Comparative detection of Cryptosporidium parvum oocysts from apple juice

Abstract

Drinking unpasteurized apple juice (or cider) has been associated with cryptosporidiosis, the diarrheal disease caused by the small protozoan parasite, Cryptosporidium parvum. This report compares detection of C. parvum oocysts from apple juice by acid-fast staining (AFS), direct immunofluorescence assay (DIFA), and polymerase chain reaction (PCR), following sample concentration by formalin–ethyl acetate sedimentation or sucrose flotation. Flotation was more efficient than sedimentation in recovering oocysts, and DIFA consistently detected lower numbers of oocysts than AFS. In combination, flotation–AFS could detect 3000 to 10 000 oocysts inoculated into 100 ml of apple juice while flotation–DIFA was able to detect as few as 100 oocysts. The highest sensitivity, 10 to 30 oocysts per 100 ml of apple juice, was achieved by DIFA following immunomagnetic capture (IC) of oocysts from samples concentrated by the flotation method. The detection limit of PCR following flotation or flotation–IC was 30 to 100 oocysts; sequence analysis of the amplicon demonstrated that the PCR amplicon was C. parvum-specific.