Effects of various physical and chemical factors on excystation of the encysted metacercariae of Echinostoma caproni

Various physical and chemical factors were studied to determine their effects on the viability of encysted metacercariae of Echinostoma caproni. Viability was equated with chemical excystation in an alkaline trypsin-bile salts (TB) medium. Control cysts showed excystation percentages of > 90% in TB. Excystation proved to be a more reliable criterion of cyst viability than observations by light microscopy. Isolated cysts and cysts left in the snail (in situ cysts) were studied. Generally, in situ cysts proved more resistant to various physical and chemical treatments than did isolated cysts. Cysts stored for 7 days at 28 C in a Locke's 1:1 solution showed 97% excystation, suggesting that cysts of this species would survive postal delays during shipment. Of numerous marinades tested, the one that was most harmful to isolated and in situ cysts was vinegar. Isolated and in situ cysts were killed by boiling (100 C) for 1 or 3 min, but freezing at -10 C did not kill all isolated or in situ cysts after 24 hr. Concentrations of potassium permanganate ranging from 300 to 1,200 mg/L killed most isolated cysts within 5 min, but in situ cysts survived these concentrations for 24 hr. Concentrated solutions of NaCl and sucrose had no effect on the viability of isolated and in situ cysts, suggesting that their use in food preparations for molluscs would not be effective in killing echinostomatid cysts in tainted snail tissues.     

Analysis of phosphorylated proteins and inhibition of kinase activity during Giardia intestinalis excystation

The parasite Giardia intestinalis undergoes a differentiation process that allows it to infect its mammal host. That process is excystation. We examined the importance of protein phosphorylation during the passage from cyst to trophozoite. Cysts obtained from patients with giardiasis were excysted in vitro and the soluble cytoplasmic proteins were analyzed during the three phases of the process, using a specific staining for phosphoproteins. We found two phosphorylated proteins and identified them with MALDI-TOF as 14-3-3 and Hsp70. Modifications were detected in both proteins, which could indicate a role in differentiation of the parasite. In addition, the inhibition of serine–threonine kinases during excystation specifically affected the cytokinesis of the excyzoite, thus inhibiting the completion of trophozoite formation.     

Effect of Chlorine on Giardia lamblia Cyst Viability

The effect of chlorine concentration on Giardia lamblia cyst viability was tested under a variety of conditions. The ability of Giardia cysts to undergo excystation was used as the criterion of viability. The experimental variables employed included temperature (25, 15, and 5°C), pH (6, 7, and 8), chlorine-cyst contact time (10, 30, and 60 min), and chlorine concentration (1 to 8 mg/liter). In the pH range studied, cyst survival generally was observed to increase as buffer pH increased. Water temperature coupled with chlorination proved to be important in cyst survival. Results of these experiments at the three temperatures studied can be summarized as follows: at 25°C, exposure to 1.5 mg/liter for 10 min killed all cysts at pH 6, 7, and 8. At 15°C, 2.5 mg of chlorine per liter for 10 min killed all cysts at pH 6, but at pH 7 and 8 small numbers of cysts remained viable after 30 min but not after 60 min. At 5°C, 1 mg of chlorine per liter for 60 min failed to kill all the cysts at any pH tested. At this temperature, 2 mg of chlorine per liter killed all cysts after 60 min at pH 6 and 7, but not at pH 8. A chlorine concentration of 4 mg/liter killed all the cysts at all three pH values after 60 min, but not after 30 min. A chlorine concentration of 8 mg/liter killed all Giardia cysts at pH 6 and 7 after contact for 10 min, and at pH 8 after 30 min. This study points up the role of temperature, pH, and chlorine demand in the halogen treatment of drinking water to destroy cysts. It also raises an epidemiological problem, namely: low water temperatures, where killing of Giardia requires relatively high chlorine concentrations and long contact times, are (i) to be expected in many areas where epidemic waterborne giardiasis has been reported and (ii) particularly conducive to the long-term survival of Giardia cysts.     

