Comparison of serum antibody responses to Giardia lamblia of symptomatic and asymptomatic patients.

The circulating anti-parasite antibody response against Giardia lamblia in symptomatic and asymptomatic Egyptian children with confirmed giardiasis was examined. Symptomatic patients were identified using the following criteria: presence of only G. lamblia cysts in the feces, and one or more of the following symptoms, diarrhea, abdominal pain, loss of weight, vomiting and/or nausea, and abdominal distention. The anti-parasite humoral response was measured using indirect immunofluorescence (IFA), ELISA, and immunoblotting. There was a significant difference in the anti-parasite antibody response measured by IFA of asymptomatic and symptomatic patients, in which more than 34% of the asymptomatic patients had a titer equal to or less than 1:500, and more that 29% of the symptomatic patients had a titer of 1:8,000 or higher. The circulating anti-parasite total IgM and IgA but not IgG, measured by ELISA, was significantly higher in symptomatic than in asymptomatic patients, and were related to higher cyst output observed in symptomatic individuals. Although total anti-parasite IgG response was similar in symptomatic and asymptomatic patients, the analysis of the IgG isotype responses revealed that both IgG1 and IgG3 were significantly higher in symptomatic patients. The antigen recognition by anti-parasite IgM, IgA, IgG1, and IgG3 of symptomatic and asymptomatic individuals, determined by immunoblotting, was heterogeneous and revealed only minor differences in the response of the two groups.

Nucleic acid stains as indicators of Cryptosporidium parvum oocyst viability.

We developed nucleic acid dye staining methodology for untreated, heat-treated and chemically inactivated C. parvum oocysts. The nucleic acid staining was compared to in vitro excystation and animal infectivity using split samples of oocysts. Among the nucleic acid stains tested, SYTO-9, hexidium and SYTO-59 stained the oocysts consistently, and the staining was related to the infectivity of the oocysts to neonatal CD-1 mice but not to in vitro excystation. The nucleic acid viability assay was used to determine log-inactivations of the oocysts after treatment with ozone, chlorine, chlorine dioxide and combinations of different chemical disinfectants, and was found to indicate log-inactivation levels similar to that of animal infectivity. A combined immunofluorescence-nucleic acid staining assay was developed for the oocysts of C. parvum and this assay will be invaluable for the detection and viability of oocysts in the laboratory and in environmental samples.

Nucleic acid stains as indicators of Giardia muris viability following cyst inactivation.

A reliable viability assay for Giardia is required for the development of disinfection process design criteria and pathogen monitoring by water treatment utilities. Surveys of single-staining nucleic acid dyes (stain dead parasites only), and double-staining vital dye kits from Molecular Probes (stain live and dead parasites) were conducted to assess the viability of untreated, heat-killed, and chemically inactivated Giardia muris cysts. Nucleic acid staining results were compared to those of in vitro excystation and animal infectivity. Nucleic acid stain, designated as SYTO-9, was considered the best among the single-staining dyes for its ability to stain dead cysts brightly and its relatively slow decay rate of visible light emission following DNA binding. SYTO-9 staining was correlated to animal infectivity. A Live/Dead BacLight was found to be the better of 2 double-staining viability kits tested. Logarithmic survival ratios based on SYTO-9 and Live/Dead BacLight were compared to excystation and infectivity results for G. muris cysts exposed to ozone or free chlorine. The results indicate that SYTO-9 and Live/Dead BacLight staining is stable following treatment of cysts with chemical disinfectants.

Comparison of assays for Cryptosporidium parvum oocysts viability after chemical disinfection.

In vitro excystation, vital dyes (4', 6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI), and infectivity in neonatal CD-1 mice were used to assess the viability of Cryptosporidium parvum oocysts after chemical disinfection. In vitro excystation and DAPI/PI staining provided similar estimates of viability in bench-scale experiments, but both of these methods significantly overestimated the viability when compared with infectivity (Pr < or = 0.01). Infectivity was the most reliable measure of the viability of C. parvum oocysts following chemical disinfection.

Three tandemly repeated 5S ribosomal RNA-encoding genes identified, cloned and characterized from Cryptosporidium parvum.

We have characterized the 5S rRNA of Cryptosporidium parvum. The gene (rDNA) encoding this 5S rRNA was identified, mapped, the primary and secondary structures determined, and the copy number estimated. Using a PCR-amplified 5S rDNA as a probe, it was shown that this gene can specifically recognize C. parvum genomic DNA, but not other intestinal and environmental organisms tested. Three repeat units of the 5S rDNA found in genomic C. parvum oocyst DNA are within the 2012-bp EcoRI-HindIII fragment and are identical in coding sequence, but differ in flanking regions. Flanking regions are A+T rich (78-89%). The termination signal for polymerase III consists of five thymidine residues at the 3' end of each of three units.

The role of humoral immunity in Cryptosporidium spp. infection. Studies with B cell-depleted mice.

Cryptosporidiosis has become an important infection in man, particularly among very young or immunocompromised individuals. Here, the protective role of antibody responses to Cryptosporidium was examined using a well established murine model of cryptosporidiosis. Although normal neonatal BALB/c mice exhibited good IgM and IgG serum antibody responses, no correlation could be drawn between the intensity of these responses and the severity or duration of cryptosporidiosis. Moreover, B cell-deficient (anti-mu-treated) neonatal BALB/c mice did not differ from untreated or normal rabbit Ig-treated, age-matched controls in the onset, peak, or duration of cryptosporidiosis. The apparent absence of a role for antibody in these self resolving infections was supported by the lack of susceptibility of anti-mu-treated adult BALB/c to attempted infection with doses of Cryptosporidium 10 times the dose required to infect 100% of normal (Ig producing) neonates. The results suggest that the role of specific in vivo antibody responses in the resolution of murine infection with this coccidian parasite is minor and that the likelihood of success for cryptosporidial vaccines aimed solely at enhancing in vivo antibody production may be limited.