Recent diarrhea is associated with elevated salivary IgG responses to Cryptosporidium in residents of an eastern Massachusetts community.

BACKGROUND: Serological data suggest that Cryptosporidium infections are common but underreported. The invasiveness of blood sampling limits the application of serology in epidemiological surveillance. We pilot-tested a non-invasive salivary anti-Cryptosporidium antibody assay in a community survey involving children and adults. MATERIALS AND METHODS: Families with children were recruited in a Massachusetts community in July; symptoms data were collected at 3 monthly follow-up mail surveys. One saliva sample per person (n = 349) was collected via mail, with the last survey in October. Samples were analyzed for IgG and IgA responses to a recombinant C. hominis gp15 sporozoite protein using a time-resolved fluorometric immunoassay. Log-transformed assay results were regressed on age using penalized B-splines to account for the strong age-dependence of antibody reactions. Positive responses were defined as fluorescence values above the upper 99% prediction limit. RESULTS: Forty-seven (13.5%) individuals had diarrhea without concurrent respiratory symptoms during the 3-month-long follow-up; eight of them had these symptoms during the month prior to saliva sampling. Two individuals had positive IgG responses: an adult who had diarrhea during the prior month and a child who had episodes of diarrhea during each survey month (Fisher's exact test for an association between diarrhea and IgG response: p = 0.0005 for symptoms during the prior month and p = 0.02 for symptoms during the entire follow-up period). The child also had a positive IgA response, along with two asymptomatic individuals (an association between diarrhea and IgA was not significant). CONCLUSION: These results suggest that the salivary IgG specific to Cryptosporidium antigens warrants further evaluation as a potential indicator of recent infections.

Interleukin-1 beta (IL-1 beta), IL-1 receptor antagonist, and TNF alpha production in whole blood.

The ability of an individual to mount defense responses to infection depend in part on the capacity to produce cytokines such as interleukin 1 (IL-1) and tumor necrosis factor (TNF). The specialized equipment, labor intensity, and sterile practice required for the standard in vitro evaluation of cytokine production can make such evaluation impractical in some clinical situations. We report a method for stimulating whole blood to produce cytokines that can be implemented in laboratories without tissue culture facilities and requires minimal sample preparation. IL-1 beta and TNF alpha production in whole blood samples was stimulated with endotoxin and/or phytohemagglutinin in standard EDTA-containing vacuum collection tubes. After incubation, plasma was removed and frozen for later assay. Comparison of this whole blood method with isolated mononuclear cell cultures indicated a significant correlation for IL-1 beta production (r = 0.746, P = 0.005). This technique also produced the newly described cytokine, IL-1 receptor antagonist. We conclude that the whole blood method is an acceptable alternative to isolated cell culture methods for measuring IL-1 beta in situations that preclude the standard in vitro approach.

Rifabutin but not clarithromycin prevents cryptosporidiosis in persons with advanced HIV infection.

BACKGROUND: Macrolides have been reported to be effective for the prevention of cryptosporidiosis in persons with HIV infection. OBJECTIVE: To evaluate the efficacy of clarithromycin and rifabutin for the prevention of cryptosporidiosis in persons with advanced HIV infection. DESIGN: Cross-protocol analysis involving 2288 individuals with a history of a CD4 cell counts of < or = 100 x 10(6) cell/l who were enrolled in two prospective clinical trials to prevent Mycobacterium avium complex (MAC) infection and cytomegalovirus (CMV) end-organ disease. INTERVENTIONS: Clarithromycin 500 mg twice daily, rifabutin 300-450 mg daily, the combination of the two or no MAC prophylaxis. MAIN OUTCOME MEASUREMENT: Laboratory-confirmed cryptosporidiosis. Subjects were analyzed in an intent-to-treat and as-treated manner using time-to-event analyses (Cox proportional hazards models). RESULTS: The median length of follow up was 463 days. The median CD4 count at entry was 29 x 10(6) cell/l (range 0-182). There were 60 cases of cryptosporidiosis during the prospective observational period, with an event rate of 2.2 per 100 person-years. In the intent-to-treat [relative risk (RR) 0.50; 95% confidence interval (CI) 0.26-0.96; P = 0.041 and as-treated (RR 0.42; 95% CI 0.20-0.91; P = 0.03) analyses, rifabutin alone was significantly associated with a lower rate of cryptosporidiosis. Clarithromycin alone was not protective in similar analyses (P = 0.98 and 0.90, respectively). CONCLUSIONS: In doses used to prevent MAC infection, rifabutin but not clarithromycin decreases the risk of developing cryptosporidiosis in persons with advanced HIV infection who are not receiving potent combination antiretroviral therapy.

