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.

scholarly journals Efficacy of Nitazoxanide againstCryptosporidium parvum in Cell Culture and in Animal Models

1998 ◽  
Vol 42 (8) ◽  
pp. 1959-1965 ◽  
Author(s):  
Cynthia M. Theodos ◽  
Jeffrey K. Griffiths ◽  
Jennifer D’Onfro ◽  
Alexandra Fairfield ◽  
Saul Tzipori
Keyword(s):  
Cell Culture ◽  
Clinical Trials ◽  
Body Weight ◽  
Animal Models ◽  
Cryptosporidium Parvum ◽  
Combined Treatment ◽  
Parasite Burden ◽  
Parasite Growth ◽  
Gnotobiotic Piglet

ABSTRACT Nitazoxanide (NTZ), a drug currently being tested in human clinical trials for efficacy against chronic cryptosporidiosis, was assessed in cell culture and in two animal models. The inhibitory activity of NTZ was compared with that of paromomycin (PRM), a drug that is partially effective against Cryptosporidium parvum. A concentration of 10 μg of NTZ/ml (32 μM) consistently reduced parasite growth in cell culture by more than 90% with little evidence of drug-associated cytotoxicity, in contrast to an 80% reduction produced by PRM at 2,000 μg/ml (3.2 mM). In contrast to its efficacy in vitro, NTZ at either 100 or 200 mg/kg of body weight/day for 10 days was ineffective at reducing the parasite burden in C. parvum-infected, anti-gamma-interferon-conditioned SCID mice. Combined treatment with NTZ and PRM was no more effective than treatment with PRM alone. Finally, NTZ was partially effective at reducing the parasite burden in a gnotobiotic piglet diarrhea model when given orally for 11 days at 250 mg/kg/day but not at 125 mg/kg/day. However, the higher dose of NTZ induced a drug-related diarrhea in piglets that might have influenced its therapeutic efficacy. As we have previously reported, PRM was effective at markedly reducing the parasite burden in piglets at a dosage of 500 mg/kg/day. Our results indicate that of all of the models tested, the piglet diarrhea model most closely mimics the partial response to NTZ treatment reported to occur in patients with chronic cryptosporidiosis.

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 Efficacy of Glutaraldehyde Disinfectant Against Cryptosporidium parvum in the Presence of Various Organic Soils

2003 ◽  
Vol 86 (1) ◽  
pp. 96-100 ◽  
Author(s):  
James Wilson ◽  
Aaron B Margolin
Keyword(s):  
Cell Culture ◽  
Cryptosporidium Parvum ◽  
Medical Equipment ◽  
Fetal Bovine Serum ◽  
Organic Soil ◽  
Organic Soils ◽  
Cell Monolayers ◽  
Glutaraldehyde Solution ◽  
Fetal Bovine

Abstract The opportunistic protozoan Cryptosporidium parvum is highly resistant to disinfectants, including those specifically used for processing reused medical equipment in hospitals. C. parvum oocysts were dried onto glass and steel grooved penicylinders and challenged with 2.5% glutaraldehyde solution in the presence of 3 types of soil with exposures at 10 min, 90 min, and 10 h. The influence of organic soils on disinfection was measured with 5% fetal bovine serum (FBS), 10% FBS, and 5 mg mucin/mL. An in vitro excystation procedure and cell culture infection assay were used to determine survivability of oocysts after the germicide challenge. In the presence of organic soil, all oocysts removed from carriers excysted and infected cell monolayers after all germicide contact times. However, excystation was observed only from oocysts that received no protection from organic soil after 10 h exposure. In these samples, no infection was observed in the cell monolayers. The results of this research demonstrate the importance of thorough cleaning of medical equipment before disinfection.

scholarly journals Quantitative-PCR Assessment of Cryptosporidium parvum Cell Culture Infection

2005 ◽  
Vol 71 (3) ◽  
pp. 1495-1500 ◽  
Author(s):  
George D. Di Giovanni ◽  
Mark W. LeChevallier
Keyword(s):  
Cell Culture ◽  
Cryptosporidium Parvum ◽  
Culture Methods ◽  
Culture Studies ◽  
Sequence Detection ◽  
Taqman Pcr ◽  
Pcr Method ◽  
The Mean ◽  
Cell Culture Methods

ABSTRACT A quantitative TaqMan PCR method was developed for assessing the Cryptosporidium parvum infection of in vitro cultivated human ileocecal adenocarcinoma (HCT-8) cell cultures. This method, termed cell culture quantitative sequence detection (CC-QSD), has numerous applications, several of which are presented. CC-QSD was used to investigate parasite infection in cell culture over time, the effects of oocyst treatment on infectivity and infectivity assessment of different C. parvum isolates. CC-QSD revealed that cell culture infection at 24 and 48 h postinoculation was approximately 20 and 60%, respectively, of the endpoint 72-h postinoculation infection. Evaluation of three different lots of C. parvum Iowa isolate oocysts revealed that the mean infection of 0.1 N HCl-treated oocysts was only 36% of the infection obtained with oocysts treated with acidified Hanks' balanced salt solution containing 1% trypsin. CC-QSD comparison of the C. parvum Iowa and TAMU isolates revealed significantly higher levels of infection for the TAMU isolate, which agrees with and supports previous human, animal, and cell culture studies. CC-QSD has the potential to aid in the optimization of Cryptosporidium cell culture methods and facilitate quantitative evaluation of cell culture infectivity experiments.

