animal giardiosis and cryptosporidiosis

emphasizes evidences on an existence of

host-adapted species and limited cross

transmission occurring among different groups of animals (Table 5 and 6) (Caccio et al.,

2005; Fayer et al., 2000; Hunter and Thompson, 2005; Xiao and Fayer, 2008; Xiao et al.,

2004). Cryptosporidium parvum has a recognized zoonotic potential and, for that, has

38

Table 8. G. duodenalis genotypes in farm animals. From Xiao and Fayer, 2008

received a major attention concerning the cross-species transmission. A few years ago, it

was thought that C. parvum could infect all mammals, although the genetic

characterization showed absence of these species in wild mammals (Feng et al., 2007a;

Zhou et al., 2004). Nowadays, it is generally accepted that C. parvum infects ruminants

and humans. Cattle, in particular young calves, aged less than 2 months, are frequently

infected by C. parvum. The prevalence in beef calves is often lower than in dairy calves

(Kvac et al., 2006). Cryptosporidium bovis and the Cryptosporidium deer-like genotype

infect mainly older dairy calves, and C. andersoni mature cattle (Fayer et al., 2006c; Feng

et al., 2007b; Langkjaer et al., 2007; Santin et al., 2004). Sheep are mostly infected with

Cryptosporidium cervine genotype and other genotypes (Ryan et al., 2005; Santin et al.,

2007). Natural C. parvum infections have been found occasionally in animals such as

mice, raccoon dogs and dogs (Giangaspero et al., 2006; Morgan et al., 1999). Companion

animals, such as dogs and cats, are most often infected with host-specific C. canis and C.

felis (Fayer et al., 2006b; Huber et al., 2007; Morgan et al., 2000; Rimhanen-Finne et al.,

2007; Santin et al., 2006; Satoh et al., 2006). The role of these animals in the

transmission of human cryptosporidiosis appears quite limited, once C. canis and C. felis

infections are infrequently reported for humans.

Giardia duodenalis is found in animals, both livestock and companion animals.

Most of these animals harbor unique G. duodenalis assemblages, although some were

also found to harbor assemblages A and B, where relies the zoonotic potential of G.

duodenalis. According to worldwide observations, majority of cattle, sheep and pigs are

infected with the assemblage E of G. duodenalis, although a significant number of cattle

39

Figure 9.

Photomicrograph of a

Giardia duodenalis cyst

seen using a Trichrome

stain. Public Health

Image Library (PHIL) #

1944 www.cdc.gov

Figure 10.

Photomicrograph of

Cryptosporidium parvum

oocysts, which had been

stained using the

modified acid-fast

method. Public Health

Image Library (PHIL) #

7829 www.cdc.gov

are also infected with assemblage A. In the opposite, assemblage B is rarely found in

cattle and other assemblages (C, D, F or G) were never found in these animals (Table 8).

There are no age-associated differences in the prevalence of assemblages A and

E in cattle. In sheep, assemblage E is much more dominant than A, and assemblage B is

rarely detected (Table 8) (Caccio et al., 2007; Castro-Hermida et al., 2007; Di Giovanni et

al., 2006; Feng et al., 2008; Geurden et al., 2008a; Geurden et al., 2008b; Lalle et al.,

2005; Langkjaer et al., 2007; Mendonca et al., 2007; Read et

al., 2002; Ryan et al., 2005; Santin et al., 2007; Souza et al.,

2007; Sulaiman et al., 2003; Traub et al., 2005; Trout et al.,

2007, 2008; Trout et al., 2004, 2005; Trout et al., 2006b). In

pigs, the pattern of assemblage A and E prevalence is

similar to that of cattle and sheep (Langkjaer et al., 2007).

Although the studies are quite limited, only assemblages A

and B were detected in horses (Traub et al., 2005).

Regarding the companion animals, for instance dogs, they

are infected by a broader range of G. duodenalis

assemblages: dogs were found to be infected with

assemblages A, B, C and D (Monis et al., 1998; Xiao and

Fayer, 2008). Cats are also infected with assemblage A and

also assemblage F: this assemblage is cat-specific and is found more frequently than

assemblage A (Xiao and Fayer, 2008). Little information is available on the prevalence of

G. duodenalis assemblages affecting wildlife. Some studies showed the presence of

assemblage A and E in wild cervids, and only assemblage A

in white-tailed deer in the USA (Trout et al., 2003), moose

and reindeer (Robertson et al., 2007), fallow deer (Lalle et

al., 2007) and fox and kangaroos (McCarthy et al., 2008).

Assemblages B, C and D are found in other wild mammals,

such as assemblage B in beavers (Fayer et al., 2006a) or

assemblage B, C and D in coyotes (Trout et al., 2006a).

2.7. Laboratory diagnosis

Diagnosis of Cryptosporidium and Giardia infections

requires, normally, the morphological identification of the

oocysts and cysts, in stool specimens, intestinal aspirates or

intestinal biopsy specimens (Alles et al., 1995). This

identification is done by microscopic examination, after

40

using microscopic staining methods and immunological-based detection methods after

concentration techniques. Molecular techniques can also be used (Fayer et al., 2000).

Diagnostic sensitivity of microscopic staining methods is often limited by the shedding of

organisms intermittently or in low numbers. This sensitivity is also dependent on the skills

of the microscope technicians. Both for Cryptosporidium and Giardia, routine staining

methods include staining with chlorazol black E or modified Kinyoun acid-fast stain (Figure

10) (Garcia et al., 1992; Ma and Soave, 1983), which can give better results than

trichrome or iron hematoxylin stain (Figure 9) (Alles et al., 1995; Bullock, 1980). These

differential staining methods also include safranin-methylene blue (Baxby et al., 1984),

Ziehl-Neelson (Henriksen and Pohlenz, 1981) and DMSO-carbol fuchsin (Pohjola et al.,

1985) which stain the parasite in red and counterstain the background. Negative staining

techniques also exists but are not routinely used (Fayer et al., 2000).

Immunological-based methods appeared with the development of polyclonal and

monoclonal antibodies (Fayer et al., 2000). These antibodies may be combined with

several molecules, for instance, fluorescent fluorochromes to develop fluorescent antibody

tests. There are also latex agglutination reactions (Pohjola et al., 1986), enzyme-linked

immunosorbent assays (ELISA) (Dagan et al., 1995), reverse passive haemagglutination

(Farrington et al., 1994) and immunochromatographic assays (Garcia and Shimizu, 2000).

The most routinely used technique is the direct immunofluorescence assay with

monoclonal antibodies (DFA) because it is a sensitive and specific technique and fast to

perform; it requires an epifluorescent microscope. Several studies compared the DFA

technique with regular staining methods and showed a great increase in sensitivity and

specificity of the reaction (Alles et al., 1995; Garcia et al., 1992; Kehl et al., 1995).

The diagnosis of Cryptosporidium and Giardia can also be extended to other

biological samples, particularly water and food samples. In these cases, the diagnostic

technology is completely different. In water samples, or water used for wash food, cysts

and oocysts need to be concentrated using methods as continuous centrifugation,

membrane filtration, flocculation with calcium carbonate, Envirocheck (Gelman) cartridge

filters and polycarbonate membrane systems (Corning Costar) (Fricker and Crabb, 1998).

After this, concentrated cysts and oocysts are separated by density gradient centrifugation

or immunomagnetic bead separation (IMS) from the remaining debris. The

immunomagnetic bead separation technique combines an antibody with magnetic

particles and has a very high recovery rate, comparing to density gradient centrifugation,

although these recovery rates are affected by many factor such as turbidity, other

physical-chemical properties of the water and cross-reaction of the antibody (Smith,

1998). For more detailed technical information see chapter IV. The previous referred

identification methods can be applied to these processed samples. The major difficulties

41

of these methods is that they are unable to distinguish the species of the parasites of

public health significance from other and to distinguish live from dead parasites (Fayer et

al., 2000).

The introduction of genetic techniques in the diagnosis of Cryptosporidium and

Giardia both in clinical and environmental samples created a great alternative to the

conventional methods and helped in get beyond difficulties associated with those methods

(Fayer et al., 2000; Morgan and Thompson, 1998; Smith, 1998). The molecular

techniques are useful in the assignment of species and on the evaluation of the respective

zoonotic potential; Polymerase chain reaction (PCR) is a rapid technique, highly sensitive

and accurate. However, the PCR may induce false positives since it may amplify naked

nucleic acids, non-viable microorganisms, suffer laboratory contamination or cross react

with other organisms that contaminates environmental samples (Fayer et al., 2000). For

more detailed technical information see chapter II to V.

2.8. Genotyping

Genotyping is defined as a process to determine the genetic differences between

individuals or cells by the use of molecular tools. The main objective of this approach is,

by using molecular based techniques, characterize or organize the organisms. The first

attempt to organize Cryptosporidium and Giardia isolates, based on molecular data, was

the analysis of zymodeme, as referred before. However, this is not a gene-based

approach, but an analysis of isozyme pattern (proteins). Nowadays, the molecular tools

used are normally PCR-based, targeting the amplification of a gene which gives

information about the species or genotype of the isolate.

These molecular tools have been helpful to enhance our knowledge and

understanding of the taxonomy, host range and transmission routes of Cryptosporidium

and Giardia and the epidemiology of human disease. Moreover, these tools are used to

understand the public health importance of different environmental routes of transmission,

leading toward improved strategies for prevention and surveillance of cryptosporidiosis

and giardiasis (Fayer et al., 2000; Jex et al., 2008; Monis and Thompson, 2003; Smith et

al., 2006). Some methods rely on the specific in situ hybridization of probes to particular

genetic loci within Cryptosporidium oocysts and Giardia cysts, whereas the majority relies

on the specific amplification of one or more loci from small amounts of genomic DNA by

polymerase chain reaction (PCR). This is particularly helpful in the environmental samples

or others where the parasite load is low (Jex et al., 2008; Smith et al., 2006). The PCR is

suitable to be combined with other post-PCR techniques, as showed in next paragraphs.

42

2.8.1. Techniques

2.8.1.1. PCR and PCR-RFLP

The amplification of a chosen target sequence by the use of specific primers under

a specific temperature cycles is the base of the PCR. PCR-restriction fragment length

polymorphism (PCR-RFLP) is a technique largely used in Cryptosporidium and Giardia

genotyping. It combines the PCR amplification with the digestion of the amplicons, or PCR

amplified fragment. The digestion is performed by the use of a restriction enzyme.

Regarding the translation of RNA to proteins, differences in the DNA sequence may cause

2 scenarios: a) the mutation does not cause a change in the aminoacid (synonymous

mutation), or b) the mutation may change the codon changing the aminoacid (non-

synonymous mutation). Even if the mutation is synonymous, it may introduce a new

restriction site for a restriction enzyme or delete it. If these mutations or polymorphisms

exist in different species and strains, the PCR-RFLP takes advance of this fact and shows

different restriction patterns in agarose gel for different species.

Several authors use PCR-RFLP to differentiate species of Cryptosporidium and

Giardia (Fayer et al., 2000; Quintero-Betancourt et al., 2002; Strong et al., 2000; Widmer,

1998; Xiao and Ryan, 2004). For Cryptosporidium species assignment, the most common

genes used are the Cryptosporidium oocyst wall protein (cowp) (Leng et al., 1996; Spano

et al., 1997b), the 70 kDa heat shock protein (hsp70) (Gobet and Toze, 2001), the

thrombospondin-related adhesive protein (trap) genes (Spano et al., 1998a) and the 60

kDa glycoprotein (gp60) gene (Wu et al., 2003). For Giardia species assignment, the most

common genes used are the Giardia lamblia open reading frame 4 (glorf-c4) (Yong et al.,

2002), the triose phosphate isomerase (tpi) gene (Amar et al., 2003), the glutamate

dehydrogenase (gdh) gene (Read et al., 2004) and the β-giardin (bg) gene (Robertson et

al., 2007).

