Excystation Method Research Articles

Evaluation of Cryptosporidium parvum oocyst inactivation following exposure to ultraviolet light-emitting diodes by in vitro excystation and dye staining assays.

Cryptosporidium spp. are protozoan parasites that are transmitted via fecal-oral routes and can exhibit chemical resistance. Chlorine resistance makes it very difficult to eliminate parasites present in contaminated drinking water. While the efficacy of ultraviolet light-emitting diodes (UV-LEDs) against microorganisms has been reported, the efficacy of UV-LEDs against Cryptosporidium spp. has not been fully evaluated. Here, we assessed the efficacy of UV-LEDs with peak wavelengths of 268, 275, 284, and 289nm against Cryptosporidium parvum at various exposure times, with a fixed exposure distance, using two in vitro methods. Consequently, the time required for 2 log10 inactivation through the excystation method by UV-LEDs of 268, 275, 284, and 289nm was estimated as 115.5, 104.1, 37.4, and 30.7min, respectively. The propidium iodide (PI) and 4',6-diamidino-2-phenylindole (DAPI) staining assays estimated the inactivation time as 311.3, 275.2, 60.6, and 39.1min, respectively. Our results showed that UV-LED irradiation at longer wavelengths produced higher inactivation activity against C. parvum, which corroborates our previously reported in vivo assay results, although further study is needed to clarify the mechanism.

Statistical comparison of excystation methods in Cryptosporidium parvum oocysts

Excystation of sporozoites of Cryptosporidium parvum from oocysts is essential for successful in vitro assays. It has also been traditionally used as a measure for oocyst viability and infectivity. Laboratories use various excystation protocols so there is a need to clarify which method is the best. In this study, six different protocols for in vitro excystation of C. parvum oocysts were compared to find the most efficient excystation method (expressed as percentage excystation). Tested protocols differed in chemical pre-incubation steps, excystation media or time of incubation.There were significant differences in percentage of excysted oocysts among groups excysted by different methods. There were also significant differences in percentage of excysted oocysts between methods using pre-incubation with sodium hypochlorite and those without. The other variables examined; the presence of trypsin, kind of excystation medium and the incubation time, did not show statistical differences in percentage excystation among groups.Pre-incubation steps which included sodium hypochlorite, enhancing the permeability of the oocysts were found to increase the excystation ratio and methods using this step were the most effective.

Development of an in vitro drug sensitivity assay based on newly excysted larvae of Echinostoma caproni

BackgroundEchinostomiasis is one of the major food-borne trematodiases and the species Echinostoma caproni serves as a useful model for trematocidal drug discovery. The current in vitro drug sensitivity assay uses adult E. caproni worms that are incubated with candidate drugs and scored microscopically for viability at 72 hrs. The aim of this study was to investigate the use of newly excysted larvae (NEL) of E. caproni for in vitro drug testing, which would be faster, more cost effective and more ethical compared to adult worm assays.MethodsLarvae were obtained by collecting metacercariae from snails and triggering their excystation using the trypsin-bile salt excystation method. Studies concerning various parameters of this chemical transformation process as well as appropriate NEL culturing conditions were carried out and findings evaluated. NEL and adult worms were incubated with praziquantel, tribendimidine, albendazole and quinine and evaluated microscopically 72 hrs post-incubation. In addition, the colorimetric markers resazurin, CellTiter-Glo® and Vybrant® were tested as an alternative assay read-out method.ResultsThe chemical excystation method successfully induced E. caproni metacercariae to excyst at a rate of about 20-60%. NEL remained viable in culture medium for 5–7 days. The results of an in vitro drug assay using NEL mirrored the results of an assay using adult worms incubated with the same drugs. None of the markers could reliably produce signals proportional to NEL viability or cytotoxicity without significant complications.ConclusionNEL are adequate for in vitro drug testing. Challenges remain in further improving the excystation yield and the practicability of the assay setup. Resolving these issues could also improve read-outs using colorimetric markers. Using NEL is in alignment with the 3 R rules of the ethical use of laboratory animals and can greatly increase the rate and affordability with which drugs are screened in vitro against this intestinal trematode.