Giardia Cyst Wall Protein 1 Is a Lectin That Binds to Curled Fibrils of the GalNAc Homopolymer

The infectious and diagnostic stage of Giardia lamblia (also known as G. intestinalis or G. duodenalis) is the cyst. The Giardia cyst wall contains fibrils of a unique β-1,3-linked N-acetylgalactosamine (GalNAc) homopolymer and at least three cyst wall proteins (CWPs) composed of Leu-rich repeats (CWP_LRR) and a C-terminal conserved Cys-rich region (CWP_CRR). Our goals were to dissect the structure of the cyst wall and determine how it is disrupted during excystation. The intact Giardia cyst wall is thin (∼400 nm), easily fractured by sonication, and impermeable to small molecules. Curled fibrils of the GalNAc homopolymer are restricted to a narrow plane and are coated with linear arrays of oval-shaped protein complex. In contrast, cyst walls of Giardia treated with hot alkali to deproteinate fibrils of the GalNAc homopolymer are thick (∼1.2 µm), resistant to sonication, and permeable. The deproteinated GalNAc homopolymer, which forms a loose lattice of curled fibrils, is bound by native CWP1 and CWP2, as well as by maltose-binding protein (MBP)-fusions containing the full-length CWP1 or CWP1_LRR. In contrast, neither MBP alone nor MBP fused to CWP1_CRR bind to the GalNAc homopolymer. Recombinant CWP1 binds to the GalNAc homopolymer within secretory vesicles of Giardia encysting in vitro. Fibrils of the GalNAc homopolymer are exposed during excystation or by treatment of heat-killed cysts with chymotrypsin, while deproteinated fibrils of the GalNAc homopolymer are degraded by extracts of Giardia cysts but not trophozoites. These results show the Leu-rich repeat domain of CWP1 is a lectin that binds to curled fibrils of the GalNAc homopolymer. During excystation, host and Giardia proteases appear to degrade bound CWPs, exposing fibrils of the GalNAc homopolymer that are digested by a stage-specific glycohydrolase.     

Viability of the encysted metacercariae of Echinostoma caproni judged by light microscopy versus chemical excystation

The purpose of this study was to determine the viability of encysted metacercariae of Echinostoma caproni stored in Locke's solution 1:1 at 4 C for 1-24 wk. Viability was judged by light microscopy (LM) based on morphological characteristics of the encysted metacercariae versus chemical excystation of the cysts in a trypsin-bile salts excystation medium. The percent viability was very similar under both methods of assessment at 4, 8, and 16 wk poststorage. At 1 and 24 wk poststorage, viability was found to be about 2x greater based on excystation than using LM. We concluded that LM alone underestimated the viability of cysts and that determination of cyst viability was more accurate under assessment by chemical excystation.     

Releasing latent Toxoplasma gondii cysts from host cells to the extracellular environment induces excystation

Toxoplasma gondii, an obligate intracellular protozoan parasite, infects a wide variety of mammals and birds. Although T. gondii infects the brain and muscles in its latent cyst form containing bradyzoite stage parasites during chronic infection, when a chronically infected host becomes immunodeficient or is preyed upon by a predator, the latent cyst undergoes excystation. However, it is not yet known how T. gondii recognises the triggers of excystation in the microenvironment surrounding the cyst. In this study, we incubated T. gondii cysts from host cells in several solutions containing a variety of ionic compositions. Excystation occurred in a solution with an ionic composition which mimicked that of the extracellular environment. However, excystation did not occur in a solution that mimicked the intracellular environment. We also found that the specific Na⁺/K⁺ ratio and the presence of Ca²⁺, mimicking the extracellular environment, are required to trigger excystation. To examine whether the stage conversion of bradyzoite to tachyzoite occurs prior to egress, we constructed a gene-modified T. gondii strain expressing a green fluorescent protein specifically in the tachyzoite stage. During the process of cyst reactivation of this strain, green fluorescence was detected prior to excystation. This suggests that stage conversion from bradyzoite to tachyzoite occurs prior to cyst disruption. These results indicate that T. gondii bradyzoites monitor the ionic composition of their surroundings to recognise their expulsion from host cells, to effectively time their excystation and stage conversion.     