One disadvantage of PCR-RFLP technique is that it uses endonuclease(s) that

only recognize a small number of variable sites, and such approach do not detect all of

the length and sequence variation within or among amplicons during analysis (Gasser,

2006). This issue bring us to the following “PCR and direct sequencing” paragraph.

2.8.1.2. PCR and direct sequencing

The direct sequencing of an amplified gene or gene portion remains the “gold-

standard” approach for detecting genetic variation or polymorphisms and, consecutively,

accurate specie assignment. It is the most common technique used throughout the world,

regarding the genotype of Cryptosporidium and Giardia. In fact, in my residence-

laboratories, both in Portugal and Italy, this technique has been used in the assignment of

43

Figure 11: Genetic relationship among named Cryptosporidium species and unnamed

genotypes inferred by a neighbor-joining analysis of the partial SSU rRNA gene. Values on

branches are percent bootstrapping using 1,000 replicates. Numbers following species or

genotypes are isolate identifications used in the construction of the phylogenetic tree,

whereas numbers in parentheses are the number of isolates sequenced. From Xiao et al.,

2004.

several other organisms, such as Toxoplasma protozoa, Echinococcus cestoda, or in

bacteria Escherichia coli.

This technique can be applied to single-copy and multi-copy genes. As an

advantage of the direct sequencing, the available data used for specie identification is

suitable for phylogenetic studies or comparative genetic investigations (Abe et al., 2003;

Berrilli et al., 2004; Geurden et al., 2009; Itagaki et al., 2005; Jex et al., 2008; Morgan et

al., 1997; Smith et al., 2006). With the use of the PCR direct sequencing more informative

is obtained from the markers (Figure 11).

2.8.1.3. PCR-SSCP

PCR-single strand conformation polymorphism (PCR-SSCP) is a particularly useful

approach based on the electrophoretic mobility of a single-stranded DNA molecule in a

44

non-denaturating gel and its dependence on the conformation and size of the molecule.

This technique may detect a single point mutation in amplicons till a size of 500 bp

(Gasser, 2006). PCR-SSCP has been particularly used for display sequence variation in

SSU and hsp70 genes of Cryptosporidium and is useful for the screen of genetic

variability and unknown mutations (Chalmers et al., 2005; Gasser, 2006; Jex et al., 2008).

2.8.1.4. Real Time PCR (qPCR)

Real Time PCR was developed in the early 1990s (Higuchi et al., 1992). It allows

the amplification in PCR to be monitored in real time. The Real Time principle consist in

the incorporation of a specific intercalating dye in the PCR and measuring the changes in

the fluorescence via a digital camera (Higuchi et al., 1993). Although it is technically very

similar to a standard PCR, the Real Time PCR has several advantages: it does not

requires handling after the amplification since it allows high throughput analysis in a

“closed-tube” format, and it can be used to differentiate amplicons of varying sequences

by melting curve analysis. The most recent improvements inside this technique,

introduced better dyes and capillary thermal cycler, enabling the quantification (Ct or cycle

threshold) of the sample by comparison with DNA standards. Also, the use of the dyes

allows the determination of the melting temperature or denaturation of the amplicons:

since this temperature depends on the sequence composition, the melting curve is used

to characterize the variation among samples. Furthermore, the range of Real Time

application became larger by the introduction of probes sequence-specific (Monis et al.,

2005).

There are Real Time PCR approaches using TaqMan probes described targeting

Cryptosporidium and Giardia genes. In 2001 and 2003 some author developed probes

targeting the Cp11 and 18S rRNA gene of Cryptosporidium (Higgins et al., 2001; Keegan

et al., 2003), and in 2002 the β-tubulin gene of Cryptosporidium (Tanriverdi et al., 2002).

In 2004 some authors targeted the SSU RNA (Verweij et al., 2004) and the elongation

factor 1 (ef1) (Bertrand et al., 2004) of Giardia. TaqMan probes are one of the most widely

used Real Time PCR chemistries mainly because the assay design is easy and the

assays are robust. TaqMan assays can be multiplexed by using probes with different

colored fluorophores (Monis et al., 2005).

2.8.1.5. DNA microarrays and microsatellites or SSR’s

Microsatellites or simple sequence repeats (SSR) or tandem repeats are very

small (1-6 bp) sequence repetitions in the genome. Minisatellites are longer than the

microsatellites (10 to 199 bp) and used for the same investigations purposes. Highly

variable loci containing microsatellite regions such as glycoprotein 60 kDa (gp60) and

45

microsatellite locus 1 (ML1) and 2 (ML2) on Cryptosporidium, have been employed in

population genetic investigations (Chalmers et al., 2005; Hunter et al., 2007; Leoni et al.,

2007; Strong et al., 2000; Sulaiman et al., 2005). Multilocus satellite has been utilized to

investigate the population structure and the role of genetic exchange in Cryptosporidium

and Giardia (Jex et al., 2008), for instance to track evidences of clonality in C. parvum

populations (Caccio et al., 2000; Mallon et al., 2003a; Widmer et al., 2004).

DNA microarrays provide a powerful tool for the simultaneous analysis of multiple

genes and gene transcripts. Microarrays are arrays of either cDNAs or oligonucleotides

spotted onto a glass microscope slide or synthesized on a silicon chip (Monis et al., 2005).

Samples may be subjected to a PCR prior to the array to improve sensitivity and to label

the sample with fluorescently labeled primers. Microarrays have been used to detect and

discriminate species of Cryptosporidium targeting polymorphisms in hsp70 gene (Straub

et al., 2002), or to detect and discriminate between a range of parasites, including

Cryptosporidium and Giardia, in a single assay (Wang et al., 2004). In this work of Wang

et al., 2004, the same targets were used for Giardia (hsp, tpi, beta giardin, gdh and orf-c4)

and Cryptosporidium (cowp, SSUrRNA, trap, hsp and p23) detection and genotyping.

2.8.2. Genes used

Central to the PCR-based identification of Cryptosporidium and Giardia specie is

the choice of the appropriate DNA target region: genetic marker or locus. Different genes

evolve at different rates and the DNA sequence target should contain a certain amount of

variability: DNA should differ enough in sequence to allow the differentiation of the species

but do not display or display minor variation within a species (Gasser, 2006; Jex et al.,

2008). A major advance made in the comprehension of the evolution of these genes and

Figure 12: EuPathDB at http://eupathdb.org/eupathdb/

46

their organization was made when the complete genome of some Cryptosporidium and

Giardia species and genotypes was released. A database has been created that contains

C. parvum and C. hominis complete genome, partial genome of C. muris, the complete

genome of two assemblages of the specie Giardia duodenalis (A and B) and partial

genome of G. duodenalis assemblage E: Eukaryotic Pathogen Database Resource

(EuPathDB) at http://eupathdb.org/eupathdb/ (Abrahamsen et al., 2004; Xu et al., 2004;

Franzen et al., 2009; Morrison et al., 2007) (Figure 12). Information regarding protein

sequences, transcripts, protein features and localization and other features is also

available for query. As refered before in this paragraph, this genetic information, now

public, allows the understanding of the organization of a gene, its localization in the

genome and may be helpful in finding other markers that may improve the genotyping

tools.

In these next paragraphs, I’m going to describe some of the genes used for

genotyping and particular features of each gene.

2.8.2.1. hsp70, cowp, β-tubulin, 18S rDNA, trap, gp60 and actin of Cryptosporidium

Heat shock protein 70 kDa (hsp70) was first described in C. parvum in 1955

(Khramtsov et al., 1995). These authors cloned the hsp70 gene describing it as an

intronless gene that codes for a protein with 674 amino acid residues, has a molecular

mass of 73403 Da and is a cytosolic protein. This protein is homologous to other

described cytoplasmic forms of 70 kDa heat shock proteins. In C. parvum this gene is

localized in chromosome 2, although it has orthologs or paralogs genes in other genome

regions, as obviously in C. hominis and C. muris. Orthologs and paralogs are two

fundamentally different types of homologous genes that evolved, respectively, by vertical

descent from a single ancestral gene and by duplication. Ubiquitous chaperones

belonging to the 70 kDa class are known to bind immature proteins or preproteins and to

facilitate their maturation and translocation across membranes into several subcellular

compartments. Recently, some authors described a C. parvum mtHSP70 that is a nuclear

gene with proto-mitochondrial origins, possesses a mitochondrial targeting sequence and

is part of the mitochondrial protein import machinery (Slapeta and Keithly, 2004). Fixed

differences on the sequence of this gene among Cryptosporidium species turn it useful in

genotyping Cryptosporidium species.

Cryptosporidium oocyst wall protein (cowp) was first described in C. parvum in

1997 (Spano et al., 1997a). These authors determine the size of cowp protein of 1622

amino acid long and localize it in a large cytoplasmic inclusion and in the wall-forming

bodies of early and late macrogametes, respectively. In oocysts, cowp is localized in the

inner layer of the oocyst wall (Spano et al., 1997a). In C. parvum this gene is localized in

47

chromosome 6, although it has orthologs or paralogs genes in other genome regions, as

obviously in C. hominis and C. muris. Fixed differences on the sequence of this gene

among Cryptosporidium species turn it useful in genotyping Cryptosporidium species.

Tubulin was first described in C. parvum in 1994 (Edlind et al., 1994). Latter, In

1997 was described by other author with greater detail (Caccio et al., 1997). These

authors described beta tubulin as a single copy gene with introns, and the protein

sequence is the most divergent among Apicomplexa, although all the beta tubulin specific

residues are conserved. In C. parvum this gene is localized in chromosome 6, although it

has orthologs or paralogs genes in other genome regions, as obviously in C. hominis and

C. muris. Microtubules are polymers of α and β tubulins found in all eukaryotic cells; they

are the major components of the mitotic spindle, the cytoskeleton and the axonemes.

Tubulins contain domains microtubules-associated protein (MAPs) and GTP binding

domains, performing a major role in microtubule-based movement of the cell, in the

structure of the cell and in many other activities. Fixed differences on the sequence of this

gene among Cryptosporidium species turn it useful in genotyping Cryptosporidium

species.

18S rRNA gene was first cloned and the sequence determined in C. parvum in

1992 (Cai et al., 1992). Latter, some authors determine the ribosomal RNA gene

organization in the same organism, based on the hypothesis that the genes encoding the

cytoplasmic ribosomal RNAs (rRNA) in most eukaryotes are organized into transcriptional

units with a small subunit rRNA gene, a 5.8S rRNA gene, and a large subunit rRNA gene

in a 5’-3’ orientation separated by internal transcribed spacers, what they called the rDNA

unit (Le Blancq et al., 1997). In this work, the authors calculated the size of the large

subunit rRNA in 3.6 kb, and concluded that the rDNA unit in C. parvum has the standard

arrangement of 5’ small subunit rRNA – internal transcribed spacer 1 – 5.8S rRNA –

internal transcribed spacer 2 – large subunit rRNA 3’, the minimum size would be 6.5 kb,

there are five copies of the rDNA unit per haploid genome in C. parvum, there are two

types of rDNA unit in C. parvum and rDNA units are dispersed through the genome of C.

parvum. From the CryptoDB database, part of the EuPathDB, these rDNA units are

localized in chromosome 1, 2, 7 and 8 of C. parvum. Fixed differences on the sequence of

this gene among Cryptosporidium species turn it useful in genotyping Cryptosporidium

species.

