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.
19 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
20 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
21 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 .
Table 1. Species of Cryptosporidium and typical hosts. From Xiao and Fayer, 2008
22 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).
Table 2. Species of Giardia and typical hosts. From Adam, 2001
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
23 distinct assemblages: A to G (Caccio et al., 2005; Hunter and Thompson, 2005; Monis and Thompson, 2003). These genotypes are represented in Table 3.
Table 3. Genotypes of Giardia duodenalis group. From Thompson, 2004
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
24 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;
25 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 Figure 1. Life cycle of shed in the environment with feces, are ready to Cryptosporidium parvum or infect a new suitable host. The prepatent period, Cryptosporidium hominis . From which means the time between the ingestion of CDC (www.cdc.gov).
26 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
Figure 2. Life cycle of Giardia duodenalis . From CDC (www.cdc.gov). approximately 7 to 10 m 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 Figure 3. The morphology of cysts is induced by the acidic nature of apicomplexan parasites. From stomachic lumen and the presence of (Morrissette and Sibley, 2002) enzymes. However, this event occurs in the
27 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 m 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.
Figure 4. Mucosal surface 2.4. Ultrastucture of the trophozoite of Giardia and of the small intestine of a sporozoite of Cryptosporidium gerbil infested with Giardia Cryptosporidium as an Apicomplexa parasite, sp. protozoa. The intestinal shares a variety of morphological traits common of this epithelial surface is almost phylum (Figure 3). These protists share the organization entirely obscured by the of unique attached Giardia organelles trophozoites. Public Health in a region Image Library (PHIL) # called the 11632 www.cdc.gov apical complex. These organelles include the rhoptires, the micronemes, the spical polar Figure 5. Trophozoite coronal section. A ring and the conoid, which are directly coronal view of a trophozoite demonstrates involved in the host cell invasion and the nuclei (N), endoplasmic reticulum (ER), interaction. Rhoptries, micronemes and flagella (F), and vacuoles (V). A mechanical dense granules are unique secretary suction is formed when the ventral disk organelles that contain products required (VD) attaches to an intestinal or glass for motility, invasion, adhesion and surface. Components of the ventral disk invasion of host cells and the include the bare area (BA), lateral crest establishment of the parsasitophorous (LC), and ventrolateral flange (VLF). From vacuole (Carruthers et al., 1999; Adam, 2001 Carruthers and Sibley, 1997; Morrissette
28 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 m long and 5 9 m 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).
29 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, Figure 6. The ultrastructural morphology of a the pathophysiology associated with Giardia protozoan’s ventral adhesive disk on these symptoms are still incompletely the left, and the circular lesion on the right, understood (Eckmann and Gillin, which can be left on the intestinal mucosal 2001; Muller and von Allmen, 2005). surface, as a result of the tight adhesion of this The intestinal colonization by the disk to the intestine’s microvillous border. parasite seems to cause villous Public Health Image Library (PHIL) # 11644 flattening or atrophy and microvillus (www.cdc.gov) 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
30 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 Figure 7. Electron microscopic evaluation of cryptosporidiosis greatly depend on HT29.74 cells infected with C. parvum . A) oocyst the immune status of the patient. In after 1 hour in culture in the process of fact, immunologically healthy excysting, which release four sporozoites (three patients usually recover are visible). B) sporozoites after 6 hours in spontaneously in a week. The culture in the process of infecting HT29.74 cell. clinical signs can range from C) mature schizont 24 hours later. The schizont asymptomatic to acute, severe and is intracellular, yet extracytoplasmic. A dense persistent diarrhea and their band and feeder layer which interface between potential for Cryptosporidium the parasite and host cell are present. Eighty transmission can persist for weeks fully developed merozoites are visible. D) mature after symptoms cease (Deng et al., schizont at 24 hours has rupted, releasing the 2004; Fayer, 2004; Hunter and merozoites. From Flanigan et al., 1991 Thompson, 2005). Diarrhea is usually watery with mucus, and without blood or
31 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).
32 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 Figure 8. The most important cycles of immune modulators. This suggests that transmission for maintaining Giardia and intestinal epithelial cells play an important Cryptosporidium. Besides direct role in initiating the mucosal immune transmission, water and food may also response to C. parvum infection (Deng et play a role in transmission. The frequency al., 2004; Riggs, 2002). of interaction between cycles is not
known. From Hunter and Thompson, 2005
33 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).
34 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).
