Infectivity of cryopreserved Giardia cysts for Mongolian gerbils (Meriones unguiculatus)

Although Giardia species trophozoites have been cryopreserved successfully, no report on the successful cryopreservation of cysts could be found. Using infectivity to Mongolian gerbils (Meriones unguiculatus) as a measure of cyst viability, we tested the viability of 4 strains of Giardia cysts that had been cryopreserved for 1-67 wk. Cysts were frozen in either Keister's medium or physiological saline, both containing 5% or 7.5% dimethylsulfoxide as the cryoprotectant. Viability of cryopreserved cysts was dependent upon the number of cysts inoculated, the length of time cysts were held at 4 C before cryopreservation, and the cryopreserving medium. Infection was established in gerbils by inoculating them with cysts that had been cryopreserved for up to 67 wk, cysts that had been held for less than 30 days before cryopreservation, and cysts frozen in Keister's medium. Saline appears to be unsuitable as a freezing medium for the cryopreservation of Giardia cysts.     

Identification of developmentally regulated Giardia lamblia cyst antigens using GCSA-1, a cyst-specific monoclonal antibody

GCSA-1, a monoclonal antibody raised against cysts generated in vitro was shown to be Giardia cyst-specific by immunoblot analysis and immunofluorescence. GCSA-1 recognized four polypeptides ranging from 29-45 kD present in the cyst wall. These antigens appeared within eight hours of exposure of trophozoites to encystation medium and were shown to be synthesized by encysting parasites by means of metabolic labelling with [35S]-cysteine. Trophozoites were not stained by the antibody. GCSA-1 also reacted with in vivo cysts obtained from faeces of infected humans, gerbils and mice. These data demonstrate that the determinants recognized by GCSA-1 are early cyst antigens which are developmentally regulated and conserved components of the cyst wall. The actual role of the antigens detected by GCSA-1 in encystation are unknown, but they represent a potential target for strategies directed at inhibiting this process.     

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.     

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.     

Prevalence of Giardia cysts and Cryptosporidium oocysts and characterization of Giardia spp. isolated from drinking water in Canada.

This study was carried out to estimate the prevalence and potential for human infectivity of Giardia cysts in Canadian drinking water supplies. The presence of Cryptosporidium oocysts was also noted, but isolates were not collected for further study. A total of 1,760 raw water samples, treated water samples, and raw sewage samples were collected from 72 municipalities across Canada for analysis, 58 of which treat their water by chlorination alone. Giardia cysts were found in 73% of raw sewage samples, 21% of raw water samples, and 18.2% of treated water samples. There was a trend to higher concentration and more frequent incidence of Giardia cysts in the spring and fall, but positive samples were found in all seasons. Cryptosporidium oocysts were found in 6.1% of raw sewage samples, 4.5% of raw water samples, and 3.5% of treated water samples. Giardia cyst viability was assessed by infecting Mongolian gerbils (Meriones unguiculatus) and by use of a modified propidium iodide dye exclusion test, and the results were not always in agreement. No Cryptosporidium isolates were recovered from gerbils, but 8 of 276 (3%) water samples and 19 of 113 (17%) sewage samples resulted in positive Giardia infections. Most of the water samples contained a low number of cysts, and 12 Giardia isolates were successfully recovered from gerbils and cultured. Biotyping of these isolates by isoenzyme analysis and karyotyping by pulsed-field gel electrophoresis separated the isolates into the same three discrete groups. Karyotyping revealed four or five chromosomal bands ranging in size from 0.9 to 2 Mb, and four of the isolates had the same banding pattern as that of the WB strain. Analysis of the nucleotide sequences of the 16S DNA coding for rRNA divided the isolates into two distinct groups corresponding to the Polish and Belgian designations found by other investigators. The occurrence of these biotypes and karyotypes appeared to be random and was not related to geographic or other factors (e.g., different types were found in both drinking water and sewage from the same community). Biotyping and karyotyping showed that isolates from this study were genetically and biochemically similar to those found elsewhere, including well-described human source strains such as WB. We conclude that potentially human-infective Giardia cysts are commonly found in raw surface waters and sewage in Canada, although cyst viability is frequently low. Cryptosporidium oocysts are less common in Canada. An action level of three to five Giardia cysts per 100 liters in treated drinking water is proposed on the basis of the monitoring data from outbreak situations. This action level is lower than that proposed by Haas and Rose (C. N. Haas and J. B. Rose, J. Am. Water Works Assoc. 87(9):81-84, 1995) for Cryptosporidium spp. (10 to 30 oocysts per 100 liters).     

