The Molecular Pathogenesis of Giardiasis

Giardia intestinalis (formerly known as Giardia lamblia) is the most common human protozoan pathogen. It is perhaps best known for the clinical diversity of the infection that they produce (1-3). In young children and previously unexposed adults, particularly travelers from low- to high-prevalence parts of the world, Giardia can produce serious diarrhoeal illness with intestinal malabsorption, marked weight loss, and in infants and young children, impairment of growth and development. In the vast majority of individuals worldwide, the parasite is apparently carried without significant clinical morbidity. There is no satisfactory explanation for the diverse clinical spectrum seen in giardiasis, and as yet no virulence factors have been identified. The failure to identify the parasite determinants responsible for the production of intestinal injury, diarrhoea, and malabsorption makes it impossible to provide a simple explanation for the molecular pathogenesis of giardiasis (4). However, there have been some important steps forward in understanding the disease mechanisms, particularly the concept of genetic diversity, within the Giardia genus. The recent description of biological variation in association with different genotypes may be the first indication that virulent and relatively avirulent isolates of Giardia intestinalis exist. This observation alone could be of great assistance in the isolation of specific virulence factors in the future. For more than two decades there has been evidence that mucosal immune responses may be involved in the development of the enteropathy associated with giardiasis (5). Repopulation of the villus with relatively immature cells may also be a factor and, according to recent evidence, could occur as a direct response to the parasite without evoking immunological pathways or epithelial cell injury. Finally, Giardia appears to be able to influence a number of important physiological events within the intestinal lumen and to interfere with the digestive process as well as the absorptive function of the small intestinal epithelium (4). GENETIC DIVERSITY OF GIARDIA INTESTINALIS The members of the Giardia genus are characterised by the three pairs of flagella, two nuclei, and ventral disc that distinguishes them from other members of the hexamitidae family (6). There are three major morphological subtypes of Giardia: G. agilis from amphibians, G. muris from mice, and G. intestinalis from humans and some other vertebrates. These different types can be distinguished by the overall shape and dimensions of the trophozoite body and by the distinctive shapes of the median bodies. Two other Giardia isolates have been described: G. psittaci, isolated from a budgerigar, and G. ardeae, from the Great Blue Heron. These can be distinguished from the other subtypes by an absence of the ventrolateral flange in the former and a single (rather than a double) caudal flagellum in the latter. However, only G. intestinalis is known to infect humans and all of these isolates are morphologically identical. Other approaches were required therefore to identify phenotypic and genotypic diversity amongst different human G. intestinalis isolates. Phenotypic Variation Isoenzyme analysis and the classification of isolates according to zymodemes have been used for many years to distinguish different isolates of protozoa. This approach has been used with some success in the identification of virulent and relatively avirulent isolates of Entamoeba histolytica; however, not all investigators accept the absolute reliability of this approach to classification. Application of this technique to the classification of Giardia isolates has resulted in the description of a variety of groupings or subclassifications that demonstrated the geographic diversity of the organism; this technique also provided some of the early evidence indicating that G. intestinalis isolates from humans and animals are similar and in some cases apparently identical (7). The main limitation of this technique, and indeed of all other approaches to classifying Giardia isolates, relates to the lack of any correlation between isoenzyme analysis and the biological behaviour of a particular isolate with respect to virulence. Phenotypic differences have also clearly been demonstrated by surface antigen analysis using immunoblotting or immunoprecipitation techniques (8,9). Although differences have been observed, it has not been possible to develop a reproducible typing system based on immunodominant antigens. This limitation may be related to the organism's ability to display a variety of surface antigens that vary within the same isolates (10). Thus, antigenic variation may be one mechanism by which this organism evades the host immune response. Genotypic Variation Giardia has been estimated to have between 8 and 50 sets of chromosomes with a total genome size of about 1.2 × 107 base pairs (11-13). Giardia nuclei are haploid, and the observed genetic diversity between isolates is thought to be based on clonal divergence. The Giardia genome appears to exhibit considerable genomic plasticity, which extends from an allelic variation of single genes to polymorphism of entire chromosomes (13). The chromosomes that have been studied most extensively in Giardia are those that encode for rRNA genes. The rRNA genes are present as short arrays close to the telomeres. Extensive and frequent polymorphisms have been noted at the end of these chromosomes, whereas the central chromosome domains are conserved. DNA analysis by restriction fragment polymorphism analysis and DNA fingerprinting has demonstrated marked genetic diversity (14-17); there is, however, close similarity between G. intestinalis isolates obtained from humans and those from domestic and wild animals, supporting the view that giardiasis is a zoonosis. However, more than one DNA fingerprint profile can be obtained from cloned isolates from the same human infection, suggesting that mixed