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.
| Edition : | Vol. - No. |
| File Size : | 1
file
, 260 KB |
| Note : | This product is unavailable in Ukraine, Russia, Belarus |
| Number of Pages : | 7 |
| Published : | 11/01/2002 |
