Infection of immunosuppressed C57BL/6N adult mice with a single oocyst of Cryptosporidium parvum

The present study was designed to determine the minimum number of Cryptosporidium parvum oocysts capable of producing patent infections in immunosuppressed C57BL/6N adult mice. Sixty-four female mice were divided into 6 groups of 8 mice each, except group 1 that contained 24 mice. Mice in groups 1-3 were immunosuppressed with dexamethasone and inoculated with 1, 5, and 10 oocysts per mouse, respectively. The accuracy of the inoculum size was microscopically confirmed. Mice in groups 4-6 served as controls: they received either only oocyst inoculation (group 4), or immunosuppression (group 5), or no treatments (group 6). Fecal oocyst shedding was monitored daily for each mouse using an indirect immunofluorescent assay. Parasite colonization in the terminal ileum of each mouse was evaluated histologically. Four of 24 mice in group 1 developed patent infections, with a prepatent period of approximately 6 days. All mice in groups 2 and 3 developed patent infections, with prepatent periods ranging from 4 to 7 days. Mice in groups 4-6 remained uninfected. Parasite colonization was observed in the terminal ilea of all mice in groups 1-3 that shed fecal oocysts. The present study experimentally demonstrates that a single viable oocyst can induce patent C. parvum infections in immunosuppressed C57BL/6N adult mice and indicates that this mouse model could be used for the parasite genotype or isolate cloning.     

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

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

Time gap between oocyst shedding and antibody responses in mice infected with Cryptosporidium parvum

We observed the time gap between oocyst shedding and antibody responses in mice (3-week-old C57BL/6J females) infected with Cryptosporidium parvum. Oocyst shedding was verified by modified acid-fast staining. The individually collected mouse sera were assessed for C. parvum IgM and IgG antibodies by enzyme-linked immunosorbent assay from 5 to 25 weeks after infection. The results showed that C. parvum oocysts were shed from day 5 to 51 post-infection (PI). The IgM antibody titers to C. parvum peaked at week 5 PI, whereas the IgG antibody titers achieved maximum levels at week 25 PI. The results revealed that IgM responses to C. parvum infection occurred during the early stage of infection and overlapped with the oocyst shedding period, whereas IgG responses occurred during the late stage and was not correlated with oocyst shedding. Hence, IgM antibody detection may prove helpful for the diagnosis of acute cryptosporidiosis, and IgG antibody detection may prove effective for the detection of past infection and endemicity.     

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.     

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.     

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.     

Cryptosporidium parvum Infection in SCID Mice Infected with Only One Oocyst: qPCR Assessment of Parasite Replication in Tissues and Development of Digestive Cancer

Dexamethasone (Dex) treated Severe Combined Immunodeficiency (SCID) mice were previously described as developing digestive adenocarcinoma after massive infection with Cryptosporidium parvum as soon as 45 days post-infection (P.I.). We aimed to determine the minimum number of oocysts capable of inducing infection and thereby gastrointestinal tumors in this model. Mice were challenged with calibrated oocyst suspensions containing intended doses of: 1, 10, 100 or 10⁵ oocysts of C. parvum Iowa strain. All administered doses were infective for animals but increasing the oocyst challenge lead to an increase in mice infectivity (P = 0.01). Oocyst shedding was detected at 7 days P.I. after inoculation with more than 10 oocysts, and after 15 days in mice challenged with one oocyst. In groups challenged with lower inocula, parasite growth phase was significantly higher (P = 0.005) compared to mice inoculated with higher doses. After 45 days P.I. all groups of mice had a mean of oocyst shedding superior to 10,000 oocyst/g of feces. The most impressive observation of this study was the demonstration that C. parvum-induced digestive adenocarcinoma could be caused by infection with low doses of Cryptosporidium, even with only one oocyst: in mice inoculated with low doses, neoplastic lesions were detected as early as 45 days P.I. both in the stomach and ileo-caecal region, and these lesions could evolve in an invasive adenocarcinoma. These findings show a great amplification effect of parasites in mouse tissues after challenge with low doses as confirmed by quantitative PCR. The ability of C. parvum to infect mice with one oocyst and to develop digestive adenocarcinoma suggests that other mammalian species including humans could be also susceptible to this process, especially when they are severely immunocompromised.     

