Ernest A. Meyer

Giardia lamblia: isolation and axenic cultivation.

Abstract Giardia lamblia trophozoites have been axenically cultured for more than a year. Initially, organisms were established in a complex liquid medium in the presence of the hosts intestinal fungi; subcultures were made of these protozoa-fungus mixtures. G. lamblia trophozoites, free of yeast, were obtained by inoculating a protozoafungus culture in one arm of a U-tube, then later removing, from the other arm of the tube, Giardia trophozoites that had migrated across the base. Medium was changed at 2- or 3-day intervals; numerous subcultures were made. Tests for the possible presence of other organisms in these axenic cultures were negative. Trophozoite cultures remained viable, after freezing in the presence of glycerol, for 14 months. This is the first reported axenic culture of this common human intestinal parasite and pathogen; its study in pure culture is now possible.

Lipid and carbohydrate metabolism of Giardia lamblia.

The lipid and carbohydrate metabolism of Giardia lamblia was studied using trophozoites isolated from a human and axenically grown in vitro in medium containing fetal bovine serum. The phospholipid, fatty acid and neutral lipid composition of the G. lamblia trophozoites was similar to that of the medium. Phosphatidylethanolamine, phosphatidylcholine and sphingomyelin were the major phospholipids detected; monoacyl-, diacyl-, triacylglycerides, sterols, and sterol esters were the major neutral lipids found. Several unidentified glycolipids were also detected. Glucose and threonine were readily incorporated by the trophozoites, but not into cellular phospholipids or sterols. However, approximately 86% of the glucose incorporated into the trophozoites was found in the nucleic acids, and 38% of the threonine incorporated was detected in the cellular proteins. Small amounts of the glucose and threonine were incorporated into glycolipid-containing fractions. Glycerol and acetate were not appreciably incorporated into trophozoites while glycerol 3-phosphate incorporation was not detected. Cholesterol was readily assimilated by the trophozoites; 98% of the incorporated was found in the sterol fraction. Radiorespirometric data suggest that the major routes of glucose metabolism in G. lamblia are via Embden- Meyerhof-Parnas and pentose phosphate pathways. However, endogenous acetate (as acetyl-CoA) formed during the metabolism of glucose is not used for lipid biosynthesis. These findings suggest that G. lamblia trophozoites are incapable of synthesizing cellular phospholipids or sterols de novo, but rather, utilize lipids already present in the medium.

Giardia sp.: Physical factors of excystation in vitro, and excystation vs eosin exclusion as determinants of viability

The effects of several factors on Giardia sp. excystation in vitro were investigated. Temperature, pH, time, and incubation medium were shown to affect the levels of excystation achieved. In general, those conditions most closely approximating the organisms in vivo environment induced the highest levels of excystation. The viability of Giardia sp. cyst suspensions was compared by eosin exclusion and excystation. Eosin exclusion consistently indicated higher cyst viability than could be demonstrated by in vitro excystation. Using excystation as the criterion of viability, the effect of storage at −13, 8, 21, and 37 C and of exposure to boiling water on Giardia sp. cyst survival was studied. Storage at 8 C permitted longest cyst survival, 77 days, at which time the cyst suspension was exhausted. Cysts stored at 21 C retained their viability for 5 to 24 days, while those at 37 C never survived longer than 4 days. Freezing and thawing cysts resulted in an almost complete loss of viability although a low level of viability (< 1%) persisted for at least 14 days. Cysts exposed to boiling water were immediately incapable of excystation.

Giardia and giardiasis.

Publisher Summary Organisms in the genus Giardia are widely distributed, flagellated protozoan parasites of all classes of vertebrates. Giardia is an intestinal protozoan parasite of humans.. Under appropriate conditions, they can cause disease. This chapter summarizes the progress in the study of Giardia and its relation with its host. Studies in this regard have resulted in a number of developments, most occurring within the past decade. These include (1) information regarding the nature of Giardia infection and disease, the host–parasite interaction, and the development of a mouse model of disease; (2) their axenic cultivation and the determination of conditions for excystment; (3) information regarding their spread via water, via sexual contact, and from one animal species to another; (4) data regarding the role of the immune system in affecting host susceptibility to these organisms and the possibility of severe disease in immunodeficient individuals; (5) recognition of the frequent difficulty in detecting this parasite, the introduction of a new method of obtaining trophozoites from the patient, and efforts to diagnose the infection immunologically; and (6) the demonstration that some Giardia infections are not eradicated with presently recommended drugs.

A comparison of isozymes of five axenic Giardia isolates.

