scholarly journals Detection of UV-Induced Thymine Dimers in Individual Cryptosporidium parvum and Cryptosporidium hominis Oocysts by Immunofluorescence Microscopy

2006 ◽  
Vol 73 (3) ◽  
pp. 947-955 ◽  
Author(s):  
B. H. Al-Adhami ◽  
R. A. B. Nichols ◽  
J. R. Kusel ◽  
J. O'Grady ◽  
H. V. Smith
Keyword(s):  
Cryptosporidium Parvum ◽  
Immunofluorescence Microscopy ◽  
Uv Light ◽  
Fluorescein Isothiocyanate ◽  
Immunofluorescence Assay ◽  
Cryptosporidium Hominis ◽  
Specific Fluorescence ◽  
Thymine Dimers ◽  
Texas Red

ABSTRACT To investigate the effect of UV light on Cryptosporidium parvum and Cryptosporidium hominis oocysts in vitro, we exposed intact oocysts to 4-, 10-, 20-, and 40-mJ�cm−2 doses of UV irradiation. Thymine dimers were detected by immunofluorescence microscopy using a monoclonal antibody against cyclobutyl thymine dimers (anti-TDmAb). Dimer-specific fluorescence within sporozoite nuclei was confirmed by colocalization with the nuclear fluorogen 4′,6′-diamidino-2-phenylindole (DAPI). Oocyst walls were visualized using either commercial fluorescein isothiocyanate-labeled anti-Cryptosporidium oocyst antibodies (FITC-CmAb) or Texas Red-labeled anti-Cryptosporidium oocyst antibodies (TR-CmAb). The use of FITC-CmAb interfered with TD detection at doses below 40 mJ�cm−2. With the combination of anti-TDmAb, TR-CmAb, and DAPI, dimer-specific fluorescence was detected in sporozoite nuclei within oocysts exposed to 10 to 40 mJ�cm−2 of UV light. Similar results were obtained with C. hominis. C. parvum oocysts exposed to 10 to 40 mJ�cm−2 of UV light failed to infect neonatal mice, confirming that results of our anti-TD immunofluorescence assay paralleled the outcomes of our neonatal mouse infectivity assay. These results suggest that our immunofluorescence assay is suitable for detecting DNA damage in C. parvum and C. hominis oocysts induced following exposure to UV light.

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 UV Inactivation of Cryptosporidium hominis as Measured in Cell Culture

2005 ◽  
Vol 71 (5) ◽  
pp. 2800-2802 ◽  
Author(s):  
Anne M. Johnson ◽  
Karl Linden ◽  
Kristina M. Ciociola ◽  
Ricardo De Leon ◽  
Giovanni Widmer ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cryptosporidium Parvum ◽  
Uv Light ◽  
Uv Disinfection ◽  
Water Industry ◽  
Cryptosporidium Hominis

ABSTRACT The Cryptosporidium spp. UV disinfection studies conducted to date have used Cryptosporidium parvum oocysts. However, Cryptosporidium hominis predominates in human cryptosporidiosis infections, so there is a critical need to assess the efficacy of UV disinfection of C. hominis. This study utilized cell culture-based methods to demonstrate that C. hominis oocysts displayed similar levels of infectivity and had the same sensitivity to UV light as C. parvum. Therefore, the water industry can be confident about extrapolating C. parvum UV disinfection data to C. hominis oocysts.

scholarly journals Dicyclic and Tricyclic Diaminopyrimidine Derivatives as Potent Inhibitors of Cryptosporidium parvum Dihydrofolate Reductase: Structure-Activity and Structure-Selectivity Correlations

2001 ◽  
Vol 45 (12) ◽  
pp. 3293-3303 ◽  
Author(s):  
Richard G. Nelson ◽  
Andre Rosowsky
Keyword(s):  
Cryptosporidium Parvum ◽  
Dihydrofolate Reductase ◽  
Drug Exposure ◽  
Immunofluorescence Microscopy ◽  
Host Cells ◽  
Indirect Immunofluorescence Microscopy ◽  
Structure Activity ◽  
Dhfr Inhibitors ◽  
Canine Kidney