Effect of disinfection of drinking water with ozone or chlorine dioxide on survival of Cryptosporidium parvum oocysts

Demineralized water was seeded with controlled numbers of oocysts of Cryptosporidium parvum purified from fresh calf feces and subjected to different treatments with ozone or chlorine dioxide. The disinfectants were neutralized by sodium thiosulfate, and neonatal mice were inoculated intragastrically and sacrificed 7 days later for enumeration of oocyst production. Preliminary trials indicated that a minimum infection level of 1,000 oocysts (0.1-ml inoculum) per mouse was necessary to induce 100% infection. Treatment of water containing 10(4) oocysts per ml with 1.11 mg of ozone per liter (concentration at time zero [C0]) for 6 min totally eliminated the infectivity of the oocysts for neonatal mice. A level of 2.27 mg of ozone per liter (C0) was necessary to inactivate water containing 5 x 10(5) oocysts per ml within 8 min. Also, 0.4 mg of chlorine dioxide per liter (C0) significantly reduced infectivity within 15 min of contact, although some oocysts remained viable.     

Effect of disinfection of drinking water with ozone or chlorine dioxide on survival of Cryptosporidium parvum oocysts.

Demineralized water was seeded with controlled numbers of oocysts of Cryptosporidium parvum purified from fresh calf feces and subjected to different treatments with ozone or chlorine dioxide. The disinfectants were neutralized by sodium thiosulfate, and neonatal mice were inoculated intragastrically and sacrificed 7 days later for enumeration of oocyst production. Preliminary trials indicated that a minimum infection level of 1,000 oocysts (0.1-ml inoculum) per mouse was necessary to induce 100% infection. Treatment of water containing 10(4) oocysts per ml with 1.11 mg of ozone per liter (concentration at time zero [C0]) for 6 min totally eliminated the infectivity of the oocysts for neonatal mice. A level of 2.27 mg of ozone per liter (C0) was necessary to inactivate water containing 5 x 10(5) oocysts per ml within 8 min. Also, 0.4 mg of chlorine dioxide per liter (C0) significantly reduced infectivity within 15 min of contact, although some oocysts remained viable.     

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

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

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.     

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.     

Cryptosporidium canis n. sp. from domestic dogs.

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

Inhibitory effect of selenium on Cryptosporidium parvum infection in vitro and in vivo

The anticryptosporidial effect of sodium selenite (selenium) was evaluated in a bovine fallopian tube epithelial (BFTE) cell culture system and an immunosuppressed C57BL/6N adult mouse model. Parasite numbers in cell culture were significantly reduced (p<0.01) following treatment with selenium (Se) at concentrations of 6, 9, and 12 μM at 48 h postinoculation (PI) and at 1.5, 3, and 6 μM at 96 h PI. Parasite reduction was greater than 50% at 48 h PI when 9 and 12 μM Se was used, and at 96 h PI when 6 μM Se was used. Such Se-induced reduction of Cryptosporidium parvum infection was significantly (p<0,0001) blocked when using free-radical scavengers such as mannitol (20 mM). A combined solution of mannitol (20 mM) and reduced glutathione (0.5 mM) enhanced the blockage to almost 100%. Adult C57BL/6N mice were immunosuppressed with dexamethasone phosphate administered ad libitum (16 μg/mL) in drinking water and inoculated with 10⁵ oocysts/mouse. Significantly fewer (p<0.001) oocysts were shed in the feces of mice treated with Se administered ad libitum (12 μM) in drinking water than in untreated mice. The survival time of mice was also significantly increased (p<0.001) following Se treatment. Collectively, these results indicate that Se plays an important role in cryptosporidiosis, and oxidative stress caused by Se is probably a major mechanism in inhibition of C. parvum infection.     

Mouse strain variation and effects of oocyst dose in infection of mice with Eimeria falciformis, a coccidian parasite of the large intestine

Stockdale P. G. H., Stockdale M. J., Rickard M. D. and Mitchell G. F. 1985. Mouse strain variation and effects of oocyst dose in infection of mice with Eimeria falciformis, a coccidian parasite of the large intestine, International Journal for Parasitology15: 447–452. Five inbred strains of mice and three hypothymic (nude) strains were infected orally with different doses of E. falciförmis oocysts. After resolution of primary infection as determined by faecal oocyst output, mice were challenged orally with a second dose of E. falciformis. Amongst the intact mice, BALB/c proved the most resistant to primary infection, while C3H/He mice were most susceptible, in terms of faecal oocyst production. Resistance was far more dramatic in BALB/c mice given high numbers of challenge oocysts. In terms of mortality at high oocyst doses, CBA/H were the most susceptible. All of the strains of mice were highly resistant to reinfection. In the case of nude mice, BALB/c. nu/nu were more susceptible than CBA/H.nu/nu or C57BL/6.nu/nu both in terms of faecal oocyst production and mortality. Thus the most resistant inbred mouse strain (BALB/c) is the least resistant in the absence of T cells. Unlike intact mice, nude mice showed no resistance to reinfection, this result being in line with previous work on this and other Eimeria spp. in nude mice.     