Optimized excystation protocol for ruminant Eimeria bovis- and Eimeria arloingi-sporulated oocysts and first 3D holotomographic microscopy analysis of differing sporozoite egress

Successful excystation of sporulated Eimeria spp. oocysts is an important step to acquire large numbers of viable sporozoites for molecular, biochemical, immunological and in vitro experiments for detailed studies on complex host cell-parasite interactions. An improved method for excystation of sporulated oocysts and collection of infective E. bovis- and E. arloingi-sporozoites is here described. Eimeria spp. oocysts were treated for at least 20 h with sterile 0.02 M _L-cysteine HCl/0.2 M NaHCO₃ solution at 37 °C in 100% CO₂ atmosphere. The last oocyst treatment was performed with a 0.4% trypsin 8% sterile bovine bile excystation solution, which disrupted oocyst walls with consequent activation of sporozoites within oocyst circumplasm, thereby releasing up to 90% of sporozoites in approximately 2 h of incubation (37 °C) with a 1:3 (oocysts:sporozoites) ratio. Free-released sporozoites were filtered in order to remove rests of oocysts, sporocysts and non-sporulated oocysts. Furthermore, live cell imaging 3D holotomographic microscopy (Nanolive®) analysis allowed visualization of differing sporozoite egress strategies. Sporozoites of both species were up to 99% viable, highly motile, capable of active host cell invasion and further development into trophozoite- as well as macroment-development in primary bovine umbilical vein endothelial cells (BUVEC). Sporozoites obtained by this new excystation protocol were cleaner at the time point of exposure of BUVEC monolayers and thus benefiting from the non-activation status of these highly immunocompetent cells through debris. Alongside, this protocol improved former described methods by being is less expensive, faster, accessible for all labs with minimum equipment, and without requirement of neither expensive buffer solutions nor sophisticated instruments such as ultracentrifuges.     

The viability and survival of sporozoites of Eimeria in vitro

Some factors affecting excystation and viability of sporozoites of several species of Eimeria from chickens were examined in vitro. Chicken embryos or cultured kidney cells were inoculated with sporozoites in order to assess viability.Sporozoites of E. tenella survived in phosphate buffer (P.B.S.) containing 0·9 per cent NaCl for 14 days. Some sporozoites survived in solutions containing up to 16 per cent NaCl for 3 days at +4°C. Sporozoites of E. maxima and E. acervulina survived for only 27 h in phosphate buffer containing 1 or 2 per cent NaCl.Sporozoites of E. brunetti, E. maxima, and E. acervulina var: mivati were released rapidly from sporocysts in vitro, but survived for relatively short periods in PBS at 4°C. However, the addition of serum or gelatine to these solutions increased survival to at least 96 h.The viability of sporozoites after freezing and storing in liquid nitrogen was best when 12 per cent dimethyl sulphoxide (DMSO) was added to the sporozoite suspensions. P.B.S. with DMSO was less suitable than the other solutions used and serum or gelatine with the DMSO, was needed to increase survival. Increasing the density of sporozoites in the frozen stabilates did not increase survival.     

Comparison of animal infectivity, excystation, and fluorogenic dye as measures of Giardia muris cyst inactivation by ozone.

Giardia muris cyst viability after ozonation was compared by using fluorescein diacetate-ethidium bromide staining, the C3H/HeN mouse-G. muris model, and in vitro excystation. Bench-scale batch experiments were conducted under laboratory conditions (pH 6.7, 22 degrees C) in ozone-demand-free phosphate buffer. There was a significant difference between fluorogenic staining and infectivity (P less than or equal to 0.05), with fluorogenic staining overestimating viability compared with infectivity estimates of viability. This suggests that viable cysts as indicated by fluorogenic dyes may not be able to complete the life cycle and produce an infection. No significant differences between infectivity and excystation and between fluorogenic staining and excystation (P less than or equal to 0.05) were detected for inactivations up to 99.9%. Only animal infectivity had the sensitivity to detect inactivations greater than 99.9%. Therefore, the animal model is the best method currently available for detecting high levels of G. muris cyst inactivation.     