infections with genetically diverse Giardia isolates occur in humans. DNA fingerprint profiles, however, have been shown to change with time and with experimental in vitro pressures, confirming the plasticity of the Giardia genome. Thus, it is unclear whether giardiasis is truly a mixed infection or the process of in vitro culture exploits the plastic genome and artificially leads to clonal divergence (14,18). Recent work from our laboratory suggests that in vitro cultured G. intestinalis isolates with distinct DNA fingerprints have different biological effects in a neonatal rat model of giardiasis with respect to both structural and functional abnormalities in the small intestine (19). This observation may lead the way to the eventual isolation of virulence factors, as it has been possible to link a possible genetic marker with a possible biotype. INTESTINAL COLONISATION: MOLECULAR MECHANISMS Infection is initiated by ingestion of Giardia cysts, generally through contaminated water or food; direct person-to-person transfer does occur. Colonisation of the small intestine is an essential component of Giardia's life cycle and a sine qua non for the production of diarrhoeal disease. Colonisation occurs as a three-step process commencing with excystation, followed by attachment to the small intestine epithelium and multiplication. However, to complete the life cycle, the organism must again encyst, following which cysts are excreted in the faeces and the cycle of faecal-oral transmission can continue. Excystation This process has been achieved in vitro for G. intestinalis and G. muris by exposure of cysts to low pH (1.3-2.7 or 4.0) followed by transfer to neutral pH (20,21). These in vitro conditions closely resemble those in vivo when cysts move from the acid environment of the stomach to the near neutral pH of duodenal and jejunal fluid. However, excystation of G. intestinalis and G. muris can occur at neutral pH (21,22), and G. muris have been shown to exist following exposure to sodium bicarbonate at pH 7.5 (22), consistent with a role for duodenal and pancreatic bicarbonate secretion in the excystation process. Excystation begins within 5-10 min of exposure to a suitable excystation environment; flagella movement is the first sign of activity. The intracellular vacuoles are thought to discharge their contents during excystation (23), suggesting a role for hydrolase activity in the process. Attachment Giardia attaches intimately to the intestinal epithelium and to a variety of other inert substrates such as glass and plastic. The ventral disc is thought to be the primary organelle of attachment. Hydrodynamic forces generated beneath the disc by continuous activity of the ventral flagella have been proposed as a possible attachment mechanism (24). It has also been suggested that flagella activity causes low pressure under the ventral disc due to fluid fluxes around the ventral and marginal grooves. However, attachment occurs with the morphologically distinct G. psittaci, in which the ventrolateral flange is incomplete; thus it is unlikely that hydrodynamic forces can be the sole explanation for disc-mediated attachment (6). The presence of contractile proteins in the peripheral regions of the ventral disc suggests that they participate in attachment (25). Inhibitors of microfilament function, such as cytachalasin-B and low calcium concentrations, inhibit attachment-again supporting a role for cytoskeletal proteins in attachment (26,27). An alternative attachment mechanism has been proposed following the discovery of a mannosebinding surface lectin on Giardia trophozoites. Lectin activity was initially described using the classic approach of a mixed-cell agglutination with mammalian erythrocytes (28). The lectin is distributed throughout the parasite surface and is not specifically localised to the ventral disc (29). The lectin is present within the trophozoite as a prolectin that can be activated by trypsin (30). Giardia lectin (also known as taglin) has been purified and found to have a molecular weight of 28-30 kDa (31). Giardia trophozoites adhere to isolated mammalian intestinal epithelial cells in vitro by mechanisms that are consistent with lectin-mediated attachments (32). Giardia also attaches to Caco-2 cells (the human colon cancer cell line that differentiates to resemble small intestinal epithelial cells) by mechanisms that incorporate both contractile proteins and surface lectin-mediated processes (33). The relative importance of mechanical and lectin-mediated attachment mechanisms has not been established. The surface lectin may operate in the initial attachment between Giardia and the epithelium, since, in contrast to ventral disc-mediated attachment, the parasite does not need to be in a specific orientation to the mucosa. Subsequently, the parasite may reorientate in a ventral surface-down position where mechanical forces may be more effective. Multiplication Giardia trophozoites divide by binary fission. Although it has been suggested that “sexual reproduction” may occur, there is no firm evidence of exchange of genetic material between organisms. Giardia is a luminal parasite and is presumed to be dependent on luminal nutrients. Bile promotes growth of Giardia in vivo and in vitro (34-37). Other related protozoa and intestinal bacteria do not exhibit a similar degree of dependence (38). It has been proposed that bile salts promote the uptake of which the organism is to and a relatively low are also required for growth In vitro culture and in vitro generally the presence of mammalian is essential for the parasite to complete life cycle and in the environment or be carried by other such as domestic and wild has been achieved in vitro by trophozoites to of bile salts and acid at neutral pH is associated with the of specific antigens during the first molecular weight antigens These antigens are to the and are involved in INTESTINAL Giardia is found in close to the surface of the the intestinal Giardia is not an there have been of