The environmentally resistant spores of Encephalitozoon intestinalis, E. cuniculi,
and E. hellem may be acquired by ingestion of contaminated food or water. These
species, along with Enterocytozoon bieneusi, may represent serious health threats for
immunocompromised populations, and can also infect immune-competent individuals.
E. bieneusi and E. intestinalis are both on the CCL-1 list, which supports investigation
for detection and viability determination procedures. Presently, in vitro spore production
is not available for E. bieneusi, but the Encephalitozoon spp. are very adaptable to cell
culture production.
Recent advances in molecular techniques have made it possible to both detect select microorganisms in water, and determine their viability. The polymerase chain
reaction (PCR) can detect a pathogen's DNA, and therefore assist in organism identification, while the reverse-transcription (RT)-PCR technique, which detects RNA,
can be used to determine an organism's viability.
RK-13 cells were seeded into individual wells of 24 well plates for the RT-PCR
CC assay and onto coverslips for the Microwell assay (Wolk et al., 2000).
Heat-inactivated or untreated spores were then inoculated onto the 24 well plates or
coverslips. The Microwell assay plates were incubated for six days, while the RT-PCR
CC plates were incubated for three days post infection. Total RNA was isolated from
the individual cell culture wells using the SNAP RNA Isolation Kit (Invitrogen). The
GeneAmp RNA PCR Kit (Perkin Elmer) was used to convert the mRNA to cDNA prior to
amplification by PCR. PCR products were detected by agarose gel electrophoresis.
The kits allow all of the process to be completed in one column.
The 16S rRNA gene sequences for the human pathogens E. cuniculi, E hellem,
E. intestinalis, and E. bieneusi have been determined and are available in the GenBank
database, and PCR primers designed to the 16S rRNA genes have been used to
identify microsporidia in clinical samples (Weiss and Vossbrinck, 1999).
The 16S rRNA primers evaluated in this study were SI500 (Weiss et al., 1994) and PMP1/VI (Fedorko et al.,1996). These primers were able to amplify products from
untreated, or live spores, in either PCR or RT-PCR CC assays, which compares well
with the in vitro Microwell plate assay. However, PCR with these primers could not be
used to determine spore viability, as they were able to amplify PCR products from both
live and dead spores.
The second primer set evaluated was designed to amplify a region within the
¿-tubulin gene.
The third primer set evaluated was designed to amplify a region within the Hsp70
gene.
From the three primer sets evaluated, the ¿-tubulin set has demonstrated an
equivalency comparable to the cell culture coverslips at 104 spores for E. intestinalis and
E. hellem, similarly with Hsp70 set for E. hellem and E. cuniculi. In order to use a RT-PCR assay for water samples
or viability testing, it may be necessary to use a nested primer approach, a multiplex
approach or to develop additional primer sets that would identify all three species with
one assay. The study,
however, demonstrated that the RT-PCR CC assay may become a useful tool for
disinfection evaluation assays.
Includes 6 references, tables.
| Edition : | Vol. - No. |
| File Size : | 1
file
, 240 KB |
| Note : | This product is unavailable in Ukraine, Russia, Belarus |
| Number of Pages : | 5 |
| Published : | 11/01/2002 |