Comparison of animal infectivity, excystation, and fluorogenic dye as measures of Giardia muris cyst inactivation by ozone.

Giardia muris cyst viability after ozonation was compared by using fluorescein diacetate-ethidium bromide staining, the C3H/HeN mouse-G. muris model, and in vitro excystation. Bench-scale batch experiments were conducted under laboratory conditions (pH 6.7, 22 degrees C) in ozone-demand-free phosphate buffer. There was a significant difference between fluorogenic staining and infectivity (P less than or equal to 0.05), with fluorogenic staining overestimating viability compared with infectivity estimates of viability. This suggests that viable cysts as indicated by fluorogenic dyes may not be able to complete the life cycle and produce an infection. No significant differences between infectivity and excystation and between fluorogenic staining and excystation (P less than or equal to 0.05) were detected for inactivations up to 99.9%. Only animal infectivity had the sensitivity to detect inactivations greater than 99.9%. Therefore, the animal model is the best method currently available for detecting high levels of G. muris cyst inactivation.     

Comparison of an in vitro method and an in vivo method of Giardia excystation.

An in vitro method and an in vivo method of excystation were compared to determine the most useful method for the retrieval of Giardia duodenalis isolates. Cysts from 11 Giardia strains were used. In vitro excystation produced motile trophozoites in 16 sets, while in vivo excystation produced trophozoites in all of the 21 comparative sets of excystations. Few cultures were lost because of contamination by either method (17% of in vitro-derived trophozoites versus 23% of in vivo-derived trophozoites; P greater than 0.05). Both methods demonstrated comparable isolate retrieval rates (15% of in vitro-derived trophozoites adapting to culture compared with 29% of in vivo-derived trophozoites; P greater than 0.05), although analysis of the strains retrieved showed that two isolates were retrieved from in vitro excystation alone, compared with four from in vivo excystation. Analysis that included results of extra in vivo cultures showed that a total of nine isolates were retrieved by using this type of excystation. Despite the disadvantages of cost and labor, in vivo excystation appears to be more useful than in vitro excystation for isolate retrieval at the present time.     

Comparison of an in vitro method and an in vivo method of Giardia excystation.

An in vitro method and an in vivo method of excystation were compared to determine the most useful method for the retrieval of Giardia duodenalis isolates. Cysts from 11 Giardia strains were used. In vitro excystation produced motile trophozoites in 16 sets, while in vivo excystation produced trophozoites in all of the 21 comparative sets of excystations. Few cultures were lost because of contamination by either method (17% of in vitro-derived trophozoites versus 23% of in vivo-derived trophozoites; P greater than 0.05). Both methods demonstrated comparable isolate retrieval rates (15% of in vitro-derived trophozoites adapting to culture compared with 29% of in vivo-derived trophozoites; P greater than 0.05), although analysis of the strains retrieved showed that two isolates were retrieved from in vitro excystation alone, compared with four from in vivo excystation. Analysis that included results of extra in vivo cultures showed that a total of nine isolates were retrieved by using this type of excystation. Despite the disadvantages of cost and labor, in vivo excystation appears to be more useful than in vitro excystation for isolate retrieval at the present time.     

Viability of Cryptosporidium parvum oocysts: correlation of in vitro excystation with inclusion or exclusion of fluorogenic vital dyes.

A viability assay for oocysts of Cryptosporidium parvum based on the inclusion or exclusion of two fluorogenic vital dyes, 4',6-diamidino-2-phenylindole (DAPI) and propidium iodide, was developed by using several different isolates of oocysts. Correlation of this assay with viability measured by in vitro excystation was highly statistically significant, with a calculated correlation coefficient of 0.997. In this research, two similar excystation protocols were utilized, and no significant difference between excystation protocols was detected. Percent excystation of oocyst suspensions could be increased or reduced by inclusion of a preincubation treatment in either excystation protocol, and this alteration was also demonstrated in the viability assay. Oocysts which excluded both dyes would not excyst in vitro unless a further trigger was provided and were more resistant to acid or alkali treatment. The results of this research provide a reproducible, user-friendly assay which is applicable to individual oocysts and also provides a useful adjunct for identification of oocysts in water and environmental samples.     

