J. Peter Bercz sent the following message and questions: 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
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:49:20 EST