Human cryptosporidiosis: epidemiology, transmission, clinical disease, treatment, and diagnosis.

Cryptosporidiosis is now recognized as one of the most common human enteric infections. In this critical review, relatively unexplored details of transmission, the interaction with malnutrition and the development of chronic diarrhea, and the need for effective treatment are highlighted. Our inability to detect small numbers of foodborne oocysts limits our understanding of this transmission route, and the possibility of respiratory transmission is yet to be rigorously studied. The toll this disease imposes on children, especially the malnourished, has not been fully appreciated. Indeed, the dynamics of the progression from acute cryptosporidiosis to chronic diarrhea and death of malnourished children is still enigmatic. Our knowledge of the intestinal pathophysiology, while limited, is increasing. The lack of effective drug therapy is both remarkable and sobering. Overall, these unknown areas demonstrate how little we truly know about this parasite.

Natural history and biology of Cryptosporidium parvum.

The taxonomy of the genus Cryptosporidium remains ambiguous, because the current criteria for speciation are insufficient to validate the 6-8 named species. Cross-transmission experiments have shown varying and conflicting results, and the limited genetic data available do not necessarily support currently proposed species designations. The reasons for this ambiguity lie with the ubiquitous nature of Cryptosporidium, probably infecting all vertebrates and variety of tissues therein, and the absence of reference strains with defined virulence attributes that can be linked to genetic markers for comparative analysis. The inability to classify oocysts or confidently to identify their origin, implicate oocysts from all sources as hazardous to humans. Another major issue is the unusual degree of resistance that Cryptosporidium has shown to antiprotozoan and antimicrobial agents. The intracellular but extracytoplasmic domain the parasite occupies is in itself a significant barrier to drug entry. In support of this we outline how the intracellular niche of this parasite differs from the related Apicomplexans, Plasmodium and Toxoplasma, and delineate why the feeder organelle membrane, rather than, or in addition to, the parasitophorous membrane, is the major portal of nutrient entry for Cryptosporidium. The broad conclusion is that anticryptosporidial agents will have to enter the parasite via the multiple apical membranes that camouflage the parasite, or via the host cell, possibly transported by vesicles to the feeder organelle membrane. This may have major implications for rational drug discovery and design.

Effects of short-term zinc supplementation on cellular immunity, respiratory symptoms, and growth of malnourished Equadorian children.

OBJECTIVE: To assess the effect of zinc supplementation on respiratory tract disease, immunity and growth in malnourished children. DESIGN: A randomized double-blind placebo-controlled trial. SETTING: A day-care center in Quito, Ecuador. SUBJECTS: Fifty children (12-59 months old) recruited by height-for-age and weight-for-age deficit. INTERVENTIONS: Twenty-five children (supplemented, S group) received 10 mg/day of zinc as zinc sulfate, and 25 (nonsupplemented, NS group) received a placebo during 60 days. All were also observed during a 60-day postsupplementation period. Two children of the S group dropped out. Daily the clinical presence of cough, respiratory tract secretions, and fever, was recorded. On days 0,60 and 120, the cutaneous delayed-type hypersensitivity (DTH) to multiple antigens, and anthropometric parameters were assessed. On days 0 and 60 serum zinc levels were also measured. RESULTS: On day 60, DTH was significantly larger (20.8 +/- 7.1 vs 16.1 +/- 9.7 mm), and serum zinc levels were significantly higher (118.6 +/- 47.1 vs 83.1 +/- 24.5 micrograms/dl) in the S group than in the NS group (P <0.05 for each). The incidence of fever [relative risk (RR): 0.30, c.i. = 0.08- 0.95, P =0.02], cough (RR): 0.52, c.i. = 0.32-0.84, P = 0.004) and upper respiratory tract secretions (RR):0.72, c.i. = 0.59-0.88, P = 0.001) was lower in the S group than in the NS group at day 60. At the end of the postsupplementation observation period (day 120), the incidence of fever and upper respiratory tract secretions was the same in both the S and NS groups. The incidence of cough was higher at day 120 in the S group than in the NS group (RR): 2.28, c.i. = 1.37-3.83, P = 0.001). CONCLUSIONS: This study supports a role for zinc in immunity, and immunity to respiratory infections, while pointing out the need for larger studies.

