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.

scholarly journals Surface Characteristics and Cell Adhesion Behaviors of the Anodized Biomedical Stainless Steel

2020 ◽  
Vol 10 (18) ◽  
pp. 6275
Author(s):  
Heng-Jui Hsu ◽  
Chia-Yu Wu ◽  
Bai-Hung Huang ◽  
Chi-Hsun Tsai ◽  
Takashi Saito ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Culture ◽  
Stainless Steel ◽  
Cell Adhesion ◽  
Oxide Layer ◽  
Photoelectron Spectroscopy ◽  
X Ray ◽  
In Vitro Cell Culture ◽  
Cell Culture Assay

In this study, an electrochemical anodizing method was applied as surface modification of the 316L biomedical stainless steel (BSS). The surface properties, microstructural characteristics, and biocompatibility responses of the anodized 316L BSS specimens were elucidated through scanning electron microscopy, X-ray photoelectron spectroscopy, X-ray diffractometry, transmission electron microscopy, and in vitro cell culture assay. Analytical results revealed that the oxide layer of dichromium trioxide (Cr2O3) was formed on the modified 316L BSS specimens after the different anodization modifications. Moreover, a dual porous (micro/nanoporous) topography can also be discovered on the surface of the modified 316L BSS specimens. The microstructure of the anodized oxide layer was composed of amorphous austenite phase and nano-Cr2O3. Furthermore, in vitro cell culture assay also demonstrated that the osteoblast-like cells (MG-63) on the anodized 316L BSS specimens were completely adhered and covered as compared with the unmodified 316L BSS specimen. As a result, the anodized 316L BSS with a dual porous (micro/nanoporous) oxide layer has great potential to induce cell adhesion and promote bone formation.

Measuring Inactivation of Cryptosporidium Parvum by In Vitro Cell Culture

2003 ◽  
pp. 225-231 ◽  
Author(s):  
Paul A. Rochelle ◽  
Alexander A. Mofidi ◽  
Karl Linden ◽  
Ricardo De Leon
Keyword(s):  
Cell Culture ◽  
Cryptosporidium Parvum ◽  
In Vitro Cell Culture

scholarly journals Determination of the Infectivity of Cryopreserved Theileria annu-lata Sporozoites in Tick Derived Stabilates Iran Ak-93 Strain, by In Vivo and In Vitro Methods

2019 ◽  
Author(s):  
Hossein MODIRROUSTA ◽  
Gholamreza HABIBI ◽  
Parviz SHAYAN ◽  
Asghar AFSHARI ◽  
Ali MIRJALILI ◽  
...  
Keyword(s):  
Cell Culture ◽  
Infected Cell ◽  
Cell Line ◽  
Challenge Test ◽  
Infected Cell Line ◽  
Rt Pcr ◽  
In Vitro Cell Culture ◽  
Microscopic Inspection

Background: The protozoan parasite Theileria annulata is the causative agent of tropical theileriosis in cattle. Vaccination is recommended by administration of attenuated schizont-infected cell lines. The expected protective immunity post-vaccination can be demonstrated by challenge test through inoculation of highly virulent infective sporozoites. The aim of this study was to produce Hyalomma anatolicum anatolicum tick infected with T. annulata (local strain) for preparation of tick-derived sporozoite stabilates for molecular characterization and infectivity test assay. Methods: A local T. annulata strain was used for experimental infection of calves. A field isolate of H. a. anatolicum was isolated, laboratory-reared and infected by blood-feeding on Theileria infected above-mentioned calves. The infectivity of calf, tick and prepared stabilate were confirmed by clinical signs of theileriosis, microscopic inspection, RT-PCR and in vitro cell culture. Results: The tick stabilate was prepared and cryopreserved in liquid nitrogen. The infectivity of the tick stabilate was verified by in vivo bioassay, in vitro cell culture infection, microscopic inspection in salivary glands and RT-PCR assay. The in vitro produced cell line in this study was characterized by T. annulata Cytochrome b gene analyzing. Conclusion: The infectivity of a new prepared tick-derived sporozoite stabilate was confirmed in susceptible calves; by microscopically, post mortem, tick microscopic and molecular assays. Moreover, naïve PBMCs were transformed and proliferated by T. annulata infected tick stabilate to immortal T. annulata schizont infected cell line. The potent infective sporozoite tick derived stabilate could be used for vaccine efficacy and challenge test as well as in vaccine development.

