Distribution of Cryptosporidium parvum sporozoite apical organelles during attachment to and internalization by cultured biliary epithelial cells

Although accumulating evidence supports an active role for host cells during Cryptosporidium parvum invasion of epithelia, our knowledge of the underlying parasite-specific processes triggering such events is limited. In an effort to better understand the invasion strategy of C. parvum, we characterized the presence and distribution of the apical organelles (micronemes, dense granules, and rhoptry) through the stages of attachment to, and internalization by, human biliary epithelia, using serial-section electron microscopy. Novel findings include an apparent organized rearrangement of micronemes upon host cell attachment. The apically segregated micronemes were apposed to a central microtubule-like filamentous structure, and the more distal micronemes localized to the periphery and apical region of the parasite during internalization, coinciding with the formation of the anterior vacuole. The morphological observations presented here extend our understanding of parasite-specific processes that occur during attachment to, and internalization by, host epithelial cells.     

The effect of lectins on Cryptosporidium parvum oocyst in vitro attachment to host cells

The influence of lectins on Cryptosporidium parvum oocyst agglutination and on attachment to both fixed Madin Darby Canine Kidney (MDCK) cells and fixed HCT-8 (human colorectal epithelial) cells was examined. Oocyst cell wall characteristics were examined by transmission electron microscopy. Lectin-free oocysts were shown to adhere equally to both MDCK cells and HCT-8 cells. In MDCK cells, the addition of 1-25 microg/ml Codium fragile lectin, 10 microg/ml Maclura pomifera lectin, 10 microg/ml Helix pomatia lectin, and 10-200 microg/ml Artocarpus integrifolia lectin significantly increased attachment to at least 1 of the cell cultures as compared to oocysts incubated without any lectin. The lectin-enhanced attachment was reversed by co-incubation of lectin treated-oocysts with 250 mM of each specific sugar (for a given lectin). In agglutination assays, concentrations as low as 0.5 microg/ml of C. fragile, M. pomifera, and A. integrifolia lectin agglutinated oocysts within 60 min. Finally, in TEM samples, colloidal gold conjugated-lectins from A. integrifolia, C. fragile, H. pomatia, and M. pomifera attached to oocysts, and this could be competitively inhibited by a lectin-specific sugar. This suggests that C. parvum oocysts are highly reactive to N-acetyl galactosamine-binding lectins and that the presence of N-acetyl-galactosamine containing molecules on oocysts can potentially help in oocyst attachment to host cells.     

Effect of bovine manure on Cryptosporidium parvum oocyst attachment to soil

The objective of this work was to assess the effect of dilute bovine manure (1.0% and 0.1%) versus that of no manure on attachment and subsequent detachment of Cryptosporidium parvum oocysts to soil. Manure enhanced the attachment of oocysts to soil particles; the maximum attachment was observed with 0.1% manure. Oocyst attachment was partially reversible; maximum detachment was observed with dilute manure. These results indicate that oocyst attachment to soil is substantially affected by bovine manure in a complex manner and should have implications for how oocysts may be transported through or over soils.     

Development of a microtitre ELISA to quantify development of Cryptosporidium parvum in vitro

Abstract An in situ enzyme-linked immunosorbent assay (ELISA) was developed to evaluate growth of Cryptosporidium parvum in vitro. Ninety-six-well tissue culture microtitre plates were each seeded with 4.0 X 104 human ileocecal adenocarcinoma (HCT-8) cells, then infected with CsCl-purified oocysts 24 h later. The growth medium consisted of RPMI 1640 supplemented with 10% fetal bovine serum, 15 mM HEPES (JV-2-hydroxyethylpiperazine N −2-ethanesulfonic acid), 50 mM glucose, 1 μg ml⁻¹ folic acid, 4 μg ml⁻¹ 4-aminobenzoic acid, 2 μg ml⁻¹ pantothenic acid and 35 μg ml⁻¹ ascorbic acid. Incubation conditions were at 37 ° C in a 5% CO₂/95% humidified air incubator. Oocysts were allowed to excyst in situ so that sporozoites could infect cells directly. Monolayers were then washed, new medium added, and infected cells re-incubated. Levels of infection were assessed 48 h later using a rat anti-C. parvum polyvalent antiserum directed against purified parasite membranes, followed by a goat anti-rat IgG conjugated to horseradish peroxidase and 3,3',5,5'-tetramethyl-benzidine as substrate. Using various parasite inoculating doses and incubation times, optimal results were obtained using a 90-min exposure of host cells to 2.5−3.0 × 10⁴ oocysts/well. Evaluation of various concentrations of four anti-microbials (monensin, lasalocid, paromomycin and sulfadimethoxine) in the system resulted in the acquisition of precise dose-response curves for each compound.     