Use of passive surveillance data to study temporal and spatial variation in the incidence of giardiasis and cryptosporidiosis.

OBJECTIVE: The goal of this study was to evaluate temporal and spatial variations in the reporting of cases of giardiasis and cryptosporidiosis to a passive surveillance system, and to assess the relationship of those variations to source of drinking water, adjusting for socioeconomic variables. METHODS: The authors analyzed temporal and spatial patterns for 4,058 cases of giardiasis and 230 cases of cryptosporidiosis reported to the Massachusetts Department of Public Health for 1993-1996. They linked each reported case to a database containing information on source of residential water supply and socioeconomic characteristics and evaluated the association between these factors and reporting rates using regression techniques. RESULTS: Reports of giardiasis and cryptosporidiosis were highest for the mixed unfiltered drinking water supply category. Reports of giardiasis were associated with income levels. Increases in reporting for both giardiasis and cryptosporidiosis were seen in summer to early fall. During a suspected outbreak of cryptosporidiosis n the city of Worcester in 1995, a significant increase in reported cases was also observed in the Boston metropolitan area. Following the suspected outbreak, weekly giardiasis rates increased slightly in Worcester and the Boston metropolitan area, while reporting of cryptosporidiosis increased dramatically. CONCLUSIONS: Consistently collected passive surveillance data have the potential to provide valuable information on the temporal variation of disease incidence as well as geographic factors. However, passive surveillance data, particularly in the initial period of surveillance, may be highly sensitive to patterns of diagnosis and reporting and should be interpreted with caution.

Host cell apoptosis impairs Cryptosporidium parvum development in vitro.

The absence of a self-sustaining in vitro propagation method for Cryptosporidium parvum is a major obstacle for research on this parasite. Conventional cell monolayers are unsuitable for long-term parasite propagation because the level of infection decreases over time and few oocysts, if any, are produced. The interaction between parasite and host cell was studied to identify factors limiting parasite development in vitro. Loss of substrate adherence and death of parasitized host cells was observed in 2 epithelial cell lines. Nuclear morphology, DNA laddering, annexin V binding, and terminal deoxytransferase-mediated dUTP nick end labeling indicated that host cell death occurred by apoptosis. At 6 hr postinfection, only a minority of infected cells remained in the monolayer, and few survived the initial phase of parasite development without losing adherence. Treatment of infected monolayers with caspase inhibitors drastically reduced cell detachment but failed to increase the number of parasites in monolayers. In contrast, cell cultures grown on laminin-coated plates showed a higher proportion of infected cells. These observations indicate that cell detachment and apoptosis in C. parvum-infected cell culture negatively affect parasite survival in vitro.

Presence of double-stranded RNAs in human and calf isolates of Cryptosporidium parvum.

We examined the occurrence of 2 virus-like double-stranded (ds)RNAs in human and calf isolates of Cryptosporidium parvum senso latu and other microorganisms, including 7 other members of the genus. A total of 32 isolates of C. parium, 16 from humans (5 from acquired immune deficiency syndrome patients) and 16 from calves, were analyzed. Ethidium bromide staining, or Northern blot analysis, or reverse transcription/polymerase chain reaction, or all 3 methods, revealed that both genotype 1 and genotype 2 isolates of C. parvum possessed these dsRNAs. No other Cryptosporidium spp. or other organisms examined possessed these dsRNAs. Comparison analysis of partial cDNA sequences of dsRNAs from human and calf isolates revealed a high degree of similarity (>92% and >93% identical nucleotides for large and small dsRNAs, respectively). Slight, consistent differences in nucleotide sequences could be seen at select sites and were associated with an isolate being either genotype 1 or 2. Because of the widespread distribution of the dsRNAs, the similarity of these molecules between isolates, and high host specificity, these nucleic acids may prove to represent species-specific molecular markers for C. parvum. Evidence also suggests that the dsRNA can be utilized for molecular genotyping of C. parvum.