Cryptosporidium-malnutrition interactions: mucosal disruption, cytokines, and TLR signaling in a weaned murine model

Cryptosporidiosis is a leading cause of persistent diarrhea in children in impoverished and developing countries and has both a short- and long-term impact on the growth and development of affected children. An animal model of cryptosporidial infection that mirrors closely the complex interaction between nutritional status and infection in children, particularly in vulnerable settings such as post-weaning and malnourishment, is needed to permit exploration of the pathogenic mechanisms involved. Weaned C57BL/6 mice received a protein-deficient (2%) diet for 3-12 days, then were infected with 5 × 10(7) excysted C. parvum oocyts, and followed for rate of growth, parasite stool shedding, and intestinal invasion/morphometry. Mice had about 20% reduction in weight gain over 12 days of malnutrition and an additional 20% weight loss after C. parvum challenge. Further, a significantly higher fecal C. parvum shedding was detected in malnourished infected mice compared to the nourished infected mice. Also, higher oocyst counts were found in ileum and colon tissue samples from malnourished infected mice, as well as a significant reduction in the villous height-crypt depth ratio in the ileum. Tissue Th1 cytokine concentrations in the ileum were significantly diminished by malnutrition and infection. mRNA for toll-like receptors 2 and 4 were diminished in malnourished infected mice. Treatment with nitazoxanide did not prevent weight loss or parasite stool shedding. These findings indicate that, in the weaned animal, malnutrition intensifies cryptosporidial infection, while cryptosporidial infection further impairs normal growth. Depressed TLR2 and 4 signaling and Th1 cytokine response may be important in the mechanisms underlying the vicious cycle of malnutrition and enteric infection.     

Methods to produce and safely work with large numbers of Toxoplasma gondii oocysts and bradyzoite cysts

Two major obstacles to conducting studies with Toxoplasma gondii oocysts are the difficulty in reliably producing large numbers of this life stage and safety concerns because the oocyst is the most environmentally resistant stage of this zoonotic organism. Oocyst production requires oral infection of the definitive feline host with adequate numbers of T. gondii organisms to obtain unsporulated oocysts that are shed in the feces for 3-10 days after infection. Since the most successful and common mode of experimental infection of kittens with T. gondii is by ingestion of bradyzoite tissue cysts, the first step in successful oocyst production is to ensure a high bradyzoite tissue cyst burden in the brains of mice that can be used for the oral inoculum. We compared two methods for producing bradyzoite brain cysts in mice, by infecting them either orally or subcutaneously with oocysts. In both cases, oocysts derived from a low passage T. gondii Type II strain (M4) were used to infect eight-ten week-old Swiss Webster mice. First the number of bradyzoite cysts that were purified from infected mouse brains was compared. Then to evaluate the effect of the route of oocyst inoculation on tissue cyst distribution in mice, a second group of mice was infected with oocysts by one of each route and tissues were examined by histology. In separate experiments, brains from infected mice were used to infect kittens for oocyst production. Greater than 1.3 billion oocysts were isolated from the feces of two infected kittens in the first production and greater than 1.8 billion oocysts from three kittens in the second production. Our results demonstrate that oral delivery of oocysts to mice results in both higher cyst loads in the brain and greater cyst burdens in other tissues examined as compared to those of mice that received the same number of oocysts subcutaneously. The ultimate goal in producing large numbers of oocysts in kittens is to generate adequate amounts of starting material for oocyst studies. Given the potential risks of working with live oocysts in the laboratory, we also tested a method of oocyst inactivation by freeze-thaw treatment. This procedure proved to completely inactivate oocysts without evidence of significant alteration of the oocyst molecular integrity.     

Susceptibility to Cryptosporidium parvum infections in cytokine- and chemokine-receptor knockout mice

To determine the role of cytokines and a chemokine receptor in the susceptibility to, and outcome of, infection, 4 different knockout mice (IL-4, IL-10, IL-12, and CCR5) were infected with Cryptosporidium parvum and monitored for infection intensity by collection of fecal pellets from individual mice. Because adult immunocompetent mice are refractory to infection, wild-type mice on the same background as the knockout mice (C57BL/6) were used as a negative control. No infection was detected over a 4-wk time period in IL-4, IL-10, and CCR5 knockout mice inoculated with 106 oocysts. IL-12 knockout mice inoculated with as little as 100 oocysts shed up to 10,000 oocysts/100 microl of feces on the peak infection day (day 8) and were able to fully recover by 2 wk after infection. IL-12 is an important inducer of IFN-gamma, which probably accounted for susceptibility to infection. Previous studies using IFN-gamma knockout mice have shown strain-related differences in infection intensity and outcome, with increased parasite loads and decreased survival among IFN-gamma knockout mice on a C57BL/6 background compared with those on a BALB/c background. Similar results were observed in IL-12 knockout mice on a BALB/c background, which exhibited little or no infection, despite higher levels of inoculation (10(6) oocysts/mouse).