Inactivation of Giardia muris cysts by free chlorine

The chlorine resistance of cysts of the flagellate protozoan Giardia muris was examined. This organism, which is pathogenic to mice, is being considered as a model for the inactivation of the human pathogen Giardia lamblia. Excystation was used as the criterion for cyst viability. Experiments were performed at pH 5, 7, and 9 at 25 degrees C and pH 7 at 5 degrees C. Survival curves were "stepladder"-shaped, but concentration-time data generally conformed to Watson's Law. Chlorine was most effective at neutral pH and was only slightly less so in acidic solutions. Comparison of inactivation data based on equivalent hypochlorous acid concentrations, which corrects for chlorine ionization, showed that the cysts have a pH-dependent resistance to inactivation. Concentration-time (C X t') products for free chlorine obtained at 25 degrees C ranged from a low of 50 mg min/liter at pH 5 to a high of 218 mg min/liter at pH 9 and were as high as 1,000 mg min/liter at 5 degrees C. It appears that G. muris cysts are somewhat more resistant to inactivation than G. lamblia cysts and rank among the microorganisms that are most resistant to inactivation by free chlorine.     

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.     

Inactivation of Giardia lamblia and Giardia canis cysts by combined and free chlorine.

Free chlorine and a combined organic N-chloramine (3-chloro-4,4-dimethyl-2-oxazolidinone, compound 1) were compared for efficacy as disinfectants against an admixture of cysts of Giardia lamblia and Giardia canis in water solution under a variety of test conditions; variables were pH, temperature, and water quality. In general, compound 1 was found to reduce the giardial excystation in the solutions at lower concentration or shorter contact time at a given total chlorine concentration than did free chlorine.     

Inactivation of Giardia lamblia and Giardia canis cysts by combined and free chlorine

Free chlorine and a combined organic N-chloramine (3-chloro-4,4-dimethyl-2-oxazolidinone, compound 1) were compared for efficacy as disinfectants against an admixture of cysts of Giardia lamblia and Giardia canis in water solution under a variety of test conditions; variables were pH, temperature, and water quality. In general, compound 1 was found to reduce the giardial excystation in the solutions at lower concentration or shorter contact time at a given total chlorine concentration than did free chlorine.     

Purification of Giardia muris cysts by velocity sedimentation.

Giardia muris cysts were separated from fecal contaminants in primary isolates by unit gravity velocity sedimentation. Crude isolates obtained by centrifugation over 1.0 M sucrose were overlaid onto a Percoll density gradient, 1.01 to 1.03 g/ml. G. muris cysts were well separated from faster-sedimenting fecal debris and slower-sedimenting Spironucleus muris and bacteria in 1.5 h.     

Purification of Giardia muris cysts by velocity sedimentation

Giardia muris cysts were separated from fecal contaminants in primary isolates by unit gravity velocity sedimentation. Crude isolates obtained by centrifugation over 1.0 M sucrose were overlaid onto a Percoll density gradient, 1.01 to 1.03 g/ml. G. muris cysts were well separated from faster-sedimenting fecal debris and slower-sedimenting Spironucleus muris and bacteria in 1.5 h.     

Carbohydrate and amino acid analyses of Giardia muris cysts.

Intact Giardia muris cysts were subjected to consecutive chloroform/methanol and 2% sodium dodecyl sulfate (SDS) extractions, and to amyloglucosidase treatment. The SDS-insoluble, amyloglucosidase-fast cyst walls (ACW) were further incubated with chymotrypsin, trypsin, papain, or pronase. Low voltage scanning electron microscopy revealed no discernible change in the ultrastructure of the filamentous layer of the cyst wall following any of these treatments. Affinity for cyst wall-specific monoclonal antibody (Meridian Diagnostics, Cincinnati, OH) was also retained after all treatments. Periodic acid-Schiff staining and gas chromatography/mass spectrometry (GC/MS) of intact and treated cyst hydrolysates showed a significant reduction in the amount of glucose associated with the cyst (72 nmoles/10(6) intact cysts vs 1.9 nmoles/10(6) ACW) as a result of amyloglucosidase treatment, indicating that glucose is stored within Giardia as an SDS-insoluble polymer. Galactosamine was identified by GC/MS as the predominant sugar associated with both the ACW and the proteinase treated ACW (42 nmoles/10(6) ACW). High performance liquid chromatographic analysis of amino acids from intact and treated cyst hydrolysates revealed a marked reduction, but not elimination, of detectable quantities of identifiable amino acid residues (255 nmoles/10(6) intact cysts vs 6.8 nmoles/10(6) proteinase treated ACW). These results suggest that the filamentous layer of the cyst wall is primarily a carbohydrate peptide complex.     

Inactivation of Giardia lamblia cysts by polychromatic UV

Aims:  Giardia lamblia is one of the most important waterborne pathogens in the world. In this study, we determined the effectiveness of a promising alternative UV technology – a polychromatic emission from a medium-pressure (MP) UV lamp – against G. lamblia cysts in phosphate buffered saline (PBS) and a filtered drinking water.
Methods and Results:  A UV collimated beam apparatus was used to expose shallow suspensions of purified G. lamblia cysts in PBS or a filtered drinking water and the UV-irradiated G. lamblia cysts were assayed in Mongolian gerbils. The inactivation of G. lamblia cysts was very rapid and reached a detection limit of >3 log₁₀ within a UV dose of 1 mJ cm⁻².
Conclusion:  The results of this study indicate that MP UV irradiation is very effective against G. lamblia cysts in both PBS and a filtered drinking water.
Significance and Impact of the Study:  It is likely that contamination of drinking water by G. lamblia cysts can be readily controlled by typical MP UV disinfection practises.     