Seasonality in six enterically transmitted diseases and ambient temperature.

We propose an analytical and conceptual framework for a systematic and comprehensive assessment of disease seasonality to detect changes and to quantify and compare temporal patterns. To demonstrate the proposed technique, we examined seasonal patterns of six enterically transmitted reportable diseases (EDs) in Massachusetts collected over a 10-year period (1992-2001). We quantified the timing and intensity of seasonal peaks of ED incidence and examined the synchronization in timing of these peaks with respect to ambient temperature. All EDs, except hepatitis A, exhibited well-defined seasonal patterns which clustered into two groups. The peak in daily incidence of Campylobacter and Salmonella closely followed the peak in ambient temperature with the lag of 2-14 days. Cryptosporidium, Shigella, and Giardia exhibited significant delays relative to the peak in temperature (approximately 40 days, P<0.02). The proposed approach provides a detailed quantification of seasonality that enabled us to detect significant differences in the seasonal peaks of enteric infections which would have been lost in an analysis using monthly or weekly cumulative information. This highly relevant to disease surveillance approach can be used to generate and test hypotheses related to disease seasonality and potential routes of transmission with respect to environmental factors.

The seasonality of human campylobacter infection and Campylobacter isolates from fresh, retail chicken in Wales.

Seasonal peaks in both human campylobacter infections and poultry isolates have been observed in several European countries but remain unexplained. We compared weekly data on human campylobacter infections with thermophilic Campylobacter isolation rates from fresh, retail chicken samples (n = 514) purchased weekly in Wales between January and December 2002. Human isolates (n = 2631) peaked between weeks 22 and 25 (early June) and chicken isolates (n = 364) between weeks 24 and 26 (late June). In the absence of a temporal association, we postulate that the seasonal rise in humans is not caused by a rise in isolation rates in poultry but that both are more likely to be associated with a common, but as yet unidentified, environmental source.

Two cases of endocarditis due to Lactobacillus species: antimicrobial susceptibility, review, and discussion of therapy.

Endocarditis caused by lactobacilli may lead to death or to relapse of infection, despite antimicrobial treatment. We report two cases of lactobacillus endocarditis in individuals with native bicuspid aortic valves who survived without relapse and review the 39 other cases reported in the literature. In only 15 previously reported cases have patients been cured with medical therapy alone. One of our patients, who was infected with Lactobacillus acidophilus, was cured by medical therapy alone, and our other patient, who was infected with Lactobacillus casei subspecies rhamnosus, required surgical replacement of his aortic valve. L. acidophilus was tolerant and L. casei subspecies rhamnosus was resistant to many antibiotics tested. Combinations of penicillin or daptomycin and gentamicin were synergistic by time-kill assay. Synergistic therapy with a penicillin and an aminoglycoside was effective clinically and appears to provide optimal medical treatment on the basis of microbiological data.

Paromomycin and geneticin inhibit intracellular Cryptosporidium parvum without trafficking through the host cell cytoplasm: implications for drug delivery.