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.

scholarly journals Bobel-24 Activity against Cryptosporidium parvum in Cell Culture and in a SCID Mouse Model

2007 ◽  
Vol 52 (3) ◽  
pp. 1150-1152 ◽  
Author(s):  
Cristina Rueda ◽  
Soledad Fenoy ◽  
Fernando Simón ◽  
Carmen del Aguila
Keyword(s):  
Cell Culture ◽  
Mouse Model ◽  
Cryptosporidium Parvum ◽  
Scid Mouse ◽  
In Vitro Growth ◽  
Scid Mouse Model ◽  
First Time

ABSTRACT The anticryptosporidial activity of Bobel-24 (2,4,6-triiodophenol) was studied for the first time, resulting in a reduction of the in vitro growth of Cryptosporidium of up to 99.6%. In a SCID mouse model of chronic cryptosporidiosis, significant differences (P < 0.05) in oocyst shedding were observed in animals treated with 125 mg/kg/day. These results merit further investigation of Bobel-24 as a chemotherapeutic option for cryptosporidiosis.

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 Comparison of Assays for Sensitive and Reproducible Detection of Cell Culture-Infectious Cryptosporidium parvum and Cryptosporidium hominis in Drinking Water

2011 ◽  
Vol 78 (1) ◽  
pp. 156-162 ◽  
Author(s):  
Anne M. Johnson ◽  
George D. Di Giovanni ◽  
Paul A. Rochelle
Keyword(s):  
Cell Culture ◽  
Drinking Water ◽  
Cryptosporidium Parvum ◽  
False Positives ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Cryptosporidium Hominis ◽  
Content Type ◽  
Detection Assay ◽  
Pcr Targeting

ABSTRACTThis study compared the three most commonly used assays for detectingCryptosporidiumsp. infections in cell culture: immunofluorescent antibody and microscopy assay (IFA), PCR targetingCryptosporidiumsp.-specific DNA, and reverse transcriptase PCR (RT-PCR) targetingCryptosporidiumsp.-specific mRNA. Monolayers of HCT-8 cells, grown in 8-well chamber slides or 96-well plates, were inoculated with a variety of viable and inactivated oocysts to assess assay performance. All assays detected infection with low doses of flow cytometry-enumeratedCryptosporidium parvumoocysts, including infection with one oocyst and three oocysts. All methods also detected infection withCryptosporidium hominis. The RT-PCR assay, IFA, and PCR assay detected infection in 23%, 25%, and 51% of monolayers inoculated with threeC. parvumoocysts and 10%, 9%, and 16% of monolayers inoculated with one oocyst, respectively. The PCR assay was the most sensitive, but it had the highest frequency of false positives with mock-infected cells and inactivated oocysts. IFA was the only infection detection assay that did not produce false positives with mock-infected monolayers. IFA was also the only assay that detected infections in all experiments with spiked oocysts recovered from Envirochek capsules following filtration of 1,000 liters of treated water. Consequently, cell culture with IFA detection is the most appropriate method for routine and sensitive detection of infectiousCryptosporidium parvumandCryptosporidium hominisin drinking water.

scholarly journals Extracts of pine bark (Pinus sylvestris) inhibit Cryptosporidium parvum growth in cell culture

2021 ◽  
Author(s):  
Berit Marie Blomstrand ◽  
Heidi Larsen Enemark ◽  
Øivind Øines ◽  
Håvard Steinshamn ◽  
Inga Marie Aasen ◽  
...  
Keyword(s):  
Cell Culture ◽  
Pinus Sylvestris ◽  
Growth Inhibition ◽  
Scots Pine ◽  
Cryptosporidium Parvum ◽  
Dry Matter ◽  
Treatment Options ◽  
Bark Extract ◽  
Dose Dependent

AbstractThe widespread apicomplexan parasite Cryptosporidium parvum is responsible for severe gastrointestinal disease in humans and animals. The treatment options are limited, and the efficacy of available drugs is low. Bark contains condensed tannins (CT), which are bioactive compounds previously shown to inhibit parasite development. Here, we examined the anti-cryptosporidial properties of bark extract of Scots pine (Pinus sylvestris) against C. parvum by means of an in vitro growth inhibition test. We hypothesised that bark extracts would have dose-dependent inhibitory effects on the development of C. parvum in cell culture.Bark extracts from Scots pine extracted with acetone, methanol, and water as solvents were investigated using human colorectal adenocarcinoma cells infected with C. parvum. Oocysts were inoculated onto the cell monolayer and bark extract was added at seven different concentrations. Parasite growth inhibition was quantified by qPCR.The acetone and methanol extracts demonstrated a sigmoid dose-dependent inhibition of C. parvum. The IC50 values were 244.6 and 279.1 µg dry matter extract/mL, and 25.4 and 24.1 µg CT/mL, for acetone and methanol extracts, respectively. The IC50 for both extracts were similar, both with regard to the dry matter concentration of each extract and to CT concentrations.Given the limited treatment options available for Cryptosporidium spp., the evidence generated in our study encourages further investigation into the in vitro and in vivo effects of pine bark extracts against C. parvum.

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