Longitudinal studies of Giardia contamination in two community drinking water supplies: cyst levels, parasite viability, and health impact.

Giardia cyst concentrations were determined in an inventory of 153 raw and 91 chlorinated drinking water samples collected at 86 sites from throughout the western Canadian province of British Columbia. Sixty-four percent of raw water samples were cyst positive (69% of sites). Cyst concentrations were lower in chlorinated than in raw water. The viability of cysts in drinking water samples assessed by infectivity in Mongolian gerbils (Meriones unguiculatus) was decreased in chlorinated water. Two rural communities using Giardia-contaminated surface drinking water sources were selected for longitudinal studies including drinking water testing and serological studies of residents. Three hundred thirty-six raw and treated samples from these communities were collected over 24 months. Cyst concentrations and viability were assessed in a 12-month study of each community. Parasite concentrations were lower in chlorinated water than in raw water in both communities. Cyst concentrations were lower in reservoir-settled water than in raw water. Viability, assessed by animal infectivity and corrected for inoculum, decreased following reservoir settling as well as after chlorination. A bolus or spiking phenomenon of cysts was observed in both community drinking water systems and deserves further study. A striking seasonal pattern was seen in one community but not in the second. The seroprevalence data and number of laboratory-confirmed cases identified in each year-long community study are consistent with the possibility that low-level endemic transmission is occurring.     

Cyst wall formation and endogenous carbohydrate utilization during synchronous encystment of Phytophthora palmivora zoospores

Summary Vigorous agitation caused the zoospores of Phytophthora palmivora to undergo rapid synchronous encystment. The rate of encystment was determined by counting the number of cells with an alkali-resistant cyst wall. 50% of the zoospores formed an alkali-resistant cyst wall within 60 sec of agitation; after 120 sec, essentially all zoospores had encysted. The rate of spontaneous encystment in nonagitated suspensions was much slower. The flagella of nearly all zoospores disappeared within 30 sec of agitation, i.e. prior to the formation of an alkali-resistant cyst wall. Zoospores depend on internal reserves for synthesizing their cyst walls. Approximately 70% of the total carbohydrate in motile zoospores was extracted with water after treating the cells with 70% éthnol. During synchronous encystment, this carbohydrate fraction composed largely of glucans decreased markedly while the insoluble carbohydrate fraction (cyst wall glucan) increased correspondingly. Clearly, the conversion of cytoplasmic glucan into wall glucan plays a major role in zoospore encystment.     

Comparative studies on the pattern of infection with Giardia spp. in mongolian gerbils

Mongolian gerbils (Meriones unguiculatus) were inoculated with known numbers of Giardia cysts isolated from humans, beavers and mice. The pattern of cyst release in the feces was studied for a period of 35 days. After a latent period of 5 days, animals infected with G. muris release cysts in their feces every day until day 14. Gerbils infected with human or beaver isolates released cysts in their feces intermittently for 30 days. These results indicated that the mode of cyst release in these animals was characteristic of the parasite, and was independent of the host. Mongolian gerbils acquire complete resistance upon homologous species challenge but demonstrate only partial protection when challenged with a different species of Giardia. We concluded that the Mongolian gerbil model could be useful in epidemiological studies for two reasons: it can be used for determination of cyst viability, and for the identification of the etiological agent.     

Detection of antigenic cyst wall elements in Colpoda inflata: an immunoelectron microscopic study and immunoblotting identification of cyst wall polypeptides

By using an antiserum against isolated cyst walls from resting cysts of the ciliate Colpoda inflata, cyst wall polypeptides have been identified by immunoblotting test. Likewise, an immunoelectron microscopical study on both complete resting cysts and isolated cyst walls to localize the cyst wall proteins recognized by the antiserum, has been carried out. The immunoblotting test showed that three main polypeptide bands were recognized by the antiserum, with tentative molecular weights of 61, 66 and 70 kDa respectively. This methodology provides a better identification of cyst wall proteins after electrophoretic separation of cyst wall samples from ciliate resting cysts.     

