Giardia muris: scanning electron microscopy of in vitro excystation

A recently developed in vitro excystation procedure results in almost total excystation of Giardia muris, an intestinal parasite of mice. The present experiment examines the G. muris cyst morphology by scanning electron microscopy and the efficacy of the excystation procedure. Untreated cysts of G. muris were elliptical and displayed a distinctive surface structure. Excystation began almost immediately after incubation had begun and most trophozoites emerged within 30 min. Excystation appears to involve flagellar action of the encysted trophozoite. A tear of the wall occurred at one pole. This opening was subsequently enlarged, presumably by flagellar action. Trophozoites emerged, posterior end first, and an associated mucoid-like material was extruded. Newly emerged trophozoites were nearly oval in shape. Trophozoites quickly became flattened, elongate, and underwent cytokinesis resulting in two daughter trophozoites. Few organisms not excysted were seen after 30 min incubation.     

Cryptosporidium: New developments in cell culture

Studies on Cryptosporidium species have been hampered by the limited amount of parasitic stages available for research. One of the major objectives of many laboratories is to develop a reproducible culture model for this important parasite. Recent research has resulted in long-term culturing of Cryptosporidium in cell culture using pH modification, sub-culturing and gamma irradiation. Further advances in the in vitro culturing of Cryptosporidium revealed that this parasite can complete its life cycle in culture medium overcoming the problem of using the host cells, as host cell overgrowth and aging resulted in the termination of the Cryptosporidium life cycle prior to its completion. Improved methods for visualizing life cycle stages in cell-free culture have also been developed. This review will discuss factors that can influence the success of Cryptosporidium culture in vitro and propose new ideas for the future optimization of the cell-free culture system.     

Comparison of assays for Cryptosporidium parvum oocysts viability after chemical disinfection

Abstract In vitro excystation, vital dyes (4', 6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI)), and infeictivity in neonatal CD-1 mice were used to assess the viability of Cryptosporidium parvum oocysts after chemical disinfection. In vitro excystation and DAPI/PI staining provided similar estimates of viability in bench-scale experiments, but both of these methods significantly overestimated the viability when compared with infectivity (Pr ≤ 0.01). Infectivity was the most reliable measure of the viability of C. parvum oocysts following chemical disinfection.     

Entamoeba invadens: Identification of ADF/cofilin and their expression analysis in relation to encystation and excystation

The differentiation processes of excystation and encystation of Entamoeba are essential for infection and completion of their life-cycle, and the processes need cell motility and its control by actin cytoskeletal reorganization. This study investigated actin depolymerizing factor (ADF)/cofilin (Cfl) family proteins, which are important molecules in actin cytoskeletal reorganization, in Entamoeba invadens in relation to the encystation and excystation. Axenic culture systems were used to induce encystation and excystation. A homology search of the E. invadens genome database and molecular cloning identified three ADF/Cfl family proteins of the parasite (named for short as EiCfl-1, EiCfl-2, and EiCfl-3). This is different from other Entamoeba species, i.e. Entamoeba histolytica and Entamoeba dispar, each of which has only one ADF/Cfl family protein. These ADF/Cfl of E. invadens do not have Ser3 (serine locates third from first methionine), similar to E. histolytica, E. dispar, Saccharomyces cerevisiae and Schizosaccharomyces pombe, although the activity of ADF/Cfl is negatively regulated by phosphorylation of the Ser3 in metazoans. Phylogenetic analysis revealed that Entamoeba Cfl formed a distinctive clade that is separate from other organisms, and the branches of the tree were separated in two consistent with the presence and absence of Ser3. Rabbit anti-EiCfl-2 serum reacted with all recombinant EiCfls and EiCfl in lysates of cysts, trophozoites and metacystic amoebae. Immunofluorescence staining with this antiserum showed co-localization of EiCfl with actin beneath the cell membrane through the life stages. Both proteins proved to be rich in pseudopodia of trophozoites and metacystic amoebae. Real-time RT-PCR showed that mRNAs of EiCfl-2 and actins were highly expressed, but there were few mRNA of EiCfl-1 and EiCfl-3. Remarkably decreased mRNA levels were observed in EiCfl-2 and actins during encystation. All three EiCfls and actins became transcribed after the induction of excystation. The mRNAs of only EiCfl-1 and EiCfl-3 increased remarkably when the excystation was induced in the presence of cytochalasin D. These findings demonstrate that EiCfl-2 and actins co-localize beneath the cell membrane in trophozoites and cysts as well as metacystic amoebae being rich in pseudopodia, that EiCfl-1 and EiCfl-3 are expressed only after the induction of excystation, and that enhanced excystation by cytochalasin D is associated with high expression of EiCfl-1 and EiCfl-3.     

