[Relationship between discontinuous sucrose gradient and viability of Cryptosporidium sp. oocysts]

Cryptosporidium sp. causes self-limiting gastroenteritis in immunocompetent individuals and can produce a life-threatening chronic diarrhea in immunodeficient patients. In order to obtain populations of selectively infective oocysts for inoculation in biological experiments, we developed an operational protocol for the enrichment of viable oocysts from crude fecal material. Using either fresh or formaldehyde-fixed feces as sources of viable and nonviable oocysts, respectively, we fractionated the samples on parallel discontinuous sucrose gradients and evaluated oocyst viability at different banding densities by an in vitro excystation assay. The formaldehyde-inactivated fecal samples formed no bands after centrifugation and 91.66% of the oocysts became concentrated in the pellet. Fresh fecal samples formed three bands at densities 1.062, 1.092, and 1.121, in addition to a sediment. Here the viability of the gradient-sedimented oocysts was 92.3% overall, and of those in the second band 100%. Modifications in oocyst permeability thus seems to alter their sedimentation characteristics so that consequent distribution on sucrose gradients can be correlated with oocyst viability. Discontinuous sucrose density gradient sedimentation would thus constitute a simple and rapid mean to obtain viable oocysts for use in biological models both in vivo and in vitro.