Development of ATP assay as a surrogate indicator of viability of Cryptosporidium parvum oocysts

2000 ◽  
Vol 41 (7) ◽  
pp. 181-188 ◽  
Author(s):  
I. Somiya ◽  
S. Fujii ◽  
N. Kishimoto ◽  
R-H. Kim
Keyword(s):  
Linear Relationship ◽  
Cryptosporidium Parvum ◽  
Simple Procedure ◽  
Ozone Treatment ◽  
Atp Assay ◽  
Cryptosporidium Oocysts ◽  
Batch Condition ◽  
Cryptosporidium Parvum Oocysts ◽  
Permeability Assay

Adenosine Triphosphate (ATP) determination was applied to evaluate viability of Cryptosporidium oocysts. Three pretreatment methods, such as incubation in acidified Hanks balanced salt solution (HBSS), excystation and sonication were investigated for ATP extraction from oocysts. Incubation in acidified HBSS was insufficient to extract ATP from oocysts, but a linear relationship between the number of oocysts and the concentration of ATP extracted was observed in the test of excystation and sonication treatments. Sonicationtreatment was able to extract ATP from oocysts more rapidly and precisely than excystation treatment. ATP amount per oocyst by sonication treatment (ATPs) was evaluated to be 2.9×10–8 μg on average, andits detection limit was 500 oocysts/100 μl. Ozone treatment experiments were conducted in batch condition to evaluate differences among ATP concentrations extracted, in vitro excystation and DAPI/PI permeability assays. ATPs assay was observed to have a linear relationship with DAPI/PI permeability assay (R2=0.98). As a result, ATP assay is applicable as a surrogate indicator of the viability of C. parvum, and is superior to in vitro excystation and DAPI/PI permeability assay, because of its rapid, accurate and simple procedure.

Effects of ozonation and chlorination on viability and infectivity of Cryptosporidium parvum oocysts

2000 ◽  
Vol 41 (7) ◽  
pp. 39-46 ◽  
Author(s):  
T. Hirata ◽  
D. Chikuma ◽  
A. Shimura ◽  
A. Hashimoto ◽  
N. Motoyama ◽  
...  
Keyword(s):  
Cryptosporidium Parvum ◽  
Experimental Studies ◽  
Free Chlorine ◽  
Log Reduction ◽  
Cryptosporidium Parvum Oocysts ◽  
Permeability Assay

Experimental studies on ozonation and chlorination were conducted to determine capacity for inactivating Cryptosporidium parvum oocysts in batch modes at pH 7, 20°C. In both experiments, the log reduction of animal infectivity was linear and clearly decreased as disinfectant CT product increased. However, the curve of reduction in viability determined by both in vitro excystation assay and DAPI/PI permeability assay exhibited a shoulder. The CT products of ozone per 1 log reduction in infectivity were 3 mg middot min/L for 0.5 mg/L and 1.5 mg · min/L for 0.3 mg/L, while viability determined by in vitro excystation was reduced by only 0.2 logs for the CT product of 3 mg · min/L. In the chlorination experiment, thereduction of animal infectivity was up to 3 logs for the CT product of 2,700 mg middot; min/L, while reduction of viability was smaller at 0.16 logs in in vitro excystation and 0.04 logs in DAPI/PI permeability (in PI exclusion) for the same CT product. The CT product of free chlorine per 1 log reduction in infectivity was estimated to be in the range of 800 to 900 mg · min/L.

Survival ofCryptosporidium parvumoocysts in source separated human urine

1999 ◽  
Vol 45 (9) ◽  
pp. 740-746 ◽  
Author(s):  
Caroline E Höglund ◽  
Thor Axel B Stenström
Keyword(s):  
Cryptosporidium Parvum ◽  
Human Urine ◽  
Ph Effects ◽  
Primary Methods ◽  
Dye Permeability ◽  
Cryptosporidium Parvum Oocysts ◽  
Microbial Risks ◽  
Permeability Assay ◽  
Made In

The survival of Cryptosporidium parvum in source separated urine was investigated as part of a broader study on microbial risks associated with the reuse of human urine for sustainable agriculture. A dye permeability assay and in vitro excystation were the primary methods used to assess viability. In the collected urine most of the nitrogen is present as ammonia and the pH is generally around 9. Parallel investigations were made in buffers to compare possible toxic effects of urine to actual pH effects. Oocysts in the untreated urine were inactivated below the detection limit (1/300) within 63 days. This inactivation rate was significantly higher (P < 0.01) than in urine adjusted to pH 5 or 7 according to the dye permeability assay. The corresponding difference between different pH values was not seen in buffers, suggesting that the antiprotozoan effect of urine was mediated by other factors besides pH. The Swedish practice of storing urine for six months before its use thus appears satisfactory for the inactivation of Cryptosporidium oocysts.Key words: Cryptosporidium parvum, oocysts, human urine, survival, source separation.