TLR4 promotes Cryptosporidium parvum clearance in a mouse model of biliary cryptosporidiosis

Cholangiocytes, the epithelial cells lining intrahepatic bile ducts, express multiple toll-like receptors (TLRs) and, thus, have the capacity to recognize and respond to microbial pathogens. In previous work, we demonstrated that TLR4, which is activated by gram-negative lipopolysaccharide (LPS), is upregulated in cholangiocytes in response to infection with Cryptosporidium parvum in vitro and contributes to nuclear factor-kappaB (NF-kB) activation. Here, using an in vivo model of biliary cryptosporidiosis, we addressed the functional role of TLR4 in C. parvum infection dynamics and hepatobiliary pathophysiology. We observed that C57BL mice clear the infection by 3 wk post-infection (PI). In contrast, parasites were detected in bile and stool in TLR4-deficient mice at 4 wk PI. The liver enzymes alanine transaminase (ALT) and aspartate transaminase (AST), and the proinflammatory cytokines tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and interleukin (IL)-6 peaked at 1 to 2 wk PI and normalized by 4 wk in infected C57BL mice. C57BL mice also demonstrated increased cholangiocyte proliferation (PCNA staining) at 1 wk PI that was resolved by 2 wk PI. In contrast, TLR4-deficient mice showed persistently elevated serum ALT and AST, elevated hepatic IL-6 levels, and histological evidence of hepatocyte necrosis, increased inflammatory cell infiltration, and cholangiocyte proliferation through 4 wk PI. These data suggest that a TLR4-mediated response is required for efficient eradication of biliary C. parvum infection in vivo, and lack of this pattern-recognition receptor contributes to an altered inflammatory response and an increase in hepatobiliary pathology.     

In vitro murine lymphocyte blastogenic responses to Cryptosporidium parvum.

Spleen and mesenteric lymph node lymphocytes from both Cryptosporidium parvum-exposed and unexposed mice were cultured with antigen (Ag) prepared from C. parvum oocysts. Spleen lymphocytes from oral-, intraperitoneal-, or oral + intraperitoneal-exposed mice did not respond significantly (P greater than 0.05) to Ag stimulation. Spleen lymphocytes from multioral-exposed mice, however, demonstrated significant (P less than or equal to 0.01) Ag-specific blastogenesis. Mesenteric lymph node lymphocytes did not respond to in vitro Ag stimulation regardless of the route of in vivo priming. These results demonstrate an in vitro cell-mediated immune response against C. parvum by lymphocytes in murine spleen.     

Host cell apoptosis impairs Cryptosporidium parvum development in vitro

The absence of a self-sustaining in vitro propagation method for Cryptosporidium parvum is a major obstacle for research on this parasite. Conventional cell monolayers are unsuitable for long-term parasite propagation because the level of infection decreases over time and few oocysts, if any, are produced. The interaction between parasite and host cell was studied to identify factors limiting parasite development in vitro. Loss of substrate adherence and death of parasitized host cells was observed in 2 epithelial cell lines. Nuclear morphology, DNA laddering, annexin V binding, and terminal deoxytransferase-mediated dUTP nick end labeling indicated that host cell death occurred by apoptosis. At 6 hr postinfection, only a minority of infected cells remained in the monolayer, and few survived the initial phase of parasite development without losing adherence. Treatment of infected monolayers with caspase inhibitors drastically reduced cell detachment but failed to increase the number of parasites in monolayers. In contrast, cell cultures grown on laminin-coated plates showed a higher proportion of infected cells. These observations indicate that cell detachment and apoptosis in C. parvum-infected cell culture negatively affect parasite survival in vitro.     

In vitro development of Cryptosporidium parvum in serum-free media

AIM: To compare commercially available serum-free media with common, standard, growth medium for their ability to support growth of Cryptosporidium parvum in HCT-8 cell cultures. METHODS AND RESULTS: Twelve serum-free media formulations with or without additional supplements were tested against a standard growth medium containing 2% FBS in HCT-8 cell cultures. After a 48-h incubation period, the level of parasite development was determined by ELISA. The extent of development in the serum-free media was determined as a percentage of infections compared with those obtained using a standard growth medium. CONCLUSIONS: Several of the serum-free media formulations, which included MDCK, UltraMDCK, PC-1, UltraCHO and UltraCulture, compared favourably with a traditional, standard growth medium. Moreover, increasing FBS concentrations to 10% actually resulted in an overall decrease in development in many cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: Several serum-free medium formulations are available which allow development of C. parvumin vitro at levels comparable with standard media employing FBS. These serum-free media are particularly useful for applications, which may require a more defined medium without the presence of FBS. Moreover, the elimination of FBS as a variable allows investigators the ability to more closely regulate their experimental systems when growing C. parvum in cell cultures.     

