Effect of orally administered monoclonal antibody on persistent Cryptosporidium parvum infection in scid mice.

Scid mice, persistently infected after exposure to 10(7) Cryptosporidium parvum oocysts, were treated daily for 14 to 17 days with 0.4 mg of monoclonal antibody (mAb) 17.41 administered by the oral route. Mice receiving mAb 17.41 shed significantly fewer (P < 0.005) C. parvum oocysts than scid mice receiving isotype control mAb. Intestinal (but not gastric) infectivity scores were also reduced for scid mice treated with mAb 17.41 (P < 0.01).     

Requirements for CD4+ cells and gamma interferon in resolution of established Cryptosporidium parvum infection in mice.

The importance of CD4+ cells and gamma interferon (IFN-gamma) in the resolution of established Cryptosporidium parvum infection was investigated with a murine model of cryptosporidiosis in severe combined immunodeficient (SCID) mice. C. parvum-infected SCID mice were reconstituted with spleen cells from immunocompetent donors. The recipients were able to resolve their C. parvum infection by 17 days postreconstitution. Treatment of reconstituted SCID mice with either anti-CD4 monoclonal antibodies to deplete them of CD4+ cells or with anti-IFN-gamma to neutralize IFN-gamma activity reduced or eliminated their ability to resolve C. parvum infection whereas treatment with either anti-CD8 monoclonal antibodies or anti-asialo-GM1 antibodies had no effect. We also found C. parvum-specific antibodies in serum samples from two of four reconstituted SCID mice killed on postreconstitution day 17 but not in unreconstituted SCID mice. Moreover, anti-CD4-treated mice had no detectable specific antibodies to C. parvum, whereas all mice treated with either anti-CD8 or anti-asialo-GM1 had substantial levels of specific antibodies in their serum. Although the role of the specific antibody is not known, these findings clearly indicate that resolution of an established C. parvum infection in immunologically reconstituted SCID mice is dependent on both CD4+ cells and IFN-gamma.     

Immune responses to Cryptosporidium muris and Cryptosporidium parvum in adult immunocompetent or immunocompromised (nude and SCID) mice.

Adult murine models of Cryptosporidium infection involving Cryptosporidium muris and C. parvum were used to study immunity to cryptosporidiosis in the mammalian host. Immunocompetent BALB/c or C57BL/6 mice developed a highly patent infection with the RN 66 strain of C. muris but overcame the infection and were immune to reinfection. In contrast, severe combined immunodeficiency (SCID) mice or nude mice had a chronic infection lasting at least 109 days. The development of the C. muris infection appeared to be confined to the gastric epithelium in immunocompetent and immunocompromised mice. SCID mice injected intraperitoneally with histocompatible spleen or mesenteric lymph node cells from uninfected BALB/c mice were able to recover from the C. muris infection. The protective effect of donor spleen cells was not reduced by depletion of the B cell population but was significantly reduced by depletion of Thy.1 cells. Treatment of C57BL/6 or BALB/c mice during infection with a gamma interferon-neutralizing monoclonal antibody, but not a tumor necrosis factor-neutralizing monoclonal antibody, resulted in a significant increase in oocyst production. In the C. parvum model, a severe and eventually fatal chronic infection with a cervine isolate was established in SCID mice, with parasitization occurring in the ileum, cecum, and colon. SCID mice injected with unprimed BALB/c spleen cells prior to inoculation of C. parvum oocysts were resistant to infection. These results suggested that the two animal models should be valuable in the study of immunity to cryptosporidial infection.     

Cryptosporidium parvum infection requires host cell actin polymerization

The intracellular protozoan parasite Cryptosporidium parvum accumulates host cell actin at the interface between the parasite and the host cell cytoplasm. Here we show that the actin polymerizing proteins Arp2/3, vasodilator-stimulated phosphoprotein (VASP), and neural Wiskott Aldrich syndrome protein (N-WASP) are present at this interface and that host cell actin polymerization is necessary for parasite infection.     