Viability of Cryptosporidium parvum oocysts: correlation of in vitro excystation with inclusion or exclusion of fluorogenic vital dyes.

A viability assay for oocysts of Cryptosporidium parvum based on the inclusion or exclusion of two fluorogenic vital dyes, 4',6-diamidino-2-phenylindole (DAPI) and propidium iodide, was developed by using several different isolates of oocysts. Correlation of this assay with viability measured by in vitro excystation was highly statistically significant, with a calculated correlation coefficient of 0.997. In this research, two similar excystation protocols were utilized, and no significant difference between excystation protocols was detected. Percent excystation of oocyst suspensions could be increased or reduced by inclusion of a preincubation treatment in either excystation protocol, and this alteration was also demonstrated in the viability assay. Oocysts which excluded both dyes would not excyst in vitro unless a further trigger was provided and were more resistant to acid or alkali treatment. The results of this research provide a reproducible, user-friendly assay which is applicable to individual oocysts and also provides a useful adjunct for identification of oocysts in water and environmental samples.     

Glucosylceramide Transferase Activity Is Critical for Encystation and Viable Cyst Production by an Intestinal Protozoan,Giardia lamblia

The production of viable cysts by Giardia is essential for its survival in the environment and for spreading the infection via contaminated food and water. The hallmark of cyst production (also known as encystation) is the biogenesis of encystation-specific vesicles (ESVs) that transport cyst wall proteins to the plasma membrane of the trophozoite before laying down the protective cyst wall. However, the molecules that regulate ESV biogenesis and maintain cyst viability have never before been identified. Here, we report that giardial glucosylceramide transferase-1 (gGlcT1), an enzyme of sphingolipid biosynthesis, plays a key role in ESV biogenesis and maintaining cyst viability. We find that overexpression of this enzyme induced the formation of aggregated/enlarged ESVs and generated clustered cysts with reduced viability. The silencing of gGlcT1 synthesis by antisense morpholino oligonucleotide abolished ESV production and generated mostly nonviable cysts. Interestingly, when gGlcT1-overexpressed Giardia was transfected with anti-gGlcT1 morpholino, the enzyme activity, vesicle biogenesis, and cyst viability returned to normal, suggesting that the regulated expression of gGlcT1 is important for encystation and viable cyst production. Furthermore, the overexpression of gGlcT1 increased the influx of membrane lipids and fatty acids without altering the fluidity of plasma membranes, indicating that the expression of gGlcT1 activity is linked to lipid internalization and maintaining the overall lipid balance in this parasite. Taken together, our results suggest that gGlcT1 is a key player of ESV biogenesis and cyst viability and therefore could be targeted for developing new anti-giardial therapies.     

The resistance of cysts of Stictodora lari (Trematoda: Heterophyidae) to encapsulation by cells of the fish host

Whereas glass beads are encapsulated by cells within 3 days after implantation into the abdominal cavity of Gambusia affinis, cysts of Stictodora lari in the same site are not encapsulated until 21–23 days after infection. The achievement of encystment in vitro demonstrated that the initial cyst wall is of parasite origin and fluorescent antibody methods showed that it does not mimic fish tissue in composition. Cysts formed in vivo have material, presumably of fish origin, associated with the cyst wall, as do living and fixed in vitro cysts following implantation.It is considered that cysts are not encapsulated for some weeks after infection because they are disguised as host tissue by material of fish origin associated with the cyst wall. An alternative explanation is proposed if the fish material does not have this functional role; the presence of spikes on the initial cyst wall may form an unsatisfactory substrate for the attachment of cells.     

Comparison of animal infectivity and excystation as measures of Giardia muris cyst inactivation by chlorine.

