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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.

Cryptosporidium parvum infection of T-cell receptor alpha (TCR-alpha)-deficient mice results in a persistent infection. In this study, treatment with a polyamine analogue (SL-11047) prevented C. parvum infection in suckling TCR-alpha-deficient mice and cleared an existing infection in older mice. Treatment with putrescine, while capable of preventing infection, did not clear C. parvum from previously infected mice. These findings provide further evidence that polyamine metabolic pathways are targets for new anticryptosporidial chemotherapeutic agents.

Mice with targeted disruptions in the T-cell receptor alpha gene (TCRalpha-/-) spontaneously develop inflammatory intestinal lesions with extensive B-cell lamina propria infiltrates. Cryptosporidium parvum infection accelerates intestinal lesion formation in TCRalpha-/- mice. In the present study, TCRalpha-/- mice were crossed with JH-/- (B-cell-deficient) mice and challenged with C. parvum to determine if B cells are required for intestinal lesion development. TCRalpha-/- x JH-/- mice challenged with C. parvum, either as neonates or adults, became persistently infected, whereas TCRalpha-/+ x JH-/+ heterozygote control mice cleared the parasite. Cryptosporidium parvum colonization of TCRalpha-/- x JH-/- mice was heaviest in the distal ileum, with fewer parasites detected in the cecum and distal colon. Despite persistent infection, TCRalpha-/- x JH-/- mice did not develop inflammatory or hyperplastic intestinal lesions as detected in C. parvum-infected TCRalpha-/- mice. These findings demonstrate that B cells are a necessary component for the development of inflammatory intestinal lesions of C. parvum-infected TCRalpha-/- mice.

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.

This study was conducted to produce a recombinant species-specific oocyst wall protein of Cryptosporidium parvum. Antigens unique to C. parvum were identified by gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of oocyst proteins from several different Cryptosporidium species. Antiserum was then prepared against a 41-kDa antigen unique to C. parvum and used to identify a recombinant DNA clone, designated rCP41. Expression of CP41 mRNA in C. parvum oocysts was confirmed by reverse transcriptase PCR (RT-PCR). Although the CP41 sequence was shown by PCR to be present in the genome of C. baileyi, CP41 mRNA was not detected in this species by RT-PCR. Immunofluorescence staining with antiserum against recombinant CP41 detected native CP41 antigen on the surface of C. parvum oocysts but failed to detect CP41 on C. baileyi oocysts. Immunoelectron microscopy demonstrated that native CP41 was distributed unevenly on the C. parvum oocyst surface and was associated with amorphous oocyst wall material. In an enzyme-linked immunosorbent assay, purified rCP41 performed as well as native C. parvum oocyst protein in measuring the serological responses of young calves and adult cows to experimental and natural C. parvum infections. These results indicate that recombinant CP41 antigen may have potential in the immunodiagnosis of cryptosporidiosis.

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.

Flora-bearing mice with targeted disruption of T cell receptor (TCR)-alpha or -beta genes spontaneously develop intestinal inflammation with features similar to ulcerative colitis in humans. TCR-alpha-deficient mice maintained germfree or colonized with a limited number of intestinal bacteria failed to develop inflammatory bowel disease (IBD)-like lesions. Evidently, inflammation in these mice does not develop spontaneously or result from a generalized antigenic stimulation, but rather requires induction by a heretofore unidentified specific stimulus. We describe the development of IBD-like lesions in germfree TCR-alpha-deficient mice monoassociated with the protozoan Cryptosporidium parvum. Lesions were seen in distal ileum, cecum, and colon and were most severe in the cecum. A prominent leukocytic infiltrate within the lamina propria was a common characteristic of the lesions observed in the C. parvum-infected germfree TCR-alpha-deficient mice. The leukocytic infiltrate was composed of aggregates of B220+ cells, the majority of which expressed surface IgD (ie, conventional B lymphocytes). It has been proposed that antigenic stimulation by a microorganism(s) is needed to initiate intestinal inflammation in TCR-alpha-deficient mice. Our results indicate that a single microbial species, C. parvum, is capable of triggering the development of IBD-like lesions in germfree TCR-alpha-deficient mice.

Objective: To determine whether periparturient cows or contact surfaces to which newborn calves are exposed are reservoirs of Cryptosporidium parvum oocysts., Animals: Periparturient cows and their calves., Procedure: Using direct fluorescent antibody (DFA) and acid-fast (AF) assays, fecal samples taken before and after calving from periparturient cows were tested for C parvum oocysts. Fecal samples from calves were collected every other day from age 7 to 21 days and were tested by use of the AF assay. Topsoil from close-up and maternity pens and scrapings from wooden walls and floors of calf hutches were tested for C parvum oocysts by use of DFA assay., Results: None of the 384 fecal samples obtained 1 to 21 days before or after calving or on the day of calving from 154 periparturient cows contained detectable C parvum oocysts. Despite this lack of detectable periparturient shedding, the period prevalence of calfhood infection was 92% (123/134) from age 7 to 21 days. Soil samples from the close-up and maternity pens where newborn calves spend the first 12 hours of life also were negative for C parvum oocysts. Wood scrap ings from the outer 2 mm of the walls and floors of empty and cleaned calf hutches that were ready to receive calves were C parvum oocyst-positive., Conclusions: Conditional on sensitivity of DFA, periparturient cows did not appear to shed detectable C parvum oocysts. In contrast, the floors and walls of wooden calf hutches contained detectable C parvum oocysts on the surface.

