Cryptosporidium parvum infection in gene-targeted B cell-deficient mice

The importance of B cells in host resistance to, and recovery from, Cryptosporidium parvum infection was examined in gene-targeted B cell-deficient (muMT-/-) mice. Neonatal muMT-/- mice infected with C. parvum at 5 days of age completely cleared the infection by day 20 PI. The kinetics of infection and clearance were similar to those seen with age-matched C57BL/6 control mice. Furthermore, B cells were not required to clear existing C. parvum infection in adult mice. Reconstitution of persistently infected Rag-1-/- adult mice with spleen cells from muMT-/- donor mice resulted in significant reduction of infection, as in the results seen with spleen cells from C57BL6 donors. These findings indicate clearly that B cells are not essential for host resistance to, and recovery from, C. parvum infection in mice.     

Treatment with agmatine inhibits Cryptosporidium parvum infection in infant mice

Cryptosporidium parvum is an intracellular protozoan parasite that causes enteric infection and diarrhea in a wide range of mammalian hosts, including humans and economically important livestock species. There are no effective vaccines or drug treatments available for cryptosporidiosis. Cryptosporidium parvum utilizes a unique metabolic pathway for the synthesis of polyamines, forming agmatine as an intermediary metabolite. We treated infant mice with oral doses of agmatine for 2 days before, the day of, and 5 days following experimental infection with C. parvum. Mice treated with agmatine were significantly less infected with C. parvum than were control mice receiving phosphate-buffered saline. Mice treated with agmatine only on the day of experimental infection with C. parvum were also significantly less infected than were control mice. These data suggest that exogenous agmatine alters the metabolism of C. parvum sufficient to interfere with its ability to colonize the mammalian intestine.     

Susceptibility dynamics in neonatal BALB/c mice infected with Cryptosporidium parvum.

BALB/c Mice were infected as neonates and at different ages to study the susceptibility dynamics in this animal model to Cryptosporidium parvum. When 4-day-old animals were infected with 10(5) C. parvum oocysts, parasites were detected in the terminal ileum when the mice became 14-25 days old (10-21 days post-infection [PI]). The percentage of animals positive for parasites was 100% up to the age of 19 days (15 days PI) but decreased immediately thereafter until no parasites were detected in 26-day-old (22 days PI) or older mice. Parasite load also decreased in these animals from 184.7 parasites per high power field in 14-day-old animals (10 days PI) to 0.22 in 25-day-old (21 days PI) mice. In a second study, some neonatal mice became resistant to C. parvum when infection was attempted at day-10 of age (day-15 of age at sacrifice). The susceptibility to C. parvum decreased until 14 days of age (19 days of age at sacrifice) when mice could no longer be infected. Parasite load also decreased in infected mice from 235.6 parasites per high power field (9 days of age at sacrifice) to 0.25 (18 days of age at sacrifice).     

Pathogenesis of Cryptosporidium parvum infection

Cryptosporidium parvum can be regarded as a minimally invasive mucosal pathogen, since it invades surface epithelial cells that line the intestinal tract but does not invade deeper layers of the intestinal mucosa. Nonetheless, infection can be associated with diarrhea and marked mucosal inflammation. This article briefly reviews in vitro and in vivo models useful for studying the pathogenesis of C. parvum infection and explores the role of innate and acquired immune responses in host defense against this protozoan parasite.     

Caspase-dependent apoptosis during infection with Cryptosporidium parvum

The protozoan parasite Cryptosporidium parvum causes persistent diarrhea and malnutrition in children and the diarrhea-wasting syndrome in AIDS. No therapy exists for eliminating the parasite in the absence of a healthy immune response. Although it had been reported that infection of intestinal cell lines with C. parvum leads to host cell death, the mechanisms of cytolysis have not been characterized. We show here that infection with C. parvum leads to typical apoptotic nuclear condensation and DNA fragmentation in host cells. Both nuclear condensation and DNA fragmentation are inhibited by a caspase inhibitor, showing that caspases are involved in this type of apoptosis. Finally, blocking apoptosis with the caspase inhibitor increases the percentage of infected cells, suggesting that parasites may use apoptosis to exit from the infected cell or that the infected cells may eliminate the parasite through apoptosis. These results suggest that apoptosis could be involved in the pathogenesis of C. parvum infections in vivo, and raise the possibility that therapeutic interference with host cell death could alter the course of the pathology in vivo.     

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.     

