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.     

A comparison of the effects of two dinitroanilines against Cryptosporidium parvum in vitro and in vivo in neonatal mice and rats

The effects of two dinitroanilines, oryzalin and trifluralin, were compared against Cryptosporidium parvum, in vitro using HCT-8 cells and in vivo using neonatal Swiss ARC mice and Wistar neonatal rats. In vitro, oryzalin and trifluralin exhibited IC(50) values (concentration necessary to cause a 50% inhibition) of 750 and 800 nM, respectively. A viability assay showed that neither compound produced a cytotoxic effect on the host cells at concentrations as high as 1 microM. The in vivo component of this study consisted of inoculation of neonatal mice and neonatal rats with 10(5) viable oocysts of C. parvum per animal and the subsequent treatment of this infection with trifluralin and oryzalin administered via gastric intubation. At doses of 100 mg kg(-1) body weight administered twice daily for 3 consecutive days, trifluralin had no statistically significant effect on the number of oocysts recovered from the gut of either rats or mice compared with controls, whereas at the same concentration, oryzalin caused 90 and 79% inhibition of oocysts recovered from mice and rats, respectively.     

Immunohistochemistry based assay to determine the effects of treatments on Cryptosporidium parvum viability

Cell culture infectivity assays can provide an accurate means of detecting viable Cryptosporidium parvum oocysts from environmental samples or to test the effects of various treatments on oocyst infectivity. Cell culture assays can also be used to test candidate chemotherapeutic agents. The use of a human cell line provides a situation close to human infection. The present assay uses an anti-Cryptospordium primary antibody, combined with a biotinylated secondary antibody, and an immunoperoxidase detection system. Cryptosporidium parvum oocysts excysted in vitro when placed on monolayers of HCT-8 cells and developmental stages including schizonts and merozoites were visualized using light microscopy of the immunoperoxidase stained slides and by transmission electron microscopy of infected HCT-8 cell cultures. Because the immunoperoxidase system used gives a permanent preparation, the cell cultures can be retained and examined later. Dose titration of oocysts indicated that as few as 50 inoculated oocysts could be detected. The activity of paromomycin was evaluated in this system and 500 microg/ml produced a 97.8% reduction in infection.     

The effect of thermal treatments on the viability and infectivity of Cryptosporidium parvum on beef surfaces

AIMS: The aim of this research was to examine the effect of thermal treatments on the viability and infectivity of Cryptosporidium parvum oocysts attached to a beef surface. METHODS AND RESULTS: This study examined the effects of heat treatment (60 or 75 degrees C) on the viability of C. parvum oocysts inoculated onto the surface of beef muscle estimated by vital dye assay. The infectivity of the oocysts was assessed against monolayers of HCT-8 cells. At 60 degrees C viability of the oocysts decreased from 100% at T0 to 64.2% at T60. At 75 degrees C the viability of the oocysts decreased from 100% at T0 to 53.7% at T15 and finally to 11.2% at T60. Oocysts were rendered noninfective against monolayers of HCT-8 cells following treatments of 60 degrees C/45 s and 75 degrees C/20 s. CONCLUSION: The washing of carcasses with hot water and standard thermal treatments is sufficient to kill C. parvum on beef. SIGNIFICANCE AND IMPACT OF THE STUDY: This study found that relatively mild heat, currently used to decontaminate and heat treat beef carcasses and to cook meat products, is capable of inactivating C. parvum.     

Immunotherapy of cryptosporidiosis in immunodeficient animal models.

Immunotherapy for persistent infection caused by Cryptosporidium parvum was attempted in two immunodeficient animal models. BALB/c Athymic (nude) mice were infected with two oral doses of 2 x 10(7) C. parvum oocysts, and subsequently treated with monoclonal antibody (MAb) 17.41 that neutralizes sporozoites and merozoites. Persistent infection was established in all exposed mice. Daily oral treatment with MAb 17.41 for 10 days significantly reduced (p less than 0.005) the number of C. parvum organisms observed by microscopic study of intestinal tracts of infected mice. Young horses with severe combined immunodeficiency (SCID) also developed persistent infection following oral exposure with 10(8) C. parvum oocysts. In contrast to nude mice, SCID foals exhibited diarrhea associated with oocyst shedding. Two foals were treated orally with MAb 18.44 and immune serum, both of which neutralized C. parvum sporozoites and merozoites. Oocyst shedding patterns did not significantly differ from those in five SCID foals treated with nonimmune reagents. The results obtained indicate that SCID foals are a useful large animal model of clinical disease associated with persistent C. parvum infection, and that nude mice are a convenient animal model for testing therapeutic potential of antibodies in persistent cryptosporidial infection.     