scholarly journals Comparison of In Vitro Cell Culture and a Mouse Assay for Measuring Infectivity of Cryptosporidium parvum

2002 ◽  
Vol 68 (8) ◽  
pp. 3809-3817 ◽  
Author(s):  
Paul A. Rochelle ◽  
Marilyn M. Marshall ◽  
Jan R. Mead ◽  
Anne M. Johnson ◽  
Dick G. Korich ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cryptosporidium Parvum ◽  
Uv Light ◽  
Mdck Cells ◽  
Response Curves ◽  
Method Of Calculation ◽  
In Vitro Cell Culture ◽  
Genotype 2 ◽  
In Vitro Cell Cultures

ABSTRACT In vitro cell cultures were compared to neonatal mice for measuring the infectivity of five genotype 2 isolates of Cryptosporidium parvum. Oocyst doses were enumerated by flow cytometry and delivered to animals and cell monolayers by using standardized procedures. Each dose of oocysts was inoculated into up to nine replicates of 9 to 12 mice or 6 to 10 cell culture wells. Infections were detected by hematoxylin and eosin staining in CD-1 mice, by reverse transcriptase PCR in HCT-8 and Caco-2 cells, and by immunofluorescence microscopy in Madin-Darby canine kidney (MDCK) cells. Infectivity was expressed as a logistic transformation of the proportion of animals or cell culture wells that developed infection at each dose. In most instances, the slopes of the dose-response curves were not significantly different when we compared the infectivity models for each isolate. The 50% infective doses for the different isolates varied depending on the method of calculation but were in the range from 16 to 347 oocysts for CD-1 mice and in the ranges from 27 to 106, 31 to 629, and 13 to 18 oocysts for HCT-8, Caco-2, and MDCK cells, respectively. The average standard deviations for the percentages of infectivity for all replicates of all isolates were 13.9, 11.5, 13.2, and 10.7% for CD-1 mice, HCT-8 cells, Caco-2 cells, and MDCK cells, respectively, demonstrating that the levels of variability were similar in all assays. There was a good correlation between the average infectivity for HCT-8 cells and the results for CD-1 mice across all isolates for untreated oocysts (r = 0.85, n = 25) and for oocysts exposed to ozone and UV light (r = 0.89, n = 29). This study demonstrated that in vitro cell culture was equivalent to the “gold standard,” mouse infectivity, for measuring the infectivity of C. parvum and should therefore be considered a practical and accurate alternative for assessing oocyst infectivity and inactivation. However, the high levels of variability displayed by all assays indicated that infectivity and disinfection experiments should be limited to discerning relatively large differences.

Evaluation of Immunomagnetic Separation for Recovery of Infectious Cryptosporidium parvum Oocysts from Environmental Samples

1999 ◽  
Vol 65 (2) ◽  
pp. 841-845 ◽  
Author(s):  
Paul A. Rochelle ◽  
Ricardo De Leon ◽  
Anne Johnson ◽  
Mic H. Stewart ◽  
Roy L. Wolfe
Keyword(s):  
Cell Culture ◽  
Reverse Transcriptase ◽  
Cryptosporidium Parvum ◽  
Environmental Samples ◽  
Environmental Water ◽  
Immunomagnetic Separation ◽  
In Vitro Cell Culture ◽  
Magnetic Capture ◽  
Cryptosporidium Parvum Oocysts