ABSTRACT A structurally diverse library of 93 lipophilic di- and tricyclic diaminopyrimidine derivatives was tested for the ability to inhibit recombinant dihydrofolate reductase (DHFR) cloned from human and bovine isolates of Cryptosporidium parvum (J. R. Vásquez et al., Mol. Biochem. Parasitol. 79:153–165, 1996). In parallel, the library was also tested against human DHFR and, for comparison, the enzyme from Escherichia coli. Fifty percent inhibitory concentrations (IC50s) were determined by means of a standard spectrophotometric assay of DHFR activity with dihydrofolate and NADPH as the cosubstrates. Of the compounds tested, 25 had IC50s in the 1 to 10 μM range against one or bothC. parvum enzymes and thus were not substantially different from trimethoprim (IC50s, ca. 4 μM). Another 25 compounds had IC50s of <1.0 μM, and 9 of these had IC50s of <0.1 μM and thus were at least 40 times more potent than trimethoprim. The remaining 42 compounds were weak inhibitors (IC50s, >10 μM) and thus were not considered to be of interest as drugs useful against this organism. A good correlation was generally obtained between the results of the spectrophotometric enzyme inhibition assays and those obtained recently in a yeast complementation assay (V. H. Brophy et al., Antimicrob. Agents Chemother. 44:1019–1028, 2000; H. Lau et al., Antimicrob. Agents Chemother. 45:187–195, 2001). Although many of the compounds in the library were more potent than trimethoprim, none had the degree of selectivity of trimethoprim for C. parvum versus human DHFR. Collectively, the results of these assays comprise the largest available database of lipophilic antifolates as potential anticryptosporidial agents. The compounds in the library were also tested as inhibitors of the proliferation of intracellular C. parvum oocysts in canine kidney epithelial cells cultured in folate-free medium containing thymidine (10 μM) and hypoxanthine (100 μM). After 72 h of drug exposure, the number of parasites inside the cells was quantitated by indirect immunofluorescence microscopy. Sixteen compounds had IC50s of <3 μM, and five of these had IC50s of <0.3 μM and thus were comparable in potency to trimetrexate. The finding that submicromolar concentrations of several of the compounds in the library could inhibit in vitro growth of C. parvum in host cells in the presence of thymidine (dThd) and hypoxanthine (Hx) suggests that lipophilic DHFR inhibitors, in combination with leucovorin, may find use in the treatment of intractable C. parvum infections.

scholarly journals Public Health Implication of Drinking Water Sources Contaminated with Giardia spp. and Cryptosporidium spp. in South Ethiopia

2021 ◽  
Author(s):  
Mengistu Damitie ◽  
Dante Santiago ◽  
Luc Leyns ◽  
Seid Tiku Mereta ◽  
Zeleke Mekonnen
Keyword(s):  
Drinking Water ◽  
Cryptosporidium Parvum ◽  
Giardia Duodenalis ◽  
Water Sources ◽  
Immunofluorescence Assay ◽  
Southern Ethiopia ◽  
Cryptosporidium Hominis ◽  
Public Health Implication ◽  
Drinking Water Sources ◽  
Genotypic Analysis

Abstract Background: Giardia duodenalis and Cryptosporidium spp. are topmost causes of gastrointestinal diseases mainly in socio-economically disadvantaged regions. Understanding the molecular diversity and distribution of these parasites in water sources and the environmental variables that influence their prevalence is important to effectively control infections in at-risk populations, however, the state of knowledge about risks of these parasites associated with source water is little in Africa. This study aimed at characterizing risks of Giardia duodenalis and Cryptosporidium spp. from water sources in southern Ethiopia.Methods: Twenty-one water samples (10L per source) were collected from five different types of drinking water sources. Sample was analyzed using immunofluorescence assay with fluorescein isothiocyanate–monoclonal antibodies and nested PCR; tpi-PCR was used for genotyping purpose in Giardia duodenalis isolates and SSU-rRNA and gp60-PCR in Cryptosporidium spp. isolates. Statistical analysis was performed using Spearman’s rho correlation and independent-samples nonparametric test.The risk was characterized using quantitative microbial risk assessment.Results: The immunofluorescence assay showed that cyst was detected in 81% (17/21) and oocyst in 71% (15/21) of the samples with mean concentrations of 64.6 cysts.10 L-1 and 62.8 oocysts.10 L-1. Genotypic analysis showed that Giardia duodenalis assemblage B was genotyped in 29.4% (5/17), assemblage A in 17.6% (3/17) and assemblages A+B in 52.9% (9/17) of the samples. Cryptosporidium spp. genotype was detected in 57.1% (12/21) of the samples; Cryptosporidium parvum in 38.1% (8/21), Cryptosporidium hominis in 14.3% (3/21) and Cryptosporidium parvum and Cryptosporidium hominis in 4.7% (1/21) of the samples. The average probability of illness from the water sources was 0.06 for Giardia duodenalis and 0.22 for Cryptosporidium spp.Conclusions: The finding highlights the significance of water for transmission of Giardia spp. and Cryptosporidium spp. in southern Ethiopia. Genotypes detected in the samples indicate multiple sources likely contaminated the sources. Health risk that is inferred from the water sources to the public are significantly higher than most other reported findings. This study recommends the need of regular follow-up, improvement in water treatment facilities and setting appropriate legislation in areas where cycles of parasitic infection is linked to water sources.