Inactivation of Giardia lamblia cysts with ozone.

Giardia lamblia cysts were inactivated in water with ozone at pH 7.0 and 5 and 25 degrees C. The concentration-time products for 99% inactivation were 0.53 and 0.17 mg-min/liter at 5 and 25 degrees C, respectively. These products were significantly lower than those reported for chlorine.     

Respiration in the cysts and trophozoites of Giardia muris

Cysts and trophozoites of the parasitic protozoon Giardia muris both showed respiratory activity but respiration in cysts was only 10 to 20% that of trophozoites. The O2 dependence of respiration in cysts and trophozoites showed O2 maxima above which respiration decreased. The O2 concentration at which the respiration rate was greatest was higher for cysts than trophozoites. The effects of various inhibitors on cyst and trophozoite respiration suggested that flavoproteins and quinones play some role in respiration. The substrate specificities and the effects of inhibitors on G. muris trophozoites were similar to those observed for Giardia lamblia. Metronidazole, the drug most commonly used in the treatment of giardiasis completely inhibited respiration and motility in trophozoites; however, it had no effect on either respiration or viability in cysts. Menadione, a redox cycling naphthoquinone, stimulated then completely inhibited respiration in cysts and trophozoites; a complete loss of cyst viability or trophozoite motility was also observed. The effects of menadione on G. muris may indicate that redox cycling compounds have potential as chemotherapeutic agents for the treatment of giardiasis.     

Inactivation of Giardia lamblia cysts with ozone

Giardia lamblia cysts were inactivated in water with ozone at pH 7.0 and 5 and 25 degrees C. The concentration-time products for 99% inactivation were 0.53 and 0.17 mg-min/liter at 5 and 25 degrees C, respectively. These products were significantly lower than those reported for chlorine.     

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.     

Immunofluorescence and morphology of Giardia lamblia cysts exposed to chlorine.

Giardia cyst-like objects detected by immunofluorescence in chlorinated water samples often cannot be positively identified by their morphological appearance. To determine the effect of chlorine on cyst immunofluorescence and morphology, Giardia lamblia cysts were exposed to chlorine for 48 h. The majority of cysts exposed to chlorine concentrations of 1 to 11 mg/liter at 5 and 15 degrees C lost their internal morphological characteristics necessary for identification, but most of them were still detectable by immunofluorescence.     

Comparison of Giardia lamblia and Giardia muris cyst inactivation by ozone.

Inactivation of Giardia lamblia and Giardia muris cysts was compared by using an ozone demand-free 0.05 M phosphate buffer in bench-scale batch reactors at 22 degrees C. Ozone was added to each trial from a concentrated stock solution for contact times of 2 and 5 min. The viability of the control and treated cysts was evaluated by using the C3H/HeN mouse and Mongolian gerbil models for G. muris and G. lamblia, respectively. The resistance of G. lamblia to ozone was not significantly different from that of G. muris under the study conditions, contrary to previously reported data that suggested G. lamblia was significantly more sensitive to ozone than G. muris was. The simple Ct value for 2 log unit inactivation of G. lamblia was 2.4 times higher than the Ct value recommended by the Surface Water Treatment Rule.     

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.     

Immunofluorescence and morphology of Giardia lamblia cysts exposed to chlorine

Giardia cyst-like objects detected by immunofluorescence in chlorinated water samples often cannot be positively identified by their morphological appearance. To determine the effect of chlorine on cyst immunofluorescence and morphology, Giardia lamblia cysts were exposed to chlorine for 48 h. The majority of cysts exposed to chlorine concentrations of 1 to 11 mg/liter at 5 and 15 degrees C lost their internal morphological characteristics necessary for identification, but most of them were still detectable by immunofluorescence.     

Inactivation of hepatitis A virus and indicator organisms in water by free chlorine residuals.

Hepatitis A virus (HAV) and selected indicator organisms were mixed together in chlorine-demand-free buffers at pH 6, 8, or 10 and exposed to free chlorine residuals, and the survival kinetics of individual organisms were compared. HAV was enumerated by a most-probable-number dilution assay, using PLC/PRF/5 liver cells for propagation of the virus and radioimmunoassay for its detection. At all pH levels, HAV was more sensitive than Mycobacterium fortuitum, coliphage V1 (representing a type of phage common in some sewage-polluted waters), and poliovirus type 2. Under certain conditions, HAV was more resistant than Escherichia coli, Streptococcus faecalis, coliphage MS2, and reovirus type 3. It was always more resistant than SA-11 rotavirus. Evidence is presented that conditions generally specified for the chlorine disinfection of drinking-water supplies will also successfully inactivate HAV and that HAV inactivation by free chlorine residuals can reliably be monitored by practical indicator systems consisting of appropriate combinations of suitable indicators such as coliform and acid-fast bacteria, coliphages, the standard plate count, and fecal streptococci.     

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.