Thrombospondin related adhesive protein of Cryptosporidium-1 (trap-c1) was first

described in C. parvum in 1998 (Spano et al., 1998b). The authors cloned and sequence

trap-c1 form C. parvum by assuming is homology with micronemal antigens of Eimeria,

and describe it has a sequence with introns localized in chromosome 6, that codes for a

76 kDa protein. It has orthologs or paralogs genes in other genome regions, as obviously

48

in C. hominis and C. muris. TRAP-C1 protein is characterized by the presence of TRM

motifs, characteristic of the thrombospondin family; this motif gives TRAP-C1 the ability to

bind to sulfated glycoconjugates, which are the constituents of the mucosal glycocalix that

covers the surface of the intestinal cells; the authors immunolocalized this protein in the

apical complex of C. parvum sporozoites; all the observations indicate that this protein is

involved in the process of host cell invasion by C. parvum sporozoites (Spano et al.,

1998a; Spano et al., 1998b). A second TSP-related protein of C. parvum, termed TRAP-

C2, is also described. Is localized in chromosome 5 and the protein of 430kDa is a

component of the sporozoites membrane, has a molecular activity of oxireductase and

iron-sulfer cluster binding, and is involved in cell differentiation process (CryptoDB,

Cryptosporidium genome resource). Fixed differences on the sequence of this gene

among Cryptosporidium species turn it useful in genotyping Cryptosporidium species.

The glycoprotein 60 kDa (gp60) was first described in C. parvum in the year 2000

(Strong et al., 2000). The authors intended to clone the sequence of an 11A5 antigen,

known to react with a monoclonal antibody that identified a 15 kDa surface glycoprotein

shed behind motile sporozoites and recognized by several lectins that neutralized parasite

infectivity in cultured epithelial cells. Surprisingly, the authors realize that the gene cloned

encoded a 330 amino acid, mucin-like glycoprotein that was predicted to contain an N-

terminal signal peptide, a homopolymeric tract of serine residues, 36 sites of O-linked

glycosylation, and a hydrophobic C-terminal peptide specifying attachment of a

glycosylphosphatidylinositol anchor. This gene is single copy, localized in the

chromosome 6, it has orthologs or paralogs genes in other genome regions, as obviously

in C. hominis and C. muris, lacked introns and was expressed during merogony to

produce a 60 kDa precursor which was proteolytically cleaved to 15 and 45 kDa

glycoprotein products that both localized to the surface of sporozoites and merozoites

(Strong et al., 2000). This gene, now named gp15/45/60, display a very high degree of

sequence diversity among C. parvum isolates, with single-nucleotide and single-amino-

acid polymorphisms defining five to six allelic classes, each characterized by additional

intra-allelic sequence variation. In this way, the gp14/45/60 gene is very useful for

haplotyping and fingerprinting isolates and for establishment meaningful relationships

between C. parvum genotypes and phenotype (O'Brien et al., 2008; Power et al., 2009;

Strong et al., 2000).

Actin is a ubiquitous and highly conserved microfilament protein first described by

in C. parvum in 1992 (Kim et al., 1992). It is hypothesized to play a mechanical, force-

generating role in the unusual gliding motility of sporozoan zoites and their active

penetration of host cells. The same authors cloned the actin sequence of C. parvum and

found an homology of 85% to Plasmodium falciparum and human γ-actin proteins (Kim et

49

al., 1992). The authors identified a 42 kDa protein containing 376 amino acids encoded by

a single-copy gene with no introns. Latter, some authors localized actin in all stages of C.

parvum development, in the pellicles and cytoplasm near the feeder organelles, all over

the cytoplasm and membranes (microvillous, parasitophorous and parasite membranes)

(Yu and Chai, 1995). According to this location, actin seems to be involved in making

shapes of parasites and in membrane surface movement such as protruding of

microspikes. The authors observed that each stage of C. parvum moved within the

epithelial cells through formation of microspikes; also, in meront stage, actin seems to be

de novo synthesized and not only polymerized; finally, actin may have an effect on

exchange and transportation of some materials between the parasite and host cell by

forming gel-like structure around the feeder organelle. Actin gene has orthologs or

paralogs genes in other genome regions, as obviously in C. hominis and C. muris. Fixed

differences on the sequence of this gene among Cryptosporidium species turn it useful in

genotyping Cryptosporidium species.

2.8.2.2. β-giardin, tpi, gdh, SSU rDNA, glorf-c4, ef-1α and vsp’s of Giardia

Giardin were first described and isolated in 1985 in Giardia (Crossley and

Holberton, 1985; Peattie et al., 1989). These authors described giardins as a family of

structural proteins found in microribbons attached to microtubules on the disc cytoskeleton

of Giardia, a family of proteins of around 30kDa with acidic isoelectric points. The genes

that code for these giardins are presented in single copy in the genome and code for

proteins very rich in α-helix conformations. To date, three classes of giardins have been

characterized: α, β and γ. These proteins have, respectively, Mr values of 33kDa (α),

29kDa (β) and 38kDa (γ) and all are localized in the ventral disk (Alonso and Peattie,

Figure 13: Search by β-giardin gene in GiardiaDB at http://eupathdb.org/eupathdb/

50

1992; Holberton et al., 1988; Nohria et al., 1992). The sequence of α-1-giardin and α-2-

giardin, proteins located on the edges of the disk microribbons, was first described in 1992

(Alonso and Peattie, 1992). γ-giardin sequence was presented in 1992 (Nohria et al.,

1992). β-giardin was first described in 1988 (Holberton et al., 1988) and the sequence

presented in the same year by other authors (Baker et al., 1988). These authors

described a 259 amino acids residues predicted to be α-helical. The molecular function of

β-giardin is defined as structural constituent of cytoskeleton, and is coded by an 819 bp

gene with no introns and the respective expression is well established in the excystation

process of Giardia. There are several α, β and γ-giardin proteins identified with orthologs

or paralogs genes in other genome regions, as obviously in other Giardia species

(GiardiaDB) (Figure 13). α-giardin are located in chromosome 3, β-giardin in chromosome

4 and γ-giardin in chromosome 3 (Adam, 2000). Fixed differences on the sequence of this

gene among Giardia species turn it useful in genotyping Giardia species.

Triose phosphate isomerase (tim or tpi) of G. duodenalis was first cloned and

sequenced in 1994 (Mowatt et al., 1994). These authors found that, similar to other

Giardia protein-coding genes, tim gene lacks introns and is transcribed to yield a

polyadenylated mRNA with an extremely short 5’ untranslated region. It codes for an

enzyme that catalyses the reversible reaction between D-glyceraldehyde 3-phosphate and

dihydroxyacetone phosphate (Adam, 2001). The gene codes for a protein with 257 amino

acid residues that functions in the cytosol. Fixed differences on the sequence of this gene

among Giardia species turn it useful in genotyping Giardia species.

NADPH-dependent glutamate dehydrogenase (gdh) of Giardia was first described

in 1992 (Yee and Dennis, 1992). Glutamate dehydrogenases are enzymes that play an

important role in carbohydrate metabolism and ammonia assimilation, amino acid

synthesis and/or catabolism. In Giardia they catalyze the interconversion between α-

ketoglutarate and L-glutamate using either NADP or NAD as coenzyme (Adam, 2001;

Park et al., 1998; Yee and Dennis, 1992). The gene in Giardia codes for a 449 amino acid

sequence. Fixed differences on the sequence of this gene among Giardia species turn it

useful in genotyping Giardia species.

Small subunit ribosomal RNA (SS rRNA or 18S rRNA) has been the most useful

gene for molecular comparisons on Giardia, because rRNA sequences are highly

conserved across life and because the function of the rRNA is very central to the biology

of the organism (Adam, 2001). Based on comparisons of SS rRNA sequences, G.

duodenalis was proposed as one of the most primitive eukaryotic organisms, an early

branching eukaryote (Sogin et al., 1989). First descriptions of SS rRNA gene showed that

is inserted within a 5.6 kb tandemly repeated DNA, as shown by Southern blot analysis

and DNA cloning (Edlind and Chakraborty, 1987). The 5.6 kb DNA contains 1300 bp and

51

2300 bp that codes for the SS rRNA and a large subunit, that are, in comparison to other

protozoa, very short (Edlind and Chakraborty, 1987). Most of these genes are contained

on a single chromosome, chromosome 1. Studies with fibrillarin, a conserved pre-

ribosomal RNA processing protein showed that the transcription and processing of rRNA

does not seem to be localized into certain regions of the nuclei (Narcisi et al., 1998).

Giardia lamblia open reading frame C4 (glorf-c4) was first described in 1992 (Nash

and Mowatt, 1992b). In this study, the authors described this gene as a 597bp in length

coding a protein of 198 amino acids characterized by a polyserine motif. Recently, other

authors characterize the coded protein which was considered to be specific of G.

duodenalis (Nores et al., 2009). The protein has 22 kDa and assembles into high-

molecular-mass complexes during the entire life cycle of the parasite. The protein

localizes in the cytoplasm of the cysts and trophozoites; it seems to interfere in the

differentiation of trophozoites and cysts. ORF-C4 protein has no orthologous proteins and

conserved domains are found in the databases. However, it contains a region structurally

similar to the alpha-crystallin domain of small heat-shock proteins that, supported by the

same study, indicates the potential role of ORF-C4 as a small chaperone involved in the

response to stress, including encystation, in G. duodenalis. Fixed differences on the

sequence of this gene among Giardia species turn it useful in genotyping Giardia species.

Elongation factor are a set of proteins involved in the regulation of the rate of

transcription elongations. The transcription elongations are the steps in protein synthesis

in peptide bond formation, either increasing (positive transcription elongation factor) or

reducing it (negative transcription elongation factor). Elongation factor 1-α (ef1-α) was first

described in 1994 (Hashimoto et al., 1994). In this work, the authors described the use of

ef1-α protein sequence to infer the phylogenetic relationship of G. duodenalis among

lower eukaryotes. These authors described the protein has having 396 amino acid

residues, although it really has 442 and no introns. Latter, the same authors described an

elongation factor 2 (ef2), the homologue of eubacterial elongation factor G (ef-G), that

catalyzes the GTP-hydrolysis dependent translocation of peptidyl-tRNA from the

aminoacyl site to the peptidyl site on the ribosome, and its importance for protein

synthesis in all organisms (Hashimoto et al., 1995). These authors also stated that ef-2 is

a single copy gene. This protein as 898 amino acids long and the gene has no introns.

Fixed differences on the sequence of this gene among Giardia species turn it useful in

genotyping Giardia species.

Giardia duodenalis trophozoites undergo antigenic variation of a repertoire of

cysteine-rich surface antigens (Adam et al., 1988; Nash et al., 1988); these antigens are

nowadays referred as variant-specific surface antigens (vsp’s). The number of vsp genes

is estimated at approximately 150, with the coding region comprising over 2% of the

52

Table 9. List of the targets, type of assay and main use of amplification-based

techniques for Cryptosporidium and Giardia. Abbreviations: cowp, Cryptosporidium

oocyst wall protein; ef-1a, elongation factor 1 a; gdh, glutamate dehydrogenase; glorf-

c4, G. lamblia open reading frame c4; gp60, glycoprotein 60; hsp70, heat shock

protein 70; RFLP, restriction fragment length polymorphism; tpi, triose phosphate

isomerase. From Caccio et al., 2005.

genome (Nash and Mowatt, 1992a; Smith et al., 1998). A great diversity of vsp genes

have been cloned and characterized (see Adam, 2000, for review). The vsp genes can be

divided into different groupings or families based on regions of similarity: this degree

varies from complete duplication to a nearby chromosomal location to lesser amounts of

identity throughout the entire coding region to regions of similarity or identity followed by

regions of greater divergence (Adam, 2000). In this way, it is reasonable to infer that the

repertoire of vsp genes has arisen by a combination of duplication with divergence and

recombination. Most of the vsp genes appear to be located on one or several regions of

chromosomes 4 and 5, with different lengths and different sequences among the species

and genotypes of Giardia, so useful for genotyping.