Cryptosporidium spp. in drinking water

In most rural areas and small communities in Switzerland the drinking water is supplied to the consumers after a minimum or even no treatment at all. However, it is just in these areas where drinking water from sources of agricultural activities can be contaminated by liquid manure and faeces of pasturing animals. The Swiss drinking water regulations are limited to the monitoring of E. coli, Enterococcus spp. and total plate counts only. Hence, resistant pathogens, as for example Cryptosporidium spp., remain unnoticed. During a drinking water survey, which lasted from June 2003 to December 2004, water samples were collected from 3 selected rural sites in Switzerland. The drinking water was investigated for Cryptosporidium spp., E. coli, Enterococcus spp., Clostridium perfringens and other parameters. In all samples oocysts of Cryptosporidium spp. were detected at elevated concentrations of up to 0.18 oocysts/l. Between 28% and 75% of the oocysts were found to be vital by the excystation method. Sampled oocysts collected from the three sites were subjected to genotyping and in one case the isolate was found to belong to the genotype of C. parvum. No evidence for increased incidents of diarrhoea in the past years was noted by local authorities.

Cryptosporidium parvum oocyst viability and behaviour of the residual body during the excystation process

This study was conducted as a comparative evaluation of time-dependent changes in the viability of purified Cryptosporidium parvum oocysts by means of different excystation methods. Oocyst samples were 2 weeks to 12 months old and were treated with bile or sodium taurocholate, partly after pretreatment with hypochlorite. Pretreatment markedly enhanced the excystation of younger oocyst samples but did not increase excystation rates of 9 or 12-month-old oocysts. A cell culture-PCR assay was used as a second indicator for oocyst viability and was most consistent with excystation trials including oocyst pretreatment. In experiments aiming at the determination of the behaviour of the oocyst residual body during excystation, it could be demonstrated that it might be involved in this process.

Quantification of Giardia transcripts during in vitro excystation: Interest for the estimation of cyst viability

The aim of the present study was to evaluate the potential of transcript quantification as an indicator of Giardia cyst viability. The variations of β-giardin, EF1A and ADHE mRNAs were quantified during excystation by real-time RT-PCR assays and compared with the percentages of viability estimated using propidium iodide staining and in vitro excystation. The first experiments were performed with purified G. duodenalis assemblage B cysts. When 55% of excysting protozoa were observed, the increase of the selected transcripts ranged from 0.40 ± 0.13 to 0.97 ± 0.11 log 10 after 1 h of incubation in excystation medium. Purified cysts were also stored at 4 °C for up to 56 days and analysed at several sampling times. Significant correlations were observed between the variations of the selected mRNAs and the percentages of viability estimated with staining and excystation methods. Among the three transcripts, β-giardin appeared to be the most appropriate to study the viability of Giardia cysts concentrated from wastewater samples.

Estimating chloramine C × T for the synergistic inactivation of Cryptosporidium with ozone followed by chloramine

A statistical model was developed to account for the error in estimating the required chloramine concentration times time (C × T) to inactivate Cryptosporidium oocysts with ozone followed by chloramine in drinking water. Generally, either a modified in vitro excystation method or an animal infectivity method is used by researchers to investigate the inactivation kinetics of Cryptosporidium in sequential inactivation studies. Published data from three sequential inactivation studies were pooled to obtain a closed‐form formula for the required chloramine C × T to achieve a specified inactivation level of Cryptosporidium. In a previously published paper, a multiple linear regression analysis was used to calculate the required chloramine C × T using the same data in which uncertainties caused by factors such as lot variability and random error were not accounted for. This article proposes a mixed linear modeling approach that accounts for these uncertainties and thus generates higher C × T values than the values reported previously.

