High-resolution immunogold localization of Giardia cyst wall antigens using field emission SEM with secondary and backscatter electron imaging.

Abstract

We describe here the ultrastructural localization of Giardia cyst antigens in the filaments associated with the outer portion of intact cysts and on developing cyst wall filaments in encysting trophozoites. Post-embedding immunogold labeling of thin sections of intact Giardia cysts with polyclonal and monoclonal antibodies specific for cyst wall antigens (major protein bands of approximately 29, 75, 88, and 102 KD on Western blots) showed strong labeling of the filamentous cyst wall, whereas no labeling was seen on the membranous portion. High-resolution field emission scanning electron microscopy (FESEM) of Giardia cysts revealed that the cyst wall-specific polyclonal rabbit antisera and monoclonal mouse antibody produced gold labeling of 20-nm filaments in the cyst wall as detected with secondary electron imaging (SEI) and backscatter electron imaging (BEI) at 10 kV, despite coating of the cells with platinum by ion sputtering. FESEM studies of encysting Giardia trophozoites demonstrated that immunostaining with antibodies to cyst wall antigens produced colloidal gold labeling of developing cyst wall filaments on the cell surface; however, the intervening membrane domains were unlabeled. Substitution of normal serum for cyst wall-specific antibodies, or preabsorption of specific antibodies with Giardia cysts, eliminated immunolabeling of the filaments.

Oral Immunization of BALB/c Mice with Giardia duodenalis Recombinant Cyst Wall Protein Inhibits Shedding of Cysts

“…These structures are usually called encystation-specific vesicles (ESVs) (20). They are seen over the entire trophozoite surface, including the adherence disk, and flagella (4,5). The localization of cyst proteins within ESVs early and late in encystation confirms that these structures may play a role in transport of these proteins to the nascent cyst wall.…”

Biology ofGiardia lamblia

“…They are approximately 5 by 7 to 10 m in diameter and are covered by a wall that is 0.3 to 0.5 m thick and composed of an outer filamentous layer and an inner membranous layer with two membranes. The outer portion of the cyst wall is covered by a web of 7-to 20-nm filaments (79,80). Four major proteins have been identified in the outer cyst wall, 29, 75, 88, and 102 kDa in size (80).…”

“…The outer portion of the cyst wall is covered by a web of 7-to 20-nm filaments (79,80). Four major proteins have been identified in the outer cyst wall, 29, 75, 88, and 102 kDa in size (80). The sugar component of the outer portion is predominantly galactosamine in the form of Nacetylgalactosamine (GalNAc) (148).…”

Immune Response to Giardia duodenalis

“…The phenomenon begins with the formation of an intracellular and extracellular phase, which requires a minimum of 14 h. The extracellular phase is initiated with the appearance of cyst wall antigens on small protusions of the trophozoite membrane, which enlarge to form "caplike structures" with progression to formation of the cyst wall. Caplike structures are detected over the entire surface of the trophozoites, including the adherence disk and flagella (57,59). Late stages in encystment include a "tailed" cyst, in which some of the flagella are not fully retracted into the cyst.…”