From Leningrad to the day-care center. The ubiquitous Giardia lamblia.

Giardiasis is recognized as a worldwide public health problem. Seroprevalence data from both the developing and developed world show high rates of carriage in populations at risk for fecal-oral transmission, such as children in day-care centers. Outbreak investigation has expanded our understanding of reservoirs for Giardia lamblia and of the routes of transmission. Various host factors have been associated with infection. The pathogenesis of giardial infections is being elucidated, in particular the role of lectin activation in producing disease. Three standard chemotherapeutic agents are available in the United States. The institution of community-wide prevention measures is equally important. Current areas of investigation including antigenic composition and enzymatic variants should result in effective forms of immunotherapy, while more effective forms of chemoprophylaxis could assist in eradicating the pathogen from institutional settings.     

Codon usage in Giardia lamblia.

A codon usage table for the intestinal parasite Giardia lamblia was generated by analysis of the nucleotide sequences of eight genes comprising 3,135 codons. Codon usage revealed a biased use of synonymous codons with a preference for NNC codons (42.1%). The codon usage of G. lamblia more closely resembles that of the prokaryote Halobacterium halobium (correlation coefficient r = 0.73) rather than that of other eukaryotic protozoans, i.e. Trypanosoma brucei (r = 0.434) and Plasmodium falciparum (r = -0.31). These observations are consistent with the view that G. lamblia represents the first line of descent from the ancestral cells that first took on eukaryotic features.     

Detection of Giardia lamblia by immunofluorescence.

High-titer immune sera to cysts of Giardia lamblia, produced in guinea pigs, were labeled with fluorescein isothiocyanate. The resulting conjugates were used to detect G. lamblia in stool specimens by fluorescence microscopy. The sera also reacted with cysts of Chilomastix mesnili, but the two organisms could be differentiated by their size.     

Frequency of variant antigens in Giardia lamblia.

Giardia lamblia undergoes antigenic variation. The rate of antigenic variation and the size of the variant antigen repertoire were estimated in clones of Giardia lamblia which reexpresses surface variant antigens that are characteristics of its parent. Calculations were based on determinations of the number of trophozoites expressing defined or nondefined epitopes as well as the total number of trophozoites in newly established clones. The rate of appearance of variant antigens containing defined epitopes was expressed as the number of generations until the first trophozoite expressing a defined epitope appeared. In clones of isolate WB, tested because their major surface variant antigens were largely nondefined, variants expressing epitopes recognized by Mabs 6E7 or 3F6 appeared after approximately 12 generations. Variants expressing epitopes recognized by Mab 5C1 appeared at about 13 generations, significantly greater than for the other epitopes. The rate of antigenic variation was studied in another isolate, GS/M, whose surface epitope repertoire differs from that of isolate WB. A single epitope recognized by Mab G10/4 was tested. Trophozoites reexpressing this epitope first appeared after about 6.5 generations, significantly less than in WB. Therefore, the single epitope studied in isolate GS/M is reexpressed much more frequently than those of WB. In isolate WB, the epitopes recognized by Mab 6E7 and 3F6 tended to appear at the same time. The median number of variant antigens in WB was estimated to lie between 20.5 and 184.     

Electron microscopy of Giardia lamblia cysts.

The flagellated protozoan Giardia lamblia is a recognized public health problem. Intestinal infection can result in acute or chronic diarrhea with associated symptoms in humans. As part of a study to evaluate removal of G. lamblia cysts from drinking water by the processes of coagulation and dual-media filtration, we developed a methodology by using 5.0-microns-porosity membrane filters to evaluate the filtration efficiency. We found that recovery rates of G. lamblia cysts by membrane filtration varied depending upon the type and diameter of the membrane filter. Examination of membrane-filtered samples by scanning electron microscopy revealed flexible and flattened G. lamblia cysts on the filter surface. This feature may be responsible for the low recovery rates with certain filters and, moreover, may have implications in water treatment technology. Formation of the cyst wall is discussed. Electron micrographs of cysts apparently undergoing binary fission and cysts exhibiting a possible bacterial association are shown.     