The relative mobilities of six enzymes from the trophozoites of five axenically-cultured isolates of Giardia from human, cat, and guinea pig hosts were compared by starch and polyacrylamide gel electrophoresis. The six enzymes compared were malate dehydrogenase (NAD+) (MDH) (EC 1.1.1.37), malate dehydrogenase (decarboxylating) (ME) (EC 1.1.1.40), hexokinase (EC 2.7.1.1), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), glucose-6-phosphate dehydrogenase (G6P) (EC 1.1.1.49), and alpha-glycerophosphate dehydrogenase (EC 1.1.1.8). The latter three enzymes have not been previously reported in Giardia. On the basis of zymogram patterns, the five Giardia isolates were divided into three zymodemes. Zymodeme I comprised human-1/England, human-1/Bethesda, and cat-1/Portland, Zymodeme II the guinea pig-1/Portland isolate, and Zymodeme III the human-1/Portland isolate. These zymodemes were further substantiated when several physical and kinetic properties of three of the enzymes, MDH, ME, and G6P, were examined. Our results, in which Giardia isolated from different mammalian hosts share multiple isoenzymes, question the validity of the practice of assigning Giardia species names on the basis of the animal host from which the protozoan was obtained.

Isolation and axenic cultivation of Giardia trophozoites from the rabbit, chinchilla, and cat☆

Abstract Monoxenic cultures of Giardia trophozoites from the rabbit, chinchilla and cat have been established with Saccharomyces cerevisiae, in medium containing yeast extract. Giardia then were separated from S. cerevisiae by inoculating the mixed culture in one arm of a U-tube; the protozoa migrated across the base of the tube and were removed from the other arm. Newly isolated trophozoites failed to grow axenically; axenic cultures were established, however, after the Giardia had been cultured for a month across a dialysis membrane from viable yeast. These cultures have now been maintained for more than a year; numerous subcultures have been made.

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

The purpose of this research was to document the formation of viable Giardia cysts in vitro. Viability staining, using fluorogenic dyes that required metabolic conversion for detection, and immunocytochemistry at the light microscopic level provided information on viability and for the identification of formed in vitro. Analysis of cysts formed in vivo and in vitro showed similar morphologic appearances by both light and electron microscopy. Cysts formed in vitro were capable of establishing infections in both mouse and gerbil models for giardiasis. Trophozoites obtained from mice experimentally infected with in vitro-formed cysts could be maintained in culture and induced a second time to form cysts in vitro. This model for the production of viable Giardia cysts in vitro should facilitate research on controlling the complete life cycle of Giardia outside an animal host.

The effect of UV light on the inactivation of Giardia lamblia and Giardia muris cysts as determined by animal infectivity assay (P-2951-01).

This study measured the effect of germicidal ultraviolet (UV) light on Giardia lamblia and Giardia muris cysts, as determined by their infectivity in Mongolian gerbils and CD-1 mice, respectively. Reduction of cyst infectivity due to UV exposure was quantified by applying most probable number techniques. Controlled bench-scale, collimated-beam tests exposed cysts suspended in filtered natural water to light from a low-pressure UV lamp. Both G. lamblia and G. muris cysts showed similar sensitivity to UV light. At 3 mJ/cm2, a dose 10-fold lower than what large-scale UV reactors may be designed to provide, > 2-log10 (99 percent) inactivation was observed. These results, combined with previously published data showing other protozoa and bacteria have similar, high sensitivity to UV light, establish that UV disinfection of drinking water is controlled by viruses which may require over 10-fold more UV dose for the same level of control.

Giardia lamblia: autoradiographic analysis of nuclear replication.

Giardia lamblia trophozoites, grown in axenic culture, were labeled for various periods of time with [3H]thymidine. After autoradiography, grains were counted over each of the two nuclei in each trophozoite. Analysis of the fraction of trophozoites labeled for each time period resulted in an estimate of a generation time of 15 hr. The DNA synthetic or S phase for a trophozoite in culture was calculated to be 1.8 hr. G1 and G2 periods were determined to be 8.5 and 3 hr, respectively. A comparison of the labeling density between the two nuclei indicated that replication takes place simultaneously in both nuclei for at least 70% of S period. The fraction of asymmetrically labeled trophozoites is consistent with a model in which the nuclei replicate out of phase by 15-30 min, but, due to the small diameter of the nuclei relative to the grain size, the possibility that replication takes place simultaneously in both nuclei of a trophozoite throughout the S phase cannot be ruled out.

Occurrence of Specific Secretory immunoglobulin A in Bile After Inoculation of Giardia lamblia Trophozoites Into Rat Duodenum

We studied the appearance of specific secretory immunoglobulin A (IgA) antibody in bile after inoculation of live Giardia lamblia trophozoites into rat intestine. Serial bile specimens collected before and after inoculation of trophozoites were assayed for IgA antibodies by indirect immunofluorescence. Secretory IgA antibodies to Giardia lamblia were first detected in bile at 3 days after inoculation, remained elevated through 12 days, and then returned to control levels. Positive immunofluorescence of trophozoites for IgA was observed at bile titers of 1:80 to 1:160, whereas control biles were usually negative at dilutions of 1:10 or less. Scanning electron microscopic examination of Giardia in conjunction with immunocytochemistry revealed IgA antibodies bound to the flagella and surfaces of the trophozoites including the adhesive disk. These data demonstrate the occurrence of a secretory IgA immune response directed against surface antigens of Giardia lamblia trophozoites in the rat.