Detection and counting of<i>Cryptosporidium parvum</i>in HCT-8 cells by flowcytometry

Mele, Rita; Gomez, Morales M; Tosini, Fabio; Pozio, Edoardo

doi:10.1051/parasite/2003104297

Cited by 7 publications

(7 citation statements)

References 5 publications

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“…While antibody-based immunoassays, such as IFA, ELISA, and FCM (Woods et al, 1995;Verdon et al, 1997;Mele et al, 2003), can detect and quantitate the rate of in vitro infection of Cryptosporidium spp., they require specific antibodies that limit the ability to follow the kinetics of the early steps of parasite attachment/invasion. Since C. parvum is surrounded by a parasitophorous membrane of cell host origin, special treatments, such as fixation and permeabilization, are needed to allow antibodies to gain access to parasite antigens, which prevents studying the dynamic interaction between parasite and host cell.…”

Section: Discussionmentioning

confidence: 99%

Quantitative Tracking of Cryptosporidium Infection in Cell Culture With Cfse

Feng

Nie

Bonilla

et al. 2006

Journal of Parasitology

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Immunofluorescence-based assays have been developed to detect and quantitate Cryptosporidium parvum infection in cell culture. Here, we describe a method that tracks and quantifies the early phase of attachment and invasion of C. parvum sporozoites using a fluorescent dye. Newly excysted sporozoites were labeled with the amine-reactive fluorescein probe carboxyfluorescein diacetate succinimidyl esters (CFSE) using an optimized protocol. The initial invasion of cells by labeled parasites was detected with fluorescent or confocal microscopy. The infection of cells was quantified by flow cytometry. Comparative analysis of infection of cells with CFSE-labeled and unlabeled sporozoites showed that the infectivity of C. parvum was not affected by CFSE labeling. Quantitative analysis showed that C. parvum Iowa and MD isolates were considerably more invasive than Cryptosporidium hominis isolate TU502. Unlike immunofluorescent assays, CFSE labeling permitted the tracking of the initial invasion of C. parvum. Such an assay may be useful for studying the dynamics of host cell-parasite interaction and possibly for drug screening.

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Section: Discussionmentioning

confidence: 99%

Quantitative Tracking of Cryptosporidium Infection in Cell Culture With Cfse

Feng

Nie

Bonilla

et al. 2006

Journal of Parasitology

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“…The various stages of C. parvum were identified by their morphological characters (i.e., size and form) in accordance with the method of Hijjawi et al (12) by using Carl Zeiss Axio-Vision software to measure sizes. At each time postinfection, the total percentage of infected cells and the percentages of cells infected with the specific stages of the parasite were evaluated (23).…”

Section: Methodsmentioning

confidence: 99%

Cryptosporidium parvumat Different Developmental Stages Modulates Host Cell Apoptosis In Vitro

et al. 2004

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We studied apoptosis in a human ileocecal adenocarcinoma tumor cell line (HCT-8) infected with Cryptosporidium parvum, from 2 to 72 h postinfection (h.p.i.). At 2 h.p.i., the percentage of annexin V-positive cells in the cell culture had increased to 10% compared to 2.5% in noninfected control culture; sorted infected cells expressed mRNA of FasL, the active form of caspase 3, and high caspase 3 activity, whereas the noninfected neighboring cells sorted from the same culture showed no signs of apoptosis. At 24 h.p.i., the percentages of early (annexin V positive) and late (DNA fragment) apoptotic cells were 13 and 2%, respectively, in the entire cell culture, and these percentages were not statistically significant in comparison with those from noninfected control cultures. At this time, sorted infected cells expressed the inactive form of caspase 3, a low caspase 3 activity, and the antiapoptotic protein Bcl-2. Noninfected cells sorted from the same culture showed expression of the active form of caspase 3, a moderate caspase 3 activity, and no Bcl-2 expression. At 48 h.p.i., the percentages of early and late apoptotic cells and caspase 3 activity had increased in the total cell culture, and both sorted infected and noninfected cells showed the active form of caspase 3. These results show that C. parvum, depending on its developmental stage, can inhibit (at the trophozoite stage) or promote (at the sporozoite and merozoite stages) host cell apoptosis, suggesting that it is able to interact with and regulate the host-cell gene expression.

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“…Using flow cytometry, the number of oocysts in the faecal sample was evaluated using an acquisition gate based on the forward-angle light scatter (FSC-H) characteristic and the fluorescence intensities of FITC (on FL1 detector) of a positive control [30, 31]. The positive control consisted of an oocyst suspension in PBS incubated with the anti- C. parvum IgG.…”

Section: Methodsmentioning

confidence: 99%

“…All samples were analysed on the day of oocyst collection. The number of oocysts was evaluated by fluorescence microscopy (Zeiss Axioplan 2; Carl Zeiss, Jena, Germany) at 400× magnification [31].…”

Section: Methodsmentioning

confidence: 99%

Delivery of SA35 and SA40 peptides in mice enhances humoral and cellular immune responses and confers protection against Cryptosporidium parvum infection

et al. 2019

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Background Cryptosporidium parvum is a major cause of diarrhea in children and ruminants at the earliest stages of life. Maternal antibodies represent the main shield of neonate mammals for most of the infections. Two recombinant antigens (SA35 and SA40), portions of two C. parvum proteins, were tested for their ability to induce immune responses in adult mice and for protection on neonate BALB/c mice born from females immunised by mucosal delivery of both peptides. Methods Adult BALB/c mice were intraperitoneally immunised with SA35 and SA40, separately or mixed, and their immune response was characterised. Furthermore, BALB/c pregnant mice were immunised by mucosal delivery with an SA35/40 mix, before and during pregnancy. Soon after birth, their offspring were infected with two doses (1 × 10 5 and 5 × 10 3 ) of C. parvum oocysts and the parasitic burden was determined at 5 and 9 days post-infection. Results Intraperitoneal immunisation with SA35 and SA40 induced specific IgG and IgG1 in serum, specific IgA in the intestinal mucosa, increase of CD3+/CD4+ and CD30+ cells in splenocytes, which produced IFN-γ. Neonates born from immunised mice and infected with 1 × 10 5 oocysts showed a significant reduction of oocysts and intestinal forms (23 and 42%, respectively). A reduction of all parasitic forms (96%; P < 0.05) was observed when neonates were infected with 5 × 10 3 oocysts. Conclusions SA35 and SA40 peptides induce specific humoral and cell-mediated immune responses to C. parvum in adult mice. Moreover, mucosal administration of the SA35/40 mix in pregnant mice reduces C. parvum burden in their litters.

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Detection and counting ofCryptosporidium parvumin HCT-8 cells by flowcytometry

Cited by 7 publications

References 5 publications

Quantitative Tracking of Cryptosporidium Infection in Cell Culture With Cfse

Quantitative Tracking of Cryptosporidium Infection in Cell Culture With Cfse

Cryptosporidium parvumat Different Developmental Stages Modulates Host Cell Apoptosis In Vitro

Delivery of SA35 and SA40 peptides in mice enhances humoral and cellular immune responses and confers protection against Cryptosporidium parvum infection

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