ABSTRACT Two commercial immunomagnetic separation (IMS) kits forCryptosporidium were compared for recovery of oocysts from environmental samples. Oocyst recovery efficiencies with the Dynal and Crypto-Scan kits ranged from 62 to 100% and 34 to 74%, respectively, for seeded environmental water concentrates (turbidity of 210 to 11,480 nephelometric turbidity units). Recovery efficiencies were dependent on the mechanism of agitation during the magnetic capture procedure. An assay combining in vitro cell culture and reverse transcriptase PCR demonstrated that oocysts recovered by IMS retained their infectivity.

Effect of Organic Acids and Hydrogen Peroxide on Cryptosporidium parvum Viability in Fruit Juices

2003 ◽  
Vol 66 (9) ◽  
pp. 1650-1657 ◽  
Author(s):  
KALMIA E. KNIEL ◽  
SUSAN S. SUMNER ◽  
DAVID S. LINDSAY ◽  
CAMERON R. HACKNEY ◽  
MERLE D. PIERSON ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Cell Culture ◽  
Organic Acids ◽  
Cryptosporidium Parvum ◽  
Most Probable Number ◽  
Oocyst Wall ◽  
Probable Number ◽  
Apple Cider ◽  
Infectivity Assay

Cryptosporidium parvum has historically been associated with waterborne outbreaks of diarrheal illness. Foodborne cryptosporidiosis has been associated with unpasteurized apple cider. Infectious oocysts are shed in the feces of common ruminants like cattle and deer in and near orchards. In this study, the ability of organic acids and hydrogen peroxide (H2O2) added to fruit juice to inhibit the survival of C. parvum was analyzed. Oocyst viability was analyzed by a cell culture infectivity assay with the use of a human ileocecal cell line (HCT-8) whose infectivity pattern is similar to that for human oral infectivity. Cell monolayers were infected with 106 treated oocysts or a series of 10-fold dilutions. Parasitic life stages were visualized through immunohistochemistry with 100 microscope fields per monolayer being counted. In vitro excystation assays were also used to evaluate these treatments. Organic acids and H2O2 were added to apple cider, orange juice, and grape juices on a weight/volume basis. Malic, citric, and tartaric acids at concentrations of 1 to 5% inhibited C. parvum's infectivity of HCT-8 cells by up to 88%. Concentrations ranging from 0.025 to 3% H2O2 were evaluated. The addition of 0.025% H2O2 to each juice resulted in a >5-log reduction of C. parvum infectivity as determined with a most-probable-number–based cell culture infectivity assay. As observed with differential interference contrast and scanning electron microscopy, reduced infectivity may be mediated through effects on the oocyst wall that are caused by the action of H2O2 or related oxygen radicals. The addition of low concentrations of H2O2 can represent a valuable alternative to pasteurization.

scholarly journals In Vitro Cell Culture Infectivity Assay for Human Noroviruses

2007 ◽  
Vol 13 (3) ◽  
pp. 396-403 ◽  
Author(s):  
Timothy M. Straub ◽  
Kerstin Höner zu Bentrup ◽  
Patricia Orosz Coghlan ◽  
Alice Dohnalkova ◽  
Brooke K. Mayer ◽  
...  
Keyword(s):  
Cell Culture ◽  
Human Noroviruses ◽  
In Vitro Cell Culture ◽  
Infectivity Assay

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.

Toxicity of South American snake venoms measured by an in vitro cell culture assay

Toxicon ◽  
2002 ◽  
Vol 40 (3) ◽  
pp. 321-325 ◽  
Author(s):  
J.C.R Oliveira ◽  
H Montes de Oca ◽  
M.M Duarte ◽  
C.R Diniz ◽  
C.L Fortes-Dias
Keyword(s):  
Cell Culture ◽  
South American ◽  
Snake Venoms ◽  
In Vitro Cell Culture ◽  
Cell Culture Assay
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