An In vitro Study of Sporulation in Cryptosporidium Species

Abstract

Oocysts of coccidian parasites complete their development either within the host (endogenous sporulation) or outside, in the environment (exogenous sporulation). Sporulation involves cell division within the oocyst, resulting in the formation of sporozoites and, in some genera, the development of internal cyst walls to form sporocysts. Sporulation may be induced in vitro by holding the oocysts at room temperature with exposure to oxygen for a period of days, usually in dilute solutions of potassium dichromate. Coccidian oocysts are believed to be infective only if they are sporulated (Hammond, 1973. In The Coccidia: Eimeria, Isospora, Toxoplasma, and related genera, D. M. Hammond and P. L. Long (eds.). University Park Press, Baltimore, pp. 45-79). Cryptosporidium sp. (Apicomplexa, Coccidiasina, Cryptosporidiidae) is a pathogenic coccidian of several animal species. It can infect various mucosal epithelia, but is most commonly found in the intestine (Tzipori, 1983, Microbiological Reviews 47: 84-96). The parasite has an oocyst which contains 4 naked sporozoites and was originally described as having endogenous sporulation (Tyzzer, 1910, Journal of Medical Research 23: 487-509). Subsequently, in vivo (Barker and Carbonell, 1974, Zeitschrift fur Parasitenkunde 44: 289-298; Iseki, 1979, Japanese Journal of Parasitology 28: 285-307) and in vitro (Current and Long, 1983, Journal of Infectious Diseases 148:1108-1113; Current and Haynes, 1984, Science 224: 603-605) studies confirmed that sporulation primarily occurs while the oocyst is within the host cell. Furthermore, calffeces containing Cryptosporidium oocysts are infective immediately after passage (Moon and Bemrick, 1981, Veterinary Pathology 18: 248-255). Anderson (1983, Laboratory Medicine 14: 5556), however, indicated that while some oocysts were sporulated at the time of passage of feces, a substantial number were not, and that sporulation of these oocysts would occur if they were exposed to sporulating conditions for 5 to 7 days. Since no objective data were presented to support these statements, it seemed worthwhile to examine this possibility experimentally. The sporulation status of most coccidian oocysts can easily be determined microscopically. While sporozoites can be seen inside some Cryptosporidium oocysts, the small size of the oocyst (4 to 5 Am) and the high refractiveness of the cyst wall can make interpretation of the internal structure of most oocysts difficult (Fig. 1). In this study, the sporulation status of Cryptosporidium oocysts was analyzed using an in vitro excystation procedure. The percentage of sporulated oocysts in a sample would have an effect either on the percentage of oocysts which would excyst (if sporulated oocysts excyst more readily than unsporulated oocysts) or on the number of sporozoites released per excysted oocyst (if sporulated and unsporulated oocysts are equally likely to excyst). Feces from infected calves were collected in metal pans over a 24-hr period beginning 1 or 3 days after the onset of oocyst shedding. Feces were mixed with 2 volumes of 2.5% potassium dichromate, and passed through a graded series of sieves (smallest sieve size 60 Am) to remove fecal debris. The resulting suspension was placed in petri dishes (fluid depth < 2 mm) and held at room temperature (about 23 C) without agitation. At various times after collection, oocysts were isolated by coverslip flotation in Sheather's sucrose solution (Sloss and Kemp, 1978, Veterinary clinical parasitology, 5th ed., Iowa State University Press, Ames, Iowa, p. 8) which had been diluted to a specific gravity of 1.177 g/ml. Oocysts were washed in phosphate buffered saline (PBS) and excysted for 1 hr at 37 C in a solution of 1.5% taurocholic acid (sodium salt, crude, Sigma) and 0.5% trypsin (1:250, Difco) in PBS. After excystation, the suspension was cooled in an ice bath, and the number of sporozoites, oocysts, and empty oocyst walls in 2 1 -mm2 areas of a hemacytometer were determined. From these data, the percent excystation [number cyst walls (number cyst walls + number oocysts)] and sporozoite/cyst wall ratios were calculated. The as-