Cryptosporidium parvum: the course of Cryptosporidium parvum infection in C57BL/6 mice co-infected with the nematode Heligmosomoides bakeri

The effects of Heligmosomoides bakeri infection on the course of a concurrent Cryptosporidium parvum infection were studied in C57BL/6 mice. Mice were initially infected with 80 L₃ of H. bakeri and then challenged with 10⁴ oocysts of C. parvum, administered during the patent period of the nematode infection (28 day post H. bakeri infection). The number of C. parvum oocysts excreted in the feces and the number of adult H. bakeri in the small intestine were monitored during the experiment. Concurrent H. bakeri infection resulted in a prolonged course of infection with C. parvum. The intensities of both parasite infections were higher in co-infections. We also investigated the cellular immune response at 14 and 42 days post infection C. parvum. During infection with C. parvum there was an increase in production of IFN-γ and IL-12 but co-infection with H. bakeri inhibited IFN-γ secretion. The present study is the first to demonstrate that infection with H. bakeri markedly exacerbates the intensity of a concurrent C. parvum infection in laboratory mice and also affects immune effectors mechanisms in co-infection with H. bakeri.     

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.     

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.     

A novel detection method of Cryptosporidium parvum infection in cattle based on Cryptosporidium parvum virus 1

Cryptosporidium parvum is an important zoonotic parasite that causes significant economic loss in the animal husbandry industry,especially the cattle industry.As there is no specific vaccine or drug against Cryptosporidium,a rapid and accurate method for the detection of C.parvum is of great significance.In this study,colloidal gold strips were developed based on Cryptosporidium parvum virus 1 (CSpV1) for the detection of C.parvum infection in cattle fecal samples.The colloidal gold solution was prepared by reducing trisodium citrate and the CSpV1 #5 monoclonal antibody was labeled with colloidal gold.A polyclonal antibody against the CSpV1 capsid protein and an anti-mouse IgG antibody were coated on the colloidal gold strips for use in the test and control lines,respectively.Our results showed that the detection sensitivity in fecal samples was up to a 1:64 dilution.There was no cross-reaction with Cryptosporidium andersoni or Giardia in the fecal samples.The different preservation conditions (room temperature,4℃,and 37℃) and preservation time (7,30,60,and 90 days) were analyzed.The data showed that the strips could be preserved for 90 days at 4℃ and for 60 days at room temperature or 37℃.The colloidal gold strips were used to detect the samples of 120 clinical fecal in Changchun,China.The results indicated that the rate of a positive test was 5%(6/120).This study provides a rapid and accurate method for detecting C.parvum infection in cattle and humans.     

NF-kappaB-mediated expression of iNOS promotes epithelial defense against infection by Cryptosporidium parvum in neonatal piglets

Cryptosporidium sp. parasitizes intestinal epithelium, resulting in enterocyte loss, villous atrophy, and malabsorptive diarrhea. We have shown that mucosal expression of inducible nitric oxide (NO) synthase (iNOS) is increased in infected piglets and that inhibition of iNOS in vitro has no short-term effect on barrier function. NO exerts inhibitory effects on a variety of pathogens; nevertheless, the specific sites of iNOS expression, pathways of iNOS induction, and mechanism of NO action in cryptosporidiosis remain unclear. Using an in vivo model of Cryptosporidium parvum infection, we have examined the location, mechanism of induction, specificity, and consequence of iNOS expression in neonatal piglets. In acute C. parvum infection, iNOS expression predominated in the villous epithelium, was NF-kappaB dependent, and was not restricted to infected enterocytes. Ongoing treatment of infected piglets with a selective iNOS inhibitor resulted in significant increases in villous epithelial parasitism and oocyst excretion but was not detrimental to maintenance of mucosal barrier function. Intensified parasitism could not be attributed to attenuated fluid loss or changes in epithelial proliferation or replacement rate, inasmuch as iNOS inhibition did not alter severity of diarrhea, piglet hydration, Cl- secretion, or kinetics of bromodeoxyuridine-labeled enterocytes. These findings suggest that induction of iNOS represents a nonspecific response of the epithelium that mediates enterocyte defense against C. parvum infection. iNOS did not contribute to the pathogenic sequelae of C. parvum infection.     

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.     

Solar UV reduces Cryptosporidium parvum oocyst infectivity in environmental waters

Aims: To determine the effect of solar radiation on Cryptosporidium parvum in tap and environmental waters. Methods and Results: Outdoor tank experiments and a cell culture infectivity assay were used to measure solar inactivation of C. parvum oocysts in different waters. Experiments conducted on days with different levels of solar insolation identified rapid inactivation of oocysts in tap water (up to 90% inactivation within the first hour). Increased dissolved organic carbon content in environmental waters decreased solar inactivation. The role of solar ultraviolet (UV) in inactivation was confirmed by long-pass filter experiments, where UV-B was identified as the most germicidal wavelength. Reductions in oocyst infectivity following solar radiation were not related to a loss of excystation capacity. Conclusions: Solar UV can rapidly inactivate C. parvum in environmental waters. Significance and Impact of the Study: This is the first study to assess natural sunlight inactivation of C. parvum oocysts in surface waters and drinking water using an infectivity measure and determines the wavelengths of light responsible for the inactivation. The findings presented here provide valuable information for determining the relative risks associated with Cryptosporidium oocysts in aquatic environments and identify solar radiation as a critical process affecting the oocyst survival in the environment.     