Inactivation of gerbil-cultured Giardia lamblia cysts by free chlorine.

Giardia lamblia cysts were harvested from Mongolian gerbils and exposed to free chlorine in buffered water at pH 5, 7, and 9 at 15 degrees C. The contact times required to obtain a 2-log reduction in cyst survival (i.e., a 99% kill) were interpolated from survival curves generated at fixed concentrations of chlorine in the range of 0.25 to about 16 mg/liter. Concentration-time (C.t') products for 99% inactivation ranged from about 120 to nearly 1,500 mg.min/liter. These values are higher than those reported previously for free chlorine using G. lamblia cysts from infected humans. The cysts isolated from gerbils, as with other Giardia cysts, were unusually sensitive to chlorine in alkaline solutions.     

Inactivation of gerbil-cultured Giardia lamblia cysts by free chlorine

Giardia lamblia cysts were harvested from Mongolian gerbils and exposed to free chlorine in buffered water at pH 5, 7, and 9 at 15 degrees C. The contact times required to obtain a 2-log reduction in cyst survival (i.e., a 99% kill) were interpolated from survival curves generated at fixed concentrations of chlorine in the range of 0.25 to about 16 mg/liter. Concentration-time (C.t') products for 99% inactivation ranged from about 120 to nearly 1,500 mg.min/liter. These values are higher than those reported previously for free chlorine using G. lamblia cysts from infected humans. The cysts isolated from gerbils, as with other Giardia cysts, were unusually sensitive to chlorine in alkaline solutions.     

Naegleria fowleri: enolase is expressed during cyst differentiation

Cysts of Naegleria fowleri present an external single-layered cyst wall. To date, little information exists on the biochemical components of this cyst wall. Knowledge of the cyst wall composition is important to understand its resistance capacity under adverse environmental conditions. We have used of a monoclonal antibody (B4F2 mAb) that specifically recognizes enolase in the cyst wall of Entamoeba invadens. By Western blot assays this antibody recognized in soluble extracts of N. fowleri cysts a 48-kDa protein with similar molecular weight to the enolase reported in E. invadens cysts. Immunofluorescence with the B4F2 mAb revealed positive cytoplasmic vesicles in encysting amebas, as well as a positive reaction at the cell wall of mature cysts. Immunoelectron microscopy using the same monoclonal antibody confirmed the presence of enolase in the cell wall of N. fowleri cysts and in cytoplasmic vesicular structures. In addition, the B4F2 mAb had a clear inhibitory effect on encystation of N. fowleri.     

The detection of Giardia muris and Giardia lamblia cysts by immunofluorescence in animal tissues and fecal samples subjected to cycles of freezing and thawing

The effects of freezing and thawing on the detection of selected Giardia spp. cysts were investigated using immunofluorescence, bright field microscopy, and low voltage scanning electron microscopy (SEM). Giardia muris cysts were obtained from either animal carcasses, fecal pellets, or isolated cyst preparations, whereas Giardia lamblia cysts were isolated from fecal samples. These samples were stained using an immunofluorescence technique after 1-3 freezing (-16 C) and thawing (20 C) cycles. Cysts were detected successfully by immunofluorescence in all samples. However, in those samples subjected to freeze-thawing, the cyst walls often became distorted and then were not detectable by bright field microscopy. Low voltage SEM demonstrated that the filaments in the distorted cyst wall underwent rearrangements of interfilament spacing. Quantitation of cyst recovery after freezing and thawing demonstrated that a substantial loss occurred after 1 cycle of alternating temperature when low concentrations of cysts were used, but not with high concentrations of cysts. Cyst recovery, after 3 freezing and thawing cycles, was dramatically lowered irrespective of the initial cyst concentration. These results demonstrated that immunofluorescence was an effective technique for the detection of Giardia spp. cysts in frozen samples and would suggest that freezing and thawing of fecal samples could prevent the detection of cysts when only bright field microscopy was employed.     