Cryptosporidium parvum, which causes intractable diarrhea and lethal wasting in people with AIDS, occupies an unusual intracellular but extracytoplasmic niche. No reliable therapy for cryptosporidiosis exists, though the aminoglycoside paromomycin is somewhat effective. We report that paromomycin and the related compound geneticin manifest their major in vitro anti-C. parvum activity against intracellular parasites via a mechanism that does not require drug trafficking through the host cell cytoplasm. We used both normal and transformed aminoglycoside-resistant Caco-2 or MDBK cells in these studies. Timed-exposure experiments demonstrated that these drugs inhibit intracellular but not extracellular parasites. Apical but not basolateral exposure of infected cells to these drugs led to very significant parasite inhibition, indicating an apical topological restriction of action. We estimated intracytoplasmic concentrations of paromomycin, using an intracellular bacterial killing assay, and found that C. parvum infection did not lead to increased paromomycin concentrations compared to those in uninfected cells. Global [3H]paromomycin uptake by Caco-2 cells was approximately 200-fold higher than the estimated intracytoplasmic paromomycin concentration, suggestive of host cell vesicular uptake and concentration (as has been reported with other cell lines). However, preinfection exposure of Caco-2 cells to paromomycin did not result in subsequent inhibition of parasite development, indicating that if exogenous paromomycin enters the infected host cell vesicular compartment, it does not effectively communicate with the parasite. Thus, the apical membranes overlying the parasite and parasitophorous vacuole may be the unsuspected major route of entry for paromomycin and may be of importance in the design and discovery of novel drug therapies for the otherwise untreatable C. parvum.

The gamma interferon gene knockout mouse: a highly sensitive model for evaluation of therapeutic agents against Cryptosporidium parvum.

Cryptosporidiosis is a serious disease in malnourished children and in people with malignancies or AIDS. Current rodent models for evaluating drug therapy against cryptosporidiosis have many limitations, including the need for a high inoculum, the absence of symptoms resembling those seen in humans, and the need to maintain exogenous immunosuppression. We have developed a gamma interferon knockout (GKO) mouse model with which to evaluate therapies against C. parvum and have used paromomycin for evaluation of this model. The GKO model offers considerable improvements over other systems, since it requires no additional immunosuppression and adult mice can be infected with as few as 10 oocysts (compared with 10(7) for SCID mice). Infected mice develop profound gastrointestinal dysfunction due to extensive infection and severe mucosal damage involving the entire small intestine. Clinical symptoms, which include depression, anorexia, weight loss, and wasting, result in death within 2 to 4 weeks. The time of death depends on the oocyst challenge dose. Paromomycin modulated parasitological and clinical parameters in highly predictable and significant ways, including prevention of death. In addition, examination of the extensively infected gut provided an important insight into the dynamics between a specific drug treatment, its impact on the extent and the site of parasite distribution, and clinical outcome. These uniform symptoms of weight loss, wasting, and death are powerful new parameters which bring this model closer to the actual disease seen in humans and other susceptible mammalian species.

Did Milwaukee experience waterborne cryptosporidiosis before the large documented outbreak in 1993?

The patterns of incidence and pathways of spread for cryptosporidiosis are poorly understood. In this study, we explored the possibility that drinking water caused significant waterborne cryptosporidiosis in Milwaukee well before the massive documented outbreak in April 1993. We generated time series of daily counts of emergency room visits and hospital admissions for gastroenteritis in Milwaukee using the billing records of the Medical College of Wisconsin for January 1, 1992, through May 3, 1993. The Milwaukee Water Works provided us with data on drinking water turbidity for the same period. The service area of the South Plant experienced a sharp rise in turbidity just before the outbreak. During the outbreak period, gastroenteritis events were most strongly associated with turbidity at a lag of 7 days in children and 8 days in adults. It is reasonable to conclude that these lag times reflect the incubation period of Cryptosporidium. During the 434 days before the outbreak, gastroenteritis events were most strongly associated with turbidity at a lag of 8 days among children and 9 days among adults in the service area of the North Plant, the plant that experienced the highest effluent turbidity during this period. These findings are consistent with the conclusion that waterborne cryptosporidiosis was occurring in Milwaukee for more than a year before the documented outbreak.

Temporal changes in permeability and structure of piglet ileum after site-specific infection by Cryptosporidium parvum.