2.9. Cryptosporidium and Giardia population structure

The understanding of a population biology and structure is a step that should be

taken before determining the usefulness of typing and sub-genotyping tools. The better

understanding of the population biology and structure is useful to define what

discrimination is required between isolates. This will decide which marker use, or which

technology use or which sampling strategy use to provide sufficient information about all

contributors to the environmental contamination or to animal/human infections (Caccio et

al., 2005; Smith et al., 2007). Table 9 shows a list of the targets and type of assays used

53

according to the level of discrimination required betweens isolates of Cryptosporidium and

Giardia.

Data generated by study and comparison of micro and mini-satellite variation

between C. hominis and C. parvum indicates that genetic exchange is frequent in C.

parvum populations and rare in C. hominis populations. In C. parvum, these genetic

exchanges leads to recombination between alleles as different loci and the generation of a

very large number of different genotypes with a high level of resolution between isolates.

The rare genetic exchange in C. hominis turns populations essentially clonal, with far

fewer combinations of alleles at different loci, resulting in a much lower resolution between

isolates with many being of the same genotype (Smith et al., 2007). The lower resolution,

or lower differences in the same population, could be solved by using more hypervariable

markers, which is an expensive and time-consuming practice. The ideal, which will

happen with the access to powerful and cheap sequencing technologies, is to sequence

large genome sequences with 10 or 20 highly variable genes, as performed by Multilocus

sequence typing (Smith et al., 2007). Due to the epidemiology and human health impact,

C. parvum and C. hominis are the most studied Cryptosporidium species regarding the

population structure. The variable genes commonly used that allow the sub-genotyping, or

intra-species differentiation in C. parvum and C. hominis are the gp60 (Smith et al., 2007;

Strong et al., 2000; Wu et al., 2003), the dsRNA element (Leoni et al., 2007) and mini and

micro-satellite (Caccio et al., 2000; Mallon et al., 2003a; Mallon et al., 2003b). The level of

variation and discrimination that the sequence of these genes provides is used for

address sources of contamination and disease tracking in public health investigations

(Glaberman et al., 2002). The mini and micro-satellite multilocus genotyping (MLG)

system developed in 2003 (Mallon et al., 2003a; Mallon et al., 2003b) differentiates C.

parvum and C. hominis into 48 and 11 sub-genotypes, respectively and, for instance, has

proven to be useful in analyzing human isolates epidemiologically implicated in a Glasgow

(Scotland) C. parvum waterborne outbreak (Smith et al., 2007).

Understanding the genetic structure of G. duodenalis population is complicated by

the fact that G. duodenalis trophozoite is binucleated and functionally tetraploid with each

nucleus being diploid (Yu et al., 2002). Also, the available evidence argues that the

genetic contents of the two nuclei are distinct: every daughter cell inherits a copy of a left

and a right nucleus during cell division, and the nuclear envelop retains its integrity during

mitosis. In this way, it is expected that the sequence divergence between two nuclei

should be high: each and isolated genome will independently evolve, accumulate

polymorphisms and substitutions (Sagolla et al., 2006). Studies on the genetic diversity of

Giardia showed that, depending on the locus and isolate studied, the results ranged from

a virtual lack of to extensive genetic variation, and it remains unclear at what level the

54

variation exists: heterozygosity within an individual versus polymorphism within a

population (Baruch et al., 1996; Meloni et al., 1995). Some authors argued for the close

relatedness of G. duodenalis isolates throughout the world (Lu et al., 2002) and others for

the evolutionary independence of clonal lineages within G. duodenalis (Meloni et al.,

1995). These authors emphasized the monophyletic character of Giardia duodenalis.

Furthermore, Giardia is considered to be strictly asexual, and the apparent lack of sex has

important implications for the taxonomy, population structure and molecular epidemiology

(Meloni et al., 1995). However, recent studies question this assumption and provide

results indicative of sex and recombination in Giardia. This question was first raised in a

study in which the authors surveyed the Giardia genome and found homologues of genes

involved in meiotic processes (Dmc1, Spo11, Mnd1, Hop1 and Hop2) (Ramesh et al.,

2005).

Recently, in an attempt to determine the amount of allelic sequence heterozygosity

(ASH), Teodorovic et al, have studied 9 strains of G. duodenalis considered to be as

assemblage A (A1 and A2) and assemblage B (Teodorovic et al., 2007). These authors

have treated the strains as distinct populations and amplified DNA fragments from six

coding regions and four non-coding regions (introns and intergenic), cloned and

sequenced 20 independent clones. Curiously, the authors found the levels of ASH

exceedingly low, confirming the observation from the genome project, suggesting the

presence of recombination. Additionally, the authors have found the presence of group A-

1 and group B-specific haplotypes in group B populations. This fact was interpreted as a

product of genetic exchange, thus suggesting that a sexual cycle exists in the parasite.

Furthermore, studies of Cooper et al, 2007, pointed to similar results (Cooper et

al., 2007). In this work, the authors sequenced large regions of chromosomes 3, 4 and 5

of 5 human isolates of G. duodenalis assemblage A2 from endemic areas to identify

single-nucleotide polymorphisms (SNPs). The results showed low level of SNP density as

expected from isolates belonging to a single genotype. Furthermore, and through a

Multilocus comparison, the authors concluded that “loci from different chromosomes

yielded significantly different phylogenetic tress, indicating that they do not share the

same evolutionary history; within individually loci, tests for recombination yielded

significant statistical support for meiotic recombination. These observations provide

genetic data supportive of sexual reproduction in Giardia”. Besides the genetic evidence

from these works, cytological evidence for nuclear fusion and transfer of genetic material

on Giardia was created by Poxleitner et al., 2008. In this work, the authors transfected

Giardia trophozoites using plasmids as episomes labeling only one nuclei and performed

fluorescent in situ hybridization (FISH) on trophozoites and cysts to determine whether the

cyst nuclei can exchange material or remain physically autonomous, as they do in

55

Figure 14: Genetic diversity in C. parvum and C. hominis from AIDS patients in

New Orleans, based on neighbor-joining analysis of the partial GP60 gene.

Values on branches are percent bootstrapping using 1,000 replicates. Five

allele families of parasite are seen: Ia, Ib, and Ie are C. hominis allele families,

and Ic and IIa are C. parvum allele families. From Xiao et al., 2004

trophozoites. With FISH, they found episomes in two or three of the four nuclei of the

cysts suggesting plasmid transfer between the nuclei during encystation. After this, and

using transmission electron microscopy (TEM), the authors demonstrated fusion of the

nuclear envelopes (karyogamy), a process which facilitates plasmid transfer, or genetic

exchanges between nuclei.

The issue of the recombination will clearly affect the taxonomy of Giardia. The

population genetics of this organism will be re-evaluated to take into account the effect of

recombination among members of the G. duodenalis species complex. As stated by

several authors, other questions needed to be addressed to understand how the

recombination questions will impact studies on the epidemiology of the infection (Caccio

and Ryan, 2008; Caccio and Sprong, 2009).

2.10. Molecular epidemiology

The epidemiology of both parasites was presented in previous sections. Also, the

molecular features of both parasites and the information that can be retrieved from the

gene sequence of the several markers were previously presented. The application of

these molecular tools to track the source of a present organism or isolate, to identify it at

56

the molecular level, to study their etiology, distribution and risk factors, is intended as

molecular epidemiology.

Numerous studies have characterized isolates of Giardia and Cryptosporidium

collected from different hosts and have demonstrated the occurrence of the same

species/genotype in humans and other animals (Monis and Thompson, 2003). Such data

is indicative of zoonotic potential but gives no information on the frequency of zoonotic

transmission. Such information can be obtained from molecular epidemiological studies

that genotype isolates of the parasites from susceptible hosts in localized foci of

transmission or as a result of longitudinal surveillance and genotyping of positive cases

(Hunter and Thompson, 2005).

As reviewed by Fayer et al., 2000 and Hunter and Thompson, 2005, an important

advantage of molecular techniques is that they allow not only for accurate and sensitive

detection of Cryptosporidium, and Giardia, but also provide information on genetic

variability of the isolates. As an example of this, and previously presented, molecular

evidences demonstrated the genetic structure or population structure of C. parvum and C.

hominis, the most important Cryptosporidium species regarding the impact for human

health (Xiao et al., 2004) (Figure 14). Interesting evidences suggests that not all C.

parvum are zoonotic (Mallon et al., 2003a; Mallon et al., 2003b). gp60 gene sequencing

has revealed C. parvum variants that are predominantly or exclusively associated with

human, but not animal, infections in defined geographic areas (Alves et al., 2003). Also,

Multilocus genotyping of C. parvum isolates based on mini and microsatellite typing,

revealed groups that are apparently human-specific (Mallon et al., 2003b). In transmission

of C. parvum, similar studies indicated that anthroponotic transmission of C. parvum is

more common (Xiao and Ryan, 2004). Cryptosporidium hominis is primarily an infection of

humans, although the zoonotic potential of this species is still under investigations due to

experimental reports and natural infections in livestock (Giles et al., 2001; Smith et al.,

2007; Smith et al., 2005). Cryptosporidium meleagridis, C. muris, C. suis, C. felis and C.

canis and the Cryptosporidium cervine and monkey genotypes also infect humans,

although further investigations (sub-genotyping) are needed to clarify the zoonotic

potential.

The most relevant Giardia species regarding the impact for human health, G.

duodenalis, has been studied regarding the genetic structure or population structure. For

G. duodenalis, the existence of zoonotic transmission has been described by the use of

molecular tools, but its importance is still not clear. Among Giardia duodenalis species,

only assemblage A and B are reported to infect humans: assemblage A-1 is generally

found in animals while the assemblage A-2 has mainly been identified in humans;

however, A-2 have also been detected in animals (Caccio and Ryan, 2008; Mendonca et

57

Figure 15: Example of

the overlapping

nucleotide peaks in a

chromatogram.

al., 2007). Assemblage B was thought to be largely restricted to humans, however, more

recently, this assemblage has been reported in a large variety of animals (cattle, dogs,

horses, monkeys,…) (Caccio and Ryan, 2008). Assemblage A and B subtypes of G.

duodenalis found in animals are not genetically identical to those found in humans (Caccio

and Ryan, 2008), and to clarify this and the issue of zoonosis, more variable loci are

required, although it is possible that the use of these loci will indicate even more genetic

differences between isolates found in animals and those in humans. Studies that

examined the transmission of Giardia duodenalis between humans and dogs showed the

possibility of assemblage C and D from dogs infect humans, although there was a lack of

concurrence on genotype assignment among the several genes analyzed (Traub et al.,

2004). Assemblage F is a cat-specific assemblage, never associated with human

infections except in a recent study in Ethiopia, although the results could not be confirmed

by the analysis of ribosomal genes (Caccio and Ryan, 2008).

As stated before, the issue of the recombination in Giardia is very important and

has clear implications in the molecular epidemiological studies of Giardia. The assumption

that the recombination is absent has driven the molecular epidemiological studies, but, i)

the appearance of intra-isolate sequence heterogeneity (mixed templates that affect

identification of subtypes with each assemblage) and ii) the fact that different markers

support the assignment of isolates to different G. duodenalis assemblages (a given isolate

cannot be unequivocally assigned to a given assemblage), indicates that the real situation

may be more complex (Caccio and Ryan, 2008).

2.11. Mixed infections

Mixed infections may be defined as the co-presence of, at least, two different

genotypes or species of Cryptosporidium and Giardia infecting the same host. This

paragraph presenting the mixed infections refers and describes situations where is

58

plausible that the referred species or genotypes may infect the host.