In vitro infection of Cryptosporidium parvum to four different cell lines

To determine a suitable condition for in vitro infection model of Cryptosporidium parvum, four different cell lines, AGS, MDCK, HCT-8 and Caco-2, were used as host cell lines which were cultured at various concentrations of added supplements. These supplement include fetal bovine serum (FBS), sodium choleate, ascorbic acid, folic acid, calcium pantothenate, para-aminobenzoic acid and pyruvate and their effects on the cell lines which were infected with C. parvum were evaluated. The results of this study showed that the AGS cell line was most susceptible to C. parvum whereas the Caco-2 cells appeared to be least susceptible to C. parvum. In regards to the serum condition, 10% FBS was suitable for the growth of AGS and HCT-8 cells, and 1% FBS was good for the growth of the MDCK cells when they were inoculated with C. parvum. Vitamins had a positive effect on the AGS cells, and pyruvate also showed positive effects on all of the cell lines except for Caco-2. Modified medium for each cell line was prepared by adding appropriate amounts of each supplement which resulted in the highest parasite infection number. Modified media increased the number of parasites infected on AGS cells to 2.3-fold higher when compared to the control media. In this study, we found that the AGS cell line was a suitable host model for evaluating C. parvum in vitro study and the media contents for the optimal infection conditions were suggested.     

Cryptosporidium parvum: in vitro cultivation in Madin-Darby canine kidney cells.

To facilitate studies of the biology of Cryptosporidium parvum, we have developed an in vitro culture system using Madin-Darby canine kidney (MDCK) cells as the host cell. Oocysts or free sporozoites were incubated 37 degrees C with monolayers of MDCK cells in supplemented RPMI 1640 medium and the cells were examined at various time intervals after initiation of the culture. High rates of infection (up to 90% of MDCK cells) were achievable. Sequential development of trophozoites, meronts, microgametocytes, and macrogametocytes was observed over a 72-h period of culture. Between 72 and 96 h we observed formation of oocyst walls, but fully sporulated oocysts were not observed. This culture system provides access to both the asexual and sexual intracellular stages of C. parvum.     

Quantification of in vitro and in vivo Cryptosporidium parvum infection by using real-time PCR

Established methods for quantifying experimental Cryptosporidium infection are highly variable and subjective. We describe a new technique using quantitative real-time PCR (qPCR) that can be used to measure in vitro and in vivo laboratory infections with Cryptosporidium. We show for the first time that qPCR permits absolute quantification of the parasite while simultaneously controlling for the amount of host tissue and correlates significantly with established methods of quantification in in vitro and in vivo laboratory models of infection.     

Ultrastructural study of asexual development of Cryptosporidium parvum in a human intestinal cell line.

The lack of a well-defined in vitro model of Cryptosporidium infection has severely hampered research on the biology of parasitic invasion of the host cell and on intracellular development of the parasite. In vitro infection of the differentiated human enterocyte cell line HT29.74 was studied by electron microscopy to detect changes in parasite and host cell morphology. Cryptosporidium oocysts obtained from AIDS patients were applied to a monolayer of cloned differentiated HT29.74 cells. Parasites and infected cells were evaluated by transmission electron microscopy at 20 min, 1 h, 6 h, 24 h and 7 days. Sporozoite invagination within the epithelial cell microvilli and subsequent penetration was evident at 1 h. At 6 h, the development of a dense band and feeder layer was visible. Development of the trophozoite into a schizont occurred over 24 h. Micronemes and dense granules were clearly visible within sporozoites and merozoites. Organization of vacuoles within the cytoplasm of the host cell was evident below the dense band. A sexual Cryptosporidium development in vitro was morphologically no different from initial development in vivo. In vitro infection of HT29.74 cells provides an excellent model to study parasite-host cell interaction and asexual parasite development.     

Detection of Cryptosporidium parvum oocysts by dot-blotting using monoclonal antibodies to Cryptosporidium parvum virus 40-kDa capsid protein

Monoclonal antibodies (MAb) were prepared against the 40-kDa capsid protein of Cryptosporidium parvum virus (CPV) by immunizing mice with purified recombinant CPV40 protein. In immunoblotting analysis, MAbCPV40-1 bound to a 40-kDa protein in extracts of C. parvum oocysts. This 40-kDa protein was localized in the sporozoite cytoplasm by immunofluorescence (IFA) staining with MAbCPV40-1. In a dot-blot assay, MAbCPV40-1 was capable of detecting 10(2) non-bleach-treated and 10(2)-10(3) bleach-treated C. parvum oocysts. MAbCPV40-1 was capable of detecting CPV40 antigen in both soluble and total C. parvum oocyst protein extracts, indicating a potential use for detecting this parasite in environmental samples.     

