Submitted to: Journal of Eukaryotic Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 6, 2001
Publication Date: October 1, 2001
Citation: Phelps, K.K., Lindsay, D.S., Sumner, S.S., Fayer, R. Immunohistochemistry based assay to determine the effects of treatments on Cryptosporidium Parvum viability. Journal of Eukaryotic Microbiology. 48:40-41.
Interpretive Summary: Cryptosporidium parvum, a prevalent protozoan parasite of calves, other ruminants, and a variety of wildlife, has been responsible for food and water borne outbreaks of diarrheal disease in humans. Methods of detecting the infectious oocyst stage in environmental specimens and tests to determine its viability have been difficult and often inaccurate. A new method, utilizing cultured human cells to grow the parasite and immune reagents to specifically stain and identify the developing stages, has been developed. Initial tests have shown that a high percentage of the parasites develop and can be detected when one million oocysts are added to a culture, but as few as fifty oocysts can also be detected. The method may be useful to screen drugs and disinfectants based on an initial trial that clearly demonstrated a reduction in developmental stages when an antimicrobial drug was added to cultures.
An assay for infectious oocysts of Cryptosporidium parvum (Cp)was developed using cultured HCT-8 cells for the bioassay portion of the method and anti- Cp primary antibody followed by a biotinylated secondary antibody in an immunoperoxidase detection system. Cp oocysts excysted in vitro on HCT-8 monolayers and developing stages were visualized and counted by bright field microscopy. An estimated 90-99% infection rate was obtained with inoculum of 1 x 106 Cp oocysts and developmental stages resulting from as few as 50 oocysts could be detected. Paromomysin added to the cultures at 500 mg/ml reduced infectivity by >97%, indicating the usefullness of this technique for testing pharmaceuticals and disinfectants against Cp.