In 1997, Hopkins and collaborators observed two overlapping nucleotide peaks at

specific position in the chromatograms from a specific amplification of SSU rRNA gene

from Giardia isolates from dogs and humans (Hopkins et al., 1997) (Figure 15). The

authors interpreted these overlapping peaks as a result of a mixed infection of Giardia,

which would be co-amplified by PCR and peaks from both genotypes should be detected

by sequencing. After this, several other studies generated similar “mixed templates” that

were submitted to GenBank, and other not even took it into account. This seems to

represent reports of mixed infections, so mixed templates, although two mechanisms may

explain these results: a real mixed infection or allelic sequence heterozygosity (ASH).

Mixed infections are reported in several studies, and may occur inter assemblages (A and

B, for instance) and intra assemblages (A-1 and A-2, for instance). What happens in these

cases is a co-amplification of genetic material from two types of cysts, genetically distinct,

and direct sequencing of PCR products and observation of heterogeneous sequencing

profiles. Recent studies, using Assemblage-specific primers showed that a relevant

percentage of mixed infections are not detected by conventional PCR (Geurden et al.,

2008a). On the other hand, ASH is considered an explanation for mixed templates

(Baruch et al., 1996). However, data from genome sequence of the WB genome

(assemblage A-1) shows very low level of ASH, less than 0.01%, and other authors found

no ASH examples in a Multilocus analysis of assemblage A-2 isolates (Cooper et al.,

2007).

In the case of Cryptosporidium, co-infections by two species are reported and tools

are developed to identify these co-infections (Kvac et al., 2009; Waldron et al., 2009).

These co-infections were caused in mice with C. parvum and C. hominis, or C. muris and

C. andersoni.

2.12. Giardia: different targets, different assignment

The assignment of isolates to specific G. duodenalis assemblages is not always

reliable, as showed in several recent studies (Caccio and Ryan, 2008; Cooper et al.,

2007; Teodorovic et al., 2007; Traub et al., 2004). This is observed using different

combination of gene markers, both in animal and human isolates, and it has very

important implications for molecular epidemiological studies: for instance, it becomes even

more relevant when the isolates may be typed as “host-specific” with one marker, but

“potentially zoonotic” with another. Several authors advanced a number of mechanisms

that could explain these results; however the strongest ones, such as the preferential

amplification of one assemblage over the other, which implies a case of mixed infections,

and the recombination events, are more actively discussed (Caccio and Ryan, 2008). The

59

issue of recombination is discussed in section 8 - Cryptosporidium and Giardia population

structure. Further investigations are needed to clarify the extent of recombination, and will

be crucial to determine the specie structure of the G. duodenalis complex.

60

In synthesis, Cryptosporidium spp. and Giardia spp. are emergent protozoa

with a worldwide distribution. Their complex life cycles associated to their

enormous resistance and biological viability under environmental conditions is

responsible for the success of both parasites in different hosts around the world.

They have been recognized as important pathogens to humans. Drinking water

has been pointed out as a main source of contamination, caused by parasite

resistance under conventional treatment conditions (chlorination and filtration). The

occurrence of cryptosporidiosis and giardiasis outbreaks associated with the

consumption of contaminated water is real, and their prevention is a concern to

Public Health Authorities.

Severe clinical complications, particularly in cryptosporidiosis, may occur as

a consequence of opportunistic infections in immunocompromised patients. The

negative impact of both diseases in Animal Production is not completely

understood. The genetic characterization of Cryptosporidium and Giardia is an

essential feature in order to understand the pathogenesis of both diseases in

animals and humans. Scientific evidences suggest limited cross transmission

occurring among different groups of animals and humans. The majority of

Cryptosporidium and Giardia species and genotypes are host-adapted in nature,

having a narrow spectrum of natural hosts.

Development of genetic markers was essential for advances in the

knowledge of Cryptosporidium and Giardia genetic diversity, host specific

interaction and zoonotic potential.

Two main objectives were defined to this work. First, we intended to evaluate the

prevalence of Cryptosporidium and Giardia in humans and bovines, and determine

the level of contamination of surface water as well as the genetic diversity in those

samples. This study was conducted in the north region of Portugal, and later

extended to Galicia, Spain, in agreement with projects under development in the

residence-laboratory. Second, we intended to improve the genotyping tools used,

particularly to introduce Real Time PCR approaches.

The development of the experimental work and respective results are

present in the following chapters under the form of research papers.

61

Chapter II

Species of Cryptosporidium and Giardia infecting humans

1. “Genotype analysis of Giardia isolated from asymptomatic children in northern

Portugal”

62

63

64

65

2. “Genetic characterization of Cryptosporidium isolates from humans in northern

Portugal”

66

67

68

69

Chapter III

Species of Cryptosporidium and Giardia infecting calves and sheep

1. “Prevalence and preliminary genetic analysis of Giardia isolated from adult sheep

in Galicia (NW, Spain)”

70

71

72

73

2. “Prevalence and preliminary genetic characterization of Cryptosporidium spp. from

asymptomatic heifers in Galicia (NW, Spain)”

74

75

76

77

3. “Occurrence of Cryptosporidium parvum and Giardia duodenalis in healthy adult

domestic ruminants”

78

79

80

81

82

83

84

85

Chapter IV

Prevalence and species of Cryptosporidium and Giardia in water

1. “Contribution of treated wastewater to the contamination of recreational river areas

with Cryptosporidium spp. and Giardia duodenalis”

86

87

88

89

90

91

92

93

94

95

96

97

98

99

2. “Presence of Cryptosporidium spp. and Giardia duodenalis through drinking water”

100

101

102

103

104

105

106

107

108

109

110

111

3. “Detection of Cryptosporidium spp. and Giardia duodenalis in surface water: a

health risk for humans and animals”

112

113

114

115

116

117

118

119

120

121

122

123

4. “Biological and genetic characterization of Cryptosporidium spp. and Giardia

duodenalis isolates from 5 hydrographical basins in northern Portugal”

Biological and genetic characterization of Cryptosporidium spp. and Giardia

duodenalis isolates from 5 hydrographical basins in northern Portugal

André Almeidaa,b, Maria João Moreiraa,c, Sónia Soaresa,c, Maria de Lurdes Delgadoa, João

Figueiredoa, Elisabete Silvaa, António Castroa, José Manuel Correia da Costaa,c

aCentro de Imunologia e Biologia Parasitária, CSPGF-INSA, Rua Alexandro Herculano, nº

321, 4000-055, Porto, Portugal

bInstituto de Ciências Biomédicas de Abel Salazar, Largo Prof. Abel Salazar, nº 2, 4099-

003, Porto, Portugal

cCentro de Estudos de Ciência Animal, CECA-ICETA, Rua D. Manuel II, 4051-401, Porto,

Portugal

Korean Journal of Parasitology 2010 Submitted

Abstract

Cryptosporidium spp. and Giardia spp. are two protozoan parasites that have been

responsible for waterborne disease outbreaks worldwide. To understand the situation in

the northern region of Portugal, we have established a long term program aimed at

pinpointing the sources of surface water and environmental contamination, working with

the water-supply industry. Here, we describe the results obtained with raw water samples

collected in rivers of the 5 hydrographical basins. A total of 283 samples were analyzed

using the Method 1623 EPA, USA. Genetic characterization was performed by PCR and

sequencing of genes 18S rRNA of Cryptosporidium spp. and β-giardin of Giardia spp.

Infectious stages of the protozoa were detected in 72.8% (206 out of 283) of the

water samples: 15.2% (43 out of 283) samples positive for Giardia duodenalis cysts, 9.5%

(27 out of 283) samples positive for Cryptosporidium spp. oocysts, and 48.1% (136 out of

283) samples positive for both stages. The most common zoonotic species found were G.

duodenalis assemblages A-I, A-II, B and E genotypes and Cryptosporidium parvum, C.

andersoni, C. hominis and C. muris. These results suggest that cryptosporidiosis and

giardiosis are important public health issues in northern Portugal.

124

Figure 1 - Geographic location of the North of

Portugal and its 5 hydrographical into Iberian

Peninsula.

Introduction

Infections by the Apicomplexa protozoa Cryptosporidium spp. and the flagellate

Giardia duodenalis are widespread in humans and animals (Hunter & Thompson, 2005).

The complex life cycle of the former includes cycles of asexual and sexual reproduction in

the enterocytes of the intestinal mucosa from the definitive host (mammals and birds)

(Fayer et al., 2000). The life cycle of Giardia spp. in the lumen of the duodenal region of

the intestine is simpler and includes two main stages: trophozoites and cysts (Adam,

2001). The infectious stages – oocysts and cysts – are released in the environment

through the hosts’ faeces. Ingestion of both stages from the environment by drinking water

or eating raw vegetables is the main transmission route to humans and animals (Caccio &

Ryan, 2008, Fayer, 2009, Hunter & Thompson, 2005, Smith et al., 2007). Among the 19

species of Cryptosporidium in vertebrate other than fish, C. parvum and C. hominis are

most commonly associated with human disease (Chappell et al., 2006, Fayer, 2009). The

specie C. parvum is recognized as a zoonotic species. The specie G. duodenalis is

comprised of several genotypes or assemblages: assemblage A and B genotypes are

infectious for humans whereas assemblages C, D and E are infectious for animal species

including livestock, in a host-specific manner (Caccio & Ryan, 2008, Thompson, 2000,

Trout et al., 2006). The above mentioned species of the genus Cryptosporidium and the

genotypes of Giardia duodenalis have been described as important pathogens in

contaminated drinking water, due to two main reasons: 1) their resistance and biological

viability under the conventional drinking water treatment conditions; 2) the occurrence of

cryptosporidiosis and giardiasis outbreaks associated with the consumption of

contaminated water (Caccio et al., 2003, Chauret et al., 1999, Fayer, 2004).

To the authors’ best knowledge no cryptosporidiosis or giardiasis outbreaks

associated with contaminated water

consumption have been described in

Portugal. However, clinical cases

among immunocompetent patients

are well known (Almeida et al.,

2006a, Almeida et al., 2006b, Alves

et al., 4 2006, Matos et al., 2004,

Sousa et al., 2006). Both clinical

situations are probably

underestimated in the country due to

the lack of systematic diagnosis of

these parasites.

125

Figure 2 - Location of the 5 hydrographical

basins in the North of Portugal. 1 - Minho;

2 - Lima; 3 - Cávado; 4 - Ave; 5 – Douro.

Significant concentrations of the infectious stages of both parasites have been

found in water samples collected from rivers in the southern region of Portugal (Alves et

al., 2006, Lobo et al., 2009). Also, studies on human and animal biological reservoirs

indicated an important presence of zoonotic species (Mendonca et al., 2007, Almeida et

al., 2006a, Almeida et al., 2006b, Matos et al., 2004). Systematic evaluation of cattle,

surface waters and humans for the presence of Cryptosporidium spp. and Giardia

duodenalis as well as its genetic characterization using molecular tools are fundamental

steps to better understand the epidemiology of the infection and to allow the

implementation of risk analysis models for those infections. The present study concerns

Cryptosporidium spp. and Giardia duodenalis infectious stages status in raw water

samples of the rivers of northern regions of Portugal.

Material and methods

The northern region of Portugal

In the north of Portugal there are 5 major hydrographical basins forming the most

important water resource of the country (Fig. 1). These hydrographical basins are named

after the main rivers: (1) Minho, (2) Lima, (3) Cávado, (4) Ave and (5) Douro (Fig. 2).

Cávado and Ave rivers run entirely inside national (Portuguese) borders, while Minho,

Lima and Douro are international rivers, with sources in Spain.

Water samples

Raw water samples were collected

twice a year between January 2004 to

December 2006 from 97 sources,

including main rivers and respective

affluents, from upriver to downriver. The

volume of each sample ranged from 25 to

100 liters. Samples were filtered through

Filta-Max filters (IDEXX 5 Laboratories,

Inc., Westbrook, ME, USA) with a pump on

the inlet side of the filter according to the

recommendation of the manufacturer.

