Sporozoite proteome analysis of Cryptosporidium parvum by one-dimensional SDS-PAGE and liquid chromatography tandem mass spectrometry

Despite the development of new technologies, new challenges still remain for large scale proteomic profiling when dealing with complex biological mixtures. Fractionation prior to liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis is usually the preferred method to reduce the complexity of any biological sample. In this study, a gel LC-MS/MS approach was used to explore the stage specific proteome of Cryptosporidium (C.) parvum. To accomplish this, the sporozoite protein of C. parvum was first fractionated using SDS-PAGE with subsequent LC-MS/MS analysis. A total of 135 protein hits were recorded from 20 gel slices (from same gel lane), with many hits occurring in more than one band. Excluding all non-Cryptosporidium entries and proteins with multiple hits, 33 separate C. parvum entries were identified during the study. The overall goal of this study was to reduce sample complexity by protein fractionation and increase the possibility of detecting proteins present in lower abundance in a complex protein mixture.

Ultrastructural Localization of Cryptosporidium parvum Antigen Using Human Patients Sera

The antigen location of Cryptosporidium parvum, which stimulates antibody formation in humans and animals, was investigated using infected human sera. Immuno-electron microscopy revealed that antigenicity-inducing humoral immunity was located at various developmental stages of parasites, including asexual, sexual stages, and oocysts. The amount of antigen-stimulating IgG antibodies was particularly high on the oocyst wall. The sporozoite surface was shown to give stimulation on IgG and IgM antibody formation. Trophozoites implicated the lowest antigenicity to humoral immunity, both IgG and IgM, by showing the least amount of gold labeling. Immunogold labeling also provided clues that antigens were presented to the host-cell cytoplasm via feeder organelles and host-parasite junctions.

Evaluation of an enzyme-linked immunoassay for the detection of Cryptosporidium antigen in fecal specimens of HIV/AIDS patients.

Cryptosporidium parvum, a protozoan parasite, causes severe diarrhea in immunodeficient hosts like HIV/AIDS patients, leading to significant morbidity and mortality. Diagnosis of the Cryptosporidium oocyst in the stool of these patients by conventional microscopy is labor intensive and time consuming. Therefore, we planned to evaluate the usefulness of a stool ELISA test in detecting Cryptosporidial antigen. About 89 stool specimens obtained from HIV-seropositive patients with diarrhea were subjected to an ELISA test and modified acid-fast staining (gold standard), on both direct and formol ether-concentrated specimens. The prevalence of Cryptosporidial diarrhea was found to be 12.4% (11/89). Other enteric pathogens detected were Isospora belli (3), Giardial cyst (3), Entamoeba coli cyst (2), and Entamoeba histolytica cyst (1). Dual infection with Cryptosporidium and Isospora belli was seen in two patients. Concentration technique improved identification by microscopy. The sensitivity and specificity for stool ELISA were found to be 90.9% and 98.7% respectively. The results of stool ELISA indicate that this simple, rapid, reliable, and standardized immunoassay test is sensitive and specific for routine diagnosis and may be useful for large-scale epidemiological studies of Cryptosporidiosis.