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.

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.

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.

Comparative effectiveness of UV wavelengths for the inactivation of Cryptosporidium parvum oocysts in water

2001 ◽  
Vol 43 (12) ◽  
pp. 171-174 ◽  
Author(s):  
K. G. Linden ◽  
G. Shin ◽  
M. D. Sobsey
Keyword(s):  
Uv Radiation ◽  
Cryptosporidium Parvum ◽  
Mercury Vapour ◽  
Wide Range ◽  
Mercury Vapour Lamp ◽  
A Cell ◽  
Cryptosporidium Parvum Oocysts ◽  
Cell Culture Assay ◽  
Uv Lamps ◽  
Collimated Beam

Cryptosporidium parvum oocysts in water were exposed to distinct wavelength bands of collimated beam ultraviolet (UV) radiation across the germicidal UV wavelength range (210-295 nm) that were emitted from a medium pressure (MP) mercury vapour lamp. The dose of UV radiation transmitted though each narrow bandpass filter was measured utilising potassium ferrioxalate actinometry. Oocyst infectivity was determined using a cell culture assay and titre was expressed as an MPN. The log10 inactivation for each band of radiation was determined for a dose of 2 mJ/cm2. Doses from all wavelengths between 250-275 nm resulted in approximately 2 log10 inactivation of Cryptosporidium parvum oocyst infectivity while doses with wavelengths higher and lower than this range were less effective. Because polychromatic radiation from MP UV lamps had about the same germicidal activity between the wavelengths of 250-275 nm for inactivation of oocyst infectivity, there was no unique advantage of MP UV over low pressure (LP) UV except for the simultaneous delivery of a wide range of germicidal wavelengths.

The effect of blueberry extracts on Giardia duodenalis viability and spontaneous excystation of Cryptosporidium parvum oocysts, in vitro

Methods ◽  
2007 ◽  
Vol 42 (4) ◽  
pp. 339-348 ◽  
Author(s):  
J.-P. Anthony ◽  
L. Fyfe ◽  
D. Stewart ◽  
G.J. McDougall ◽  
H.V. Smith
Keyword(s):  
Cryptosporidium Parvum ◽  
Giardia Duodenalis ◽  
Cryptosporidium Parvum Oocysts

scholarly journals A simple, scalable approach to building a cross-platform transcriptome atlas

2020 ◽  
Author(s):  
Paul W Angel ◽  
Nadia Rajab ◽  
Yidi Deng ◽  
Chris M Pacheco ◽  
Tyrone Chen ◽  
...  
Keyword(s):  
Blood Cell ◽  
Single Cell ◽  
New Technologies ◽  
New Technology ◽  
Cell Types ◽  
Variance Partitioning ◽  
Primary Data ◽  
Batch Correction

ABSTRACTGene expression atlases have transformed our understanding of the development, composition and function of human tissues. New technologies promise improved cellular or molecular resolution, and have led to the identification of new cell types, or better defined cell states. But as new technologies emerge, information derived on old platforms becomes obsolete. We demonstrate that it is possible to combine a large number of different profiling experiments summarised from dozens of laboratories and representing hundreds of donors, to create an integrated molecular map of human tissue. As an example, we combine 850 samples from 38 platforms to build an integrated atlas of human blood cells. We achieve robust and unbiased cell type clustering using a variance partitioning method, selecting genes with low platform bias relative to biological variation. Other than an initial rescaling, no other transformation to the primary data is applied through batch correction or renormalisation. Additional data, including single-cell datasets, can be projected for comparison, classification and annotation. The resulting atlas provides a multi-scaled approach to visualise and analyse the relationships between sets of genes and blood cell lineages, including the maturation and activation of leukocytes in vivo and in vitro.In allowing for data integration across hundreds of studies, we address a key reproduciblity challenge which is faced by any new technology. This allows us to draw on the deep phenotypes and functional annotations that accompany traditional profiling methods, and provide important context to the high cellular resolution of single cell profiling. Here, we have implemented the blood atlas in the open access Stemformatics.org platform, drawing on its extensive collection of curated transcriptome data. The method is simple, scalable and amenable for rapid deployment in other biological systems or computational workflows.Graphical abstractRecursive approach to generating a multi-scaled atlas. Top panel: The method integrates data from all cell types in the Stemformatics database, and shows clear division of samples into global categories of stromal, pluripotent or blood (inset) cell types. Bottom panel: Integration of only the blood cell subsets provides a blood atlas. Projection of external samples (green) onto the blood atlas. Samples are coloured by curated annotations derived from the original studies, and can be viewed at Stemformatics.org

Applications of in-vitro cell culture for measuring infectivity and inactivation of Cryptosporidium parvum

2004 ◽  
Vol 4 (2) ◽  
pp. 87-92
Author(s):  
P.A. Rochelle
Keyword(s):  
Cell Culture ◽  
Cryptosporidium Parvum ◽  
Rt Pcr ◽  
In Vitro Cell Culture ◽  
Infectivity Assay ◽  
Public Health Officials ◽  
Practical Tool ◽  
Cell Culture Assay ◽  
Evaluating Methods

Cryptosporidium parvum presents a significant problem for the water industry and public health officials because of its prevalence in sources of drinking water and its resistance to chlorine-based disinfectants; there is an urgent need for alternative, more effective disinfection strategies. Therefore, developing and evaluating methods for assessing the infectivity and inactivation of C. parvum oocysts are of paramount importance. Infectivity assays based on in-vitro cell culture have been developed as alternatives to human and animal-based assays to overcome ethical, cost, and practicality issues. Data obtained over a two-year period with an HCT-8 cell culture/RT-PCR infectivity assay generated an ID50 of 99 oocysts (95% CI: 84-117) and demonstrated that the cell culture assay was equivalent to the standard CD-1 mouse model for measuring infectivity of C. parvum oocysts. Aggregate data generated over two years using the HCT-8 cell culture/RT-PCR assay to measure UV disinfection of C. parvum demonstrated that 2.4 mJ/cm2 and 4.9 mJ/cm2 were necessary to achieve 1-log10 and 2-log10 inactivation, respectively. This work demonstrated that an HCT-8 cell culture-based infectivity coupled with RT-PCR for detecting C. parvum infections is a practical tool that can provide valuable information about the efficacy of disinfectants and the infectivity of oocysts in environmental waters.

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