Table 4. Prevalence of five common Cryptosporidium species in humans. From Xiao et al., 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
35 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 Table 5. Species and assemblages of infect animals, which imply that the Giardia . From Xiao and Fayer, 2008 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,
36 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 Table 6. Cryptosporidium spp. and genotypes that infects humans and emphasizes evidences on an existence of other hosts. From Xiao and Fayer, host adapted species and limited cross 2008. 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
Table 7. Prevalence of Giardia duodenalis assemblage A and B in humans. From (Caccio and Ryan, 2008)
37 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
Table 8. G. duodenalis genotypes in farm animals. From Xiao and Fayer, 2008 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
38 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). Figure 9. Although the studies are quite limited, only assemblages A Photomicrograph of a and B were detected in horses (Traub et al., 2005). Giardia duodenalis cyst Regarding the companion animals, for instance dogs, they seen using a Trichrome are infected by a broader range of G. duodenalis stain . Public Health assemblages: dogs were found to be infected with Image Library (PHIL) # assemblages A, B, C and D (Monis et al., 1998; Xiao and 1944 www.cdc.gov 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 Figure 10. assemblage B, C and D in coyotes (Trout et al., 2006a). Photomicrograph of
Cryptosporidium parvum 2.7. Laboratory diagnosis oocysts, which had been Diagnosis of Cryptosporidium and Giardia infections stained using the requires, normally, the morphological identification of the modified acid fast oocysts and cysts, in stool specimens, intestinal aspirates or method . Public Health intestinal biopsy specimens (Alles et al., 1995). This Image Library (PHIL) # identification is done by microscopic examination, after 7829 www.cdc.gov
39 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
40 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.
41 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
42 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
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.
43 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
44 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/
45 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
46 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
47 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
48 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.
Figure 13: Search by β giardin gene in GiardiaDB at http://eupathdb.org/eupathdb/
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,
49 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
50 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
51 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
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.
52 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
53 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
54 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
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
55 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
56 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
Figure 15: Example of the overlapping nucleotide peaks in a chromatogram.
57 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
58 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.
59
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.
60 Chapter II Species of Cryptosporidium and Giardia infecting humans
1. “Genotype analysis of Giardia isolated from asymptomatic children in northern Portugal”
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64 2. “Genetic characterization of Cryptosporidium isolates from humans in northern Portugal”
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68 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)”
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72 2. “Prevalence and preliminary genetic characterization of Cryptosporidium spp. from asymptomatic heifers in Galicia (NW, Spain)”
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76 3. “Occurrence of Cryptosporidium parvum and Giardia duodenalis in healthy adult domestic ruminants”
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84 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 ”
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110 3. “Detection of Cryptosporidium spp. and Giardia duodenalis in surface water: a health risk for humans and animals”
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122 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 Almeida a,b , Maria João Moreira a,c , Sónia Soares a,c , Maria de Lurdes Delgado a, João Figueiredo a, Elisabete Silva a, António Castro a, José Manuel Correia da Costa a,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.
123 Introduction Infections by the Apicomplexa protozoa Cryptosporidiu m 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 Figure 1 Geographic location of the North of underestimated in the country due to Portugal and its 5 hydrographical into Iberian the lack of systematic diagnosis of Peninsula. these parasites.
124 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 Figure 2 Location of the 5 hydrographical the laboratory. The filter was taken from basins in the North of Portugal. 1 Minho; the container and processed with the aid 2 Lima; 3 Cávado; 4 Ave; 5 – Douro. of a Filta Max Manual Wash Station (IDEXX Laboratories, Inc., Westbrook, ME, USA) for further elution and concentration
125 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 l 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 100 l 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
126 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
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.
127 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 Table 1 Results from the genetic amplification was performed over 80 characterization of Cryptosporidium spp. and samples. Giardia duodenalis and the respective number Genetic characterization was of cysts and oocysts.
128 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
129 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
130 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.
References Adam, R. D., 2001: Biology of Giardia lamblia. Clin Microbiol Rev, 14, 447 475. Agency, U. S. E. P., 2005: Method 1623: Cryptosporidium and Giardia in water by filtration/IMS/FA. In: U. S. E. P. A. Office of Water (ed.) . Washington, DC. Almeida, A. A., M. L. Delgado, S. C. Soares, A. O. Castro, M. J. Moreira, C. M. Mendonca, N. B. Canada and J. M. Da Costa, 2006a: Genotype analysis of Giardia isolated from asymptomatic children in northern Portugal. J Eukaryot Microbiol, 53 Suppl 1, S177 178. Almeida, A. A., M. L. Delgado, S. C. Soares, A. O. Castro, M. J. Moreira, C. M. Mendonca, N. B. Canada, J. M. Da Costa and H. G. Coelho, 2006b: Genetic characterization of Cryptosporidium isolates from humans in northern Portugal. J Eukaryot Microbiol, 53 Suppl 1, S26 27. Alves, M., L. Xiao, F. Antunes and O. Matos, 2006: Distribution of Cryptosporidium subtypes in humans and domestic and wild ruminants in Portugal. Parasitol Res, 99, 287 292. Alves, M., L. Xiao, I. Sulaiman, A. A. Lal, O. Matos and F. Antunes, 2003: Subgenotype analysis of Cryptosporidium isolates from humans, cattle, and zoo ruminants in Portugal. J Clin Microbiol, 41, 2744 2747. Caccio, S. M., M. De Giacomo, F. A. Aulicino and E. Pozio, 2003: Giardia cysts in wastewater treatment plants in Italy. Appl Environ Microbiol, 69, 3393 3398.
