FORMATION OF THE GIARDIA CYST WALL - STUDIES ON EXTRACELLULAR ASSEMBLY USING IMMUNOGOLD LABELING AND HIGH-RESOLUTION FIELD-EMISSION SEM

Encystment of the intestinal protozoan, Giardia, is a key step in the life cycle that enables this parasite to be transmitted From host to h ost via either fecal oral, waterborne, or foodborne transmission. The process of encystment was studied by localizing cyst wall specific ant igens with immunofluorescence for light microscopy and immunogold stai ning for field emission scanning electron microscopy. Chronological sa mpling of Giardia cultures stimulated with endogenous bile permitted i dentification of an intracellular and extracellular phase in cyst wall formation, a process which required a total of 14-16 h. The intracell ular phase lasted for 8-10 h, while the extracellular phase, involved the appearance of cyst wall antigen on the trophozoite membrane, and t he assembly of the filamentous layer, a process requiring an additiona l 4-6 h for completion of mature cysts. The extracellular phase was in itiated with the appearance of cyst wall antigen on small protrusions of the trophozoite membrane (similar to 15 nm), which became enlarged with time to caplike structures ranging up to 100 nm in diameter. Capl ike structures involved with filament growth were detected over the en tire surface of the trophozoite including the adhesive disc and flagel la. Encysting cells rounded up, lost attachment to the substratum, and became enclosed in a layer of filaments. Late stages in encystment in cluded a ''tailed'' cyst in which flagella were not fully retracted in to the cyst. Clusters of cysts were seen in which filaments at the sur face 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 morphologicall y different stages (intracellular and extracellular) which requires 16 h for completion. Further investigation of the extracellular stage wi th regard to assembly of the filamentous layer of the cyst wall may le ad to innovative methods for interfering with production of an intact functional cyst wall, and thereby, regulation of viable Giardia cyst r elease from the host.