Cryptosporidium parvum is a protozoan parasite that is now recognized as one of the leading causes of diarrhea in young calves. To date, there are no drugs or preventive measures available for the control of this disease. We have developed an oral vaccine that, when given to calves at birth, protects against experimental challenge with C. parvum. However, when field tested on a large dairy operation with heavy endemic C. parvum infection, the vaccine failed to provide protection. The difference in these results is most likely due to uncontrolled early (probably within hours of birth) exposure to C. parvum on the farm versus controlled exposure at 1 wk of age in the experimental trials. The successful control of C. parvum in the field may require vaccines that generate a rapid (within the first few days of life) cell-mediated immune response in the calf. Successful use of such a vaccine will also require improved hygiene and management practices to minimize the exposure of calves to C. parvum in the initial days of life, thus allowing time for protective immune responses to be generated. Careful attention to hygiene in the management of sick calves is also critical to minimize the spread of the parasite to other animals.

We examined the efficacy of oral administration of putrescine (a byproduct of arginine metabolism) in the prevention of Cryptosporidium parvum infection of neonatal C57BL-6 mice. Mice were challenged with the parasite at 7 days of age. Mice receiving putrescine from 3 through 10 days of age had a delayed pattern of infection as compared with control mice. Mice receiving putrescine from 3 through 21 days of age did not become infected, whereas control mice were heavily infected. We also tested the hypothesis that putrescine inhibited C. parvum infection by enhancing nitric oxide (NO) production. Mice receiving the NO inhibitor N omega-L-arginine methyl ester (L-NAME) parenterally and putrescine orally did not become infected. Thus, it appears that putrescine inhibits C. parvum infection in an NO-independent manner.

TCR-alpha-deficient mice spontaneously develop inflammatory bowel disease (IBD) at 8-9 mo old. This study characterizes an accelerated form of IBD induced by Cryptosporidium parvum infection. Cryptosporidium parvum-infected TCR-alpha-deficient mice developed IBD as early as 4 wk old when challenged at 1 wk old. The lesions of this accelerated IBD resembled the lesions of spontaneous IBD in TCR-alpha-deficient mice and consisted of a mononuclear cell infiltrate within the intestinal lamina propria and an increased proliferation of enterocytes. The mononuclear cells within the lamina propria consisted of B cells and gamma delta T cells. The distal ileum, cecum, and colon were grossly thickened due to a hyperplastic mucosa and edematous submucosa. The mechanism by which C. parvum infection accelerates development of IBD is presently unclear.

Objective: To test the ability of oral vaccination or probiotic treatment with lactic acid-producing bacteria to protect calves from Cryptosporidium parvum infection under field conditions., Animals: 134 Holstein calves born on a dairy farm where cryptosporidiosis was endemic., Procedure: Calves were randomly assigned to 1 of 3 treatment groups at birth. Calves in the vaccine group received an oral dose of C parvum vaccine within several hours of birth. Calves in the bacteria group received an oral dose of lactic acid-producing bacteria daily for the first 10 days after birth. Control calves were not treated. All calves were monitored for diarrhea and fecal shedding of C parvum oocysts for 3 weeks., Results: There were no significant differences in the incidence of diarrhea and oocyst shedding among the 3 groups., Conclusions: Neither vaccination nor probiotic treatment was effective in preventing C parvum infection in calves under field conditions. High numbers of C parvum in the environment may have overwhelmed any potential benefits of these regimens. Further work is necessary to develop effective prophylaxis against C parvum under field conditions.

Cryptosporidium parvum is a major cause of diarrheal disease in humans and has been identified in 78 other species of mammals. The oocyst stage, excreted in feces of infected humans and animals, has been responsible for recent waterborne outbreaks of human cryptosporidiosis. High temperature and long exposure time have been shown to render oocysts (suspended in water) noninfectious, but for practical purposes, it is important to know if high-temperature--short-time conditions (71.7 degrees C for 15 s) used in commercial pasteurization are sufficient to destroy infectivity of oocysts. In this study, oocysts were suspended in either water or whole milk and heated to 71.7 degrees C for 15, 10, or 5 s in a laboratory-scale pasteurizer. Pasteurized and nonpasteurized (control) oocysts were then tested for the ability to infect infant mice. No mice (0 of 177) given 10(5) oocysts pasteurized for 15, 10, or 5 s in either water or milk were found to be infected with C. parvum on the basis of histologic examination of the terminal ileum. In contrast, all (80 of 80) control mice given nonpasteurized oocysts were heavily infected. These data indicate that high-temperature--short-time pasteurization is sufficient to destroy the infectivity of C. parvum oocysts in water and milk.

