Problem Statement: In wanting to
determine the minimally infective dose
(oral route in a neonatal mouse model as
well as in a human colon tumor cell line) of
cryptosporidium oocysts, we must obtain
highly accurately sorted and calibrated
pure cyst suspensions from crude density
gradient preps. Main contaminants in
crude are yeasts.
We have a highly specififc FITC-MAB,
fluor tagging of cysts has been well
worked out.
We have a Coulter Epics Elite equipped
with the SPE fast sorter and autoclone,
we are experienced sorters. ( We also
have a Meridian ULTIMA confocal scope
as an adjunct tool)
My inquiry is about:
1. Does anyone have experience with flow
sorting and/or confocal mapping of
protozoan oocysts, about potential
problems and technical tricks, etc?
2. Would FITC-MAB tagging and flow
sorting of oocysts have an adverse
impact on cyst viability, infectivity
and virulence?
3. Do I need to worry about aerosol-borne
infectious control.
Any advice or dialogue will be greatly
appreciated.
In reply, we have not sorted oocysts but see no reason why this can not be
done. We also have a MAb reactive with oocysts that gives a very good FITC
signal. It would seem that sorting on MAb-FITC labeled oocysts would yield
a very pure population. However, we have no information on the effects of
bound MAb-FITC on viability or infectivity. If viability is not affected,
then I would think virulence is not affected. This question may be answered
by inoculating neonatal mice or by using simple excystation procedures.
Possibly another option is to sort the oocysts using FSC and SSC parameters
in log mode. Since Cryptosporidium parvum oocysts are 4 - 6 microns, they
are separated from bacteria, microsporidia, and some yeast. We have no FSC
& SSC profiles on yeast that are the same size as oocysts, but hopefullly
their SSC profiles would be different than oocysts. If this is the case,
you would still obtain a pure population. I'm sure that most personnel do
not want infectious oocysts in aerosol form in their laboratory.
Good Luck
Delynn M. Moss
Centers for Disease Control
Parasitic Diseases Division