Intestinal epithelial cell apoptosis following Cryptosporidium parvum infection

Cryptosporidium parvum induces moderate levels of apoptosis of cultured human intestinal epithelial cells, which are maximal at 24 h after infection. Apoptosis is further increased in C. parvum-infected cells by inhibition of NF-kappaB. C. parvum infection also attenuates epithelial apoptosis induced by strongly proapoptotic agents. The data suggest C. parvum has developed strategies to limit apoptosis in order to facilitate its growth and maturation in the early period after epithelial cell infection.     

Cell phenotypic change due to Cryptosporidium parvum infection in immunocompetent mice

Cryptosporidium parvum is an intracellular parasite causing enteritis which can become life-threatening in immunocompromised host. Immunoregulatory T cells play a central role in the regulatory network of the host. Here, we proposed to characterize the populations of immune cells during infection and reinfection with C. parvum. Four-week-old BALB/C mice were inoculated with oocysts of C. parvum at days 0 and 22. Fecal and blood samples, spleens, and small intestines were collected for analysis. Peripheral blood and spleen cell populations were characterized by flow cytometry. After infection (days 0 to 21), mice presented higher values of neutrophils, eosinophils, NK cells and CD4⁺CD25^high T cells in peripheral blood. After reinfection, this upward trend continued in the following days for all four populations in infected mice. At day 35, infected mice presented similar values to the control group, except for CD4⁺CD25^high T cells, which remained higher in infected mice. A possible correlation between alterations in blood and spleen cell populations was also studied, but no consistent association could be established. Small intestine sections were screened for intracellular stages of the parasite but no evidence of pathology was observed. Here, we report information which may be important for the understanding of the specific cell-mediated response in immunocompetent mice to C. parvum infection. Although some questions remain unanswered and complementary studies are needed, our results are expected to contribute to a better understanding of innate and Treg cells role in the clearance process of this parasite.     

Cryptosporidium parvum infection in T-cell receptor (TCR)-alpha- and TCR-delta-deficient mice.

Mice deficient in either alpha beta or gamma delta T cells were more susceptible to infection with the protozoan parasite Cryptosporidium parvum than were control mice. Both neonatal and adult alpha beta-T-cell-deficient mice developed chronic infection. Gamma delta-T-cell-deficient neonatal mice were more susceptible than control mice but were able to clear the infection. Adult gamma delta-T-cell-deficient mice were not susceptible to infection. These data indicate that alpha beta T cells are important for resistance to C. parvum while gamma delta T cells have a less critical role.     

Eradication of Cryptosporidium parvum infection by mice with ovalbumin-specific T cells

CD154 is necessary for mice to clear a Cryptosporidium parvum infection, but whether this ligand has to be expressed on T cells with specificity for C. parvum has not been determined. We infected DO11.10 (ovalbumin specific) T-cell receptor transgenic mice that had been bred to a RAG(-/-) background with C. parvum and found that the infection was cleared within 6 weeks, while RAG(-/-) controls were unable to clear C. parvum infection. Recovery was accompanied by an increase in the number of splenic T cells with the CD44(high) phenotype that characterizes memory cells. To determine whether a C. parvum-infected environment sufficed to activate transgenic T cells, we reconstituted C. parvum-infected BALB/c SCID mice with DO11.10 RAG(-/-) splenocytes. Fecal excretion of C. parvum antigen ceased in the 12 weeks following the adoptive transfer, unless the mice were also injected with tolerizing doses of ovalbumin. DO11.10 T cells were found in the submucosa of C. parvum-infected, but not uninfected, BALB/c SCID hosts within 48 h of injection. The transferred DO11.10 T cells divided and acquired a CD44(high) memory phenotype in C. parvum-infected, but not uninfected, recipients. DO11.10 splenocytes from CD154 knockout donors failed to clear a C. parvum infection, confirming a requirement for CD154 in recovery. In vitro, the DO11.10 cells did not proliferate in response to C. parvum antigen, and a tBlast GenBank search revealed no matches between the ovalbumin peptide and C. parvum DNA sequences. C. parvum-infected SCID mice given RAG(-/-) CD8(+) T cells with a Listeria-specific transgene did not recover from C. parvum infection. Our data suggest that antigen-nonspecific CD4(+) T-cell effector mechanisms in combination with the innate arm of the immune system are sufficient for the eradication of C. parvum infection.     

Experimental Cryptosporidium parvum infections in immunosuppressed adult mice.