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.

Lymphocyte phenotypes and cellular immune responses (blastogenesis and production of cytokines) to Cryptosporidium parvum were determined for spleen cells taken from BALB/c mice. These parameters were measured in mice at 1, 2, 3, and 4 wk of age, either exposed or not exposed to C. parvum in vivo. The percentage of T cells and the T-helper subset increased from weeks 2 to 4; B cells reached a peak percentage at 2 wk. Blastogenic responses were elevated at 1 wk and declined to a low level during weeks 2 to 4. Interferon-gamma production was maximal at 4 wk. No interleukin-5 production was seen. Data obtained were similar for cells from mice either exposed or not exposed to C. parvum in vivo. These data indicate that age-related changes, particularly the increased percentage of T cells and increased interferon-gamma production, are temporally related to the acquisition of resistance to colonization of mice with C. parvum. The data also indicate that these age-related changes occur in the absence of specific exposure to parasite antigens.

Cryptosporidium parvum causes enteric infection and diarrhea in calves, other species of economically important livestock, and humans. There are no effective treatments currently licensed for this parasite, and preventive measures are difficult. In addition to direct economic losses to the cattle industry, infected calves may contaminate water supplies with oocysts and contribute to human cryptosporidiosis. We have developed a vaccine offering partial protection against C. parvum infection in calves. Nine calves received an oral preparation of lyophilized C. parvum oocysts shortly after birth, and 10 calves served as nonvaccinated controls. All calves received colostrum. At 1 wk of age, all calves were administered 10(4) viable C. parvum oocysts orally. Clinical disease and oocyst shedding were monitored daily. Mean duration of diarrhea was 4 days for control calves and 1.7 days for vaccinated calves. Mean duration of oocyst shedding was 5.3 days for control calves and 2 days for vaccinated calves. These differences were statistically significant and suggest that this vaccine has the potential to reduce diarrhea and oocyst shedding caused by C. parvum.

Cryptosporidium parvum is a protozoan parasite now recognized as a significant cause of neonatal diarrhea in calves, and infection is also widespread in both immunocompetent and immunocompromised humans. No effective treatment or preventive measures against C. parvum infection are available, owing largely to the lack of understanding of immunologic mechanisms of resistance to and recovery from this parasite. In the present study, we compared phenotypes of lymphocytes from peripheral blood, spleen, mesenteric, and prescapular lymph nodes of calves infected or not infected with C. parvum. We also compared reactivity of these lymphocytes to mitogens and C. parvum antigen in vitro. There were more non-T, non-B (null) lymphocytes in all tissues of infected compared with control calves. The percent of CD8+ lymphocytes was significantly increased in spleens of infected compared with control calves, and there were markedly less CD4+ than CD8+ cells in spleens of both groups (i.e. low CD4/CD8 ratios). Splenic lymphocytes showed significantly decreased in vitro proliferation to pokeweed mitogen and C. parvum antigen stimulation compared with lymphocytes from other tissues. These findings suggest that null lymphocytes and CD8+ lymphocytes may be important in the expression and regulation of bovine immune responses to C. parvum in vivo.

Spleen cells from mice immunized with Cryptosporidium parvum were enriched for T cells by passage over an affinity chromatography column. The proliferative response of these cells was > 2-fold higher than the response of unenriched cells. T-enriched cells were enriched further for either CD4+ cells or CD8+ cells. The proliferative response of CD4-enriched cells was > 4-fold higher than the response by unenriched cells. CD8+ cells were essentially nonresponsive to C. parvum antigen. Culture supernatant fractions from these variously enriched splenocyte populations were assayed for cytokine production. Cultures containing CD4+ cells produced gamma interferon and interleukin-2 following incubation with C. parvum antigen. None of the cultures produced interleukin-4. Production of gamma interferon and interleukin-2, but not interleukin-4, is characteristic of the previously described Th1 helper cell subset. Our data indicate that a subset of murine lymphocytes consistent with the Th1 helper cell phenotype proliferates following in vitro stimulation with C. parvum antigen.

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.

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.

Cryptosporidium parvum is a protozoan parasite which colonizes intestinal epithelium, causing transient diarrheal illness in immunocompetent hosts and severe chronic disease in immunocompromised hosts. We examined the resistance of severe combined immunodeficient mice, either bearing intestinal flora or germfree, to intestinal infection with C. parvum. Infection was not readily detected in flora-bearing adult severe combined immunodeficient mice until 5 to 7 weeks following oral challenge with C. parvum. In contrast, germfree adult severe combined immunodeficient mice were heavily infected 3 weeks following challenge. These data support the hypothesis that resistance of adult mice to C. parvum infection does not require a specific immune response but can be mediated by nonspecific mechanisms associated with the presence of intestinal flora.