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Notes: . The nucleotide sequence of the 16S rRNA gene, part of the 23S rRNA gene and the spacer DNA region was determined for Giardia duodenalis, obtained from humans in The Netherlands (AMC-4) and Washington State (CM). These rDNA sequences differ from other G. duodenalis isolates (Portland-1 and BRIS/83/HEPU/106) both of which have virtually identical rDNA sequences. The most characteristic feature was found close to the 5’end of the 16S rRNA. The Portland-1 - Bris/83/HEPU/106 type has GCG in position 22–24, while AMC-4 and CM have AUC in this position. These two sequences, present in an otherwise conserved region of the 16S rRNA, are “signature” sequences, which divide Giardia isolates into two different groups.

Notes: Encystment of the intestinal protozoan, Giardia, is a key step in the life cycle that enables this parasite to be transmitted from host to host via either fecal oral, waterborne, or foodborne transmission. The process of encystment was studied by localizing cyst wall specific antigens with immunofluorescence for light microscopy and immunogold staining for field emission scanning electron microscopy. Chronological sampling of Giardia cultures stimulated with endogenous bile permitted identification of an intracellular and extracellular phase in cyst wall formation, a process which required a total of 14-16 h. The intracellular phase lasted for 8-10 h, while the extracellular phase, involved the appearance of cyst wall antigen on the trophozoite membrane, and the assembly of the filamentous layer, a process requiring an additional 4-6 h for completion of mature cysts. The extracellular phase was initiated with the appearance of cyst wall antigen on small protrusions of the trophozoite membrane (-15 nm), which became enlarged with time to caplike structures ranging up to 100 nm in diameter. Caplike structures involved with filament growth were detected over the entire surface of the trophozoite including the adhesive disc and flagella. Encysting cells rounded up, lost attachment to the substratum, and became enclosed in a layer of filaments. Late stages in encystment included a “tailed” cyst in which flagella were not fully retracted into the cyst. Clusters of cysts were seen in which filaments at the surface of one cyst were connected with the surface of adjacent cysts or the “tailed” processes of adjacent cysts, suggesting that the growth of cyst wall filaments may be at the terminal end. In conclusion, the process of encystment has been shown to consist of two morphologically different stages (intracellular and extracellular) which requires 16 h for completion. Further investigation of the extracellular stage with regard to assembly of the filamentous layer of the cyst wall may lead to innovative methods for interfering with production of an intact functional cyst wall, and thereby, regulation of viable Giardia cyst release from the host.

Notes: . In vitro excystation of Spironucleus muris cysts, purified by sequential sucrose and Percoll gradients from mouse feces, was studied. Three in vitro excystation procedures, used for Giardia, were assessed to determine the most useful method. Excystation was monitored by light microscopy and subsequently characterized by transmission and scanning electron microscopy. Spironucleus muris excysted routinely at a level greater than 90% when induced in Hanks’ balanced salt solution containing sodium bicarbonate at pH 2.0 and transferred to Tyrodes’ salt solution as an excystation medium. Similarly, high rates of excystation were recorded after induction of S. muris cysts in 0.1 M potassium phosphate buffer (pH 7.0) with sodium bicarbonate and excystation in trypticase-yeast extract-iron medium (TYI medium) or phosphate-buffered saline. A lower rate and percentage of excystation were observed after induction of S. muris cysts in an aqueous hydrochloric acid solution (pH 2.0) followed by excystation in TYI medium. All excystation methods produced extremely active S. muris trophozoites with normal morphology. Nonexcysting S. muris cysts have a wall composed of an outer fibrous and an inner membranous portion. Following induction, numerous vesicles appeared in the peritrophic space. Excystation began by the cyst wall opening at one pole, and the anterior part of the trophozoite protruding from the cyst wall. The trophozoite emerged progressively from the cyst wall and the empty cyst wall appeared to collapse. Excysted trophozoites exhibited normal morphological features of S. muris trophozoites isolated from the mouse intestine.

Notes: Giardia lamblia, an aerotolerant anaerobe, respires in the presence of oxygen by a flavin, iron-sulfur protein-mediated electron transport system. Glucose appears to be the only sugar catabolized by the Embden-Meyerhof-Pamas and hexose monophosphate pathways, and energy is produced by substrate level phosphorylation. Substrates are incompletely oxidized to CO2, ethanol and acetate by nonsedimentable enzymes.The lack of incorporation of inosine, hypoxanthine, xanthine, formate or glycine into nucleotides indicates an absence of de novo purine synthesis. Only adenine, adenosine, guanine and guanosine are salvaged, and no interconversion of these purines was detected. Salvage of these purines and their nucleosides is accomplished by adenine phosphoribosyltransferase, adenosine hydrolase, guanosine phosphonbosyltransferase and guanine hydrolase.The absence of de novo pyrimidine synthesis was confirmed by the lack of incorporation of bicarbonate, orotate and aspartate into nucleotides, and by the lack of detectable levels of the enzymes of de novo pyrimidine synthesis. Salvage appears to be accomplished by the action of uracil phosphoribosyltransferase, uridine hydrolase, uridine phosphotransferase, cytidine deaminase, cytidine hydrolase, cytosine phosphoribosyltransferase and thymidine phosphotransferase. Nucleotides of uracil may be converted to nucleotides of cytosine by cytidine triphosphate synthetase, but thymidylate synthetase and dihydrofolate reductase activities were not detected.Uptake of pyrmidine nucleosides, and perhaps pyrimidines, appears to be accomplished by carrier-mediated transport, and the common site for uptake of uridine and cytidine is distinct from the site for thymidine. Thymine does not appear to be incorporated into nucleotide pools. Giardia trophozoites appear to rely on preformed lipids rather than synthesizing them de novo. Major lipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, sphingomyelin, sterol (probably cholesterol) and mono-, di- and triacylglycer-ides. The lipid composition of the cysts of G. lamblia isolated from gerbils and G. muris isolated from mice are similar to those obtained from the trophozoites of G. lamblia grown in vitro.The activities of several hydrolases of G. lamblia have been shown to be confined to a single lysosome-like particle population with an equilibrium density of approximately 1.15 in sucrose. Contrary to the trophozoites of Entamoeba and the trichomonads, Giardia trophozoites appear to lack most carbohydrate splitting hydrolases.Calmodulin has been reported in G. lamblia trophozoites, and it appears to have properties similar to the calmodulin isolated from other eucaryotic cells.