In this study, in vitro excystation and mouse infectivity were compared as methods for quantitatively determining the viability of Giardia muris cysts before and after exposure to free residual chlorine. The mouse infectivity results show that very few cysts (1 to 15) constitute an infectious dose. The results of the inactivation studies indicate that in vitro excystation is an adequate indication of G. muris cyst infectivity for the host and can be used to determine the effects of disinfectants on cyst viability.     

Low pressure ultraviolet studies for inactivation of Giardia muris cysts

AIMS: The research was initiated to confirm earlier ultraviolet (u.v.) light inactivation studies performed on Giardia cysts using excystation as the viability indicator. Following this, a comparison of in vitro excystation and animal infectivity was performed for assessing cyst viability after exposure to low-pressure u.v. irradiation. METHODS AND RESULTS: Cysts of Giardia muris were inactivated using a low-pressure u.v. light source. Giardia muris was employed as a surrogate for the human pathogen Giardia lamblia. Cyst viability was determined by both in vitro excystation and animal infectivity. Cyst doses were counted using a flow cytometer for the animal infectivity experiments. Using in vitro excystation as the viability indicator, fluences as high as approximately 200 mJ cm(-2) did not prevent some cysts from excysting, thus verifying earlier work. Using animal infectivity, u.v. fluences of 1.4, 1.9 and 2.3 mJ cm(-2) yielded log10 reductions ranging from 0.3 to >or= 4.4. CONCLUSIONS: Results indicate that in vitro excystation is not a reliable indicator of G. muris cyst viability after u.v. disinfection. Very low doses of u.v. light rendered G. muris cysts non-infective in the mouse model employed. SIGNIFICANCE AND IMPACT OF THE STUDY: Data presented represent the only complete u.v. inactivation curve for G. muris. This research provides evidence that u.v. can be an effective barrier against Giardia spp. in the treatment of drinking water supplies.     

Comparison of animal infectivity and excystation as measures of Giardia muris cyst inactivation by chlorine

In this study, in vitro excystation and mouse infectivity were compared as methods for quantitatively determining the viability of Giardia muris cysts before and after exposure to free residual chlorine. The mouse infectivity results show that very few cysts (1 to 15) constitute an infectious dose. The results of the inactivation studies indicate that in vitro excystation is an adequate indication of G. muris cyst infectivity for the host and can be used to determine the effects of disinfectants on cyst viability.     

Modeling Long-Term Host Cell-Giardia lamblia Interactions in an In Vitro Co-Culture System

Globally, there are greater than 700,000 deaths per year associated with diarrheal disease. The flagellated intestinal parasite, Giardia lamblia, is one of the most common intestinal pathogens in both humans and animals throughout the world. While attached to the gastrointestinal epithelium, Giardia induces epithelial cell apoptosis, disrupts tight junctions, and increases intestinal permeability. The underlying cellular and molecular mechanisms of giardiasis, including the role lamina propria immune cells, such as macrophages, play in parasite control or clearance are poorly understood. Thus far, one of the major obstacles in ascertaining the mechanisms of Giardia pathology is the lack of a functionally relevant model for the long-term study of the parasite in vitro. Here we report on the development of an in vitro co-culture model which maintains the basolateral-apical architecture of the small intestine and allows for long-term survival of the parasite. Using transwell inserts, Caco-2 intestinal epithelial cells and IC-21 macrophages are co-cultured in the presence of Giardia trophozoites. Using the developed model, we show that Giardia trophozoites survive over 21 days and proliferate in a combination media of Caco-2 cell and Giardia medium. Giardia induces apoptosis of epithelial cells through caspase-3 activation and macrophages do not abrogate this response. Additionally, macrophages induce Caco-2 cells to secrete the pro-inflammatory cytokines, GRO and IL-8, a response abolished by Giardia indicating parasite induced suppression of the host immune response. The co-culture model provides additional complexity and information when compared to a single-cell model. This model will be a valuable tool for answering long-standing questions on host-parasite biology that may lead to discovery of new therapeutic interventions.