Codon Usage in Giardia lamblia

ABSTRACT A codon usage table for the intestinal parasite Giardia lamblia was generated by analysis of the nucleotide sequences of eight genes comprising 3,135 codons. Codon usage revealed a biased use of synonomous codons with a preference for NNC codons (42.1%). The codon usage of G. lamblia more closely resembles that of the prokaryote Halobacterium halobium (correlation coefficient r = 0.73) rather than that of other eukaryotic protozoans, i.e. Trypanosoma brucei ( r = 0.434) and Plasmodium falciparum ( r =–0.31). These observations are consistent with the view that G. lamblia represents the first line of descent from the ancestral cells that first took on eukaryotic features.     

Antigenic variation in Giardia lamblia

Clones of the WB isolate of Giardia lamblia were exposed to cytotoxic mAb 6E7 which reacts with a 170-kDa surface Ag. Surviving progeny occurred at a frequency of about 1 in 1000 and were resistant to the effects of mAb 6E7. Analysis of progeny and clones of these progeny by surface radiolabeling, surface immunofluorescence, and Western blotting failed to detect the 170-kDa Ag. Loss of this Ag was associated with the appearance of a series of new surface Ag. A cytotoxic mAb (5C1) was produced to one of the newly appearing antigens (approximately equal to 64 kDa) and Giardia resistant to the cytotoxic effects of 5C1 isolated. Neither the approximately equal to 64 kDa nor the 170 kDa Ag were present and were replaced by a second series of new Ag. These studies clearly establish the loss and subsequent replacement of two antigenically distinct epitopes on Giardia derived from a single organism.     

Antigenic variation in Giardia lamblia

Giardia lamblia undergo surface antigenic variation in vitro and in vivo. The presence of variant trophozoites can be detected in clones after exposure to cytotoxic monoclonal antibodies. Surviving Giardia (progeny) no longer possess the initial major surface antigen which is replaced by new antigens. Exposure of a clone from one progeny to another cytotoxic mAb specific to one newly appearing surface antigen resulted in the loss of this antigen and replacement by another set of antigens. The frequency of change was rapid (1:100-1:1000) and was dependent upon the isolate. The presence of variant populations in clones was confirmed by direct and indirect immunofluorescence using mAbs to major surface antigens of subsequent progeny. The putative amino acid sequence of a portion of one antigen revealed a cysteine-rich composition which was confirmed in this variant protein as well as others by preferential uptake of [35S]cysteine. The mechanism(s) responsible most likely involves genomic rearrangements since Southern blots revealed a family of related genes which changed frequently compared to other areas of the genome. However, expression-linked copies have not been detected. Loss and gain of surface antigens have also been found in gerbils and humans infected with defined clones, but there does not appear to be cyclical appearance of variant populations. The biological importance of antigenic variation is not known but it may contribute to chronic and/or repeated infections.     

Chromosome rearrangements in Giardia lamblia

Recent studies have shown that the genome of Giardia lamblia is plastic. Clinical isolates exhibit extensive karyotypic heterogeneity and chromosome rearrangements occur frequently, in vitro. In this review, Sylvie Le Blancq looks at genome organization and the impact of DNA rearrangement events.     

