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.

scholarly journals Cryptosporidium infection in non-human hosts in Malawi

2009 ◽  
Vol 76 (4) ◽  
Author(s):  
Z. Banda ◽  
Rosely A.B. Nichols ◽  
A.M. Grimason ◽  
H.V. Smith
Keyword(s):  
Cryptosporidium Parvum ◽  
Rainy Season ◽  
18S Rrna ◽  
Cryptosporidium Oocyst ◽  
Dry Season ◽  
Cool Season ◽  
Cryptosporidium Hominis ◽  
Adult Cattle ◽  
Faecal Samples ◽  
Cryptosporidium Oocysts

Of 1 346 faecal samples from the Chikwawa and Thyolo districts of Malawi, analysed for the presence of Cryptosporidium oocysts between October 2001 and May 2003, 61.3 % were from cattle (29.8 % of these were from calves < 6 months old). Cryptosporidium oocysts were detected during all three seasons studied in Chikwawa and Thyolo. In Chikwawa, 13.6 % of adult cattle and 11.7 % of calves were infected, compared to 28.9 % of adult cattle and 36.7 % of calves in Thyolo. Dependent on season, between 7.8 % and 37.7 % (Chikwawa) and 16.7 % and 39.3 % (Thyolo) of cattle samples contained oocysts. In Chikwawa, the highest percentage of infections occurred in the cool season, whereas in Thyolo, the highest percentage of infections occurred in the dry season. Faecal samples from goats [n = 225], pigs [n = 92], sheep [n = 6]), rabbits, guinea pigs, chickens, ducks, turkeys, doves and guinea fowls were also analysed. Up to 5.6 % of goat samples contained oocysts in Chikwawa, compared to between 16.7 % and 39.3 % in Thyolo. Again, in Chikwawa, the highest percentage of infections occurred in the cool season and the lowest in the rainy season, whereas, in Thyolo, the highest percentage of infections occurred in the dry season and the lowest in the cool season. In pigs, more infections were detected in the dry season in Chikwawa, but infections in the cool season were similar (17.7 %), whereas in Thyolo, infections occurred in all three seasons (17.9 % in the rainy season, 25 % in the cool season and 60 % in the dry season). Often diarrhoeic, oocyst positive cattle faecal samples collected from Chikwawa and subjected to PCR-RFLP, four oocyst positive samples (two from heifers, one from a cow and one unknown) were amplified at an 18S rRNA and Cryptosporidium oocyst wall protein (COWP) loci. RFLP of the 18S rRNA locus indicated that Cryptosporidium parvum, Cryptosporidium hominis, Cryptosporidium bovis and / or Cryptosporidium ryanae DNA, or a mixture of them was present. Cryptosporidium parvum DNA was identified in one sample that amplified at the COWP locus, indicating the presence of the major zoonotic Cryptosporidium species in Malawi.

scholarly journals Field scale quantification of microbial transport from bovine faeces under simulated rainfall events

2006 ◽  
Vol 5 (1) ◽  
pp. 83-95 ◽  
Author(s):  
Christobel M. Ferguson ◽  
Cheryl M. Davies ◽  
Christine Kaucner ◽  
Nicholas J. Ashbolt ◽  
Martin Krogh ◽  
...  
Keyword(s):  
Cryptosporidium Oocyst ◽  
Experimental Treatment ◽  
Bare Soil ◽  
Simulated Rainfall ◽  
Rainfall Events ◽  
E Coli ◽  
Artificial Rainfall ◽  
Cryptosporidium Oocysts ◽  
Cryptosporidium Parvum Oocysts ◽  
Experimental Soil

The dispersion and transport of Cryptosporidium parvum oocysts, Escherichia coli and PRD1 bacteriophage seeded into artificial bovine faecal pats was studied during simulated rainfall events. Experimental soil plots were divided in two, one sub-plot with bare soil and the other with natural vegetation. Simulated rainfall events of 55 mm.h-1 for 30 min were then applied to the soil plots. Each experimental treatment was performed in duplicate and consisted of three sequential artificial rainfall events (‘Runs’): a control run (no faecal pats); a fresh faecal pat run (fresh faecal pats); and an aged faecal pat run (one week aged faecal pats). Transportation efficiency increased with decreasing size of the microorganism studied; Cryptosporidium oocysts were the least mobile followed by E. coli and then PRD1 phage. Rainfall events mobilised 0.5 to 0.9% of the Cryptosporidium oocysts, 1.3‒1.4% of E. coli bacteria, and 0.03‒0.6% of PRD1 bacteriophages from the fresh faecal pats and transported them a distance of 10 m across the bare soil sub-plots. Subsequent rainfall events applied to aged faecal pats only mobilised 0.01‒0.06% of the original Cryptosporidium oocyst load, between 0.04 and 15% of the E. coli load and 0.0006‒0.06% of PRD1 bacteriophages, respectively.

