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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 (approximately 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.

The oxygen uptake rate and metronidazole (MTZ) sensitivity in Giardia spp. cysts is greatly reduced from that in trophozoites. Thus, this project was undertaken to assess when in the encystation process these phenomena occur. Oxygen uptake rates approximately doubled (from approximately 4.9 to 8.3 microM O2 min(-1) 10(-6) cells) during the first 5 hr into encystation. This increase was followed by a marked decrease to 2.3 microM O2 min(-1) 10(-6) by 12 hr. By 50 hr into encystation, oxygen uptake was 0.7 microM O2 min(-1) 10(-6) cells. Glucose stimulated oxygen uptake by 89% in trophozoites but did not demonstrably stimulate oxygen uptake in cells after 12 hr into encystment. Deoxy-D-glucose uptake dropped by more than an order of magnitude in encysting cells compared to nonencysting cells. In contrast, aspartate uptake remained relatively constant regardless of whether cells were encysting or not. This suggests that there is a change in the parasite's ability to transport glucose during cyst formation; a similar change in the parasite's ability to transport aspartate was not observed after 40 hr into encystation. MTZ inhibited oxygen uptake by 77% in trophozoites, but there was no detectable inhibition of oxygen uptake 8 hr after trophozoites were transferred to encystation medium. We propose that this resistance to MTZ may be due to a change in metabolic flux away from the pyruvate ferredoxin oxidoreductase pathway. Oxygen uptake by noninduced cysts increased exponentially during the 30 min following the induction of excystation. Likewise, MTZ sensitivity returned within 15 min after the induction of excystation, and by 30 min into excystation full sensitivity had returned.

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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 gpi1 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" (beta 1 --> 3 GalNAc transferase) into a highly insoluble beta 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.

Using various sizes and dilutions of hapten-conjugated DNA probes, we compared catalyzed reporter deposition (CARD) to fluorochrome-conjugated antibody layering (immunological method) for amplifying FISH signals. Cosmid and phage probes that contained human DNA inserts of 40 KB and 15 KB, respectively, and were mapped to chromosome 15q11.2 were used to evaluate these amplification methods. The probes were used either at standard concentrations (10 ng/microliter) or at dilutions up to 1:40 (0.25 ng/microliter). Detection of FISH signals using either immunological (three antibody layers) or CARD methods were comparable when the undiluted (10 ng/microliter) or 1:4 dilution (2.5 ng/microliter) of the cosmid probe was used. Use of a single fluorochrome-conjugated antibody layer produced very weak FISH signals. However, addition of an unlabeled secondary antibody followed by a third antibody conjugated to the same fluorochrome (i.e., two rounds of amplification) produced a strong signal that was detected at a 1:4 probe dilution but was not successfully detected at probe dilutions of 1:10 or greater. In contrast, intense probe signals were produced with the CARD method at all probe dilutions, particularly when coupled to extended hapten-antibody incubation times. The 15-KB phage probe was difficult to detect at a 1:4 dilution with the standard immunologic amplification methods but was readily detected with the CARD method. These data suggest that CARD may be useful for FISH in that (a) less probe may be needed and therefore valuable probe reagents may be conserved, and (b) smaller targets may be detected, thus extending the range of this technique.

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