131 Caccio, S. M., M. De Giacomo and E. Pozio, 2002: Sequence analysis of the beta giardin gene and development of a polymerase chain reaction restriction fragment length polymorphism assay to genotype Giardia duodenalis cysts from human faecal samples. Int J Parasitol, 32, 1023 1030. Caccio, S. M. and U. Ryan, 2008: Molecular epidemiology of giardiasis. Mol Biochem Parasitol, 160, 75 80. Castro Hermida, J. A., A. Almeida, M. Gonzalez Warleta, J. M. Correia da Costa, C. Rumbo Lorenzo 15 and M. Mezo, 2007: Occurrence of Cryptosporidium parvum and Giardia duodenalis in healthy adult domestic ruminants. Parasitol Res, 101, 1443 1448. Castro Hermida, J. A., I. Garcia Presedo, A. Almeida, M. Gonzalez Warleta, J. M. Correia Da Costa and M. Mezo, 2008: Contribution of treated wastewater to the contamination of recreational river areas with Cryptosporidium spp. and Giardia duodenalis. Water Res, 42, 3528 3538. Chappell, C. L., P. C. Okhuysen, R. Langer Curry, G. Widmer, D. E. Akiyoshi, S. Tanriverdi and S. Tzipori, 2006: Cryptosporidium hominis: experimental challenge of healthy adults. Am J Trop Med Hyg, 75, 851 857. Chauret, C., S. Springthorpe and S. Sattar, 1999: Fate of Cryptosporidium oocysts, Giardia cysts, and microbial indicators during wastewater treatment and anaerobic sludge digestion. Can J Microbiol, 45, 257 262. Fayer, R., 2004: Cryptosporidium: a water borne zoonotic parasite. Vet Parasitol, 126, 37 56. Fayer, R., 2009: Taxonomy and species delimitation in Cryptosporidium. Exp Parasitol , 124, 90 97. Fayer, R., U. Morgan and S. J. Upton, 2000: Epidemiology of Cryptosporidium: transmission, detection and identification. Int J Parasitol, 30, 1305 1322. Hunter, P. R. and R. C. Thompson, 2005: The zoonotic transmission of Giardia and Cryptosporidium. Int J Parasitol, 35, 1181 1190. Jiang, J., K. A. Alderisio, A. Singh and L. Xiao, 2005: Development of procedures for direct extraction of Cryptosporidium DNA from water concentrates and for relief of PCR inhibitors. Appl Environ Microbiol, 71, 1135 1141. Lobo, M. L., L. Xiao, F. Antunes and O. Matos, 2009: Occurrence of Cryptosporidium and Giardia genotypes and subtypes in raw and treated water in Portugal. Lett Appl Microbiol, 48, 732 737. Matos, O., M. Alves, L. Xiao, V. Cama and F. Antunes, 2004: Cryptosporidium felis and C. meleagridis in persons with HIV, Portugal. Emerg Infect Dis, 10, 2256 2257.
132 McCuin, R. M. and J. L. Clancy, 2003: Modifications to United States Environmental Protection Agency methods 1622 and 1623 for detection of Cryptosporidium oocysts and Giardia cysts in water. Appl Environ Microbiol, 69, 267 274. Mendonca, C., A. Almeida, A. Castro, M. de Lurdes Delgado, S. Soares, J. M. da Costa and N. Canada, 2007: Molecular characterization of Cryptosporidium and Giardia isolates from cattle from Portugal. Vet Parasitol, 147, 47 50. Read, C. M., P. T. Monis and R. C. Thompson, 2004: Discrimination of all genotypes of Giardia duodenalis at the glutamate dehydrogenase locus using PCR RFLP. Infect Genet Evol, 4, 125 130. Robertson, L. J., L. Hermansen and B. K. Gjerde, 2006: Occurrence of Cryptosporidium oocysts and Giardia cysts in sewage in Norway. Appl Environ Microbiol, 72, 5297 5303. Smith, H. V., S. M. Caccio, N. Cook, R. A. Nichols and A. Tait, 2007: Cryptosporidium and Giardia as foodborne zoonoses. Vet Parasitol, 149, 29 40. Sousa, M. C., J. B. Morais, J. E. Machado and J. Poiares da Silva, 2006: Genotyping of Giardia lamblia human isolates from Portugal by PCR RFLP and sequencing. J Eukaryot Microbiol, 53 Suppl 1, S174 176. Thompson, R. C., 2000: Giardiasis as a re emerging infectious disease and its 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 of Cryptosporidium and Giardia and assessment of zoonotic transmission. Int J Parasitol, 38, 1239 1255. Xiao, L., U. M. Morgan, J. Limor, A. Escalante, M. Arrowood, W. Shulaw, R. C. Thompson, R. Fayer and A. A. Lal, 1999: Genetic diversity within Cryptosporidium parvum and related 17 Cryptosporidium species. Appl Environ Microbiol, 65, 3386 3391.
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162 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).
164 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 .
165 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
166 (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.
167 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,
168 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
169 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.
170 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.
171 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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