The suppressive effect of Mekabu fucoidan on an attachment of Cryptosporidium parvum oocysts to the intestinal epithelial cells in neonatal mice

The present study was done to investigate the effects of fucoidan and de-sulfated fucoidan isolated from the sporophyll of Undaria pinnatifida on the C. parvum adhesion to the cultured human intestinal cells and on the C. parvum infection in neonatal mice. The C. parvum adhesion to human Intestinal 407 cells was significantly suppressed by a low dose (1 micro g/ml) of Mekabu fucoidan (1 micro g/ml) (approx. 20.5 oocysts, p<0.0001), but not by de-sulfated fucoidan (approx. 138.2 oocysts), as compared with that (approx. 121.0 oocysts) of phosphate-buffered saline (PBS). The in vivo experiments presented here revealed that C. parvum oocysts in the fucoidan-treated mice was reduced nearly one fifth (approx. 5.4x10(4) oocysts, p<0.02) of the total number of oocysts (approx. 3.0x10(5)) in mice treated with PBS, but no significant effect of de-sulfated fucoidan was observed. These results show that (i) fucoidan effectively inhibits the growth of C. parvum in mice; and (ii) the ester sulfate of fucoidan is an active site to prevent the adhesion of C. parvum to the intestinal epithelial cells. Finally, we concluded that fucoidan might inhibit cryptosporidiosis through the direct binding of fucoidan to the C. parvum-derived functional mediators in the intestinal epithelial cells in neonatal mice.     

Quantitative-PCR assessment of Cryptosporidium parvum cell culture infection

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.     

Inhibition of Cryptosporidium parvum infection of a mammalian cell culture by recombinant scFv antibodies

Two phage display antibody libraries (Tomlinson I and J) were screened against the whole oocysts of Cryptosporidium parvum to select for scFv (single chain variable fragment) antibodies. Three scFv antibodies were selected that bound to C. parvum oocysts as determined by monoclonal phage ELISA. DNA sequencing revealed that clone A11 lacked the majority of its V (H) chain. Clone B10 had a stop codon in the first framework region of the V (H) chain. We changed this stop codon to Gly by site-directed mutagenesis, and designated the variant mutB10. Clone B9 had a complete scFv gene with no internal stop codons. These antibody genes were individually subcloned into the pET-20b expression vector for soluble scFv antibody production. C. parvum infectivity was determined by infection of HCT-8 tissue culture monolayers and quantified by the foci detection method. By incubating C. parvum oocysts with individual scFv antibodies for 1 h at 37 degrees C prior to infecting the HCT-8 cells with the oocyst-scFv mixture, the infectivity of C. parvum was reduced in a dose-dependant manner. At the highest soluble scFv concentration tested (4 nmol), the mean number of infectious foci was reduced by 82%, 73% and 94% for the A11, B9 and mutB10 scFv, respectively. This inhibition of oocyst infectivity was abolished when the scFvs were exposed to boiling water. The results showed that the 3 selected scFvs bound to C. parvum oocysts, and their ability to neutralize infectivity may have potential therapeutic potential against cryptosporidiosis.     

Cytokine expression and specific lymphocyte proliferation in two strains of Cryptosporidium parvum-infected gamma-interferon knockout mice

Differences in the immune response between 2 strains of interferon-gamma knockout mice (BALB/c-GKO and C57BL/6-GKO) infected with Cryptosporidium parvum were examined because the course of infection among these 2 strains is markedly different. Infection of the BALB/c-GKO with C. parvum (2 X 10(6) oocysts/mouse) resulted in slight weight loss, oocyst shedding, and recovery from infection by 2 wk postinfection (PI). Infection with 100 oocysts in the C57BL/6-GKO mice resulted in significant weight loss, oocyst shedding, and death by day 10 PI. Splenocytes from infected mice were able to proliferate in a dose-dependent manner to soluble C. parvum-sporozoite antigen (SAg). In vitro stimulation with SAg resulted in an increase in interleukin (IL)-2, IL-4, IL-5, and tumor necrosis factor-alpha mRNA cytokine expression from splenocytes of infected BALB/cGKO mice. In contrast, only IL-5 mRNA expression was increased in the splenocytes from C. parvum-infected C57BL/6-GKO mice. Phenotypic analysis indicated no significant differences in the splenic cell populations. Previous studies indicated that susceptibility to C. parvum is dependent on CD4+ T cells and interferon-gamma production. The present study indicates that although both of these strains of knockout mice become infected with C. parvum, resolution of infection may be in part dependent on the expression of Th2 cytokines.     