Comparing the Efficacy of Chlorine, Chlorine Dioxide, and Ozone in the Inactivation of Cryptosporidium parvum in Water from Parana State, Southern Brazil

In the present work, assays were performed to compare the efficacy of hypochlorous acid, chlorine dioxide, and ozone in the inactivation of Cryptosporidium oocyst in public water supply from Brazilian South conditions. Experiments were carried out in samples containing 2 x 10(4) oocysts/ml of C. parvum purified from feces of experimentally contaminated calves. An in vitro excystation method was used to evaluate oocysts' viability and to determine the inactivation rates of hypochlorous acid at 2 ppm, chlorine dioxide at 1, 2, and 5 ppm, and ozone at the doses of 0.18, 0.24, 0.36, 0.48, and 1.44 mg/l. By using hypochlorous acid, the maximum inactivation rate obtained was 49.04% after 120 min. Chlorine dioxide at 5 ppm inactivated 90.56% of oocysts after 90 min of contact. Ozone was the most effective product, rendering an inactivation of 100% with the concentration of 24 mg/l. Resistance of Cryptosporidium to the usual disinfectants and the need for more effective water treatments to prevent waterborne diseases in Brazil are discussed in this manuscript.

Efficacy of Two Peroxygen-Based Disinfectants for Inactivation of Cryptosporidium parvum Oocysts

Two commercial peroxygen-based disinfectants containing hydrogen peroxide plus either peracetic acid (Ox-Virin) or silver nitrate (Ox-Agua) were tested for their ability to inactivate Cryptosporidium parvum oocysts. Oocysts were obtained from naturally infected goat kids and exposed to concentrations of 2, 5, and 10% Ox-Virin or 1, 3, and 5% Ox-Agua for 30, 60, and 120 min. In vitro excystation, vital dyes (4',6'-diamidino-2-phenylindole and propidium iodide), and infectivity in neonatal BALB/c mice were used to assess the viability and infectivity of control and disinfectant-treated oocysts. Both disinfectants had a deleterious effect on the survival of C. parvum oocysts, since disinfection significantly reduced and in some cases eliminated their viability and infectivity. When in vitro assays were compared with an infectivity assay as indicators of oocyst inactivation, the excystation assay showed 98.6% inactivation after treatment with 10% Ox-Virin for 60 min, while the vital-dye assay showed 95.2% inactivation and the infectivity assay revealed 100% inactivation. Treatment with 3% Ox-Agua for 30 min completely eliminated oocyst infectivity for mice, although we were able to observe only 74.7% inactivation as measured by excystation assays and 24.3% with vital dyes (which proved to be the least reliable method for predicting C. parvum oocyst viability). These findings indicate the potential efficacy of both disinfectants for C. parvum oocysts in agricultural settings where soil, housing, or tools might be contaminated and support the argument that in comparison to the animal infectivity assay, vital-dye and excystation methods overestimate the viability of oocysts following chemical disinfection.

Inactivation of Cryptosporidium parvum oocysts with ozone and free chlorine

The objective of this study is to investigate the synergy involved in the sequential inactivation of C. parvum oocysts with ozone followed by free chlorine at 1–20°C. Primary ozone and free chlorine inactivation curves are characterized by an initial lag-phase, followed by one or two post-lag-phase segments, the first segment at a faster rate than the second, of pseudo-first-order inactivation. The kinetics of primary inactivation with ozone and free chlorine has a relatively strong temperature dependence, and vary both with oocyst lot and oocyst age. Synergy is observed for the sequential inactivation of C. parvum oocysts with ozone/free chlorine. Ozone pre-treatment results in the disappearance of the lag-phase and the occurrence of a secondary free chlorine inactivation curve with generally two pseudo-first-order segments, the first segment at a faster rate than the second. The kinetics of both secondary segments is significantly faster than the post-lag-phase rate of inactivation with free chlorine alone. The temperature dependence for both phases of the secondary free chlorine inactivation kinetics is weaker compared to that for primary inactivation with ozone or free chlorine. As a result, the level of synergy in sequential disinfection with ozone/free chlorine increases with decreasing temperature within the range relevant to drinking water utilities. Good agreement is found between the kinetics determined using the modified in-vitro excystation method of viability assessment and animal infectivity data recently reported in the literature for both primary inactivation with ozone, and sequential disinfection with ozone/free chlorine.

