numbers ofoocysts shed per gram of feces per day and the patterns of ... to Cryptosporidium infection, the total number of oocysts shed (a thousand times the ...
Vol. 28, No. 2
JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1990, p. 356-357
0095-1137/90/020356-02$02.00/0 Copyright CD 1990, American Society for Microbiology
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Experimental Cryptosporidiosis in Hamsters P. ROSSI, E. POZIO,* M. G. BESSE, M. A. GOMEZ MORALES, AND G. LA ROSA
Laboratory of Parasitology, Istituto Superiore di Sanità, viale Regina Elena 299, 00161 Rome, Italy Received 31 May 1989/Accepted 10 October 1989
A new laboratory animal model for experimental cryptosporidiosis is described. Adult immunosuppressed hamsters were infected per os with 0.5 x 105 and 1 x 105 Cryptosporidium oocysts of calf origin. The mean numbers of oocysts shed per gram of feces per day and the patterns of infection are described. The susceptibility to Cryptosporidium infection, the total number of oocysts shed (a thousand times the infective dose), and the ease of handling in laboratory conditions make hamsters a good animal model for cryptosporidiosis.
In recent years, the increasing interest of scientists in cryptosporidia has led to the development of different methods of cultivating these parasites under laboratory conditions. Several experiments have been done in vitro on different cell culture systems, e.g., human fetal lung cells (4) and chicken embryos (5), etc., which enable the parasite to develop completely. It is often difficult to use these systems for the evaluation of antiprotozoan drugs and the study of biological aspects of the parasite life cycle, owing to the lack of standardization of the methods. Moreover, these systems cannot be used for propagation of infection, i.e., for oocyst production, since autoinfection of cultured cells has not been observed and the number of released oocysts does not increase as it does in in vivo infections. Thus, several laboratory animals have been screened to provide a good in vivo model for cryptosporidiosis. Calves are the most suitable hosts for oocyst production, owing to the large amount of infected feces produced and continued infection (9). However, the problems related to maintaining a large-sized host make calves unsuitable for all laboratories. Suckling mice and rats have been tested successfully for cryptosporidiosis (6, 7), but the handling of newborn animals and the low production of oocysts make them unsuitable for many research purposes as well. Adult immunosuppressed Sprague-Dawley rats fed a lowprotein diet acquire the infection, which can last until day 100 postinfection (p.i.) (2). We tried to establish the infection in adult immunosuppressed laboratory rodents. In a preliminary study (data not shown), four female Wistar rats (200 g), eight female Swiss CD1 mice (20 g), and four female Syrian golden hamsters (80 to 100 g) (Charles River Breeding Laboratories, Inc.) were immunosuppressed by 100 mg of hydrocortisone acetate per kg of body weight twice a week, starting 1 week before infection, and by an X-ray dose (500 rads) on the day of infection. The animals were infected with 2 x 103 oocysts per g of body weight. All the animals became infected between days 5 and 7 p.i. The hamsters proved to be more receptive to infection that lasted, and the animals shed a greater number of oocysts. A more detailed study was then performed on this animal model. *
Female outbred white hamsters weighing 80 to 100 g were used in this experiment. The animals weré immunosuppressed by subcutaneous injection of 8 to 10 mg of hydrocortisone acetate administered on days 7, 6, 3, 2, and 0 before challenge and by a dose of X rays (500 rads) on the day of infection. The hydrocortisone treatment was also given on days 1 and 4 p.i., and then it was stopped. Oocysts were obtained from an experimentally infected holstein-friesian 2-day-old calf challenged with 7.5 x 107 oocysts of an isolate of calf origin (supplied by C. Genchi, University of Milan, Milan, Italy). After collection, the feces were passed through sieves (297-, 125-, and 53-,um pore size), suspended in an equal volume of 5% K2Cr2O7, and stored at 4°C. The oocysts were partially purified with a discontinuous sucrose gradient by the technique described by Arrowood and Sterling (1). The oocysts, suspended in phosphate-buffered saline (100 mM), were administered per os to the hamsters. Two groups of 12 animals each were infected (group 1, 0.5 x 105 oocysts per animal; group 2, 1 x 105 oocysts per animal). Six other animals (control group) were immunosuppressed but not infected. The animals were housed one per cage provided with a grating on the bottom to avoid contact between animal and feces. The animals were fed a normal diet. The feces were collected daily, weighed, and stored in 10% Formalin at room temperature. Each animal produced 3 to 4 g of feces per day. Detection of oocysts was performed by an indirect immunofluorescence procedure (Merifluor; Meridian Diagnostics, Inc., Cincinnati, Ohio). The number of oocysts per gram of feces was then measured as follows. The stored feces were homogenized in a ground-glass homogenizer, filtered through a 297-,um-pore-size sieve to exclude larger particles, washed three times, and suspended in phosphate-buffered saline (0.1 g of dry feces per ml). Two milliliters of this suspension was added to 10 ml of phosphate-buffered saline and 5 ml of ethyl ether in 50-ml conical centrifuge tubes, mixed, and centrifuged at 500 x g for 10 min (10). The supernatant was discarded, and the pellet was suspended in 4 ml of phosphate-buffered saline. Counting was performed in triplicate with a hemacytometer. In group 1, oocyst shedding began on days 3 to 5 p.i. and reached a peak on day 9 p.i., on which a mean production of 3.28 x 107 oocysts per g of feces was observed (Fig. 1). Group 2 showed the same pattern of oocyst shedding, with a
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