A Light Microscopic Comparison of The Cysts of Four Species of Sarcocystis Infecting The Domestic Reindeer (Rangifer Tarandus) in Northern Norway

Fresh preparations of micro-isolated sarcocysts from skeletal and cardiac muscle of 12 reindeer were examined by light microscopy. On the basis of cyst structure and cyst wall structure 4 Sarcocystis spp. could be differentiated. New names have been proposed for 2 previously unnamed Sarcocystis spp. of reindeer, and S. grueneri has been redefined. S. rangiferi n. sp. had macroscopic cysts in skeletal muscle measuring 2106×403 µm. The cyst wall protrusions were finger-like and measured 13.2×6.7 µm. The cysts were surrounded by a layer of fibrillar material. S. tarandi n. sp. had micro- to macroscopic cysts primarily in skeletal muscle, but a few cysts were found in the heart of one animal. In skeletal muscle the cysts measured 999×75µm; in the heart the cysts were shorter and wider. The cyst wall protrusions were fingerlike and measured 9.2×2.2 µm. S. grueneri had micro- to macroscopic cysts in cardiac muscle measuring 581×137 µm. The cyst wall was thin and relatively smooth with no visible protrusions. Sarcocystis sp. had micro- to macroscopic, slender cysts in skeletal muscle measuring 916×64 µm. The cyst wall had tightly packed, short, knob-like protrusions. The cysts of this species were previously classified as cysts of S. grueneri.     

Prediction of viability of porcine neurocysticercosis with proton magnetic resonance spectroscopy: correlation with histopathology

Neurocysticercosis (NCC) is the most frequent parasitic disease of central nervous system. In our earlier study, we had observed creatine [(creatine + phosphocreatine); (tCr)] on ex vivo proton MR spectroscopy (1H MRS) in some of the cysticercus cyst fluid samples obtained from swine's brain parenchyma. In current study, swine brains of freshly slaughtered animals naturally infected with NCC were subjected to ex vivo magnetic resonance (MR) imaging on a 1.5Tesla MR system. Cysticercus cysts (n = 12) were removed from these brains and were labeled depending upon presence or absence of edema around cysts as observed on imaging. Cysticercus cyst fluid (100 microl) was subjected to different 1H MRS experiments and results were compared with histopathological examinations to look for any relationship between tCr and parameters like quantification of musculature, and cellular infiltration in wall of the parasite. Histopathology of cyst wall was categorized into two groups based on cellular characteristics and the amount of musculature. Grade I cysts (n = 5) with no or minimal inflammation and large amount of musculature showed tCr on 1H MRS. However, grade II cysts (n = 7) with profuse inflammation and less amount of musculature in the cyst wall lacked tCr. Higher amount of musculature in grade I cysts was associated with higher concentration of tCr in the cyst fluid (r2 = 0.93, P = 0.007). Creatine appears to be a marker of innocuous and viable NCC.     

A comparison of Giardia microti and Spironucleus muris cysts in the vole: an immunocytochemical, light, and electron microscopic study

We have shown that cysts of the genus Spironucleus share many common morphological features with Giardia cysts including: 2-4 nuclei, flagellar axonemes, a distinct cyst wall, and they even display the same immunostaining as Giardia cysts when labeled with antibodies specific for Giardia cyst wall. A direct comparison of Spironucleus muris and Giardia microti cysts have revealed that cysts of S. muris are significantly smaller than cysts of G. miroti. At the ultrastructural level, the cyst walls are similar in fibrillar appearance, but the width of the S. muris cyst wall is significantly less than that of G. microti. The cysts of S. muris also differ from G. microti in that they contain a striated rootlet fiber, flagellar sheath, and numerous glycogen rosettes. Characteristic features of Giardia include the adhesive disc and median body. Although the cysts of Spironucleus and Giardia are similar in appearance, these unique morphological features can be used to distinguish between the 2 protozoa and should be employed in the detection of Giardia cysts in water samples.     