Sequential inactivation of Cryptosporidium parvum oocysts with chlorine dioxide followed by free chlorine or monochloramine

The main objective of this study was to assess the effect of temperature (4–30°C) on the inactivation kinetics of Cryptosporidium parvum oocysts with sequential disinfection schemes involving the use of chlorine dioxide as the primary disinfectant and free or combined chlorine as the secondary disinfectant in synthetic water. The synergy previously reported for sequential inactivation of C. parvum oocysts with ozone/free chlorine or ozone/combined chlorine did not occur when chlorine dioxide was used, instead of ozone, as the primary disinfectant within the temperature range (4–30°C) and the pre-treatment levels investigated. Sequential ozone/chlorine dioxide and chlorine dioxide/ozone experiments revealed that the lower level or absence of synergy for chlorine dioxide/free chlorine and chlorine dioxide/monochloramine was likely the result of chlorine dioxide reacting with oocyst chemical groups that are mostly different from those reacting with ozone, free chlorine, or monochloramine. The CT concept was found to be valid for the primary inactivation kinetics of C. parvum oocysts with chlorine dioxide, thus allowing the use of the simpler CT approach for the development of C. parvum inactivation requirements with chlorine dioxide. General consistency was found between the secondary inactivation kinetics of C. parvum oocysts with free chlorine and monochloramine after chlorine dioxide pretreatment obtained in this study with oocyst viability determined by a modified in vitro excystation method and those reported in the literature for the same sequential disinfection schemes based on an animal infectivity assay.

Inactivation of Cryptosporidium parvum oocysts with chlorine dioxide

The effect of solution pH and temperature on the rate of inactivation of Iowa strain Cryptosporidium parvum ( C. parvum) oocysts with chlorine dioxide was determined. Oocyst viability was assessed with a modified in vitro excystation method consistent with animal infectivity data. Inactivation curves were characterized by a lag phase followed by Chick–Watson first order kinetics. The rate of inactivation was essentially the same at pH 6 and 8. CT requirements at pH 10 were approximately 20–30% lower than those at pH 6–8. The magnitude of the lag phase and the inactivation rate constant were both found to obey Arrhenius law for the experimental temperature range of 4–30°C. CT requirements for C. parvum oocyst inactivation were found to increase by an average factor of approximately 3.4 for every 10°C decrease in temperature.

Inactivation of Cryptosporidium parvum oocysts with ozone

Ozone inactivation rates for Cryptosporidium parvum (C. parvum) oocysts were determined with an in vitro excystation method based on excysted sporozoite counts. Results were consistent with published animal infectivity data for the same C. parvum strain. The inactivation kinetics for C. parvum were characterized by a lag phase followed by pseudo-first order kinetics. Both the magnitudes of the lag phase and the pseudo-first order rate constant were found to be different for C. parvum oocysts from two different sources, and to obey Arrhenius law for the experimental temperature range of 5–30°C. CT requirements for C. parvum inactivation were found to increase by an average factor of approximately three for every 10°C decrease in temperature.

In vitro excystation of Spironucleus muris.

In vitro excystation of Spironucleus muris cysts, purified by sequential sucrose and Percoll gradients from mouse feces, was studied. Three in vitro excystation procedures, used for Giardia, were assessed to determine the most useful method. Excystation was monitored by light microscopy and subsequently characterized by transmission and scanning electron microscopy. Spironucleus muris excysted routinely at a level greater than 90% when induced in Hanks' balanced salt solution containing sodium bicarbonate at pH 2.0 and transferred to Tyrodes' salt solution as an excystation medium. Similarly, high rates of excystation were recorded after induction of S. muris cysts in 0.1 M potassium phosphate buffer (pH 7.0) with sodium bicarbonate and excystation in trypticase-yeast extract-iron medium (TYI medium) or phosphate-buffered saline. A lower rate and percentage of excystation were observed after induction of S. muris cysts in an aqueous hydrochloric acid solution (pH 2.0) followed by excystation in TYI medium. All excystation methods produced extremely active S. muris trophozoites with normal morphology. Nonexcysting S. muris cysts have a wall composed of an outer fibrous and an inner membranous portion. Following induction, numerous vesicles appeared in the peritrophic space. Excystation began by the cyst wall opening at one pole, and the anterior part of the trophozoite protruding from the cyst wall. The trophozoite emerged progressively from the cyst wall and the empty cyst wall appeared to collapse. Excysted trophozoites exhibited normal morphological features of S. muris trophozoites isolated from the mouse intestine.