Intact filters were kept in refrigerated

containers and transported immediately to

the laboratory. The filter was taken from

the container and processed with the aid

of a Filta-Max Manual Wash Station

(IDEXX Laboratories, Inc., Westbrook, ME, USA) for further elution and concentration

126

process, which consisted of decompress the filter, pass the sample through a membrane

and centrifugation. A sample pellet (around 2 mL) was obtained and transferred to a

Leighton tube for subsequent immunomagnetic separation (IMS).

Parasite detection

The IMS procedure was performed according the USEPA method 1623 (Agency,

2005). Briefly, anti-Giardia and anti-Cryptosporidium magnetic beads were mixed with SL

Buffer A and SL Buffer B in each Leighton tube containing the sample concentrate

(Dynabeads GC-Combo, Invitrogen Dynal, A. S., Oslo, Norway) and incubated 1h at room

temperature. Then, using two magnetic particle concentrators, beads were collected,

washed and transferred into a 1.5ml tube. Fifty microliters of 0.1N HCl were added to

each sample to dissociate beads from the target organisms, the beads were rejected and

the suspension was transferred to the wells of the slides containing 5 µl of 1.0N NaOH.

The samples were air dried overnight and stained with FITC-conjugated anti-

Cryptosporidium spp. and anti-Giardia spp. monoclonal antibodies, according the

manufacturer instructions (Crypto/Giardia Cells, Cellabs, Australia). Slides were examined

by epifluorescence microscope. Giardia cysts and Cryptosporidium oocysts were

indentified and counted based on their shape and size using a Nikon Optiphot

fluorescence microscope (Nikon Corporation, Tokyo, Japan). The number of cysts and

oocysts per each well was recorded and concentrations extrapolated per 10 liters of

sample. Positive and negative controls were performed as indicated by the manufacturer

and recommended in the Method 1623. 6 The mean recovery percentages of oocysts of

Cryptosporidium spp. and cysts of Giardia spp. using Filta-Max system and IMS

procedures from water samples is, according to the manufacturer, 50±13% and 41±79%,

respectively (McCuin & Clancy, 2003).

DNA extraction, PCR and sequencing

PCR analysis was performed in the samples with the highest density of infectious

stages of both parasites detected by DFA. The criterion utilized was the detection of a

minimum of 100 cyst/oocyst stages of any parasite in the total sample volume. In this

context, the genetic characterization was executed in 80 samples. The cover slip was

separated from the slide and with the aid of cotton swab soaked with 100µl of distillated

water, the surface of the slide was scraped in order to collect the sample. It was

confirmed, under microscope observation that the slide had no remaining cysts or

oocysts. The tip of the cotton swab was cut and placed in a 1.5mL tube for subsequent

DNA extraction with a QIAamp DNA Mini Kit (QIAGEN GmbH, Germany), according to the

manufacturer’s instructions. For determining the species of Cryptosporidium spp. and

Giardia spp. present in the samples, a PCR analysis was performed. A two-step nested

PCR was performed to amplify a portion of the small subunit (SSU) ribosomal RNA gene

127

Figure 3 - Distribution of the results obtained by Method 1623 EPA-USA for infectious

stages of Cryptosporidium spp. and Giardia duodenalis in water samples collected in

the 5 hydrographical basins.

of Cryptosporidium spp. (Xiao et al., 1999). For the molecular typing of Giardia spp., a

semi-nested PCR was performed to amplify a portion of the β-giardin gene (Caccio et al.,

2002). For all PCR reactions, negative and positive controls were performed, with sterile

water and reference DNA, respectively. The PCR products were analyzed in agarose gel

(1.4%) stained with ethidium bromide under UV light. Images were captured with a gel

documentation system (GelDoc2000, BioRad). The PCR products of the successful

reaction were purified (Wizard SV Gel and PCR Clean-up System, Promega) and

sequenced in both strands by an external laboratory (EUROFINS MWG OPERON,

Germany). Chromatograms were examined with the software 7 ChromasPro

(http://www.technelysium.com.au/ChromasPro.html) and the sequences with the software

ProSeq

(http://www.biology.ed.ac.uk/research/institutes/evolution/software/filatov/proseq.htm).

Sequences were compared with the GenBank database with the tool BLAST

(http://blast.ncbi.nlm.nih.gov/Blast.cgi) and deposited in the database Zoop-Net of the

Med-Vet-Net network (http://www.medvetnet.org/cms/).

Results

IMS and DFA detection of infectious stages of Cryptosporidium spp. and

Giardia duodenalis

The number of validated raw water samples in this study was 283. Environmental

stages of the protozoa were detected in 72.8% (206 out of 283) of the water samples,

being 15.2% (43 out of 283) cysts of Giardia duodenalis, 9.5% (27 out of 283) oocysts of

Cryptosporidium spp. and 48.1% (136 out of 283) both parasites. In Figure 3 the

128

Table 1 - Results from the genetic

characterization of Cryptosporidium spp. and

Giardia duodenalis and the respective number

of cysts and oocysts.

percentages of positive and negative samples from the 5 hydrographical basins are

shown individually. The Ave basin shows the highest percentage of positive samples:

90.2% of the samples are positive. Minho basin shows the lowest percentage of positive

samples, even though this value is more than 64%. In all the 5 hydrographic basins, the

co-presence of Cryptosporidium and Giardia counts for the majority of positive samples,

with the exception of Minho basin in which Giardia positive samples are slightly more than

Cryptosporidium and Giardia positive samples. In the cases where both parasites were

present in the same sample, the number of Giardia duodenalis cysts always outnumbered

Cryptosporidium spp. oocysts. We also found no correlation between the concentrations

of both parasites, meaning that when the concentration of Giardia cysts is high not

necessarily Cryptosporidium oocysts are also high (data not shown). Furthermore, the

range of the concentrations of Giardia duodenalis cysts was much higher than the

Cryptosporidium spp. oocysts (0.17-50000 cysts per 10 liters and 0.2-726.1 oocysts per

10 liters, respectively).

In all cases, it was possible to

observe an increase of parasite load

from upriver to downriver. The

majority of water samples from the

international rivers (Minho, Lima and

Douro) collected at the border with

Spain was negative.

Genetic characterization of

species and genotype isolated

PCR was not able to amplify

DNA extracted from slides containing

less than 100 oocysts of

Cryptosporidium and 100 cysts of

Giardia. Furthermore, positive

amplifications over 3 replicates were

never obtained when the number of

cysts and oocysts was less than 1000

per slide. With this criterion, of all the

positive IMS samples, PCR

amplification was performed over 80

samples.

Genetic characterization was

129

successful in 8 samples for Giardia duodenalis and 20 samples for Cryptosporidium spp.

In 59 samples PCR amplification was not successful. A summary of the PCR results is

shown is table 1: Cryptosporidium andersoni was found in 16 samples, C. parvum in 2

samples, C. hominis in one sample and C. muris in one sample. Giardia duodenalis

assemblage A-II was found in 4 samples, assemblage B in one sample and in the

remaining 3 samples assemblages A, B and E was found.

Discussion

The results of the present study indicate that the infectious stages of

Cryptosporidium spp. and Giardia duodenalis are widely distributed in the rivers of

northern Portugal in very significant concentrations. Curiously, Giardia duodenalis cysts

always outnumbered Cryptosporidium spp. oocysts (data not shown). The region has a

high density of livestock farms favoring the cycle of parasites amplification. The surface

water collected from the rivers is used as drinking water for the animals or used for

agricultural purposes and the feces are directly released into the rivers or reach it by

runoff waters.

Systematic studies on the genetic characterization of both protozoa indicates that

the genus Cryptosporidium includes species that are infective for humans only

(anthroponotic), humans and animals (zoonotic) and other pathogenic species that are not

infective for humans (Fayer, 2009). Also, several Giardia duodenalis genotypes are

infective to human - zoonotic genotypes, and others are nonpathogenic (Caccio & Ryan,

2008). Amplification and sequencing genes 18S SSU rRNA for Cryptosporidium spp. and

β-giardin for G. duodenalis has been used to identify the zoonotic species and genotypes

of the parasites (Hunter & Thompson, 2005, Read et al., 2004). Obviously, it is largely

recognized a lack of consensus about genetic markers for the correct assignment of the

species and subspecies of Cryptosporidium and Giardia. The gene markers (18S SSU

rRNA and β-giardin) are generally accepted as good markers mainly because they are

multicopy genes (18S SSU rRNA), restricted to these parasites (β-giardin) and with fixed

differences among Cryptosporidium and Giardia species and sub-species (both genes). In

an attempt to produce relevant and comparable results, the choice of 18S SSU rRNA and

β-giardin genes, frequently used by the most recognized researchers, was considered.

C. parvum (zoonotic) and C. hominis (anthroponotic) are the most common

human-infecting species reported in river water samples in Europe, and C. andersoni is

the animal-infecting species (Xiao & Fayer, 2008, Caccio & Ryan, 2008, Castro-Hermida

et al., 2008). The same studies indicated G. duodenalis assemblage A as the most

common zoonotic genotype, and G. duodenalis assemblage E as the most common non-

130

zoonotic genotype (Almeida et al., 2006a, Caccio et al., 2003, Xiao & Fayer, 2008, Sousa

et al., 2006, Castro-Hermida et al., 2008).

Our results suggest that the contamination of the surface waters in the north of

Portugal is highly significant. We have found the zoonotic species of the genus

Cryptosporidium described by other authors (Lobo et al., 2009). In addition, there was

higher concentration of Giardia duodenalis detected by genotyping, with a greater genetic

diversity. Assemblage A was found in seven PCR positive 10 samples (one A-I and 6 A-

II). The presence of the assemblage A-I has been suggested as an indicator of water

contamination by livestock, while assemblage A-II has been considered a potential

indicator of water contamination by humans (Caccio & Ryan, 2008, Almeida et al., 2006a).

Nevertheless, in the northern part of the country assemblage A-I have been found in

human samples (Sousa et al., 2006), and assemblage A-II in bovine samples (Mendonca

et al., 2007, Castro-Hermida et al., 2007). Also, G. duodenalis assemblage B was

detected in four out of eight samples. Assemblage B has been reported as a zoonotic

genotype. The presence of infectious stages of this genotype in water samples has been

attributed to water contamination by humans (Caccio & Ryan, 2008). Assemblage E was

detected in two samples associated with assemblage A and B, suggesting a mixed human

and animal source of contamination.

Cryptosporidium parvum was detected in 2 of the 20 processed samples. This

species has a great zoonotic potential, and may have an animal or human source of

contamination. A few studies concerning the biological reservoir (human and bovine) in

the north of Portugal have indicated C. parvum as an important pathogen infecting the

great majority of bovine as well as immunocompromised human patients (Almeida et al.,

2006b, Mendonca et al., 2007). Recent data suggest that sub-genotyping tools may

generate more information about the zoonotic potential of C. parvum isolates, although

there is still lack of evidences on the useful of the generated data for risk assessment

(Fayer, 2009). C. hominis, considered an anthroponotic (human restricted) species, was

detected in one sample. This species was also reported in water samples and in human

stool samples from Portugal (Lobo et al., 2009, Alves et al., 2003, Almeida et al., 2006b).

C. andersoni a strictly bovine pathogen was detected in 16 samples; C. muris was

detected in only one sample suggesting water contamination by rodents.

Curiously, as previously mentioned, no PCR amplification was obtained from 59

samples. This problem has been described by other authors (Jiang et al., 2005). We have

no clear explanation for that. 11 Our sensitivity analysis indicates simultaneously a lack of

reproducibility in the PCR analysis and the difficulty to achieve amplification in samples

with low levels of contamination. It has been suggested that DNA may not be present in

sufficient amounts (empty (oo) cysts after excystation) or the lack of PCR amplification

131

may be due to the presence of inhibitors of PCR in the samples (Robertson et al., 2006).