Notes: . Intact Giardia muris cysts were subjected to consecutive chloroform/methanol and 2% sodium dodecyl sulfate (SDS) extractions, and to amyloglucosidase treatment. The SDS-insoluble, amyloglucosidase-fast cyst walls (ACW) were further incubated with chymotrypsin, trypsin, papain, or pronase. Low voltage scanning electron microscopy revealed no discernible change in the ultrastructure of the filamentous layer of the cyst wall following any of these treatments. Affinity for cyst wall-specific monoclonal antibody (Meridian Diagnostics, Cincinnati. OH) was also retained after all treatments. Periodic acid-Schiff staining and gas chromatography/mass spectrometry (GC/MS) of intact and treated cyst hydrolysates showed a significant reduction in the amount of glucose associated with the cyst (72 nmoles/106 intact cysts vs 1.9 nmoles/106 ACW) as a result of amyloglucosidase treatment, indicating that glucose is stored with in Giardia as an SDS-insoluble polymer, Galactosamine was identified by GC/MS as the predominant sugar associated with both the ACW and the proteinase treated ACW (42 nmoles/106 ACW). High performance liquid chromatographic analysis of amino acids from intact and treated cyst hydrolysates revealed a marked reduction, but not elimination, of detectable quantities of identifiable amino acid residues (255 nmoles/106 intact cysts vs 6.8 nmoles/106 proteinase treated ACW). These results suggest that the filamentous layer of the cyst wall is primarily a carbohydrate peptide complex.

Notes: . Oxygen uptake in cysts and trophozoites of the parasitic protozoan Giardia lamblia was examined. Both showed oxygen uptake activity, but that of cysts was only 10% to 20% that of trophozoites. Oxygen dependence of oxygen uptake in cysts and trophozoites showed oxygen maxima above which oxygen uptake decreased. the oxygen concentration at which the oxygen uptake rate was greatest was higher for trophozoites than for cysts. the effect of various inhibitors on cyst and trophozoithe oxygen uptake suggested that flavoproteins and quinones play some role in oxygen uptake. the substrate specificities and the effect of inhibitors on G. lamblia trophozoites were similar to those observed for G. muris. Metronidazole, the drug most commonly used in treatment of giardiasis, inhibited oxygen uptake and motility in trophozoites; however, it had no obvious effect on either oxygen uptake or excystation in cysts. Menadione, a redox cycling naphthaquinone, first stimulated, then completely inhibited, oxygen uptake in cysts and trophozoites; a complete loss of cyst viability and trophozoite motility was also observed. the effect of menadione on G. Iamblia may indicate that redox cycling compounds have potential as chemotherapeutic agents for the treatment of giardiasis.

Notes: The biosynthesis of the carbohydrate component of the cyst wall of the protozoan parasite Giardia lamblia, a polymer of N-acetylgalactosamine (GalNac). is by a pathway that is initiated with the conversion of fructose 6-phosphate to glucosamine 6-phosphate by an aminating isomerase. glucose 6-phosphate isomerase. This enzyme appears only after Giardia trophozoites an: induced to start the production of cyst wall components after bile is added. To investigate whether induction of glucosamine 6-phosphate isomerase is by protein modification or by transcription activation, its gene was cloned and sequenced. Two genes, gpi1 and gpi2, encoding putative glucosamine 6-phosphate isomerases were identified but only one, gpi1 was expressed. The transcript for gpi1 appeared not earlier than 6 h after cells were induced with bile salts. These results show that the first enzyme in the pathway leading to GalNac synthesis in encysting Giardia cyst wall biosynthesis is under transcriptional control.

Notes: . Giardia intestinalis trophozoites encyst when they are exposed to bile. During encystment, events related to the inducible synthesis of a novel N-acetyl-d-galactosamine (GalNAc) homopolymer, occur. Within the first 6 h of encystment, mRNA for glucosamine 6-P isomerase (GPI), the first inducible enzyme unique to this pathway appears, oxygen uptake rates double from non-encysting levels, and metronidazole (MTZ) inhibits oxygen uptake. Within 12 h, GPI and its activity are detectable and OU decreases 50% from non-encysting levels; glucose's stimulation and MTZ's inhibition of oxygen uptake cease. In contrast, aspartate uptake remained constant throughout the 40 h monitored. Two genes, gpi 1 and 2 encode for GPI, but only gpi l is expressed during encystment. Glucosamine 6-P (GlcN6P), the synthetic product of GPI, activates UDP-N-acetylglucosamine (UDP-GlcNAc) pyrophosphorylase, a downstream enzyme, 3 to 5-fold in the direction of UDP-GlcNAc synthesis. UDP-GlcNAc is epimerized to UDP-GalNAc and UDP-GalNAc is polymerized by “cyst wall synthase” (β 1 → 3 GalNAc transferase) into a highly insoluble β 1,3-linked homopolymer. This GalNAc polysaccharide, the major component of cyst wall filaments, forms, in conjunction with polypeptides, the outer cyst wall of Giardia.