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