Dietary nucleosides and nucleotides reduce Cryptosporidium parvum infection in dexamethasone immunosuppressed adult mice.

Numerous studies have demonstrated that dietary sources of nucleosides and nucleotides are important for the maintenance of cellular and humoral immune responses. To determine the immunological effects of feeding a nucleoside-nucleotide mixture to dexamethasone-immunosuppressed C57BL/6 adult mice infected with Cryptosporidium parvum, we examined fecal oocyst shedding, lymphoproliferative responses to concanavalin (Con) A, and C. parvum antigen, interleukin (IL-2), and gamma-interferon (IFN-gamma) production by cultured spleen cells. Mice were fed a nucleotide-free 20% casein diet (control group) or this diet supplemented with a 0. 5% nucleoside-nucleotide mixture before and after inoculation with C. parvum. Spleens from mice receiving the supplemented diet had higher (P < 0.05) Con A and antigen-specific induced cell proliferation than those from control mice. In addition to the increased cell proliferation, the spleen cells from the supplemented mice produced significantly more IL-2 (P < 0.002) and significantly more IFN-gamma (P <; 0.004) than cells from the control mice. Mice fed the supplemented diet excreted fewer (P < 0.05) C. parvum oocysts in the feces than control mice. The cumulative survival rate in the nucleoside-nucleotide mixture-fed group was higher compared with the control group (P < 0.05). We conclude that nucleosides and nucleotides may partially counteract the immunosuppressive effects of dexamethasone in C. parvum-challenged mice.     

A factor derived from adult rat and cow small intestine reduces Cryptosporidium parvum infection in infant rats

Cryptosporidium parvum is an intracellular protozoan parasite of the mammalian intestine. In rats, C. parvum infection is age related; infants are susceptible, whereas adults are resistant. The transition from susceptibility to resistance usually takes place around the age of weaning. In the present study, infant rats were orally inoculated with a preparation of intestinal scrapings taken from adult rats or cows. Infant rats received the scrapings daily from 3 to 14 days of age, were inoculated with C. parvum oocysts at 9 days of age, and killed at 15 days of age. Fecal samples and intestinal tissues were examined for the presence of C. parvum. Significantly fewer rats were infected in the groups that received intestinal scrapings compared with controls. In addition, infected rats in the treatment groups shed significantly fewer oocysts than those in the control group. Scrapings from the intestinal mucosa of adult cows were also able to protect infant rats from infection, whereas scrapings from intestines of calves were not protective. In sum, these data indicate the presence of a factor in the intestines of adult rats and cows that can transfer protection against C. parvum infection to susceptible infant rats.     

Effect of sodium hypochlorite exposure on infectivity of Cryptosporidium parvum oocysts for neonatal BALB/c mice.

Oocysts of Cryptosporidium parvum suspended in 5.25, 2.63, or 1.31% aqueous sodium hypochlorite (Clorox laundry bleach) for 10, 30, 60, or 120 min at 21 degrees C were administered by gastric intubation to neonatal BALB/c mice. Microscopic examination of intestinal tissue sections revealed developmental stages of C. parvum in all of the mice.     

Unexpected activity of beta-cyclodextrin against experimental infection by Cryptosporidium parvum

An unexpected activity of beta-cyclodextrin, an excipient used in pharmaceutical technology, was observed against Cryptosporidium parvum. The viability and infectivity of purified oocysts, exposed for 24 hr to beta-cyclodextrin (2.5% suspension), were evaluated by inclusion/exclusion of 2 fluorogenic vital dyes and a suckling murine model, respectively. Results of the viability assay showed a high proportion of nonviable oocysts (81.5%). The intensity of experimental infection, determined 7 days postinoculation by examination of intestinal homogenates, was significantly lower (P < 0.05) than in the control litters. The preventive and curative efficacies of beta-cyclodextrin suspension were also evaluated in experimentally infected neonatal mice. Infection was prevented when the suspension was administered 2 hr before inoculated oocysts and on days 1 and 2 postinoculation.     

Infectivity of preserved Cryptosporidium parvum oocysts for immunosuppressed adult mice

Abstract The present study was undertaken to determine the infectivity of Cryptosporidium parvum oocysts for immunosup-pressed adult C57BL/6N mice after the oocysts had been stored from 1–48 months at 4°C in 2.5% potassium dichromate. All mice inoculated with oocysts 1–18 months old developed patent infections, while mice inoculated with older oocysts remained uninfected. The prepatent period was extended from 2 to 6 or 7 days as the storage time for oocysts increased. The finding that C. parvum oocysts remain infective for mice for at least 18 months offers important economic and time-saving advantages for investigators who frequently require large numbers of oocysts that must be painstakingly purified from calf manure.