Five strains of adult mice were immunosuppressed with the synthetic glucocorticosteroid dexamethasone (DEX), administered either orally or intraperitoneally. The strains of mice used were C57BL/6N, DBA/2N, CBA, C3H/HeN, and BALB/cAnN. All mice were evaluated for susceptibility to Cryptosporidium parvum after intragastric inoculation with 10(6) oocysts per mouse. The DBA/2N, CBA, C3H/HeN, and BALB/cAnN mice given 0.25 micrograms of DEX per g per day orally (the dose and route previously used to infect rats with C. parvum) failed to develop chronic infections. However, the C57BL/6N mice sustained light infections during the entire 28-day experiment. The five strains of mice were also administered DEX intraperitoneally at concentrations ranging from 62.5 to 500 micrograms/day. Only the C57BL/6N mice given DEX at 125 micrograms/day developed chronic infections which persisted over 10 weeks, suggesting that the genetic background of the mouse plays a role in determining susceptibility to cryptosporidosis following immunosuppression with DEX. We believe that the C57BL/6N mouse model will prove to be superior to other animal models for evaluating potential anticryptosporidial agents, as well as for elucidating the immunological defects that allow C. parvum to establish chronic infections, because of cost effectiveness and ease in maintenance, breeding, and handling. We also evaluated the C3H/HeJ/beige mouse (lacks natural killer cell activity) and the C57BL/6N mouse maintained on a low-protein diet to induce immunosuppression. Neither of these mice exhibited heavy cryptosporidial infections.     

Microtubule inhibitors block Cryptosporidium parvum infection of a human enterocyte cell line.

No effective therapy exists for Cryptosporidium parvum, a coccidial protozoan parasite that causes severe diarrhea in patients with AIDS. The role of microtubules in parasite invasion of host cells was investigated by incubating 10(7) oocysts with a HT 29.74 cell line for 24 h in the presence of microtubule-disrupting drugs. The number of parasites per 1,000 cells was reduced by 77% (P < 0.001, n = 4) from 182 +/- 3 in untreated cells to 42 +/- 4 in cells treated with 10(-4) M colchicine. Inhibition of C. parvum infection was concentration dependent. Similar results were seen with a second microtubular depolymerization agent, vinblastine. These data suggest that microtubules are important in host cell invasion by C. parvum and may represent targets for development of new therapeutic drugs for treatment of cryptosporidiosis.     

Gamma interferon functions in resistance to Cryptosporidium parvum infection in severe combined immunodeficient mice.

Severe combined immunodeficient (SCID) adult mice are relatively resistant to Cryptosporidium parvum infection, even though they are deficient in both T- and B-cell function. The requirement for gamma interferon (IFN-gamma) in this resistance was examined by treatment of these mice with monoclonal antibody to IFN-gamma. SCID mice injected intraperitoneally with monoclonal anti-IFN-gamma 4 h before and three times weekly after challenge with C. parvum had heavy intestinal infections 3 weeks postchallenge. SCID mice similarly injected with irrelevant antibody were not infected. Furthermore, SCID mice receiving a single injection of anti-IFN-gamma either 2 h before or 18 h after challenge were also susceptible to infection. Although IFN-gamma was not detected in SCID mouse intestinal samples, it was found in the supernatant of SCID mouse splenocyte cultures after stimulation with C. parvum antigens. On the other hand, SCID mice receiving multiple injections of antibodies against tumor necrosis factor remained resistant to infection. These data indicate that the resistance of SCID mice to C. parvum infection is IFN-gamma dependent, whereas tumor necrosis factor appears not to play a significant role.     

MyD88-dependent pathways mediate resistance to Cryptosporidium parvum infection in mice

