Protection of calves against cryptosporiosis by oral inoculation with gamma-irradiated Cryptosporidium parvum oocysts

The purpose of this study was to determine whether gamma-irradiated Cryptosporidium parvum oocysts could elicit protective immunity against cryptosporidiosis in dairy calves. Cryptosporidium parvum Iowa strain oocysts (1 x 10(6) per inoculation) were exposed to various levels of gamma irradiation (350-500 Gy) and inoculated into 1-day-old dairy calves. The calves were examined daily for clinical signs of cryptosporidiosis, and fecal samples were processed for the presence of C. parvum oocysts. At 21 days of age, the calves were challenged by oral inoculation with 1 x 10(5) C. parvum oocysts and examined daily for oocyst shedding and clinical cryptosporidiosis. Calves that were inoculated with C. parvum oocysts exposed to 350-375 Gy shed C. parvum oocysts in feces. Higher irradiation doses (450 or 500 Gy) prevented oocyst development, but the calves remained susceptible to C. parvum challenge infection. Cryptosporidium parvum oocysts exposed to 400 Gy were incapable of any measurable development but retained the capacity to elicit a protective response against C. parvum challenge. These findings indicate that it may be possible to protect calves against cryptosporidiosis by inoculation with C. parvum oocysts exposed to 400-Gy gamma irradiation.     

Quantitative flow cytometric evaluation of maximal Cryptosporidium parvum oocyst infectivity in a neonate mouse model

The importance of waterborne transmission of Cryptosporidium parvum to humans has been highlighted by recent outbreaks of cryptosporidiosis. The first step in a survey of contaminated water currently consists of counting C. parvum oocysts. Data suggest that an accurate risk evaluation should include a determination of viability and infectivity of counted oocysts in water. In this study, oocyst infectivity was addressed by using a suckling mouse model. Four-day-old NMRI (Naval Medical Research Institute) mice were inoculated per os with 1 to 1,000 oocysts in saline. Seven days later, the number of oocysts present in the entire small intestine was counted by flow cytometry using a fluorescent, oocyst-specific monoclonal antibody. The number of intestinal oocysts was directly related to the number of inoculated oocysts. For each dose group, infectivity of oocysts, expressed as the percentage of infected animals, was 100% for challenge doses between 25 and 1,000 oocysts and about 70% for doses ranging from 1 to 10 oocysts/animal. Immunofluorescent flow cytometry was useful in enhancing the detection sensitivity in the highly susceptible NMRI suckling mouse model and so was determined to be suitable for the evaluation of maximal infectivity risk.     

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.     

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.     

A comparative study on the biology of Cryptosporidium sp. from guinea pigs and Cryptosporidium parvum (Apicomplexa).

Cryptosporidium sp. from guinea pigs and C. parvum were compared morphologically, electrophoretically, and for the ability to infect suckling mice. Oocysts from guinea pigs measured 5.4 x 4.6 (4.8-5.6 x 4.0-5.0) microns and had a shape index (length/width) of 1.17 (1.04-1.33). Oocysts of C. parvum were similar and measured 5.2 x 4.6 (4.8-5.6 x 4.2-4.8) microns with a shape index of 1.16 (1.04-1.33). All suckling mice inoculated with oocysts of C. parvum became infected, whereas most, but not all, mice fed oocysts of the guinea pig isolate also became infected. However, mice inoculated with oocysts from guinea pigs produced on average 100-fold fewer oocysts by day 7 postinoculation than did mice infected with C. parvum, and the resulting infections were sparse and patchy along the ileum. Electrophoretic profiles were similar, but 125I surface labeling of outer oocyst wall proteins revealed striking differences between the two isolates. Cryptosporidium parvum had a wide molecular size range of 125I-labeled bands, whereas C. sp. from guinea pigs had a banding pattern clustered between 39 and 66 kDa, with a smaller number of bands greater than 100 kDa.     