Long-term survival of Cryptosporidium parvum oocysts in seawater and in experimentally infected mussels (Mytilus galloprovincialis).

Transmission of infectious oocysts of Cryptosporidium parvum via surface- and drinking-water supplies has been reported and many surface waters flow into the sea, potentially causing runoff of animal-infected faeces. Eating raw mussels is a common practice in many countries, increasing the public's risk of acquiring enteric pathogens. The aims of the present study were to estimate how long C. parvum oocysts remain infectious in artificial seawater, to determine if the oocysts are retained in mussel tissues (Mytilus galloprovincialis), and how long they maintain their infectivity. Oocysts were incubated in artificial seawater at 6-8 degrees C under moderate oxygenation and the infectivity of oocysts was tested five times, over a 12 month period after incubation in seawater, in BALB/c mice. Each pup was inoculated per os with 10(5) oocysts and killed 5 days p.i. Oocysts remained infectious for 1 year. Forty mussels held in an aquarium containing artificial seawater filtered out more than 4 x 10(8) oocysts in a 24 h period. Oocysts were detected in the gill washing up to 3 days p.i., in the haemolymph up to 7 days p.i., and in the intestinal tract up to 14 days p.i. Oocysts collected from the gut of mussels 7 and 14 days p.i. were observed to have infected mice. These results suggest that C. parvum oocysts can survive in seawater for at least 1 year and can be filtered out by benthic mussels, retaining their infectivity up to 14 days, so seawater and molluscs are a potential source of C. parvum infection for humans.     

Cryptosporidium canis n. sp. from domestic dogs.

Oocysts of Cryptosporidium, from the feces of a naturally infected dog and from an HIV-infected human, were identified as the previously reported canine genotype of Cryptosporidium parvum, hereafter referred to as Cryptosporidium canis n. sp. Also among the oocysts from the dog, a trace amount of C. parvum bovine genotype was detected. Cryptosporidium canis oocysts from both the dog and human were infectious for calves. Oocysts excreted by calf 1 (dog source) were approximately 90% C. canis and 10% C. parvum, whereas those excreted by calf 3 (human source) were 100% C. canis. Oocysts from calf 1 infected calf 2 resulting in excretion by calf 2 of oocysts approximately 90% C. parvum and 10% C. canis. Oocysts of C. canis were not infectious for BALB/c neonatal mice or immunosuppressed C57 juvenile mice, although all control mice became infected with the C. parvum Beltsville isolate. Oocysts of C. canis from calf 1 and the human were structurally indistinguishable from oocysts of the C. parvum Beltsville isolate (bovine). However, C. canis oocysts differed markedly at the molecular level from all known species of Cryptosporidium based on sequence data for the 18S rDNA and the HSP 70 gene. The differences in genetics and host specificity clearly differentiate C. canis as a new species.     

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.     

Dose response of Cryptosporidium parvum in outbred neonatal CD-1 mice.

Cryptosporidium parvum infectivity in a neonatal CD-1 mouse model was used to determine the dose needed to infect 50% of the population. The 50% infective dose was estimated to be 79 oocysts. It was observed that a mean oral inoculum of 23 oocysts produced infection in 2 of 25 neonatal mice 7 days postinoculation. All animals became infected when the mean oral dose exceeded 310 oocysts per animal. The dose response of C. parvum was modeled with a logit dose-response model suitable for use in water disinfection studies.     

The utilization of sodium taurocholate in excystation of Cryptosporidium parvum and infection of tissue culture.