Efficacy of measuring cellular ATP levels to determine the inactivation of pulsed UV treated Cryptosporidium parvum oocysts suspended in water

2013 ◽  
Vol 13 (2) ◽  
pp. 202-213 ◽  
Author(s):  
Mary Garvey ◽  
Jennifer Hayes ◽  
Eoghan Clifford ◽  
Dominik Kirf ◽  
Neil Rowan
Keyword(s):  
Cell Culture ◽  
Cryptosporidium Parvum ◽  
Quantitative Polymerase Chain Reaction ◽  
In Vitro Cell Culture ◽  
Atp Assay ◽  
Interesting Insight ◽  
Novel Approach ◽  
Cryptosporidium Parvum Oocysts ◽  
Uv Dose

This constitutes the first study to report on the use of a novel approach to determine inactivation in PUV-irradiated Cryptosporidium parvum oocysts suspended in water based on the measurement of cellular adenosine triphosphate (ATP) concentration. This study also compares the efficiency of a novel ATP assay to that of using the combined in vitro HCT-8 cell culture – quantitative polymerase chain reaction (qPCR) method for determining the inactivation in the waterborne pathogen C. parvum after exposure to pulsed UV (PUV) treatments. Findings were compared with using the combined cell culture-qPCR approach for determining oocyst viability in similarly treated samples. PUV effectively killed C. parvum with a 5.4 log10 loss in oocyst viability after exposure to a UV dose of 8.5 μJ/cm2 as determined by the in vitro cell culture – qPCR assay. The ATP assay was shown to be significantly less effective in measuring loss of oocyst viability in similarly PUV-irradiated samples for all combination of treatment regimes studied. Measurement of cellular ATP is not suitable as an indicator of the disinfection efficiency of PUV-irradiated C. parvum oocysts. The levels of ATP present post PUV-irradiated samples suggests that significant cellular activity remained in treated oocysts that are unable to invade human HCT-8 cells. However, further studies are merited to investigate factors that might aid repair post PUV treatments in this water-borne human parasite. Use of this ATP assay offers an interesting insight into loss of infectivity in PUV-treated C. parvum. This rapid assay does not appear suitable for investigating or optimizing treatment efficiency under varying operational settings as it detects PUV-treated oocysts at levels significantly higher compared with using the in vitro cell culture-qPCR infectivity assay. Overestimation of survivors by the ATP assay may suggest that a sub-population of C. parvum oocysts may exist in a viable but non-infectious state or may require a period of resuscitation to facilitate photo-repair (if possible) that may lead to regained ability to infective human hosts.

Microwave Inactivation of Cyclospora cayetanensis Sporulation and Viability of Cryptosporidium parvum Oocysts

2006 ◽  
Vol 69 (8) ◽  
pp. 1957-1960 ◽  
Author(s):  
YNES R. ORTEGA ◽  
JYEYIN LIAO
Keyword(s):  
Cryptosporidium Parvum ◽  
Propidium Iodide ◽  
Cryptosporidium Oocyst ◽  
Cooking Time ◽  
Cyclospora Cayetanensis ◽  
The Third ◽  
Infectivity Assay ◽  
Cryptosporidium Oocysts ◽  
Cryptosporidium Parvum Oocysts ◽  
Cooking Times

The efficacy of microwave heating on the viability of Cryptosporidium parvum oocysts and on the sporulation of Cyclospora cayetanensis oocysts for various periods of cooking times (0, 10, 15, 20, 30, and 45 s) at 100% power was determined. Cyclospora oocysts were stored in 2.5% dichromate at 23°C for 2 weeks, and sporulation rates were then determined. The 4′,6-diamidino-2-phenylindole and propidium iodide vital stain and the neonate animal infectivity assay determined Cryptosporidium oocyst viability. Cryptosporidium oocysts could be completely inactivated with as little as 20 s of cooking time, whereas Cyclospora sporulation was observed up to 45 s. Two of the examined microwave ovens were more effective at reducing sporulation and viability than the third one. Because of the variability of temperature achieved by the various ovens, cooking time was not an accurate parameter for parasite inactivation. Cryptosporidium oocysts could be inactivated only when temperatures of 80°C or higher were reached in the microwave ovens.