The IMS procedure applied over the samples for parasite isolation does not guarantee a

complete purity of the sample. Thus, optimization of DNA extraction and amplification

protocols is warranted.

The results obtained in the present study suggest that cryptosporidiosis and

giardiosis should be considered very important public health issues in the north of

Portugal. Also, the genetic characterization of Cryptosporidium spp. and Giardia

duodenalis support the possibility that there is a greater risk of infection by Giardia

duodenalis for humans, while Cryptosporidium spp. poses a greater risk for animals.

Thus, systematic monitoring of drinking water, livestock and human biological samples are

needed for risk assessment of both diseases. National Health Authorities should consider

the urgent implementation of a national monitoring program for microbiological quality of

drinking water that includes Cryptosporidium spp. and Giardia duodenalis analyses.

These activities are fundamental steps to understand better the epidemiology of the

infection and to allow the implementation of risk analysis models for those infections.

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Parasitol, 124, 90-97.

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transmission, detection and identification. Int J Parasitol, 30, 1305-1322.

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Jiang, J., K. A. Alderisio, A. Singh and L. Xiao, 2005: Development of procedures

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zoonotic potential. Int J Parasitol, 30, 1259-1267.

Trout, J. M., M. Santin, E. C. Greiner and R. Fayer, 2006: Prevalence and

genotypes of Giardia duodenalis in 1-2 year old dairy cattle. Vet Parasitol, 140, 217-222.

Xiao, L. and R. Fayer, 2008: Molecular characterization of species and genotypes

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5. “Presence of Cryptosporidium spp. and Giardia duodenalis in drinking water

samples in the north of Portugal”

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Chapter V

PCR Direct sequencing and Real Time PCR (qPCR) – new targets and approaches

1. “Genotyping Giardia duodenalis cysts by new real-time PCR assays: detection of

mixed infections in human samples”

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2. “Identification of Giardia species and Giardia duodenalis assemblages by

sequence analysis of the 5.8S rDNA gene and internal transcribed spacers”

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Chapter VI

General discussion

Cryptosporidium and Giardia parasites have emerged in the last two decades as

intriguing microbes with an enormous impact on Human and Animal Health. Both can

cause mild to severe diarrhea. Cryptosporidiosis may cause chronic and debilitating

illness in immunocompromised individuals, which may even cause death. Both protozoa

have been recognized as important pathogens in contaminated drinking water. However,

Cryptosporidium was not recognized as an important cause of human diarrheal illness

until 1982, while Giardia was first recognized as a source of waterborne illness in the

1970s. Neither Giardia nor Cryptosporidium are "new" parasites. In fact, we were unable

to detect them, due to the absence of specific clinical signs and symptoms and of a lack of

appropriate laboratorial tools. Techniques to identify these organisms and their associated

diseases have only been developed relatively recently. Moreover, genotyping is crucial for

the understanding of pathogenesis in both diseases. We are deeply convinced that our

work was an important contribution to answer the initial questions we had defined:

How prevalent are giardiasis and cryptosporidiosis in humans in Portugal?

According to Adam, 2001, Giardia infections have a higher incidence in children

aged less than 5 years because their immune systems are still not fully developed and

because of lower personal hygiene. So, our approach has elected a population with these

characteristics. Data reported by other European countries indicated prevalence rates

ranging from 2% to 7%. Our data estimated the prevalence rate of G. duodenalis

infections in a population of 177 healthy school children of 4%. Genotyping Giardia cysts

isolated from positive samples revealed the presence of G. duodenalis assemblage B in

five samples (four assemblage B, one assemblage B-1) and G. duodenalis assemblage A

in two samples (one A-2 and one A-3).

We have done the first study on Cryptosporidium spp. infections in

immunocompromised individuals in northern Portugal. Our data estimates a prevalence of

4% of cryptosporidiosis in HIV patients living in this region of the country. Other authors

have suggested a prevalence of 8% of cryptosporidiosis among HIV patients in other

regions of Portugal (Matos et al. 2004). In the present survey, HIV patients were more

commonly infected with C. parvum than with C. hominis and C. meleagridis. Briefly,

Portugal has a Public Health profile similar to other European countries. However, Health

Authorities should not forget that Portuguese population has a high risk of infection, and a

monitoring program should be implemented.

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How prevalent are giardiasis and cryptosporidiosis in domestic ruminants of

Portugal and Galicia?

Galicia. Although infections by Cryptosporidium spp. and G. duodenalis have been

reported for calves, sheep and goats in many parts of the world, the prevalences have

varied markedly. In the present study, the prevalence and intensity of infection for both C.

parvum and G. duodenalis in cows, sheep and goats samples were relatively low, but

widespread. The results obtained reflect a serious situation, taking into account that the

study was carried out on healthy adult animals and that the farms were selected at

random and the only possible restriction was whether the farmers consented to the study.

Both the prevalence and intensity of infection by G. duodenalis were significantly higher

than those for C. parvum, as has also been observed in other studies (Fayer et al. 2000b;

Castro-Hermida et al. 2005b; Maddox-Hyttel et al. 2006). One possible reason for this is

that G. duodenalis infections usually last longer than C. parvum infections, often becoming

chronic, whereas C. parvum infections may be acute, but usually with spontaneous

recovery over a relatively short time period. Therefore, G. duodenalis infections are more

likely to be identified in single samples.

When animal cryptosporidiosis and giardiosis is discussed in a public health

context, it is necessary to identify clearly the species and genotypes involved. In the

present study, IFAT was more sensitive than PCR for detecting Cryptosporidium oocysts

and G. duodenalis cysts in faecal samples from cows, sheep and goats. Thus, after DNA

extraction from samples containing low numbers of parasites (<800 OPG or CPG), we

were not able to amplify Cryptosporidium spp. and Giardia DNA by PCR. This may have

been due to inhibitors present in the faeces, the small volumes used or to loss of parasites

during the concentration and purification steps that anticipate PCR. Although PCR is a

highly sensitive and useful technique, so far, we have only been able to apply it

successfully to samples with a high parasite load, which makes purification of the

oocysts/cysts easier. There are very few reports about Cryptosporidium spp. in cows older

than 3 years, nevertheless, in the present study, only C. parvum was identified in ten

PCR-positive cows, which indicates that these animals are also sources of zoonotic

cryptosporidiosis for humans. Genetic analysis for G. duodenalis showed that assemblage

E was the most prevalent genotype detected in cows, sheep and goats. Thus,

assemblage E was detected in four fecal samples from cows, in 11 fecal samples from

sheep and in one goat fecal sample. This genotype is common in domestic ruminants,

and there are no epidemiological or genetic data that support its zoonotic potential

(Becher et al. 2004; Fayer et al. 2004; Hunter and Thompson, 2005; Trout et al. 2005).

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The present results are consistent with those of Ryan et al. (2005), who suggested that

sheep might not constitute an important zoonotic reservoir for G. duodenalis, and those of

Langkjaer et al. (2007) who concluded that cows are only infected by isolates of the

livestock group, assemblage E. Other studies in Australia and North America have also

indicated a minimal risk to public health related to the prevalence of G. duodenalis in

livestock (O’Handley et al. 2000; Hoar et al. 2001). However, domestic ruminants are

reported as susceptible to infection by zoonotic genotypes of G. duodenalis and that

assemblage A is the most commonly reported zoonotic genotype (Thompson et al. 2000).

Moreover, it has been suggested that the patent periods may differ for genotype A

(zoonotic) and genotype E (nonzoonotic; Trout et al. 2005). Interestingly, in the present

study, only one isolate of G. duodenalis assemblage B (zoonotic genotype) was detected

in a sheep fecal sample. Because of the small number of animals sampled, it is not clear

whether or not the results represent differences in the geographical distribution of

assemblage A. Further studies are required to confirm this hypothesis.

Portugal. Similar studies have been carried out in northern Portugal, which

borders with Galicia (the two areas together represent a large area of animal production

and is denominated the “Galicia– Northern Portugal Euroregion”), and have shown that

the prevalent G. duodenalis genotypes in livestock in the area were assemblages E, A

and B. The results of the present study suggest that healthy adult domestic ruminants

may not be an important zoonotic reservoir of G. duodenalis, although these animals may

harbor some G. duodenalis assemblages that are infective to humans. Nevertheless, the

variation in the gene assemblages reduces our ability to estimate the risk of these animals

as a reservoir of G. duodenalis infectious to humans. The results of the current study

show a low but widespread prevalence of C. parvum and G. duodenalis in healthy adult

domestic ruminants. The genotyping data suggests that cows are a potential source of

environmental contamination with C. parvum. The G. duodenalis genotypes harbored by

these animals infect mainly other domestic ruminants and wild animals, although humans

can also become infected by exposure to infected sheep.

In Portugal, from the Cryptosporidium positive samples we obtained 63 isolates

from calves’ samples and 7 isolates from adult samples. Additionally, Giardia was isolated

in 13 out of 41 positive samples from calves and it was also possible to isolate Giardia

from a positive adult sample. Molecular characterization of the Cryptosporidium and

Giardia isolates showed that C. parvum and G. duodenalis assemblage E were the

prevalent species. C. parvum may infect humans, representing a potential public health

risk. On the other hand, assemblages B and A2 of Giardia, previously described in

humans, were here identified in calves. Further studies are needed to determine the

importance of calves as carriers of zoonotic assemblages of G. duodenalis.

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In summary, our data suggests that calves may represent an important biological

reservoir and a potential risk for environmental contamination with Cryptosporidium and

Giardia. Genetic characterization of parasites isolated from surface waters used in bovine

production areas is critical for risk assessment. Also, our data indicates that

Cryptosporidiosis and Giardiasis should be considered as real issues for animal

production and public health by the Veterinarians who work on buiatrics, in a first step,

and local and national health authorities.

How prevalent are Giardia and Cryptosporidium in raw, treated and wastewater

samples in Portugal and Galicia?

Galicia. During our work, wastewater, drinking water and surface water samples

were tested for the presence of Giardia and Cryptosporidium. The methodology used is

based on the Method 1623 EPA, USA, followed by immunofluorescence identification of

the parasites and molecular characterization. We also performed DAPI staining to

evaluate the viability of cysts and oocysts. With this approach, 12 wastewater treatment

plants in Galicia were studied and contamination by Cryptosporidium and Giardia

evaluated. The concentration of these parasites was higher in spring and summer,

although both parasites were present in 100% of the wastewater treatment plants studied.

Cryptosporidium parvum, C. andersoni, C. hominis and G. duodenalis assemblages A-I,

A-II, and E were detected. The conclusion of this study is that the risk of contamination of

water courses by Cryptosporidium spp. and G. duodenalis is considerable, and

wastewater treatment authorities should develop adequate countermeasures to reduce

contamination levels.

Regarding drinking water samples, a second study was performed, using the same

methodology, over 16 drinking water treatment plants covering 128 water samples in 16

water plants. In this study, the mean concentration of parasites was much lower in the

effluent, showing the effectiveness of the treatment plants in reducing the parasite load.

However, both parasites were present in almost all plants: C. parvum, C. andersoni, C.

hominis and G. duodenalis assemblages A-I, A-II, and E were detected, even in effluent

samples. These results suggest a considerable risk for human and animal infection.