scholarly journals Effect of Batch-Process Solar Disinfection on Survival of Cryptosporidium parvum Oocysts in Drinking Water

2005 ◽  
Vol 71 (3) ◽  
pp. 1653-1654 ◽  
Author(s):  
F. Méndez-Hermida ◽  
J. A. Castro-Hermida ◽  
E. Ares-Mazás ◽  
S. C. Kehoe ◽  
K. G. McGuigan
Keyword(s):  
Drinking Water ◽  
Standard Deviation ◽  
Cryptosporidium Parvum ◽  
Propidium Iodide ◽  
Batch Process ◽  
Simulated Sunlight ◽  
Solar Disinfection ◽  
Suckling Mice ◽  
Cryptosporidium Parvum Oocysts

ABSTRACT The results of batch-process solar disinfection (SODIS) of Cryptosporidium parvum oocysts in water are reported. Oocyst suspensions were exposed to simulated sunlight (830 W m−2) at 40°C. Viability assays (4′,6′-diamidino-2-phenylindole [DAPI]/propidium iodide and excystation) and infectivity tests (Swiss CD-1 suckling mice) were performed. SODIS exposures of 6 and 12 h reduced oocyst infectivity from 100% to 7.5% (standard deviation = 2.3) and 0% (standard deviation = 0.0), respectively.

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

2005 ◽  
Vol 71 (5) ◽  
pp. 2479-2483 ◽  
Author(s):  
Joaquin Quilez ◽  
Caridad Sanchez-Acedo ◽  
Catalina Avendaño ◽  
Emilio del Cacho ◽  
Fernando Lopez-Bernad
Keyword(s):  
Cryptosporidium Parvum ◽  
Deleterious Effect ◽  
In Vitro Assays ◽  
Infectivity Assay ◽  
Vital Dyes ◽  
Vital Dye ◽  
Potential Efficacy ◽  
Infected Goat ◽  
Cryptosporidium Parvum Oocysts

ABSTRACT 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.

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.

Inactivation of Cryptosporidium parvum oocysts and other microbes in water and wastewater by electrochemically generated mixed oxidants

2000 ◽  
Vol 41 (7) ◽  
pp. 127-134 ◽  
Author(s):  
M. J. Casteel ◽  
M. D. Sobsey ◽  
M. J. Arrowood
Keyword(s):  
Escherichia Coli ◽  
Cell Culture ◽  
Cryptosporidium Parvum ◽  
Propidium Iodide ◽  
Treated Wastewater ◽  
E Coli ◽  
Water And Wastewater ◽  
Cryptosporidium Parvum Oocysts ◽  
Cell Culture Assay ◽  
Escherichia Coli B

Alternative disinfectants of water and wastewater are needed because conventional chlorination is ineffective against C. parvum oocysts. Reliable indicators of disinfection efficacy against C. parvum also are needed. Mixedoxidants (MO) electrochemically generated from brine were evaluated in batch disinfection experiments for inactivation of C. parvum oocysts and Cl. perfringensspores in both oxidant demand-free (ODF) water and treated wastewater. Coliphage MS2 and Escherichia coli B were also tested under some conditions. C. parvum oocyst infectivity was quantified by cell culture assay, and the dyes DAPI (4′,6-diamidino-2-phenylindole) and propidium iodide (PI) were used to assess oocyst viability in wastewater experiments. In treated wastewater dosed with 10–13 mg/L MO, inactivation after 90 minutes was about 3 log10 for C. parvum and about 2.5 log10 for Cl. perfringens spores; MS2 and E. coli were rapidly inactivated by &gt; 5 log10. In ODF water, a 4 mg/L dose of MO inactivated ∼3 log10 of C. parvum oocysts and ∼1.5 log10 of Cl. perfringens spores. Inactivation of C. parvum oocysts and Cl. perfringensspores was less extensive at a lower MO dose of 2 mg/L. The use of DAPI and PI to determine viability of oocysts treated with MO did not correlate with, and greatly overestimated, cell culture infectivity. At practical doses and contact times, MO disinfection of water and wastewater achieves appreciable inactivation of both C. parvum oocysts and Cl. perfringens spores. Cl. perfringens spores reliably indicated oocyst inactivation by MO, but E. coli and coliphage MS2 were inactivated much too rapidly to indicate C. parvum inactivation.

