Production of viable Giardia cysts in vitro: Determination by fluorogenic dye staining, excystation, and animal infectivity in the mouse and Mongolian gerbil

Schupp, Daniel G.; Januschka, Mary M.; Sherlock, L A; Stibbs, Henry H.; Meyer, Ernest A.; Bemrick, William J.; Erlandsen, Stanley L.

doi:10.1016/0016-5085(88)90283-1

Cited by 70 publications

(34 citation statements)

References 30 publications

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“…After 4-5 days, trophozoites were harvested by centrifugation, washed three times in 0?25 M sucrose and stored at 280 uC. To obtain 24 or 48 h induced cells (2?3610 9 and 2?0610 9 cells, respectively), trophozoites were grown identically to 0 h cells and transferred into encystment medium (pH 7?6 with 10 % bovine bile) (Schupp et al, 1988) and harvested after an additional 24 or 48 h of encystment at 37 uC. Harvested cells were washed three times with 0?25 M sucrose, and stored at 280 uC until analysis, typically from 2 to 6 weeks.…”

Section: Methodsmentioning

confidence: 99%

Amino sugar phosphate levels in Giardia change during cyst wall formation

et al. 2004

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The parasite Giardia intestinalis exists as a trophozoite (vegetative) that infects the human small intestine, and a cyst (infective) that is shed in host faeces. Cyst viability in the environment depends upon a protective cyst wall, which consists of proteins and a unique b(1-3) GalNAc homopolymer. UDP-GalNAc, the precursor for this polysaccharide, is synthesized from glucose by an enzyme pathway that involves amino sugar phosphate intermediates. Using a novel method of microanalysis by capillary electrophoresis, the levels of amino sugar phosphate intermediates in trophozoites before encystment, during a period of active encystment and after the peak of encystment were measured. These levels were used to deduce metabolic control of amino sugar phosphates associated with encystment. Levels of amino sugar phosphate intermediates increased during encystment, and then decreased to nearly non-encysting levels. The most pronounced increase was in glucosamine 6-phosphate, which is the first substrate unique in this pathway, and which is the positive effector for the pathway's putative rate-controlling enzyme, UDP-GlcNAc pyrophosphorylase. Moreover, more UDP-GalNAc than UDP-GlcNAc, its direct precursor, was detected at 24 h. It is postulated that the enhanced UDP-GalNAc is a result of enhanced synthesis of UDP-GlcNAc by the pyrophosphorylase, and its preferential conversion to UDP-GalNAc. These results suggest that kinetics of amino sugar phosphate synthesis in encysting Giardia favours the direction that supports cyst wall synthesis. The enzymes involved in synthesis of UDP-GalNAc and its conversion to cyst wall might be potential targets for therapeutic inhibitors of Giardia infection.

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Section: Methodsmentioning

confidence: 99%

Amino sugar phosphate levels in Giardia change during cyst wall formation

et al. 2004

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“…As trophozoites replicate and colonize the intestinal surface, some encyst in the jejunum after exposure to biliary secretions. Encystation has been performed in vitro by exposing trophozoites to an environment that mimics that of the jejunum (114,115,301). Specific conditions that promoted encystation included a mildly alkalotic pH of 7.8 and conjugated bile salts plus fatty acids (114).…”

Section: Encystationmentioning

confidence: 99%

Biology ofGiardia lamblia

2001

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Giardia lamblia is a common cause of diarrhea in humans and other mammals throughout the world. It can be distinguished from other Giardia species by light or electron microscopy. The two major genotypes of G. lamblia that infect humans are so different genetically and biologically that they may warrant separate species or subspecies designations. Trophozoites have nuclei and a well-developed cytoskeleton but lack mitochondria, peroxisomes, and the components of oxidative phosphorylation. They have an endomembrane system with at least some characteristics of the Golgi complex and encoplasmic reticulum, which becomes more extensive in encysting organisms. The primitive nature of the organelles and metabolism, as well as small-subunit rRNA phylogeny, has led to the proposal that Giardia spp. are among the most primitive eukaryotes. G. lamblia probably has a ploidy of 4 and a genome size of approximately 10 to 12 Mb divided among five chromosomes. Most genes have short 5′ and 3′ untranslated regions and promoter regions that are near the initiation codon. Trophozoites exhibit antigenic variation of an extensive repertoire of cysteine-rich variant-specific surface proteins. Expression is allele specific, and changes in expression from one vsp gene to another have not been associated with sequence alterations or gene rearrangements. The Giardia genome project promises to greatly increase our understanding of this interesting and enigmatic organism

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“…Several conditions are known to trigger this encystation in vitro. For instance, an increase in the concentration and/or the source of bile or bile salts in the culture medium and a shift in pH from 7 to 7.8 have been implicated in the induction of encystation; however, encystation also has been achieved under conditions of bile starvation (4)(5)(6). Once encystation is initiated, cyst wall components are rapidly synthesized and sorted to characteristic secretory granules, the encystationspecific secretory vesicles (ESVs) that transport the cyst wall components to the plasma membrane for release to the cell exterior (7)(8)(9)(10)(11).…”

mentioning

confidence: 99%

“…At 24 h, the number of water-resistant cysts from the supernatant media was determined. Water-resistant cysts free of trophozoites were obtained as described (16), and their viability was determined by fluorescein diacetate/propidium iodine staining (6,29).…”

mentioning

confidence: 99%

Cholesterol starvation induces differentiation of the intestinal parasite Giardia lamblia.

Luján

Mowatt

Byrd

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

127

109

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Giardia lamblia, like most human intestinal parasitic protozoa, sustains fundamental morphological and biochemical changes to survive outside the small intestine of its mammalian host by differentiating into an infective cyst. However, the stimulus that triggers this differentiation remains totally undefined. In this work, we demonstrate the induction of cyst formation in vitro when trophozoites are starved for cholesterol. Expression of cyst wall proteins was detected within encystation-specific secretory vesicles 90 min after the cells were placed in lipoprotein-deficient TYI-S-33 medium. Four cloned lines derived from two independent Giardia isolates were tested, and all formed cysts similarly.

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Production of viable Giardia cysts in vitro: Determination by fluorogenic dye staining, excystation, and animal infectivity in the mouse and Mongolian gerbil

Cited by 70 publications

References 30 publications

Amino sugar phosphate levels in Giardia change during cyst wall formation

Amino sugar phosphate levels in Giardia change during cyst wall formation

Biology ofGiardia lamblia

Cholesterol starvation induces differentiation of the intestinal parasite Giardia lamblia.

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