[Cryptosporidium parvum (Apicomplexa: Sporozoa, Coccidia)--the optimization of a technic for isolating a large number of oocysts].

The Waldman e. a. (1986) method of separation of Cryptosporidium spp. oocysts from feces by using a percoll discontinuous density gradient appeared a method of choice for obtaining large numbers of oocysts of C. parvum free of fecal contamination. Feces of 7-12 day old calves, spontaneously infected with C. parvum, were concentrated and purified by the above technique. The purified oocysts were shown to be infectious by inoculation of 6-9 day old rats with an average dose of 20,000 oocysts per animal. The rats shed oocysts after 4 days. At necropsy on day 4 postinoculation, the pattern of endogenous development appeared normal, when examined on frozen sections of fresh tissue, using the Bright cryostat, stained with hematoxylin and eosin. Samples of the clean sediment, presumably containing only oocysts of C. parvum, were smeared and stained with carbol fuchsin after Ziehl-Neelsen, and with gentian violet after Sidorenko (1988). With the latter technique, an intense gentian violet staining screened all the constituents of the smear, except the oocysts, which being "negatively stained" looked as small transparent spheres 4-5 mkm in diameter. But of special interest was the reaction of the smeared organisms with carbol fuchsin. Some organisms stained dark red and had a variable number of dark granules, seemingly on the surface; whereas others stained light reddish, if at all, and appeared as transparent spheres. It does not seem unlikely that the sediment, resulting from the final step of percoll separation, may contain, besides oocysts, some other endogenous stages (meronts, gamonts, thin-walled oocysts) that appeared in the lumen of the intestine because of an intense flow of diarrheal fluid during cryptosporidiosis. Unlike the thick walled oocysts, other endogenous stages are not covered with protective walls and thus fail to absorb acid fast staining. Segmented meronts were obviously observed on the rat fecal smears 96 hours after infection. This observation enables us to propose that newly infected hosts-recipients may obtain, with diarrheal fecal masses of infected donors, not only sporulated oocysts, but also some earlier developmental stages. Merozoites, released from the segmented meronts, could start in the intestine asexual rounds, thus shortening the resulting prepatent period. Fluctuations in prepatent period duration are characteristic of Cryptosporidium spp., and the above observation may be one of its explanations.