BACKGROUND/AIMS: Cryptosporidiosis is an important enteric infection associated with diarrhea in humans. The structural and functional basis for diarrhea is poorly understood. The aim of the study was to determine the structural and functional basis of diarrhea in cryptosporidiosis during evolving host cell-parasite interactions in the intestine. METHODS: We used the piglet model for temporal studies of alterations in intestinal epithelial structure and function that occurred 12-48 hours postinoculation. Segments of intestine were directly inoculated in vivo, harvested, and studied in vitro using Ussing chamber techniques. RESULTS: Villus architectural alterations corresponded to the extent of infection. Increased numbers of lamina propria inflammatory cells were evident at 36 hours postinoculation. Solute and macromolecular permeability was not increased. Glucose-responsive short-circuit current was diminished at 48 hours postinoculation. The short-circuit current response to theophylline was the same in control and infected tissues. CONCLUSIONS: We conclude that passive solute and macromolecular permeability in infected tissues is not significantly increased during parasite-host cell interactions 12-48 hours postinoculation. Electrogenic glucose stimulated Na+ absorption, a function principally of villus absorptive cells, is impaired, and electrogenic Cl- secretion, a function of crypt epithelial cells, remains the same. These findings parallel structural observations that include loss of the Na+/glucose-transporting villus epithelium without loss of crypt epithelium.

Shigellosis in neonates and young infants.

To determine the clinical features and outcome of shigellosis in young infants, we reviewed the hospital records of 159 infants < or = 3 months of age (including 30 neonates) and 159 children 1 to 10 years of age with shigellosis who were admitted to the Diarrhoea Treatment Centre in Dacca, Bangladesh. Infants more commonly had a history of nonbloody diarrhea (82.8% vs 42.7%; p < 0.001), moderate or severe dehydration (59.9% vs 32.1%; p < 0.001), or bacteremia (12.0% vs 5.0%; p = 0.027) and less commonly had fever (32.7% vs 58.6%; p < 0.001), abdominal tenderness (1.9% vs 12.6%; p < 0.001), or rectal prolapse (0% vs 8.3%; p = 0.001). Infections caused by Shigella boydii (20.8% vs 6.3%; p < 0.001) and Shigella sonnei (7.5% vs 1.3%; p = 0.006) were more common, and Shigella dysenteriae type 1 (9.4% vs 31.4%; p < 0.001) infections were less common in infants than in older children; the proportion of Shigella flexneri infections was equivalent in the two groups (59.1% vs 60.4%). Infants were twice as likely to die as older children (16.4% vs 8.2%; p = 0.026). Only 17 infants (14.3%) were being exclusively breast fed at the onset of their illness. In a multiple logistic regression analysis, independent predictors of death in infants were gram-negative bacteremia, ileus, decreased bowel sounds, hyponatremia, hypoproteinemia, and a lower number of erythrocytes detected on microscopic examination of stool specimens. Diarrhea management algorithms that rely only on clinical findings of dysentery to diagnose and treat shigellosis are likely to be unreliable in this high-risk age group.

PIP: Findings are reported from a study conducted to determine the clinical features and outcome of shigellosis in young infants. The authors reviewed the hospital records of 159 infants of no greater than age 3 months and those of 159 children aged 1-10 years with shigellosis who were admitted to the Diarrhea Treatment Center in Dacca, Bangladesh. 82.8% of infants had a history of nonbloody diarrhea, 59.9% moderate or severe dehydration, 12% bacteremia, 32.7% fever, 1.9% abdominal tenderness, and 0% rectal prolapse. 42.7% of children had a history of nonbloody diarrhea, 32.1% moderate or severe dehydration, 5.0% bacteremia, 58.6% fever, 12.6% abdominal tenderness, and 8.3% rectal prolapse. Infections caused by Shigella boydii and Shigella sonnei were more common in infants, while Shigella dysenteriae type 1 infections were less common in infants than in older children. There was an equivalent proportion of Shigella flexneri infections in the two groups. Infants were twice as likely to die as older children. Only 17 infants were being exclusively breastfed at the onset of their illness. Multiple logistic regression analysis identified the independent predictors of death among infants to be gram-negative bacteremia, ileus, decreased bowel sound, hyponatremia, hypoproteinemia, and a lower number of erythrocytes detected on the microscopic examination of stool specimens. Diarrhea management algorithms which rely exclusively upon clinical findings of dysentery to diagnose and treat shigellosis are likely to be unreliable in this high-risk age group.