scholarly journals The in vitro and ex vivo effect of Auranta 3001 in preventing Cryptosporidium hominis and Cryptosporidium parvum infection

Gut Pathogens ◽  
2017 ◽  
Vol 9 (1) ◽  
Author(s):  
Alexandros Ch Stratakos ◽  
Filip Sima ◽  
Patrick Ward ◽  
Mark Linton ◽  
Carmel Kelly ◽  
...  
Keyword(s):  
Cryptosporidium Parvum ◽  
Ex Vivo ◽  
Cryptosporidium Hominis

scholarly journals Detection of Cryptosporidium parvum oocysts in calf fecal samples by direct immunofluorescence assay

2011 ◽  
Vol 20 (4) ◽  
pp. 269-273 ◽  
Author(s):  
Weslen Fabricio Pires Teixeira ◽  
Willian Marinho Dourado Coelho ◽  
Cáris Maroni Nunes ◽  
Marcelo Vasconcelos Meireles
Keyword(s):  
Cryptosporidium Parvum ◽  
Polyclonal Antibodies ◽  
Deae Cellulose ◽  
Fluorescein Isothiocyanate ◽  
Cross Reactivity ◽  
Immunofluorescence Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Direct Immunofluorescence ◽  
Fecal Samples ◽  
Direct Immunofluorescence Assay

The aim of this study was to produce a conjugate containing anti-Cryptosporidium parvum polyclonal antibodies and standardize a Direct Immunofluorescence Assay (DIF) for detecting C. parvum oocysts in fecal samples from calves. In order to obtain anti-C. parvum polyclonal antibodies, two New Zealand rabbits were immunized with a purified solution of C. parvum oocysts and Freund's adjuvant. Purification of the immunoglobulin G (IgG) fraction was performed by means of precipitation in ammonium sulfate and chromatography using a DEAE-cellulose column. The anti-C. parvum polyclonal antibody titer was determined by means of the enzyme-linked immunosorbent assay (ELISA). The rabbit anti-C. parvum IgG fraction was conjugated with fluorescein isothiocyanate and standardization of the DIF was performed using various dilutions of conjugate on slides positive for C. parvum oocysts. The cross-reactivity of the anti-C. parvum conjugate was tested using oocysts of Cryptosporidium serpentis, Cryptosporidium andersoni, Escherichia coli, Eimeria sp., and Candida sp. An anti-C. parvum conjugate was successfully produced, thus allowing standardization of DIF for detection of Cryptosporidium oocysts in fecal samples. Cross-reactivity of anti-C. parvum polyclonal antibodies with C. andersoni and C. serpentis was also observed.

scholarly journals Defining Stage-Specific Activity of Potent New Inhibitors of Cryptosporidium parvum Growth In Vitro

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Lisa J. Funkhouser-Jones ◽  
Soumya Ravindran ◽  
L. David Sibley
Keyword(s):  
Life Cycle ◽  
Cryptosporidium Parvum ◽  
Specific Activity ◽  
Trna Synthetase ◽  
Mechanisms Of Action ◽  
Cryptosporidium Hominis ◽  
Content Type