Highly Active AntiRetroviral Therapy and cryptosporidiosis

In HIV infected persons, Cryptosporidium parvum causes chronic diarrhoea, which can be life-threatening in persons with AIDS and with a low CD4+ T cell count. However, a specific and effective therapy for this opportunistic infection does not yet exist. Since the use of a combination therapy with a highly active antiretroviral therapy (HAART), the prevalence of C. parvum infection in persons with AIDS has been strongly reduced. This favorable outcome was usually attributed to the recovery of the host immunity, however improvements from this opportunistic infection have been demonstrated even in the absence of immunological recovery. The aim of the present study was to determine whether HIV protease inhibitors (PIs) exert an anti-C. parvum activity. We selected the indinavir (an aspartyl protease inhibitor included in HAART) for our experiments, since a resolution of cryptosporidial enteritis in a person with AIDS after treatment with this drug has been reported. Human ileocecal adenocarcinoma tumor cells (HCT-8) were used as in vitro model. To determine whether or not indinavir had an effect on the parasite attachment to, or invasion of the HCT-8 cells, indinavir was added to the cultures at the same time as the infective dose (3 oocysts/cell) at one of the following concentrations: 0.1, 0.5, 5, 10, 20, and 50 microM (maximum DMSO content 0.5% vol/vol). To determine whether or not indinavir had an effect on established C. parvum infection, HCT-8 cells were infected with excysted oocysts at a ratio of 3 oocysts/cell at day 0, and then indinavir at a concentration of 50 microM was added to the cultures every 24 h for 4 days. The infection level was evaluated at 2, 3, 4 and 5 days p.i. using a flowcytometric assay. Three-day-old Balb/c mice were used as animal model, animals were infected per os with 50 microl of PBS containing 10(5) oocysts. The infected mice were divided into two groups (Group A and Group B), both of which received per os indinavir diluted in PBS with 0.1% DMSO at a concentration of 10 microM (24 mg/kg). For Group A, which consisted of 15 mice (3 litters), indinavir was administered at the same time that experimental infection was performed and then every day until the mice were sacrificed (i.e., 5 days p.i.), to determine the effect of indinavir on the attachment/invasion of the enterocytes. For Group B, which also consisted of 15 mice (3 litters), indinavir was administered after the infection was established (i.e., 72 h p.i.) and every day until being sacrificed, to determine the effect of indinavir on established infection. The mice of Group B were sacrificed 7, 10, 11 and 13 days p.i., corresponding to 4, 7, 8, and 10 days of treatment with indinavir. In vitro, the treatment of the excystated oocysts with different concentrations of indinavir reduced the percentage of HCT-8 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. Treatment for 48 h resulted in a 40.1% reduction in infected cells (from 90% to 53%). After 72 h of treatment, the percentage of infected cells did not substantially differ from that observed after 48 h. Treatment for 96 h resulted in a 57.8% reduction (from 90 to 38%). 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 demonstrate that PIs directly exert an inhibitory effect on C. parvum and the extent of this effect depended on the specific dose and the duration of treatment. Although there are no reports of aspartyl proteases in C. parvum, the inhibitory effect of PIs on C. parvum growth in vitro suggests that aspartyl proteases could have some important functions for this parasite. In fact, proteolytic activities have been demonstrated during peak periods of excystation in C. parvum oocysts and cysteine and serine protease classes have been functionally associated with this process. Moreover, we identified several different C. parvum sequences that showed homology with a protein family related to aspartyl proteases. In prospect, PIs could be valuable for the chemotherapy of cryptosporidiosis.     

Comparison of the host ranges and antigenicity of Cryptosporidium parvum and Cryptosporidium wrairi from guinea pigs.

Oocysts of a Cryptosporidium isolate from guinea pigs were not infectious for adult mice, but were infectious for two of three newborn calves and for suckling mice. However, oocysts isolated from calves or mice infected with guinea pig Cryptosporidium were not infectious for guinea pigs. Four isolates of C. parvum from calves were incapable of infecting weanling guinea pigs. Microscopic examination of tissue from the colon and cecum of suckling guinea pigs inoculated with C. parvum revealed sparse infection of some pups. These host range studies and previously described differences in 125I-labeled oocyst surface protein profiles between Cryptosporidium sp. from guinea pigs and C. parvum suggest they are distinct species. We propose the name Cryptosporidium wrairi be retained. Studies with monoclonal antibodies indicate that C. wrairi and C. parvum are antigenically related.     

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.     

