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The primary objective of this study was to develop a method for the recovery and identification of human pathogenic microsporidia from natural waters. Spores of Encephalitozoon species were maintained in culture flasks of rabbit kidney (RK-13) cells grown to confluency and maintained in RPMI media at 35C. Spores were harvested from infected cells after several weeks of incubation and were purified from the spent cell culture media by differential density gradient centrifugation. Pellets were washed and resuspended in sterile 18 M. resistivity water. The 50% infective cell culture dose (TCID50) was determined by seeding spores onto coverslips in 24 well plates and were incubated for up to 72 hours. Serial 10-fold dilutions of fresh spores (enumerated by multiple hemacytometer counts or sorted directly using a flow cytometry equipped with a cell sorter) were introduced into plate wells and incubated at 35 degrees C. Six days post-infection, coverslips were methanol-fixed, stained with Giemsa, mounted on glass slides and the entire surface of the coverslip was examined by light microscopy to score for evidence of parasitophorous vacuoles containing mature microsporidian spores. Coverslips showing one or more infected cells were considered "positive" and infection percentages were computed as the quotient of positive wells and the total number of infected wells. The response logit was computed for each spore dose as the natural log of the proportion of wells infected over (1.0 - the proportion of wells inoculated) and was regressed against the log of the spore number per dose. TCID50 values were read directly from each regression line. In order to improve the sensitivity of the assay method and shorten the time required for analytic results, an application of the reverse transcription polymerase chain reaction (RT-PCR) application was integrated into the diagnostic procedure. For this modification, RK-13 cells grown on cover slips were seeded with serial 10-fold dilutions of fresh spores. Seventy two hours post-infection cover slips were lysed, total RNA was recovered using commercial spin columns and then subjected to reverse transcription. Chromosomal DNA was then subjected to nucleic acid amplification (polymerase chain reaction) and samples were scored for viability by identifying microsporidial DNA following agarose gel electrophoresis. Other topics covered include: antibody production; immunomagnetic separation; flow cytometry; recovery from seeded laboratory waters by filtration/elution and continuous flow centrifugation; capture of spores from 10 liter samples; and, recovery of spores/enumeration by epifluorescence microscopy. Includes 22 references, table.

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Edition: Vol. - No. Published: 11/01/2002 Number of Pages: 7 File Size: 1 file , 260 KB