Influence of US EPA 1622 method successive steps on the viability of Cryptosporidium oocysts

2000 ◽  
Vol 41 (7) ◽  
pp. 189-196 ◽  
Author(s):  
K. Guyot ◽  
M. F. Gireaudot-Liepmann ◽  
A. Cabon ◽  
I. Riveau-Ricard ◽  
M. Lange ◽  
...  
Keyword(s):  
Water Samples ◽  
Tap Water ◽  
High Recovery ◽  
The Us ◽  
Cryptosporidium Oocysts ◽  
Cartridge Filter ◽  
Us Epa ◽  
Cryptosporidium Parvum Oocysts ◽  
Elution Step

Viable Cryptosporidium parvum oocysts were processed by the US EPA 1622 method to determine if the procedure that requires successive filtration, elutionand centrifugation alters their integrity and viability (determined by in vitro excystation). Oocyst seeded in tap water samples were also used to evaluate recovery efficiencies and impact of the whole procedure on oocyst viability. Filtration through Envirochek Gelman cartridge was found not to damage oocysts. The use of Laureth-12 buffer during the elution step was shown to lead to greater spontaneous oocysts excystation than other phosphate buffers containing between 80 and/or SDS (like the Gelman buffer). However, this drawback was widely balanced against the best efficiency of this buffer to elute oocysts captured by the cartridge filter and therefore against its high recovery efficiency. Thus, in water samples in which the oocyst concentration is expected to be low, it is more advantageous to employ the Laureth-12 buffer for the elution through it can influence viability. Centrifugation speeds (1,000–5,000 g) did not alter oocysts.

Aging of Cryptosporidium parvum oocysts in river water and their susceptibility to disinfection by chlorine and monochloramine

1998 ◽  
Vol 44 (12) ◽  
pp. 1154-1160 ◽  
Author(s):  
Christian Chauret ◽  
Kerry Nolan ◽  
Ping Chen ◽  
Susan Springthorpe ◽  
Syed Sattar
Keyword(s):  
Cryptosporidium Parvum ◽  
Environmental Fate ◽  
Water Environment ◽  
Water Disinfection ◽  
Ottawa River ◽  
In Situ Conditions ◽  
Cryptosporidium Parvum Oocysts ◽  
St Lawrence River

Cryptosporidium parvum oocysts were aged in waters from both the St. Lawrence River and the Ottawa River. In situ survival experiments were carried out by incubating the oocysts in either dialysis cassettes or microtubes floated into an overflow tank. A significant portion of the oocysts survived in the test waters for several weeks. Oocyst survival in the St. Lawrence River was better in membrane-filtered (0.2-µm-pore diameter) water than in unfiltered water, suggesting that biological antagonism may play a role in the environmental fate of the parasite. Oocysts aged in river waters under in situ conditions and control oocysts kept refrigerated in synthetic water (100 ppm as CaCO3; pH 7.0) were subjected to the same disinfection protocol. Aged oocysts were at least as resistant as, if not more resistant than, the control oocysts to disinfection. This indicates that the oocysts surviving in the water environment may be just as difficult to inactivate by potable water disinfection as freshly shed oocysts. Therefore, water treatment should not be based on the assumption that environmental oocysts may be more easily inactivated than freshly shed oocysts. First-order kinetics die-off rates varied from one river to another (from 0.013 to 0.039 log10·day-1) and from one experiment to another with water from the same river collected at different times. Calculation of the die-off rates based on either in vitro excystation or in vitro excystation in combination with total counts (overall die-off rates) showed that the assessment of oocyst viability by microscopic methods must account for the total oocyst loss observed during long-term inactivation assays of river waters.Key words: Cryptosporidium, survival, disinfection, biological antagonism.

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