trophozoites found within the this be as a of the in the and of giardiasis relates to the spectrum of disease which from of the organism to and This clinical variation may to host factors, which may be genetically or or to the of the infection by parasite within the small there is evidence to that Giardia isolates may vary in which could be a major of disease variety of structural abnormalities of the small intestinal have been in humans and in of giardiasis In giardiasis is associated with the complete of abnormalities of from to or The majority of individuals have or relatively associated with an in However, in human small intestine are relatively and by cases that are in a which the more end of the giardiasis clinical In human giardiasis, there may be no abnormalities in the small in of this than of with giardiasis have There is an in cells in the and in the epithelium in human infections in mice, and can produce similar abnormalities of mucosal the and abnormalities are relatively The a particularly model with which to small intestinal and function, as develop and have significant morphological abnormalities by of infection, with in in the and in in the and In the there is a small significant in These early in and occur in the absence of any in the and with no in the of have similar in a neonatal rat model of giardiasis (19). Two of three in no significant in In there was no in epithelial cell an in the number of or In human giardiasis, when appears by such as and of are present The model has the of these a marked in surface in both and these abnormalities were This in the of was found was not specifically related to of trophozoite attachment. There is evidence from human to that the of the mucosal relates to the of the In human giardiasis, morphological abnormalities have been associated with a in and in the have been in experimental infections in mice, and in is when and morphological abnormalities are most In the neonatal rat model of giardiasis, of was demonstrated at in the three distinct isolates that were (19). Although in the small intestine has not previously been in a infection, it is in other where there is mucosal or other a response. has been associated with both giardiasis and immune have the of giardiasis in with and found it to occur in of However, in one from were described with giardiasis and no Thus, the and giardiasis unclear it that any two can occur in without any direct indication of There are also no as to the pathogenesis of have shown a of cells within the and suggesting there be immune to a luminal with failure to from to production within the in that there are functional of the structural abnormalities and the in activity. In of sodium and was not different in sodium was in the not in the of in using small intestinal in in vivo in animals have also shown and in response to was similar to that in In a neonatal rat model of infection, of and was with some animals in a for sodium and (19). of a these with from provided evidence that there is impairment of and acid Although phenotypic and genotypic differences have been different Giardia isolates for some the absence of any clearly virulence factors has it to whether any variation in or has a in the of diverse virulence experimental infection in however, have shown that different isolates can produce immune responses in the host and that these differences can in to and of infection studied the biological of experimental infection in neonatal with three distinct G. intestinalis genotypes as by DNA fingerprinting (19). The isolates in their ability to fluid and and in the response to a in virulence with respect to activity and These differences in could not be to differences in parasite or of infection, these were identical for all three isolates. In there were no major differences in the intestinal immune response to Giardia as by indicating that these differences in virulence could not be to differences in These in neonatal be to human infection they that Giardia isolates vary in for at some of the clinical diversity seen in this MOLECULAR MECHANISMS OF The mechanisms by which Giardia causes and intestinal malabsorption on pathogenesis suggested that Giardia trophozoites as a mechanical to or for host the functional of the small intestine and the relatively small of the parasite these There is evidence, however, that Giardia can produce of mucosal and at the same time influence conditions in the intestinal lumen that could and Giardia trophozoites to the epithelium and have been shown by to and at the where the ventral disc with the It is possible therefore that factors involved in this for at of There is some evidence to that Giardia and the intestinal lumen yet no parasite has been to for the morphological observed in the small Giardia however, a number of which surface and In the surface lectin of Giardia may to epithelial can intestinal epithelial cells and produce abnormalities similar to those seen in giardiasis It to be whether any of these parasite are to the host the mechanism by which Giardia villus epithelial cells and epithelial cell loss, there to be a cell response with an in and cell In other conditions in which this response such as there is of the villus by relatively immature with absorptive intestinal has been in the model of giardiasis using activity as a marker of there is no evidence in the or that the cells the are than those in these it that the structural and functional abnormalities observed in the to direct than to a mechanism cell in our laboratory has however, that Giardia can the a in and cell in Caco-2 cells in vitro and in neonatal rat small intestine in vivo of during the attachment process. trophozoites or of Giardia have no this mechanism could have a role in a in the small intestinal to a simple explanation for epithelial has that immunological mechanisms may be involved to those thought to be responsible for the villus of disease. In human giardiasis there is a immune response within the infection is associated with an in the number