Giardia lamblia -- a model organism for eukaryotic cell differentiation

Giardia lamblia is a binucleated, flagellated protozoan parasite that inhabits the upper small intestine of its vertebrate hosts. The entire life cycle, which can be completed in vitro, is simple with cycling between a vegetative trophozoite and a highly resistant cystic form. The parasite is one of the earliest diverging eukaryotes known and more than 95% of the genome is sequenced. This makes Giardia an excellent model system for studies of basic eukaryotic processes like cell differentiation. In this review we will discuss recent data concerning Giardia differentiation with a focus on DNA replication and cytokinesis.     

Anaerobiosis-induced differentiation of Acanthamoeba castellanii

Acanthamoeba castellanii is a free living amoeba ubiquitous in soil and also commonly found in aquatic environments. In waterlogged soils, anoxia is quickly established as the dissolved oxygen is consumed by the organisms present. We were interested in the effects of anoxic conditions upon this organism. Batch cultures degassed with N₂ during mid-exponential growth, induced encystation within 12 h of anoxia, and mature cysts were formed within 2–3 days. Excystation (99%) was achieved by subsequent aeration of these cultures after 3–6 days. Anoxia-induced cysts, maintained in anoxic conditions for up to four months, remained viable. Difference spectra, during anaerobiosis, revealed that cytochromes were not lost, suggesting that the organism retains its respiratory components. The growth rate of trophozoites, grown in a chemostat, was dependent on the concentration of O₂ in the head space and glucose uptake increased at lower dissolved O₂ tensions. The results obtained suggest that A. castellanii has a complex adaptive strategy enabling it to cope with microaerobic and anoxic conditions which may be experienced in the environment.     

Compounds of the upper gastrointestinal tract induce rapid and efficient excystation of Entamoeba invadens

The infective stage of Entamoeba parasites is an encysted form. This stage can be readily generated in vitro, which has allowed identification of stimuli that trigger the differentiation of the parasite trophozoite stage into the cyst stage. Studies of the second differentiation event, emergence of the parasite from the cyst upon infection of a host, have been hampered by the lack of an efficient means to excyst the parasite and complete the life cycle in vitro. We have determined that a combination of exposures to water, bicarbonate and bile induces rapid excystment of Entamoeba invadens cysts. The high efficiency of this method has allowed the visualization of the dynamics of the process by electron and confocal microscopy, and should permit the analysis of stage-specific gene expression and high-throughput screening of inhibitory compounds.     

In vitro excystation of Echinostoma paraensei (Digenea: Echinostomatidae) metacercariae assessed by light microscopy,morphometry and confocal laser scanning microscopy

Trypsin and bile salts have been identified as important triggers for excystation of Echinostoma metacercariae. Although excystation in trematodes is a well-known phenomenon, some morphological developmental changes remain to be elucidated. In order to gain further insight into the in vitro development of metacercariae, we assayed different cultivating conditions: 0.5% trypsin and 0.5% bile salts; 1% trypsin and 1% bile salts; 1% trypsin and 0.5% bile salts; 0.5% bile salts; or 0.5% trypsin. By means of light microscopy and confocal microscopy, we characterized each encysted, activated, breached and excysted stage based on the morphological features. However, breached and excysted stages were not revealed in both bile salts and trypsin-free medium. Excretory concretions (25 ± 3.9) were visualized within excretory tubules, close to the ventral sucker and genital anlage. The oral sucker armed with spines and digestive system was similar to those of adult worms. The reproductive system is composed of a genital anlage and the cirrus sac primordium. In short, trypsin and bile salts associated were fundamental for the in vitro metacercariae excystation of Echinostoma paraensei. This article presents the first detailed information of all stages of metacercariae excystation obtained through light and confocal microscopy.     

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 organism's 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.