The Giardia lamblia genome

Giardia lamblia is a protozoan parasite of humans and other mammals that is thought to be one of the most primitive extant eukaryotic organisms. Although distinctly eukaryotic, it is notable for its lack of mitochondria, nucleoli, and perixosomes. It has been suggested that Giardia spp. are pre-mitochondriate organisms, but the identification of genes in G. lamblia thought to be of mitochondrial origin has generated controversy regarding that designation. Giardi lamblia trophozoites have two nuclei that are identical in all ways that have been studied. They are polyploid with at least four, and perhaps eight or more, copies of each of five chromosomes per organism and have an estimated genome complexity of 1.2x10(7)bp of DNA, and GC content of 46%. There is evidence for recombination at the telomeres of some of the chromosomes, and multiple size variants of single chromosomes have been identified within cloned isolates. However, the internal regions of the chromosomes demonstrate no evidence of recombination. For example, there is no evidence for control of vsp gene expression by DNA recombination, and no evidence for rapid mutation in the vsp genes. Single pass sequences of approximately 9% of the G. lamblia genome have already been obtained. An ongoing genome project plans to obtain approximately 95% of the genome by a random approach, as well as a complete physical map using a bacterial artificial chromosome library. The results will facilitate a better understanding of the biology of Giardia spp. as well as their phylogenetic relationship to other primitive organisms.     

Protein trafficking in Giardia lamblia

Giardia, a protozoan parasite of humans and other vertebrates, is a common cause of intestinal disease worldwide. Besides its medical importance, Giardia is considered an excellent system to study the evolution of fundamental cellular processes because it belongs to the earliest branches of the eukaryotic lineage of descent. Giardia trophozoites lack organelles typical of higher eukaryotes such mitochondria, peroxisomes and compartments involved in intracellular protein trafficking and secretion, such as the Golgi apparatus and secretory granules. Nevertheless, the minimal machinery for protein transport and sorting is present in this parasite. When Giardia undergoes encystation, the biogenesis of secretory organelles necessary to transport cyst wall constituents to the cell surface takes place. Recent studies in both vegetative and encysting trophozoites have provided interesting information regarding the secretory pathway of this important human pathogen.     

Overlapping genes in parasitic protist Giardia lamblia.

The parasitic protist Giardia lamblia lacks mitochondria and peroxisomes, as well as many typical membrane-bound organella characteristics of higher eukaryotic cells, together with extremely economized usage of DNA sequence, as demonstrated by the lack of introns. We describe here the presence of overlapping genes in G. lamblia, in which a part of the protein coding sequence of one mRNA exists in a region corresponding to the 3′-noncoding region of another mRNA transcribed from a gene on the opposite strand. Recently we isolated 13 kinesin-related cDNAs from G. lamblia. Nine of these cDNAs contain long 3′-noncoding sequences in which long open reading frames (ORFs) exist (in the remaining four cDNAs, the lengths of the 3′-noncoding sequences are very short). The predicted amino acid sequences of these ORFs were subjected to a search for homologies with sequences in databases. The amino acid sequences of the six ORFs exhibited significant sequence similarities with known sequences. These lines of evidence suggest the frequent occurrence of gene overlap in Giardial genome.     

Isoenzyme comparison of axenic Giardia lamblia strains.

We obtained isoenzyme patterns by polyacrylamide gradient gel electrophoresis (PGGE) of water-soluble protein fractions prepared from trophozoites of 11 axenic G. lamblia strains. The strains were isolated from animals and humans (both symptomatic and asymptomatic) from various geographic locations. Isoenzymes were also separated by isoelectric focusing. Of 12 enzymes attempted, eight exhibited well-defined and reproducible isoenzyme patterns by PGGE, based on which the strains were grouped into four zymodemes. Although the 11 strains were grouped into four zymodemes based on PGGE, no correlation between zymodeme and the known characteristics of the strains existed. Thus, a high degree of characteristic sharing appears to occur among genetically different G. lamblia strains.     

Frequent rearrangements of rRNA-encoding chromosomes in Giardia lamblia.

The ribosomal RNA (rRNA) genes in Giardia lamblia are present as short tandem arrays of a 5.6 Kb repeat unit on at least six telomeres. Four of these telomeres have the same overall organisation comprising a domain ranging in size from 25 to 300 Kb, delineated chromosome internally by a conserved island of restriction enzyme sites. Cloned lines of G. lamblia derived from the WB strain contain polymorphic subsets of chromosomes encoding rRNA genes. However, changes in the size of the rRNA telomere domains of these polymorphic chromosomes alone cannot account for the total size changes in the chromosomes. The rearrangement events are very frequent: 60% of subcloned lines had discrete rearranged karyotypes that differed from each other, suggesting either an estimated rearrangement rate that may be as high as 3% per division or a cloning-induced rearrangement event. The extreme plasticity of the genome has obvious implications for the maintenance of a functional genome and the control of gene expression in Giardia.     