Studies of in vitro excystation of Cryptosporidium parvum from calves

Studies of in vitro excystation of Cryptosporidium parvum from calves showed that sporozoite yields were optimum when oocysts were treated with sodium hypochlorite, then incubated at 37 degrees C for 60 min in the presence of taurocholic acid solutions at pH about 7.0. Trypsin was not required for excystation and high concentrations were inhibitory. Studies using protease inhibitors and direct assays for proteolysis failed to implicate proteolytic enzymes as effectors of excystation. The results suggest that Cryptosporidium uses excystation mechanisms that are different from those used by Eimeria spp.     

Intact Cryptosporidium parvum oocysts isolated after in vitro excystation are infectious to neonatal mice

In vitro excystation is often used as a measure of viability of encysted protozoan parasites. Parasites that do not excyst in vitro are assumed to be non-viable and non-infectious, whereas those that do excyst are assumed viable. To test the validity of these assumptions, Cryptosporidium parvum oocysts were excysted in vitro using two different excystation protocols, and the non-excysted intact oocysts were isolated using flow cytometry. Non-excysted sorted oocysts readily infected neonatal CD-1 mice. Increasing the duration of the excystation assays from 1 h to 3 h resulted in a higher percent of excysted oocysts, but the remaining non-excysted parasites were still capable of infecting neonatal CD-1 mice. Our results suggest that in vitro excystation is not an accurate measure of the viability or infectious potential of C. parvum oocysts.     

Indinavir reduces Cryptosporidium parvum infection in both in vitro and in vivo models

The use of highly active antiretroviral therapy in persons with acquired immunodeficiency syndrome has reduced the prevalence of infection with Cryptosporidium parvum and the length and severity of its clinical course. This effect has in most cases been attributed to the recovery of the host immunity; however, some works suggest that human immunodeficiency virus protease inhibitors, indinavir in particular, which is one of the human immunodeficiency virus protease inhibitors used in highly active antiretroviral therapy, may be capable of controlling Microsporidia and Cryptosporidium infections, which are refractory to other treatments. The objective of the present study was to investigate the effect of human immunodeficiency virus protease inhibitors on C. parvum infections. Since preliminary experiments using ritonavir, saquinavir, and indinavir showed a drastic reduction of C. parvum infection both in vivo (neonatal Balb/c mice) and in vitro (human ileocecal adenocarcinoma tumour cell line) models, indinavir alone was tested in successive experiments. In vitro, the treatment of the sporulated oocysts with different concentrations of indinavir reduced the percentage of human ileocecal adenocarcinoma tumour cell line infected cells in a dose-dependent manner. For established infection, the treatment with 50 microM of indinavir decreased the percentage of infected cells in a time-dependent manner. In vivo, mice treated with indinavir at the same time they were infected with the oocysts showed a 93% reduction in the number of oocysts present in the entire intestinal contents and a 91% reduction in the number of intracellular parasites in the ileum. For established infection, indinavir treatment reduced the number of oocysts in the entire intestinal content by about 50% and the number of intracellular parasites in the ileum by about 70%. These data show that indinavir directly interferes with the cycle of C. parvum, resulting in a marked reduction in oocyst shedding and in the number of intracellular parasites. Protease inhibitors could be considered as good candidates for the treatment of cyptosporidiosis in immunosuppressed persons.     

Inhibitory effect of selenium on Cryptosporidium parvum infection in vitro and in vivo

The anticryptosporidial effect of sodium selenite (selenium) was evaluated in a bovine fallopian tube epithelial (BFTE) cell culture system and an immunosuppressed C57BL/6N adult mouse model. Parasite numbers in cell culture were significantly reduced (p<0.01) following treatment with selenium (Se) at concentrations of 6, 9, and 12 μM at 48 h postinoculation (PI) and at 1.5, 3, and 6 μM at 96 h PI. Parasite reduction was greater than 50% at 48 h PI when 9 and 12 μM Se was used, and at 96 h PI when 6 μM Se was used. Such Se-induced reduction of Cryptosporidium parvum infection was significantly (p<0,0001) blocked when using free-radical scavengers such as mannitol (20 mM). A combined solution of mannitol (20 mM) and reduced glutathione (0.5 mM) enhanced the blockage to almost 100%. Adult C57BL/6N mice were immunosuppressed with dexamethasone phosphate administered ad libitum (16 μg/mL) in drinking water and inoculated with 10⁵ oocysts/mouse. Significantly fewer (p<0.001) oocysts were shed in the feces of mice treated with Se administered ad libitum (12 μM) in drinking water than in untreated mice. The survival time of mice was also significantly increased (p<0.001) following Se treatment. Collectively, these results indicate that Se plays an important role in cryptosporidiosis, and oxidative stress caused by Se is probably a major mechanism in inhibition of C. parvum infection.