A third study, performed in the Galicia region, focused on surface water samples,

and, in parallel, fecal samples from neonatal calves, cows and heifers. We analyzed 116

water samples and 1316 dairy calves fecal samples from 18 dairy farms. Cryptosporidium

spp. oocysts were detected in 40 neonatal calves (28.8%), 20 heifers (4.2%) and 25 cows

(3.6%) from 18 dairy farms (100%). Giardia duodenalis cysts were identified in 29

neonatal calves (20.9%), 45 heifers (9.4%) and 49 cows (7.0%) from 18 dairy farms

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(100%). In water samples, Cryptosporidium spp. oocysts were detected in 62/116 (53.4%)

samples from 27/29 (93.1%) sampling points throughout the year, whereas G. duodenalis

cysts were detected in 78/116 (67.2%) samples from 29/29 (100%) sampling points. After

molecular characterization, C. parvum was detected in 7 (41.2%) samples of surface

water, C. andersoni and C. hominis in 6 (35.3%) and 4 (23.5%) of the samples,

respectively. Giardia duodenalis cysts of assemblage A-I and A-II were detected in

samples from 2 (11.8%) to 4 (23.5%) of the points, respectively. Moreover, in another 4

samples (23.5%) both assemblages AI and E were detected, and in another 5 samples

(29.4%), both assemblages A-II and E were detected. Assemblage E alone was detected

in 2 water samples (11.8%). The presence of the same species and genotypes in calves

and in water samples suggests that calves are the source of water contamination. The

presence and viability of both protozoa should be monitored in calves, in sources of water

used for recreational purposes and in artificial waterways used by farmers (water

channels, animal drinking water and drainage systems).

Portugal. The north region of Portugal was analyzed for the prevalence of

Cryptosporidium and Giardia according to the methodology described above. In this case,

drinking water samples and surface water samples were investigated according to Method

1623 EPA, USA, followed by immunofluorescence identification, DAPI staining, and

molecular characterization. As stated before, the northern region of Portugal borders with

Galicia and represents a large area of animal production. The fact that Portugal and

Galicia constitutes a Euroregion that shares river watercourses drove our work in a

direction of assembly both regions. Two main types of water samples were studied during

a program supported by Portuguese Calouste Gulbenkian Foundation: surface water and

drinking water samples in the north of Portugal. This program was divided in two studies:

surface water samples and drinking water samples. This was mainly due to the difficulties

in collecting surface water samples from the respective treated and drinking water sample.

To study the prevalence of these pathogens in the surface water in the north of

Portugal, several collection points were selected in the 5 hydrographical basins in this

region. A total of 283 water samples were collected and infectious stages of the protozoa

were detected in 72.8% (206 out of 283) of the water samples: 15.2% (43 out of 283)

samples were positive for Giardia duodenalis cysts, 9.5% (27 out of 283) samples were

positive for Cryptosporidium spp. oocysts, and 48.1% (136 out of 283) samples were

positive for both parasites. As demonstrated by molecular typing, the most common

species found were G. duodenalis assemblages A-I, A-II, B and E and Cryptosporidium

parvum, C. andersoni, C. hominis and C. muris. The main conclusion from this work is

that cryptosporidiosis and giardiosis are important public health issues in northern

Portugal.

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Regarding drinking water samples, 167 samples taken in the same region were

analyzed. Environmental stages of the protozoa were detected in 25.7% (43 out of 167) of

the water samples, 8.4% (14 out of 167) with cysts of Giardia, 10.2% (17 out of 167) with

oocysts of Cryptosporidium and 7.2% (12 out of 167) with both stages. These results

suggest that treatment procedures in these drinking water plants must be improved to

reduce the levels of contamination. An implementation of systematic monitoring programs

for both protozoa has been suggested.

The work presented in these paragraphs has shown contamination of water

samples both in Galicia and the North of Portugal. There are no doubts about the

widespread occurrence of the protozoa in the water, their high prevalence and, most

importantly, the risk of human and animal infection. With the aid of molecular tools, it was

possible to detect several genotypes of G. duodenalis and species of Cryptosporidium

that are of human and bovine origin. The species and genotypes, both zoonotic and non

zoonotic, which were detected in human and bovine fecal samples, are all present in

water samples. This indicates that consumption of this water, or indirect exposure via

contaminated food or recreational areas that use this water, may represent a risk for

human infection. In conclusion, the water authorities should consider these parasites as

important pathogens that pose a risk for human health, and need to improve the efficiency

of treatment procedures with the aim of reducing contamination levels.

Molecular typing: new molecular approaches on Giardia genotyping.

Several aspects regarding the molecular typing of the two protozoa under study

need to be clarified, in particular those concerning Giardia. The molecular typing scheme

for Cryptosporidium is well established. One important point is the use of highly

polymorphic genes in which the level of variation and discrimination provides very useful

information in tracking the sources of contamination and disease. One good example of

this kind is the gp60 gene. However, we did not perform sub-genotyping of C. hominis and

C. parvum, mainly due to the lack of an adequate technology in our laboratory.

The genetic structure of Giardia duodenalis is less understood. Opinions differ

about the fact that sexuality exists or not in this organism. If there is no recombination,

then the nuclei of this polyploid organism should accumulate mutations in an independent

way, and this should generate high levels of ASH. Sequencing of the WB genome, an

assemblage A strain, demonstrated that ASH is very low. However, sequencing of the GS

genome, an assemblage B strain, showed much higher levels of ASH. On the other hand,

if some form of sex is present, then some predictions may be done. It has been shown

that the Giardia genome contains genes that in other eukaryotes are involved in meiosis,

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a finding compatible with the occurrence of sex. Another study has provided evidence of

nuclear fusion and of an exchange of a plasmid between the nuclei, again in favour of

some form of sexuality. Genetic analysis of field isolates from humans has been used to

provide evidence for recombination within assemblage A (AII) and between assemblages

A and B.

The interpretation of genotyping data in the context of molecular epidemiologic

studies, usually obtained by direct sequencing of PCR products, is affected by two

observations: 1) the presence of double peaks in the sequence chromatograms, and 2)

the assignment of the same isolate to different assemblages by the analysis of several

loci. Thus, the crucial question is: Are these findings a consequence of a mixed infection

or the result of recombination? Looking for a practical approach to investigate this

question, we designed an experience based on assemblage specific primers of Giardia

duodenalis assemblage A and B and, using a sensitive and quantitative technique to

detect the amplification of DNA, real time PCR. We applied these primers over pre-typed

G. duodenalis cysts purified from human fecal samples. In a second experiment, a

method was developed, based on sequence analysis of the 5.8S rDNA and ITS

sequences, with the objective to identify of all Giardia species in a reliable manner.

In the first experiment, assemblage A and B specific primers were designed to

target the gdh, tpi and orf-c4 genes. These assays were designed to allow the specific

identification of assemblages A and B based on the melting curve, size and sequence of

the amplification products, and were applied to 30 human samples. Primers targeting the

β-giardin gene, previously published, were also used in this study. Both fecally-extracted

DNA and purified cyst from each sample were tested. The results obtained on fecally-

extracted DNA indicate that a large number of human stools contained DNA of both

assemblages A and B. Further experiments on the cysts purified from the same samples

showed that this finding is essentially attributable to mixed infections. Indeed, only one

assemblage was detected when dilutions of cysts, down to a nominal level of one cyst,

were tested. In a few cases, however, detection of both assemblages was observed even

when single cysts were tested. In short, the qPCR assays are useful: a) to investigate the

occurrence of mixed infections in clinical samples; b) to detect Giardia cysts infectious to

humans in samples from animals, in water and food, c) to trace recombination event

between (or within) assemblages, provided that accurate methods for cyst separation and

enumeration are used.

In the second experiment, a PCR-based approach was developed with the

objective to identify all Giardia species and G. duodenalis assemblages. Primers were

designed to match strongly conserved regions in the 3’ end of the small subunit ribosomal

gene (forward primer) and in the 5’ end of the large subunit ribosomal gene (reverse

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primer). The target region comprises the 5.8S gene and the two flanking internal

transcribed spacers (ITS1 and ITS2). The assay was tested over 49 isolates of human

and animal origin. Sequence analysis of the target region showed that G. ardeae, G.

muris, G. microti and the seven G. duodenalis assemblages could be easily distinguished.

Therefore, the 5.8S-ITS assay represents a versatile tool for molecular epidemiologic

investigations, as it combines an excellent robustness with a high level of genetic

variability both among Giardia species and G. duodenalis assemblages. This

characteristic is of particular relevance when water samples and samples from wild

animals are investigated.

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Chapter VII

Conclusion and perspectives

Parasites in the genera Cryptosporidium and Giardia are responsible for severe

gastrointestinal infections and are reported worldwide as waterborne diseases with a

serious impact on Human and Animal health. Our approach, integrating a multidisciplinary

and national program, has produced important contributions towards: a) a better

knowledge of epidemiological and transmission aspects of cryptosporidiosis and

giardiasis; b) improvements in laboratorial tools with the ability to discriminate Giardia

species based on genotyping.

According to our data, and taking into account the underestimated character of

both diseases in the country, two aspects seems to be relevant: a) the prevalence rates of

the human diseases in the north of Portugal are similar to those described for other

European countries; b) the prevalence and intensity of infection by both C. parvum and G.

duodenalis in calves, sheep and goats are relatively low, but widespread. Also, our data

suggests that calves may represent an important biological reservoir and a potential risk

for environmental contamination by Cryptosporidium and Giardia.

Cryptosporidium and Giardia are also recognized as important pathogens in

contaminated water, particularly drinking water. Our data shows a widespread distribution

of oocysts and cysts in the water, and, more importantly, an actual risk of human and

animal infection by drinking water.

The understanding of transmission dynamics is limited. A better comprehension of

taxonomy and zoonotic potential is necessary. The molecular analysis at informative loci

is necessary to distinguish the species and genotypes involved in infection. In this respect,

Giardia was our major challenge. As previously described, we have performed an original

PCR-based protocol able to detect and to discriminate G. ardeae, G. muris, G. microti and

G. duodenalis assemblages by sequence analysis of the region of the ribosomal unit that

spans the 5.8 S and the two ITS. Our approach represents a versatile tool for molecular

epidemiologic investigations, combining an excellent robustness, with a high level of

genetic variability among Giardia species and G. duodenalis assemblages. In order to

clarify the issue of mixed infections in humans, we have observed: a) high level of mixed

infections with both G. duodenalis A and B assemblages in human stools; b) evidences

suggesting that recombination can occur between G. duodenalis assemblage A and B.

Finally, for the first time, Giardia cysts were used directly in qPCR protocols for

assemblage-specific amplification, with excellent results.

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Perspectives:

This work allowed the clarification of several issues regarding (a) the levels of

water contamination by Giardia and Cryptosporidium; (b) the prevalence of these

parasites in human and animal in the north of Portugal and Galicia; and (c) some

molecular features of Giardia. This work is not closed in itself and several future

perspectives emerged from its realization. Three main future research lines derived from

this work: a) the implementation of molecular tools to perform Cryptosporidium sub-

genotyping; b) a need to increase the number of human samples studied; c) the

application of the new assemblage-specific primers in qPCR to environmental, human and

animal samples to determine the level of mixed infections by Assemblage A and B of G.

duodenalis. In the first case, by the application of sub-genotyping tools, particularly the

gp60 gene, to human, animal and environmental samples, a deeper understanding of

transmission dynamics of C. parvum and C. hominis could be obtained. In the second

case, the study of a larger number of human fecal samples should clarify the actual

prevalence of these infections, even in other age groups. Finally, the application of the

assemblage-specific primers with qPCR approaches in environmental, human and animal

samples would be informative to estimate the number of mixed infections by G.

duodenalis assemblage A and B. Furthermore, the ability of this assemblage-specific

primer approach to detect recombination events in G. duodenalis should be verified. In

turn, this requires an adequate technology to isolate and enumerate single cysts, such as

micromanipulation or flow cytometry. These research lines should be considered in future

projects.

In order to develop ideas produced during our thesis work, a new research

program has received a positive decision for financing from the Portuguese Foundation for

Science and Technology. As leader investigator it is my commitment to develop new

systems for prophylaxis and diagnostic of Cryptosporidium infections. A similar research

project on Giardia was prepared and recently submitted for evaluation.

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