Cell Culture Infectivity to Assess Chlorine Disinfection of Cryptosporidium Oocysts in Water

2019 ◽  
pp. 283-302
Author(s):  
Jennifer L. Murphy ◽  
Michael J. Arrowood
Keyword(s):  
Cell Culture ◽  
Chlorine Disinfection ◽  
Cryptosporidium Oocysts

scholarly journals A Spore Counting Method and Cell Culture Model for Chlorine Disinfection Studies of Encephalitozoon syn.Septata intestinalis

2000 ◽  
Vol 66 (4) ◽  
pp. 1266-1273 ◽  
Author(s):  
D. M. Wolk ◽  
C. H. Johnson ◽  
E. W. Rice ◽  
M. M. Marshall ◽  
K. F. Grahn ◽  
...  
Keyword(s):  
Cell Culture ◽  
Quantitative Measure ◽  
Rabbit Kidney ◽  
In Vitro Growth ◽  
Spectrophotometric Methods ◽  
Log Reduction ◽  
Chlorine Disinfection ◽  
Infective Dose ◽  
Before And After

ABSTRACT The microsporidia have recently been recognized as a group of pathogens that have potential for waterborne transmission; however, little is known about the effects of routine disinfection on microsporidian spore viability. In this study, in vitro growth ofEncephalitozoon syn. Septata intestinalis, a microsporidium found in the human gut, was used as a model to assess the effect of chlorine on the infectivity and viability of microsporidian spores. Spore inoculum concentrations were determined by using spectrophotometric measurements (percent transmittance at 625 nm) and by traditional hemacytometer counting. To determine quantitative dose-response data for spore infectivity, we optimized a rabbit kidney cell culture system in 24-well plates, which facilitated calculation of a 50% tissue culture infective dose (TCID50) and a minimal infective dose (MID) for E. intestinalis. The TCID50 is a quantitative measure of infectivity and growth and is the number of organisms that must be present to infect 50% of the cell culture wells tested. The MID is as a measure of a system's permissiveness to infection and a measure of spore infectivity. A standardized MID and a standardized TCID50 have not been reported previously for any microsporidian species. Both types of doses are reported in this paper, and the values were used to evaluate the effects of chlorine disinfection on the in vitro growth of microsporidia. Spores were treated with chlorine at concentrations of 0, 1, 2, 5, and 10 mg/liter. The exposure times ranged from 0 to 80 min at 25°C and pH 7. MID data for E. intestinalis were compared before and after chlorine disinfection. A 3-log reduction (99.9% inhibition) in the E. intestinalis MID was observed at a chlorine concentration of 2 mg/liter after a minimum exposure time of 16 min. The log10 reduction results based on percent transmittance-derived spore counts were equivalent to the results based on hemacytometer-derived spore counts. Our data suggest that chlorine treatment may be an effective water treatment for E. intestinalis and that spectrophotometric methods may be substituted for labor-intensive hemacytometer methods when spores are counted in laboratory-based chlorine disinfection studies.

scholarly journals Removal of Cryptosporidium by wastewater treatment processes: a review

2015 ◽  
Vol 14 (1) ◽  
pp. 1-13 ◽  
Author(s):  
Abidelfatah M. Nasser
Keyword(s):  
Animal Species ◽  
Protozoan Parasite ◽  
High Rate ◽  
Environmental Stability ◽  
Infectious Dose ◽  
Chlorine Disinfection ◽  
Untreated Wastewater ◽  
Treatment Processes ◽  
Cryptosporidium Oocysts ◽  
Suitable Treatment

Cryptosporidium is a protozoan parasite that infects humans and various animal species. The environmental stability and the low infectious dose of Cryptosporidium facilitate its transmission by water and food. Discharge of untreated wastewater may result in waterborne or foodborne Cryptosporidium outbreaks, therefore a suitable treatment may prevent its dissemination. Most studies on the prevalence of Cryptosporidium oocysts in wastewater have reported a concentration range between 10 and 200 oocysts/L and a prevalence of 6 to 100%. Activated sludge has been found to be ineffective for the removal of Cryptosporidium oocysts. Stabilization ponds and constructed wetlands are efficient for the reduction of Cryptosporidium from wastewater, especially when the retention time is longer than 20 days at suitable sunlight and temperature. High rate filtration and chlorine disinfection are inefficient for the reduction of Cryptosporidium from effluents, whereas ultrafiltration and UV irradiation were found to be very efficient for the reduction of Cryptosporidium oocysts. Adequate tertiary treatment may result in high quality effluent with low risk of Cryptosporidium for unrestricted irrigation and other non-potable applications.

