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Immunohistochemistry Based Assay to Determine the Effects of Treatments on Cryptosporidium parvunt Viability
Author(s) -
PHELPS KALMIA K.,
LINDSAY DAVID S.,
SUMNER SUSAN S.,
PAYER RON
Publication year - 2001
Publication title -
journal of eukaryotic microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.067
H-Index - 77
eISSN - 1550-7408
pISSN - 1066-5234
DOI - 10.1111/j.1550-7408.2001.tb00447.x
Subject(s) - cryptosporidium parvum , biology , infectivity , immunoperoxidase , cryptosporidium , cell culture , microbiology and biotechnology , antibody , biotinylation , virology , immunology , monoclonal antibody , feces , virus , genetics
SUMMARY Cell culture infectivity assays can provide an accurate means of detecting viable Cryptosporidium parvum oocysts from environmental samples or to test the effects of various treatments on oocyst infectivity. Cell culture assays can also be used to test candidate chemotherapeutic agents. The use of a human cell line provides a situation close to human infection. The present assay uses an anti ‐Cryptospordium primary antibody, combined with a biotinylated secondary antibody, and an immunoperoxidase detection system. Cryptosporidium parvum oocysts excysted in vitro when placed on monolayers of HCT‐8 cells and developmental stages including schizonts and merozoites were visualized using light microscopy of the immunoperoxidase stained slides and by transmission electron microscopy of infected HCT‐8 cell cultures. Because the immunoperoxidase system used gives a permanent preparation, the cell cultures can be retained and examined later. Dose titration of oocysts indicated that as few as 50 inoculated oocysts could be detected. The activity of paromomycin was evaluated in this system and 500 μg/ml produced a 97.8% reduction in infection.