Formation of the Giardia Cyst Wall: Studies on Extracellular Assembly Using Immunogold Labeling and High Resolution Field Emission SEM

Encystment of the intestinal protozoan, Giardia, is a key step in the life cycle that enables this parasite to be transmitted from host to host via either fecal oral, waterborne, or foodborne transmission. The process of encystment was studied by localizing cyst wall specific antigens with immunofluorescence for light microscopy and immunogold staining for field emission scanning electron microscopy. Chronological sampling of Giardia cultures stimulated with endogenous bile permitted identification of an intracellular and extracellular phase in cyst wall formation, a process which required a total of 14-16 h. The intracellular phase lasted for 8-10 h, while the extracellular phase, involved the appearance of cyst wall antigen on the trophozoite membrane, and the assembly of the filamentous layer, a process requiring an additional 4-6 h for completion of mature cysts. The extracellular phase was initiated with the appearance of cyst wall antigen on small protrusions of the trophozoite membrane (-15 nm), which became enlarged with time to caplike structures ranging up to 100 nm in diameter. Caplike structures involved with filament growth were detected over the entire surface of the trophozoite including the adhesive disc and flagella. Encysting cells rounded up, lost attachment to the substratum, and became enclosed in a layer of filaments. Late stages in encystment included a “tailed” cyst in which flagella were not fully retracted into the cyst. Clusters of cysts were seen in which filaments at the surface of one cyst were connected with the surface of adjacent cysts or the “tailed” processes of adjacent cysts, suggesting that the growth of cyst wall filaments may be at the terminal end. In conclusion, the process of encystment has been shown to consist of two morphologically different stages (intracellular and extracellular) which requires 16 h for completion. Further investigation of the extracellular stage with regard to assembly of the filamentous layer of the cyst wall may lead to innovative methods for interfering with production of an intact functional cyst wall, and thereby, regulation of viable Giardia cyst release from the host.     

High-resolution immunogold localization of Giardia cyst wall antigens using field emission SEM with secondary and backscatter electron imaging

We describe here the ultrastructural localization of Giardia cyst antigens in the filaments associated with the outer portion of intact cysts and on developing cyst wall filaments in encysting trophozoites. Post-embedding immunogold labeling of thin sections of intact Giardia cysts with polyclonal and monoclonal antibodies specific for cyst wall antigens (major protein bands of approximately 29, 75, 88, and 102 KD on Western blots) showed strong labeling of the filamentous cyst wall, whereas no labeling was seen on the membranous portion. High-resolution field emission scanning electron microscopy (FESEM) of Giardia cysts revealed that the cyst wall-specific polyclonal rabbit antisera and monoclonal mouse antibody produced gold labeling of 20-nm filaments in the cyst wall as detected with secondary electron imaging (SEI) and backscatter electron imaging (BEI) at 10 kV, despite coating of the cells with platinum by ion sputtering. FESEM studies of encysting Giardia trophozoites demonstrated that immunostaining with antibodies to cyst wall antigens produced colloidal gold labeling of developing cyst wall filaments on the cell surface; however, the intervening membrane domains were unlabeled. Substitution of normal serum for cyst wall-specific antibodies, or preabsorption of specific antibodies with Giardia cysts, eliminated immunolabeling of the filaments.     

Resting cysts of Parentocirrus hortualis Voß, 1997 (Ciliophora,Hypotrichia), with preliminary notes on encystation and various types of excystation

Resting cysts of Parentocirrus hortualis were investigated, using live observation, SEM and TEM. Processes during encystation and excystation were observed in vivo under the light microscope. During encystation, the trophic body becomes globular, the ciliature is resorbed in an anterior direction, the macronuclear nodules fuse into an elongated mass, and finally a cyst wall develops. As typical for oxytrichids, the resting cysts of P. hortualis are of the kinetosome-resorbing type and their wall is made of four layers: ectocyst, mesocyst, endocyst, and metacyst. The beginning of excystation is indicated by the formation of an excystation vacuole that helps the regenerating specimen to break the cyst wall. The excysting specimen leaves the resting cyst in a thin membrane that is gradually resorbed in the outer environment. Also two other excystation modes were observed. During the rare mode, the excystation vacuole breaks the thin membrane instead of the cyst wall that ruptures under the pressure of the body of the regenerating specimen. During the reproduction mode, the regenerating specimen divides within the resting cyst, producing two to four tomites. This is the first report of division in resting cysts of oxytrichids, but reproduction in division cysts was already described in keronopsids.     

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.