Spectrum and significance of bacteremia due to Moraxella catarrhalis.

Fifty-eight cases of bacteremia due to Moraxella catarrhalis, including seven that occurred in patients treated at our facilities, are analyzed. The host's medical history plays a major role in the presentation and outcome of M. catarrhalis bacteremia. Bacteremia is typically accompanied by pneumonia in adults with underlying respiratory disease. Many neutropenic patients do not manifest a focus of infection; in contrast, the source identified in healthy, immunocompetent patients is usually the upper airway or the ears. In the recent literature, it has been reported that a rash is typically absent in adults with bacteremic pneumonia and in immunocompetent hosts and that only some neutropenic patients have a rash. The prognosis is grave for patients with endocarditis and for patients with immunoglobulin deficiency or neutropenia not related to a hematologic malignancy. In addition, mortality is substantial among bacteremic patients with respiratory conditions or other chronic debilities, especially when respiratory copathogens are present. The prognosis is good for febrile neutropenic patients with underlying leukemia or lymphoma when the neutropenia resolves. When healthy, immunocompetent individuals are affected with M. catarrhalis bacteremia, their presentations range from self-limited febrile illness to life-threatening disease.

Cryptosporidium parvum infection of Caco-2 cell monolayers induces an apical monolayer defect, selectively increases transmonolayer permeability, and causes epithelial cell death.

Caco-2 cells were grown on permeable filters and infected with Cryptosporidium parvum. Infection rates exceeded 50% of target cells with a sufficient inoculum dose of parasites. Infection induced a dose- and time-dependent fall in transmonolayer resistance, which was closely related to both the inoculum dose and the number of parasites detected by immunofluorescence. Caco-2a, MDBK, and MDBK subclone F5D2 evidenced similar declines in resistance when grown and infected under similar circumstances. Caco-2 monolayers became permeable to molecules of < or = 1,000 Da but continued to remain impermeable to exogenously added, or endogenously produced, proteins of > or = 1,881 Da. We found that infected monolayers released up to 50% of the total cellular lactase dehydrogenase into apical media, but not basal media, and that the vital dye propidium iodide avidly stained infected cells, and often parasites, when added to the apical reservoir. Cryptosporidium infection of Caco-2 monolayers increases transmonolayer permeability, induces an apical cellular and monolayer defect, and causes cell death.

Profiles of healing and nonhealing Cryptosporidium parvum infection in C57BL/6 mice with functional B and T lymphocytes: the extent of gamma interferon modulation determines the outcome of infection.

This study describes healing and nonhealing models of Cryptosporidium parvum infection with adult mice that have functional T and B lymphocytes. In our nonhealing model, mice on a C57BL/6 background which have a targeted disruption in the gamma interferon (IFN-gamma) gene (GKO mice) are utilized. C. parvum-infected GKO mice shed extremely high levels of oocysts and displayed overwhelming infection of the entire small intestine. The majority of these mice succumbed within 2 to 3 weeks due to severe acute infection and profound mucosal destruction. In our healing murine model, C57BL/6J mice treated with a single injection of the neutralizing anti-IFN-gamma monoclonal antibody XMG 1.2 prior to infection were used. These mice developed two peaks of oocyst shedding but were ultimately free of parasites on day 30 of infection. Again, the small intestine was the primary site of infection. Mesenteric lymph node (MLN) cells isolated from C. parvum-infected nonhealing GKO mice proliferated and secreted interleukin 2 (IL-2) but not IFN-gamma or IL-4 in response to ex vivo restimulation with intact C. parvum sporozoites or a C. parvum sporozoite antigen preparation. In contrast, parasite-specific MLN cells isolated from healing C57BL/6J mice secreted IL-2 and IFN-gamma but not IL-4. These results suggest that IFN-gamma, either directly or indirectly, is important for resistance to and resolution of cryptosporidiosis. Moreover, these models now allow the analysis of parasite-specific cell-mediated and humoral mucosal immune responses to determine what constitutes protective immunity to C. parvum.