Combination of cell culture and quantitative PCR (cc–qPCR) to assess disinfectants efficacy on Cryptosporidium oocysts under standardized conditions

2010 ◽  
Vol 167 (1) ◽  
pp. 43-49 ◽  
Author(s):  
M. Shahiduzzaman ◽  
V. Dyachenko ◽  
J. Keidel ◽  
R. Schmäschke ◽  
A. Daugschies
Keyword(s):  
Cell Culture ◽  
Quantitative Pcr ◽  
Cryptosporidium Oocysts

An in vitro method for detecting infectious Cryptosporidium oocysts with cell culture.

1997 ◽  
Vol 63 (9) ◽  
pp. 3669-3675 ◽  
Author(s):  
T R Slifko ◽  
D Friedman ◽  
J B Rose ◽  
W Jakubowski
Keyword(s):  
Cell Culture ◽  
In Vitro Method ◽  
Cryptosporidium Oocysts

scholarly journals Use of Integrated Cell Culture-PCR To Evaluate the Effectiveness of Poliovirus Inactivation by Chlorine

2000 ◽  
Vol 66 (5) ◽  
pp. 2267-2268 ◽  
Author(s):  
Felisa Blackmer ◽  
Kelly A. Reynolds ◽  
Charles P. Gerba ◽  
Ian L. Pepper
Keyword(s):  
Cell Culture ◽  
Contact Time ◽  
Free Chlorine ◽  
Chlorine Concentration ◽  
Chlorine Disinfection ◽  
Complete Inactivation ◽  
Cell Culture Assay ◽  
Current Standards

ABSTRACT Current standards, based on cell culture assay, indicate that poliovirus is inactivated by 0.5 mg of free chlorine per liter after 2 min; however, integrated cell culture-PCR detected viruses for up to 8 min of exposure to the same chlorine concentration, requiring 10 min for complete inactivation. Thus, the contact time for chlorine disinfection of poliovirus is up to five times greater than previously thought.

A chemiluminescent immunofocus assay (CIFA) for non-microscopic enumeration of Cryptosporidium parvum infectivity in cell culture

2003 ◽  
Vol 47 (3) ◽  
pp. 137-142 ◽  
Author(s):  
O.D. Simmons ◽  
M.D. Sobsey
Keyword(s):  
Cell Culture ◽  
Drinking Water ◽  
Cryptosporidium Parvum ◽  
Microscopic Examination ◽  
Detection System ◽  
Detection Methods ◽  
Biomedical Analysis ◽  
Mammalian Cell Lines ◽  
Cryptosporidium Oocysts

As Cryptosporidium parvum continues to cause waterborne disease, despite extensive efforts by drinking water suppliers and regulators, it is important to have reliable and convenient methods for detection of this pathogen in wastewater discharges, environmental source waters and finished drinking water supplies. In order to better understand the health risks of this organism, it is necessary that detection methods be able to distinguish between infectious and non-infectious Cryptosporidium oocysts in these environmental samples. Cryptosporidium infectivity assay systems based on infections in mice and on in vitro infections in continuous mammalian cell lines are available. Currently, these methods are impractical for routine analysis of water samples because they are tedious, lengthy and costly. These methods rely on careful microscopic examination or further analysis by PCR and then characterisation of the amplified DNA. Practical and affordable non-microscopic methods to determine Cryptosporidium infectivity are much needed for environmental analysis. A cell culture infectivity detection system was developed for infectious Cryptosporidium oocysts that does not rely on microscopic examination of samples to score results, is applicable to a variety of samples and has the potential to be used for routine water monitoring and other environmental or biomedical analysis. Using a chemiluminescent immunoassay, the discrete foci of developmental stages of Cryptosporidium in cell cultures are clearly visible as discrete objects in an image of the entire cell culture layer in a dish or on a slide. These objects are directly countable with the unaided eye and their identity can be further confirmed or verified by microscopic examination.