Comparison of the Host Ranges and Antigenicity of Cryptosporidium parvum and Cryptosporidium wrairi from Guinea Pigs

ABSTRACT. Oocysts of a Cryptosporidium isolate from guinea pigs were not infectious for adult mice, but were infectious for two of three newborn calves and for suckling mice. However, oocysts isolated from calves or mice infected with guinea pig Cryptosporidium were not infectious for guinea pigs. Four isolates of C. parvum from calves were incapable of infecting weanling guinea pigs. Microscopic examination of tissue from the colon and cecum of suckling guinea pigs inoculated with C. parvum revealed sparse infection of some pups. These host range studies and previously described differences in ¹²⁵I-labeled oocyst surface protein profiles between Cryptosporidium sp. from guinea pigs and C. parvum suggest they are distinct species. We propose the name Cryptosporidium wrairi be retained. Studies with monoclonal antibodies indicate that C. wrairi and C. parvum are antigenically related.     

Supplementation with Lactobacillus reuteri or L. acidophilus reduced intestinal shedding of cryptosporidium parvum oocysts in immunodeficient C57BL/6 mice.

The effect of L. acidophilus supplementation to reduce fecal shedding of Cryptosporidium parvum oocysts was compared to L. reuteri using C57BL/6 female mice immunosuppressed by murine leukemia virus (strain LP-BM5) inoculation. After 12 weeks post LP-BM5 inoculation, 15 immunosuppressed mice each were randomly assinged to one of the following treatment groups: historical control (group A), LP-BM5 control (group B), C. parvum (group C), L. reuteri plus C. parvum (group D) or L. acidophilus plus C. parvum (group E). Mice were pre-fed the L. reuteri or L. acidophilus bacteria strains daily for 13 days, challenged with C. parvum oocysts and thereafter fed the specified Lactobacillus regimens daily during the experimental period. Animals supplemented with L. reuteri shed fewer (p<0.05) oocysts on day-7 post C. parvum challenge compared to controls. Mice supplemented with L. acidophilus also shed fewer (p<0.05) oocysts on days 7 and 14 post-challenge compared to controls. Overall, Lactobacillus supplementation reduced C. parvum shedding in the feces but failed to suppress the production of T-helper type 2 cytokines [interleukin-4 (IL-4), IL-8)] which are associated with immunosuppression. Additionally, Lactobacillus supplementation did not restore T-helper type 1 cytokines (interleukin-2 (IL-2) and gamma interferon (IFN-gamma), which are required for recovery from parasitic infections. Altered T-helper types 1 and 2 cytokine production as a consequence of immunodysfunction permitted the development of persistent cryptosporidiosis while mice with intact immune system were refractory to infection with C. parvum. Reduction in shedding of oocysts observed in the Lactobacillus supplemented mice during deminished IL-2 and IFN-gamma production may be mediated by factors released into the intestinal lumen by the Lactobacillus and possibly other host cellular mechanisms. These observations suggest that L. reuteri or L. acidophilus can reduce C. parvum parasite burdens in the intestinal epithelium during cryptosporidiosis and may serve potential benefits as probiotics for host resistance to intestinal parasitic infections. L. acidophilus was more efficacious in reducing fecal shedding than L. reuteri and therefore may also have implication in the therapy of cryptosporidiosis during immunosuppressive states including human AIDS.     

Viability and infectivity of Cryptosporidium parvum oocysts are retained upon intestinal passage through a refractory avian host.

Six Cryptosporidium-free Peking ducks (Anas platyrhynchos) were each orally inoculated with 2.0 x 10(6) Cryptosporidium parvum oocysts infectious to neonatal BALB/c mice. Histological examination of the stomachs jejunums, ilea, ceca, cloacae, larynges, tracheae, and lungs of the ducks euthanized on day 7 postinoculation (p.i.) revealed no life-cycle stages of C. parvum. However, inoculum-derived oocysts extracted from duck feces established severe infection in eight neonatal BALB/c mice (inoculum dose, 2.5 x 10(5) per mouse). On the basis of acid-fast stained direct wet smears, 73% of the oocysts in duck feces were intact (27% were oocyst shells), and their morphological features conformed to those of viable and infectious oocysts of the original inoculum. The fluorescence scores of the inoculated oocysts, obtained by use of the MERIFLUOR test, were identical to those obtained for the feces-recovered oocysts (the majority were 3+ to 4+). The dynamics of oocyst shedding showed that the birds released a significantly higher number of intact oocysts than the oocyst shells (P < 0.01). The number of intact oocysts shed (87%) during the first 2 days p.i. was significantly higher than the number shed during the remaining 5 days p.i. (P < 0.01) and significantly decreased from day 1 to day 2 p.i. (P < 0.01). The number of oocyst shells shed during 7 days p.i. did not vary significantly (P > 0.05). The retention of infectivity of C. parvum oocysts after intestinal passage through an aquatic bird has serious epidemiological and epizootiological implications. Waterfowl may serve as mechanical vectors for the waterborne oocysts and may enhance contamination of surface waters with C. parvum. As the concentration of Cryptosporidium oocysts in source waters is attributable to watershed management practices, the watershed protection program should consider waterfowl as a potential factor enhancing contamination of the source water with C. parvum.     