Cryptosporidium spp. cause diarrheal disease worldwide. Innate immune responses mediating resistance to this parasite are not completely understood. To determine whether MyD88-dependent pathways play a role in resistance to Cryptosporidium parvum, we compared the course of infection in MyD88(-/-) mice to that in their wild-type (WT) littermate controls. Three- to 4-week-old mice were infected with C. parvum, and infection was monitored by quantifying fecal oocyst shedding. Twelve days postinfection, the histology of the intestines was examined to quantify intestinal parasite burden and to determine if there were any pathological changes. Fecal oocyst shedding and intestinal parasite burden were significantly greater in MyD88(-/-) mice than in littermate controls. Nonetheless, both WT and MyD88(-/-) mice cleared the infection within 3 weeks. These results indicate that MyD88-dependent pathways are involved in mediating initial resistance to C. parvum. Since gamma interferon (IFN-gamma) is known to mediate resistance to C. parvum, we also studied infection in MyD88(-/-) mice and WT controls in which this cytokine was temporarily neutralized. Fecal oocyst shedding, as well as intestinal parasite burden, intestinal inflammation, and mortality, was significantly greater in MyD88(-/-) mice in which IFN-gamma was neutralized than in IFN-gamma-neutralized WT mice or in MyD88(-/-) mice in which this cytokine was active. These results suggest that MyD88 and IFN-gamma had an additive effect in conferring protection from C. parvum infection. While this study confirms the importance of IFN-gamma in conferring resistance to infection with C. parvum, it suggests that MyD88-mediated pathways also play a role in innate immunity to this parasite.     

An intestinal xenograft model for Cryptosporidium parvum infection.

Paired segments of near-term fetal rabbit small intestine were transplanted subcutaneously into athymic nude mice. At 5 weeks postsurgery, the xenografts were inoculated intraluminally with Cryptosporidium parvum sporozoites. Parasites rapidly and reliably infected the xenograft mucosal epithelium. Lesions typical of cryptosporidiosis were readily apparent by light microscopy and scanning and transmission electron microscopy. Xenografts are well suited to the study of the early events of C. parvum infection and are of potential value in the evaluation of anticryptosporidial chemotherapeutic agents.     

Bovine antibody against Cryptosporidium parvum elicits a circumsporozoite precipitate-like reaction and has immunotherapeutic effect against persistent cryptosporidiosis in SCID mice.

Control of cryptosporidiosis is currently hampered by the absence of drugs or vaccines proven consistently effective against Cryptosporidium parvum. On the basis of observations that anti-C. parvum antibody has therapeutic effect against cryptosporidiosis, cows were immunized with C. parvum to produce hyperimmune colostral antibody. An antibody-rich fraction was prepared and differentiated from control (nonhyperimmune) antibody by enzyme-linked immunosorbent assay, immunofluorescence assay, immunoelectron microscopy, and in vitro neutralizing titer against DEAE-cellulose-isolated C. parvum sporozoites. Oocyst, purified sporozoite, and merozoite antigens recognized by hyperimmune antibody were defined by Western blot (immunoblot). Hyperimmune antibody recognized antigens common to oocysts, sporozoites, and merozoites, as well as stage-specific antigens. Upon incubation with hyperimmune antibody, sporozoites underwent distinct morphologic changes characterized by progressive formation and eventual release of membranous sporozoite surface antigen-antibody complexes, similar to the malaria circumsporozoite precipitate reaction. The infectivity of sporozoites having undergone this reaction was neutralized. The reaction was minimal or absent on sporozoites incubated with control antibody. To determine therapeutic effect in vivo, persistent C. parvum infection was established in adult severe combined immune-deficient (SCID) mice by oral inoculation with 10(7) oocysts. At 5 weeks postinfection, infected mice were treated for 10 days with hyperimmune or control antibody by inclusion in drinking water and daily gavage. Fecal oocyst shedding and infection scores in the gastrointestinal tract and gall bladder/common bile duct in hyperimmune antibody-treated mice were significantly lower than those in the control antibody-treated mice. Hyperimmune bovine antibody prepared against C. parvum may provide a first-generation therapy for control of cryptosporidiosis. Additionally, the defined antigens can be evaluated as subunit immunogens to produce better-characterized polyclonal antibody for control of cryptosporidiosis or as targets for monoclonal antibody-based immunotherapy.     

Susceptibility of germfree or antibiotic-treated adult mice to Cryptosporidium parvum.