Excretory-secretory products of Giardia lamblia

The surface of Giardia lamblia strain WB was radioiodinated with either 1,3,4,6-tetrachloro-3 alpha, 6 alpha-diphenylglycoluril (IODOGEN) or lactoperoxidase, and the labeled membrane components as well as the released excretory-secretory (E-S) products were identified. The surface of G. lamblia was easily labeled, and G. lamblia excretory-secretory (E-S) products were identified in the medium. Over 70% of the label on the cell surface was released over 24 hr. The major E-S product released was polydisperse (m.w. 225 to 94,000), protease VI and periodate-sensitive, chloroform-methanol insoluble, and failed to adhere to a series of carbohydrate-binding lectins or to diethylaminoethyl (DEAE) cellulose. Hydrophobicity was suggested by adherence to phenyl-Sepharose. The major E-S product of another G. lamblia isolate, Portland-1 (P-1), was antigenically different. A previous study showed that strain P-1 lacked a major antigenic component of strain WB. In the present study, this material was identified as the major secretory product of WB by crossed immunoelectrophoresis.     

Circannual incidence of Giardia lamblia

A circannual rhythm of Giardia lamblia positive stools was found by examination of records from three clinical laboratories in central Arkansas for the period 1980-1986. Cosinor analysis of monthly Giardia incidence based on stool specimen records from approximately 12,000 patients over the 7-year period revealed a circannual rhythm (P less than 0.001) on the basis of percent positive patients/month, with a computive acrophase occurring in late summer and minimum values in the winter. Patients involved in the study were primarily from the central Arkansas metropolitan areas, southern delta regions and northern mountainous regions of the state. Analysis of the data on the basis of total positive Giardia patients/month also revealed a circannual rhythm with the acrophase again occurring in late summer. The overall mean for percent positive stool specimens for the 7-year period was 5.3%, compared with the national average of 3.8% for G. lamblia positive stools. The data indicate that there may be a "Giardia season" in Arkansas since they could not be explained on the basis of day-care age distribution, or geographic origin. Awareness by epidemiologists, public health officials and other health care professionals of this circannual incidence of giardiasis is important for the prevention, diagnosis and treatment of this infectious disorder.     

Mechanisms of Giardia lamblia differentiation into cysts.

Microbiologists have long been intrigued by the ability of parasitic organisms to adapt to changes in the environment. Since most parasites occupy several niches during their journey between vectors and hosts, they have developed adaptive responses which allow them to survive under adverse conditions. Therefore, the life cycles of protozoan and helminthic parasites are excellent models with which to study numerous mechanisms involved in cell differentiation, such as the regulation of gene expression, signal transduction pathways, and organelle biogenesis. Unfortunately, many of these studies are very difficult because the conditions needed to elicit developmental changes in parasites remain undetermined in most cases. Recently, several interesting findings were reported on the process of differentiation of Giardia lamblia trophozoites into cysts. G. lamblia is a flagellated protozoan that inhabits the upper small intestine of its vertebrate host and is a major cause of enteric disease worldwide. It belongs to the earliest identified lineage among eukaryotes and therefore offers a unique insight into the progression from primitive to more complex eukaryotic cells. The discovery of a specific stimulus that induces trophozoites to differentiate into cysts, the identification and characterization of encystation-specific molecules, the elucidation of novel biochemical pathways, and the development of useful reagents and techniques have made this parasite an excellent model with which to study differentiation in eukaryotic cells. In this review, we summarize the most recent fundings on several aspects of Giardia differentiation and discuss the significance of these findings within the context of current knowledge in the field.