The present work deals with optimization of excystation of Cryptosporidium parvum oocysts and the infection process of tissue culture cells by the parasite. It was shown that presence of the bile salt sodium taurocholate in the incubation medium expedited excystation of the tested GCH1 isolate and enhanced it, as compared with bleaching of the oocysts. This bile salt had no effect on the viability of tissue culture cell lines MDBK and HCT-8 at the tested concentration of 0.375% for up to 2 hr of coincubation. Infection studies conducted on tissue culture cells showed higher infection rates in the presence of sodium taurocholate than with bleached oocysts in the absence of this bile salt. It may be concluded that, at least as regards the GCH1 strain of C. parvum, the whole infection process can be performed in the presence of sodium taurocholate, and does not require separation and cleaning of the excysted sporozoites before their application to tissue culture cells.     

Improving the rate of infectivity of Cryptosporidium parvum oocysts in cell culture using centrifugation.

Centrifugation was evaluated as a method to improve infectivity assays of Cryptosporidium parvum in cell culture using the focus detection method, an immunofluorescence-based method for detecting infectious C. parvum oocysts in vitro. Human ileocecal adenocarcinoma (HCT-8) cells were grown for 48 hr on 13-mm cover slips in 24-well microtiter plates and infected with bleach-treated C. parvum oocysts. Plates were centrifuged at 228 g for 10 min and incubated at 37 C for 5, 12, 18, 24, and 48 hr. Foci of infection were stained by immunofluorescence and enumerated using epifluorescent microscopy. Results were compared to noncentrifuged controls. Foci in centrifuged samples could be enumerated after 18 hr. According to most probable number (MPN) analysis, the number of infectious oocysts estimated at 48 hr (13,326 infectious oocysts) was reached by 18 hr in centrifuged samples. After 48 hr, there was no significant difference (P < 0.05) between centrifuged and noncentrifuged samples enumerated by number of foci or the MPN of infectious oocysts. Centrifugation may expedite detection during C. parvum infectivity assays. Furthermore, multiwell plate formats are more cost effective than traditional chamber slides.     

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.     

Detection and counting of Cryptosporidium parvum in HCT-8 cells by flowcytometry

The objective of the present study was to evaluate flowcytometry analysis (FCA) as a tool for rapidly and objectively estimating the percentage of cells infected with Cryptosporidium parvum in an in vitro model. We compared the results to those obtained with immunofluorescence assay (IFA) and evaluated the intra-assay variability of both assays and the inter-assay variability of IFA. Human ileocecal adenocarcinoma cells (HCT-8) were infected with different doses of excysted oocysts. After 24 hours, cells were analysed by FCA and by IFA using a monoclonal antibody that recognises a C. parvum antigenic protein and a lectin that binds with glycoproteins present in the parasitophorous vacuoles. The coefficient of variability in terms of the percentage of infected cells was lower for FCA (i.e., 13-14%) than for IFA (i.e., 27-38% when performed by a single operator and 19-22% when performed by three operators), suggesting that FCA is more accurate, in that it is not subject to operator expertise. FCA also has the advantage of allowing the entire culture to be examined, thus avoiding problems with heterogeneity among microscopic fields. In light of these results, this method could also be used to test new anti-Cryptosporidium drugs.     

Role of intraepithelial lymphocytes in mucosal immune responses of mice experimentally infected with Cryptosporidium parvum

In order to investigate the role of intestinal intraepithelial lymphocytes (IELs) in host defense against Cryptosporidium parvum infection, conventionally bred immunocompetent (ImCT) ICR mice and immunosuppressed (ImSP) littermates were infected orally with 10(6) C. parvum oocysts. Then fecal oocyst excretion, the number and location of IELs, and their T lymphocyte subsets were observed on days 4, 7, 10, 13, 16, and 20 postinfection (PI). Uninfected ImCT and ImSP mice were used as controls. The starting point of oocyst excretion was day 4 PI in both ImCT- and ImSP-infected mice. The highest oocyst excretion occurred on day 7 PI in both groups, though the number of oocysts excreted was 3 times greater in ImSP than in ImCT mice. In ImCT mice, IELs greatly increased in number on days 16 and 20 PI (P < 0.05), but the increase was minimal in ImSP mice. IELs changed their location from the basal area to intermediate and apical areas of villous epithelial cells during the early stage of infection. In ImCT-infected mice, IEL phenotypes also changed; whereas CD4+ cells increased temporarily on day 7 PI (P < 0.05), CD8+ cells increased significantly on days 16 and 20 PI (P < 0.05). The results strongly suggest that IELs play a significant role in host defense against C. parvum infection, with helper T cells initiating control of the infection and cytotoxic T cells eliminating the parasites.