Protocols for in vitro excystation of oocysts of Cryptosporidium parvum, including different chemical pre-incubation steps, were compared to examine some of the biochemical triggers involved in excystation and to define an in vitro excystation protocol of a reproducibly high efficiency. Pre-incubation steps which increased the permeability of the oocysts were found to enhance excystation dynamics and pre-treatment of oocysts with saliva was found to decrease the permeability and reduce excystation. Although excystation was maximal after incubation for 4 h, sporozoites tended to lyse over this period, and maximum sporozoite recovery occurred after 30 min. The results obtained are discussed in relation to excystation protocols adopted by different research groups and a number of recommendations are given for in vitro excystation of C. parvum oocysts.
"However, there are publications reporting different outcomes of pretreatment of oocysts varying from a rather lethal impact on oocyst viability to amplifying effects on the excystation process. There are also reports on differences between the strains and isolates used (Robertson et al. 1993). "
[Show abstract][Hide abstract] ABSTRACT: SUMMARY Cryptosporidium host cell interaction remains fairly obscure compared with other apicomplexans such as Plasmodium or Toxoplasma. The reason for this is probably the inability of this parasite to complete its life cycle in vitro and the lack of a system to genetically modify Cryptosporidium. However, there is a substantial set of data about the molecules involved in attachment and invasion and about the host cell pathways involved in actin arrangement that are altered by the parasite. Here we summarize the recent advances in research on host cell infection regarding the excystation process, attachment and invasion, survival in the cell, egress and the available data on omics.
"Giardia excystment is particularly responsive to host-supplied low pH conditions and proteases, whereas Cryptosporidium responds primarily to low pH and elevated temperature (37 °C) (Fayer et al., 1998). For each of these parasites, in vitro conditions have been described that support the efficient excystation of culture-and in vivo-derived cysts or oocysts (Bingham and Meyer, 1979; Robertson et al., 1993), and in the case of Giardia the inclusion of other components found in the upper GI tract enhances the rates of in vitro excystation (Rice and Schaeffer, 1981; Boucher and Gillin, 1990; Feely et al., 1991). Trophozoites of certain strains of Entamoeba invadens, whose natural hosts are reptiles, will efficiently (>95%) form cysts (encyst) in vitro (McConnachie, 1955; Rengpien and Bailey, 1975; Sanchez et al., 1994). "
[Show abstract][Hide abstract] ABSTRACT: 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.
International journal for parasitology 12/2009; 40(6):751-60. DOI:10.1016/j.ijpara.2009.11.012 · 3.87 Impact Factor
"Assessment of oocyst viability using the maximized in vitro excystation assay The viability of stock oocysts was also determined using the maximized in vitro excystation assay (Robertson et al. 1993). Stock solutions of bile [1% bovine bile in Hanks' minimum essential medium (HMEM)] and sodium hydrogen carbonate (0AE4% NaHCO 3 in distilled water) were prepared. "
[Show abstract][Hide abstract] ABSTRACT: To evaluate individual and combined effects of temperature (4, 18 and 25 degrees C), pH (7 and 10), ammonia (5 and 50 mg l(-1)) and exposure time (1, 2, 4 and 6 days) on the viability of Cryptosporidium parvum oocysts in water.
The viability of oocysts was evaluated using the fluorogenic vital dyes assay (4',6-diamidino-2-phenylindole and propidium iodide). All the factors analysed (temperature, pH, ammonia and exposure time) and their interaction were statistically significant (P < 0.005). Exposure of oocysts to pH 10 for 6 days at 25 degrees C reduced oocyst viability from approximately 80% to 51%. Similarly, the exposure of C. parvum oocysts to 5 mg NH(3) l(-1) and 50 mg NH(3) l(-1) for 4 days reduced their viability from between approximately 80% to 41.5% and 14.8%, respectively.
The interaction between pH, temperature and exposure time may have adverse effects on the survival of C. parvum oocysts in water. Low concentrations of ammonia, as commonly found in alga-based wastewater systems, over a long period of time can produce high C. parvum oocyst inactivation rates.
This study provides relevant data on the inactivation of C. parvum oocysts in alga-based wastewater-treatment systems in the northwest of Spain.
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