of and These have been more to in experimental animals However, there is evidence that cell alone can produce villus in intestinal disease and of intestinal is characterised by cell and a human small intestinal it has been possible to the mechanisms involved by cells with or both approaches villus and confirming cell for this has been obtained from in experimental G. muris infection in infection, the of the cell is in than in from the of were mice, abnormalities in the small intestine more However, in the cell not occur in the and it that cell mechanisms are also involved In in in more effects on in animals, indicating that epithelial is not dependent on immune function are in number in giardiasis and have been in in experimental G. muris infection, villus when the in evidence to mechanisms however, provided in in which G. were trophozoite the primary infection This approach resulted in a in mucosal which was thought to be The antigen was most marked in the which is genetically to G. muris Giardia lectin can as a and cells to be established. However, the lectin is a which a similar role for the Giardia for In to the variation in the impairment of and in neonatal rat small have shown similar effects on small intestinal and activity (19). in animals with the Giardia and was by and the isolate to produce a significant in villus a lack of morphological the most on and and significant in and all three isolates of in common with other or in the small evidence of variation was with more activity than or Thus, it that there are parasite determinants that may be by different isolates and that to influence the degree of morphological and functional in the small In to the morphological abnormalities in the small intestine that may to the of and malabsorption, a variety of luminal factors have been that operate on digestive processes that to and There is some evidence to that giardiasis is associated with of bacteria in the small In one from 8 of with more than bacteria cultured from duodenal whereas no from a giardiasis without of the with also found of bacteria in of with giardiasis to the two also present can produce abnormalities in the small intestine similar to those seen in giardiasis and it may thus have a role in mucosal injury. However, in both of these one to of bacteria in the small intestine of the presence of the of these There have been no in with giardiasis in the to whether associated occurs in this of and whether it is an mechanism of in Bile The of the or from bile salts their in and thus their in within the intestinal In bile salts are and can intestinal thus to the pathogenesis of found evidence of bile in all of their with and in of giardiasis without malabsorption other have not these (38). Giardia does not have the ability to bile salts Bile by Giardia Bile and bile salts to important to the life cycle of The presence of bile at low has been shown to parasite growth and time and thus may be an important factor for this parasite The can be by the of bile salts which to the uptake of and that the parasite is to it has been shown that in bile are than those in a and that the presence of bile antigenic by the parasite of bile salts parasite the of bile it to that the parasite was bile salts that this was relatively specific for Giardia and that uptake to be by a process Bile a and was dependent on pathways in the Bile salts were and found to be in the following The of bile uptake for the parasite has not been of host bile salts during could the bile and to malabsorption by of and the of pancreatic the of which is bile of of and have been shown to be in with giardiasis There is no evidence that this is due to a failure of pancreatic it could be related to the more recent observation that Giardia trophozoites and trophozoite inhibit trypsin activity and in vitro The mechanisms by which the parasite these has not been could be related to the direct between a parasite such as and the host However, the has a functional and the of the observed in clinical is unlikely to for However, the could to the of abnormalities that and to and the it is that no single virulence factor or mechanism the pathogenesis of to that infection can be associated with small enteropathy with an associated in and impairment of and However, epithelial and in humans can occur in the absence of abnormalities of as by the observation that of individuals with giardiasis to have no small by and the in our laboratory that one Giardia isolate not change a on small intestinal absorptive function (19). Although the cell is for the there is no evidence as yet that this is important in human infection and the evidence in is However, factors that digestive processes could well be of a it unlikely that they alone can the and malabsorption associated with this Thus, are with the of to in our present of a and a process of approach to the development of a model for pathogenesis is to the three common clinical of the infection, and with intestinal For the one must that the host is with a relatively avirulent that virulence factors or that host immune and mechanisms are to parasite and infection It is whether the begins with diarrhoeal illness or whether the entire of The for giardiasis is about This be consistent with a relatively for and of infection and the relatively process of parasite multiplication. This time is of different from that of such as that due to which generally begins within of the There is a time for and also a of For Giardia the time to be consistent with the failure to the activity in the The be with the of In infection with malabsorption, the process a of mucosal and luminal events is to the organism lead to of epithelial and function, and there is some evidence to that the degree of and is closely related to the of the intestinal malabsorption the which in of intestinal in time this In infection, one that the parasite that trophozoite be to a significant on the activity of luminal and to in bile Thus, there be a cycle that not be was initiated with an model of