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).     

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.     

Inactivation of Cryptosporidium parvum oocysts in fresh apple cider by UV irradiation

This study evaluated the efficacy of UV irradiation on the inactivation of Cryptosporidium parvum oocysts in fresh apple cider. Cider was inoculated with oocysts and exposed to 14.32 mJ of UV irradiation/cm(2). Oocyst viability was assessed with the gamma interferon gene knockout (GKO) mouse and infant BALB/cByJ mouse models. All GKO mice challenged with UV-treated cider demonstrated no morbidity or mortality, and infant BALB/c mice challenged with treated cider were negative for the presence of C. parvum. In contrast, the GKO mice challenged with non-UV-treated inoculated cider died and the parasite was detected in the ileums of all challenged infant mice. This study shows that UV irradiation can be used to inactivate C. parvum in fresh apple cider.     

Anticryptosporidial activity is associated with specific sulfonamides in immunosuppressed rats

Dexamethasone-immunosuppressed rats infected with Cryptosporidium parvum were used to assess 23 sulfonamides for anticryptosporidial activity. Five of the compounds administered before the animals were inoculated with C. parvum oocysts reduced the severity of cryptosporidial infections in the rat model. Two of the 5 agents with prophylactic activity, sulfadimethoxine and sulfamethazine, were effective also against an established infection, indicating that some sulfonamides may have therapeutic value in immunosuppressed patients with cryptosporidiosis. The findings also suggest that sulfonamide treatment of cryptosporidiosis in the immunocompromised host may not be successful unless the compound is administered continuously or over several weeks.     

High-yield amplification of Cryptosporidium parvum in interferon gamma receptor knockout mice

To date, large-scale production of Cryptosporidium parvum oocysts has only been achieved by amplification in neonatal calves and sheep. Many laboratories currently depend on supplies from external sources and store oocysts for prolonged periods which results in progressive loss of viability. Six to 8-week-old interferon gamma receptor knockout (IFN gamma R-KO) mice on a C57BL/6 background were inoculated by gavage (2000 oocysts/animal). Fecal pellets were collected daily from 7 days post-infection (p.i.) up to 2 weeks p.i. Intestinal oocyst yield was assessed at days 11, 12 and 14 p.i. by homogenization of intestinal tissues. Ether extraction and one or more NaCl flotations were used to purify oocysts. Total recoveries averaged 2.6 x 10(6) oocysts/mouse from fecal material and 3.8 x 10(7) oocysts/mouse from intestinal tissues. Overall, 2.3 x 10(9) purified oocysts were obtained from 60 mice. Recovered oocysts were capable of sporulation and were shown to be infectious both in vitro and in vivo. Oocyst amplification was achieved in only 11-14 days with minimal expense. The simplicity of this method presents a practical alternative for the routine passage, maintenance and storage of C. parvum in biomedical laboratories.     

Oral dosing of neonatal mice with sucrose reduces infection with Cryptosporidium parvum

Cryptosporidium parvum is a significant cause of diarrheal disease in humans and economically important livestock species. There is no effective treatment available for this protozoan parasite. Mechanisms of intestinal colonization by C. parvum are not well understood, but it has been suggested that the parasite may utilize a lectin-like receptor. We used an infant mouse model to test whether high sugar concentrations in the intestine would affect in vivo colonization with C. parvum. We found that a single oral dose of sucrose, administered to mice at the time of, or 24 hr before, challenge with C. parvum significantly reduced infection. Significant reduction of infection was also seen in mice given isomaltose. Histologic examination of intestinal sections of mice treated with sucrose or isomaltose, but not other sugars, showed marked vacuolation of the small intestinal epithelium 1 day after treatment. Three days after treatment, tissue appeared normal. Thus, sucrose and, to a lesser extent, isomaltose reduced in vivo colonization with C. parvum and altered epithelial cell morphology in intestines of mice.     

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.