A T.E.M. study of mouse mammary epithelial cell secretory differentiation cultured on collagen and Matrigel

1991 ◽  
Vol 49 ◽  
pp. 188-189
Author(s):  
W.N. Bentham ◽  
V. Rocha
Keyword(s):  
Cell Culture ◽  
Mammary Epithelial ◽  
Secretory Process ◽  
Cell Culture System ◽  
Protein Maturation ◽  
Cell Culture Techniques ◽  
Culture Techniques ◽  
Mouse Mammary Epithelial Cell ◽  
Secretory Differentiation

It has been an interest of our lab to develop a mammary epethelial cell culture system that faithfully duplicates the in vivo condition of the lactating gland. Since the introduction of collagen as a matrix on which cells are cultivated other E.C.M. type matrices have been made available and are used in many cell culture techniques. We have previously demonstrated that cells cultured on collagen and Matrigel do not differentiate as they do in vivo. It seems that these cultures often produce cells that show a disruption in the secretory process. The appearance of large ribosomal studded vesicles, that specifically label with antibody to casein, suggest an interruption of both protein maturation and secretion at the E.R. to golgi transition. In this report we have examined cultures on collagen and Matrigel at relative high and low seeding densities and compared them to cells from the in vivo condition.

Solid-phase immunoelectron microscopy for rapid diagnosis of enteroviruses

1984 ◽  
Vol 42 ◽  
pp. 226-227 ◽  
Author(s):  
K. Pegg-Feige ◽  
F. W. Doane
Keyword(s):  
Electron Microscopy ◽  
Cell Culture ◽  
Immunoelectron Microscopy ◽  
Detection Method ◽  
Solid Phase ◽  
Protein A ◽  
Plant Viruses ◽  
Rapid Diagnosis ◽  
Virus Diagnosis ◽  
Antiviral Antibody

Immunoelectron microscopy (IEM) applied to rapid virus diagnosis offers a more sensitive detection method than direct electron microscopy (DEM), and can also be used to serotype viruses. One of several IEM techniques is that introduced by Derrick in 1972, in which antiviral antibody is attached to the support film of an EM specimen grid. Originally developed for plant viruses, it has recently been applied to several animal viruses, especially rotaviruses. We have investigated the use of this solid phase IEM technique (SPIEM) in detecting and identifying enteroviruses (in the form of crude cell culture isolates), and have compared it with a modified “SPIEM-SPA” method in which grids are coated with protein A from Staphylococcus aureus prior to exposure to antiserum.

Automated cell counting of astrocytes on patterned substrates containing aliphatic and charged properties

1995 ◽  
Vol 53 ◽  
pp. 974-975
Author(s):  
W. Shain ◽  
H. Ancin ◽  
H.C. Craighead ◽  
M. Isaacson ◽  
L. Kam ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Attachment ◽  
Culture Method ◽  
Cell Counting ◽  
Data Sets ◽  
Nuclear Staining ◽  
Double Positive ◽  
A Cell ◽  
Wafer Test ◽  
Cell Densities

Neural protheses have potential to restore nervous system functions lost by trauma or disease. Nanofabrication extends this approach to implants for stimulating and recording from single or small groups of neurons in the spinal cord and brain; however, tissue compatibility is a major limitation to their practical application. We are using a cell culture method for quantitatively measuring cell attachment to surfaces designed for nanofabricated neural prostheses.Silicon wafer test surfaces composed of 50-μm bars separated by aliphatic regions were fabricated using methods similar to a procedure described by Kleinfeld et al. Test surfaces contained either a single or double positive charge/residue. Cyanine dyes (diIC18(3)) stained the background and cell membranes (Fig 1); however, identification of individual cells at higher densities was difficult (Fig 2). Nuclear staining with acriflavine allowed discrimination of individual cells and permitted automated counting of nuclei using 3-D data sets from the confocal microscope (Fig 3). For cell attachment assays, LRM5 5 astroglial cells and astrocytes in primary cell culture were plated at increasing cell densities on test substrates, incubated for 24 hr, fixed, stained, mounted on coverslips, and imaged with a 10x objective.

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