ABSTRACT Cryptosporidium parvum and Cryptosporidium hominis have emerged as major enteric pathogens of infants in the developing world, in addition to their known importance in immunocompromised adults. Although there has been recent progress in identifying new small molecules that inhibit Cryptosporidium sp. growth in vitro or in animal models, we lack information about their mechanism of action, potency across the life cycle, and cidal versus static activities. Here, we explored four potent classes of compounds that include inhibitors that likely target phosphatidylinositol 4 kinase (PI4K), phenylalanine-tRNA synthetase (PheRS), and several potent inhibitors with unknown mechanisms of action. We utilized monoclonal antibodies and gene expression probes for staging life cycle development to define the timing of when inhibitors were active during the life cycle of Cryptosporidium parvum grown in vitro. These different classes of inhibitors targeted different stages of the life cycle, including compounds that blocked replication (PheRS inhibitors), prevented the segmentation of daughter cells and thus blocked egress (PI4K inhibitors), or affected sexual-stage development (a piperazine compound of unknown mechanism). Long-term cultivation of C. parvum in epithelial cell monolayers derived from intestinal stem cells was used to distinguish between cidal and static activities based on the ability of parasites to recover from treatment. Collectively, these approaches should aid in identifying mechanisms of action and for designing in vivo efficacy studies based on time-dependent concentrations needed to achieve cidal activity. IMPORTANCE Currently, nitazoxanide is the only FDA-approved treatment for cryptosporidiosis; unfortunately, it is ineffective in immunocompromised patients, has varied efficacy in immunocompetent individuals, and is not approved in infants under 1 year of age. Identifying new inhibitors for the treatment of cryptosporidiosis requires standardized and quantifiable in vitro assays for assessing potency, selectivity, timing of activity, and reversibility. Here, we provide new protocols for defining which stages of the life cycle are susceptible to four highly active compound classes that likely inhibit different targets in the parasite. We also utilize a newly developed long-term culture system to define assays for monitoring reversibility as a means of defining cidal activity as a function of concentration and time of treatment. These assays should provide valuable in vitro parameters to establish conditions for efficacious in vivo treatment.

scholarly journals Interaction of Cryptosporidium hominis and Cryptosporidium parvum with Primary Human and Bovine Intestinal Cells

2006 ◽  
Vol 74 (1) ◽  
pp. 99-107 ◽  
Author(s):  
Amna Hashim ◽  
Grace Mulcahy ◽  
Billy Bourke ◽  
Marguerite Clyne
Keyword(s):  
Cryptosporidium Parvum ◽  
Cell Model ◽  
Host Cells ◽  
Intestinal Cells ◽  
Cryptosporidium Hominis ◽  
Kinase C ◽  
Adenocarcinoma Cells ◽  
Transduction Mechanisms ◽  
Different Origins

ABSTRACT Cryptosporidiosis in humans is caused by the zoonotic pathogen Cryptosporidium parvum and the anthroponotic pathogen Cryptosporidium hominis. To what extent the recently recognized C. hominis species differs from C. parvum is unknown. In this study we compared the mechanisms of C. parvum and C. hominis invasion using a primary cell model of infection. Cultured primary bovine and human epithelial intestinal cells were infected with C. parvum or C. hominis. The effects of the carbohydrate lectin galactose-N-acetylgalactosamine (Gal/GalNAc) and inhibitors of cytoskeletal function and signal transduction mechanisms on entry of the parasites into host cells were tested. HCT-8 cells (human ileocecal adenocarcinoma cells) were used for the purpose of comparison. Pretreatment of parasites with Gal/GalNAc inhibited entry of C. parvum into HCT-8 cells and primary bovine cells but had no effect on entry of either C. parvum or C. hominis into primary human cells or on entry of C. hominis into HCT-8 cells. Both Cryptosporidium species entered primary cells by a protein kinase C (PKC)- and actin-dependent mechanism. Staurosporine, in particular, attenuated infection, likely through a combination of PKC inhibition and induction of apoptosis. Diversity in the mechanisms used by Cryptosporidium species to infect cells of different origins has important implications for understanding the relevance of in vitro studies of Cryptosporidium pathogenesis.

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