Adult mice are more resistant than neonatal mice to intestinal colonization with the protozoan parasite Cryptosporidium parvum. Development of a mature intestinal flora may play a role in this resistance. We compared susceptibilities to colonization with C. parvum in adult conventional mice, adult germfree mice, and adult conventional mice treated with oral antibiotics to deplete the intestinal flora. Germfree mice of both CD1 and BALB/c strains were colonized at day 7 following inoculation with C. parvum oocysts isolated from the feces of an infected, diarrheic calf. Age-matched conventional mice of the same strains were comparatively resistant to colonization. Conventional mice treated with antibiotics remained resistant to colonization. These results suggest that the microflora in the intestine was not the sole determinant of resistance or susceptibility to colonization. The germfree adult mouse as an experimental model of cryptosporidiosis is discussed.     

Identification of genotypically mixed Cryptosporidium parvum populations in humans and calves

Genotypic analyses of Cryptosporidium parvum oocysts have divided the species into two genotypes, referred to as type 1 and type 2. Although humans are susceptible to both types, mixed type 1/type 2 infections have rarely been identified. The paucity of mixed infections could be explained by the predominance of one type over the other in mixed infections, or by the poor sensitivity of restriction fragment length polymorphism (RFLP) analyses for detecting subpopulations. Using a type-specific real-time PCR assay capable of detecting type 1 or type 2 constituting as little as 0.01% of the population, archived and new isolates of human, bovine, and mouse origin were genotyped. Mixed type 1/type 2 infections were identified in humans and calves, including in samples previously found to be homogeneous by RFLP. Isopycnic fractionation of mixed isolates revealed that type 1 and type 2 oocysts differ in their sedimentation properties. The detection of a type 1 subpopulation in serially-propagated bovine isolates indicates that type 1 and type 2 are stably maintained during long-term passage. Together with recently reported experimental bovine and ovine type 1 infections, the persistence of type 1 subpopulation in experimentally infected animals suggests that animals may play a previously unrecognized role in the maintenance of C. parvum type 1.     

Monoclonal antibody immunotherapy in nude mice persistently infected with Cryptosporidium parvum.

Three groups of congenitally athymic nude mice were persistently infected following oral administration of 2 x 10(7) Cryptosporidium parvum oocysts. Two groups were treated once daily for 10 days with either neutralizing monoclonal antibody (MAb) 17.41 or an isotype control MAb. The third group received no treatment. Intestinal-infection scores were significantly decreased in nude mice treated with MAb 17.41 compared with isotype control MAb-treated and nontreated control groups (P less than 0.005). Biliary and pancreatic cryptosporidial-infection scores were similar for the MAb 17.41-treated and isotype control MAb-treated groups (P greater than 0.05).     

Marrow-derived CD40-positive cells are required for mice to clear Cryptosporidium parvum infection

To clear a Cryptosporidium parvum infection, mice need CD4+ T cells, major histocompatibility complex class II, and an intact CD40-CD154 signaling pathway. CD40 is constitutively expressed on marrow-derived cells such as dendritic cells and B lymphocytes and is induced by gamma interferon (IFN-gamma) on most somatic cells. To determine whether the CD40 needed to clear a C. parvum infection has to be on marrow-derived mononuclear cells or on the epithelial cells that normally harbor the parasite, we transplanted CD40-/- mice with CD40+/- bone marrow and then infected them with C. parvum. These chimeras cleared the C. parvum infection, while CD40+/- controls transplanted with CD40-/- marrow cells remained infected. CD40 expression on marrow-derived cells therefore suffices for a C. parvum infection to be cleared, while CD40 expression on intestinal epithelial cells is not sufficient. There was no difference between the acquisition of CD69 and CD154 by mesenteric lymph node T cells of C. parvum-infected animals with intact or disrupted CD40-CD154 pathways. CD4 T cells entered the intestinal laminae propriae of C. parvum-infected animals whether or not the CD40 genes of these recipients were intact. These results suggest that, for a C. parvum infection to be cleared, CD40 is not necessary for T-cell activation but may instead contribute to an effector pathway of marrow-derived cells.     

Increased susceptibility of beta7-integrin-deficient neonatal mice in the early stage of Cryptosporidium parvum infection

Numerous inflammatory cells are recruited in response to Cryptosporidium parvum infection. These cells include interferon gamma-producing T lymphocytes, which are of major importance for the resolution of infection. Here, we show that beta7 integrin is not essential for the control of infection in mice but that beta7-deficient neonatal mice are more susceptible during the early stages of infection.