Effect of Chlorine, Blanching, Freezing, and Microwave Heating on Cryptosporidium parvum Viability Inoculated on Green Peppers

2012 ◽  
Vol 75 (5) ◽  
pp. 936-941 ◽  
Author(s):  
G. L. M. C. DUHAIN ◽  
A. MINNAAR ◽  
E. M. BUYS
Keyword(s):  
Microwave Heating ◽  
Cryptosporidium Parvum ◽  
Propidium Iodide ◽  
Control Point ◽  
Minimally Processed ◽  
Critical Control Point ◽  
Propidium Iodide Staining ◽  
Cryptosporidium Parvum Oocysts ◽  
Minimally Processed Vegetables ◽  
Effect Of Chlorine

Cryptosporidium parvum oocysts have been found on the surface of vegetables in both developed and developing countries. C. parvum can contaminate vegetables via various routes, including irrigation water. This study investigated the effect of individual treatments of chlorine, blanching, blast freezing, and microwave heating, as well as combined treatments of chlorine and freezing, and chlorine and microwave heating on the viability of C. parvum oocysts inoculated on green peppers. The viability of the oocysts after the treatments was assessed using propidium iodide and a flow cytometer. Based on the propidium iodide staining, the chlorine treatments did not affect the viability of the oocysts. Blast freezing significantly inactivated 20% of the oocysts. Microwave heating and blanching significantly inactivated 93% of oocysts. Treatment with chlorine followed by blast freezing did not affect the viability of the oocysts significantly. Treatment with chlorine and microwave heating was significantly more effective than microwave heating alone and inactivated 98% of the oocysts. The study indicates that C. parvum oocysts are sensitive to heat and, to some extent, to blast freezing, but are resistant to chlorine. Therefore, the use of chlorine during vegetable processing is not a critical control point for C. parvum oocysts, and the consumption of raw or minimally processed vegetables may constitute a health risk as C. parvum oocysts can still be found viable on ready-to-eat, minimally processed vegetables.

Impact of purification procedures on the viability and infectivity of Cryptosporidium parvum oocysts

2000 ◽  
Vol 41 (7) ◽  
pp. 23-29 ◽  
Author(s):  
T. R. Slifko ◽  
A. Coulliette ◽  
D. E. Huffman ◽  
J. B. Rose
Keyword(s):  
Cryptosporidium Parvum ◽  
New Technologies ◽  
New Technology ◽  
Cryptosporidium Oocyst ◽  
Cesium Chloride ◽  
Considerable Variability ◽  
Before And After ◽  
Cryptosporidium Parvum Oocysts ◽  
Cell Culture Assay

The development of new technologies for Cryptosporidium oocyst detection as well as inactivation and removal is at the forefront of research objectives for the drinking and wastewater industries. One of the major issues associated with testing new technology is determining oocyst viability and infectivity before and after treatment. Because oocysts must be isolated from feces, preparation and pretreatment procedures may affect oocyst infectivity and potentially confound results obtained during survival and disinfection studies. The principal objective of this study was to evaluate the effects of preparation and pretreatment on C. parvum oocyst (Ames, Iowa isolate) infectivity in an in vitro cell culture assay. In vitro excystation, sporozoite yield, and vital dye exclusion using DAPI and PI were used to test viability. A matrix of purification procedures using two defatting agents (ethyl acetate and ethyl ether), cesium chloride (CsCl) and Sheather's solution was evaluated. Effects of immuno-magnetic separation (IMS) and bleach treatment were also assessed. Oocysts purified using CsCl alone showed the most consistent infection from experiment to experiment, compared to preparations that were purified using a defatting agent. Defatting agents, IMS and bleach treatment had no detrimental effects on oocyst infectivity, however, considerable variability between oocyst lots was observed. This study determined that both purification processes and age affect oocyst infectivity.

Novel Methodology for the Detection of Cryptosporidium parvum: A Comparison of Cooled Charge Couple Devices (CCD) and Flow Cytometry

1993 ◽  
Vol 27 (3-4) ◽  
pp. 89-92 ◽  
Author(s):  
Andrew Campbell ◽  
Lucy Robertson ◽  
Huw Smith
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Cryptosporidium Parvum ◽  
Propidium Iodide ◽  
Three Dimensional ◽  
Flow Cytometer ◽  
Cryptosporidium Oocysts ◽  
Exact Size

Flow cytometry and CCD were assessed for their usefialness in the detection of oocysts of Cryptosporidium. Oocysts were labelled with FITC-monoclonal antibody and with the nuclear stains 4’6-diamidino-2-phenyl indole (DAPI) and propidium iodide (PI) before analysis by flow cytometer and CCD. Although the flow cytometer tested was able to concentrate particles and place them on a slide for subsequent viewing, readily sorting oocysts from contaminating debris, confirmation by fluoresence microscopy was still essential, even when additional parameters such as the inclusion of DAPI is used. Initial observations from the use of CCD, however, suggested that screening samples for oocysts was a possibility. Three dimensional visualisation of individual oocysts can map precisiely both the detailed morphology and the exact size of oocysts, thereby making confirmation by fluoresence microscopy unneccesary.

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