Structure of the Oocyst and Sporocyst Walls and Excystation of Sporozoites of Isospora canis

Abstract

Excystation of sporozoites from oocysts of Isospora canis occurred 50 to 100 min after these were treated at about 22 C with trypsin and a bile salt (sodium taurocholate). Treatment with the bile salt alone resulted in excystation in 60 to 120 min, but no excystation was observed in oocysts treated with trypsin alone. Excystation occurred in 10 to 35 min when the oocysts were pretreated with Clorox before exposure to trypsin and the bile salt. Usually, the oocyst wall became flattened or indented at one or both ends; the sporocyst wall ruptured suddenly, and broke into several pieces, allowing the sporozoites to escape. The sporocyst wall consisted of 2 layers. The inner layer, about 4 times as thick as the outer, was made up of 4 separate plates. During excystation, the wall evidently breaks at the sites of apposition between the plates. Thus, the mechanism of excystation in I. canis differs considerably from that previously reported for Eimeria species. Excystation of various coccidian oocysts has been described previously (Doran and Farr, 1962; Hammond, Ernst, and Chobotar, 1970; Roberts, Speer, and Hammond, 1970). Bunch and Nyberg (1970) reported that Isospora canis differed from various Eimeria species in that excystation occurred readily without a CO2 stimulus in the former. Our findings with the electron microscope as to the structure of the oocyst and sporocyst walls of Isospora canis are described herein, as well as observations with the light microscope of excystation of I. canis sporozoites. MATERIALS AND METHODS Oocysts of Isospora canis obtained from experimentally infected beagle puppies were cleaned of fecal debris, sporulated (Nyberg and Hammond, 1964), and then stored in 2.5% K2CR207 at 5 C for 8 weeks. For light microscope studies, some oocysts were concentrated by centrifugation and resuspended in distilled water or saline A, whereas others were exposed to 5.25% sodium hypochlorite solution (Clorox) for 20 min, washed twice in saline A, concentrated, and then resuspended in saline A. One drop of the suspension of Cloroxtreated oocysts was placed on each of 5 slides, and to this drop was added a drop of 0.5% trypsin (1 to 300 Nutritional Biochemical) in saline A, Received for publication 29 August 1972. * This investigation was supported by PHS Research Grant No. AI-07488 to Utah State University from the NIAID. Published as Journal Paper No. 1301, Utah Agricultural Experiment Station. t Present address: Department of Histology, Dental Branch, University of Texas, Texas Medical Center, Houston 77025. t On leave from Department of Zoology, University of Alberta, Edmonton, Canada. 1.5% sodium taurocholate (Nutritional Biochemical) in saline A, 0.5% trypsin and 1.5% sodium taurocholate in saline A, saline A, and distilled water, respectively. In order to prevent drying, a cover slip was placed on each of the above preparations and sealed with Vaseline. The slides were then examined at room temperature (about 22 C) with bright-field and Zeiss-Nomarski interferencecontrast microscopy for observation of the excystation process. On each slide, 10 to 20 oocysts were observed to determine the timing of each event occurring during the excystation process. Oocysts not treated with Clorox were studied similarly. For electron microscope studies, sporocysts from oocysts which had been ground in a tissue grinder to break the walls were suspended for 10 min in a 0.25% trypsin and 0.75% sodium taurocholate excysting medium and then added to a cell suspension which had been harvested with a trypsin-versene solution from cell monolayers in two 2-ounce Brockway culture flasks. The suspension was centrifuged at 750 g for 5 min. The pellet was fixed in Karnovsky's fixative (Karnovsky, 1965) with cacodylate buffer for 2 hr at 22 C. The pellet was then dehydrated in 35 and 50% ethanol for 5 min each, and prestained with 1% uranyl acetate and 1% phosphotungstic acid in 70% ethanol at 4 C for 15 hr. It was further dehydrated in ethanol and 2 changes of propylene oxide, embedded in Dow epoxy resin (Lockwood, 1964), sectioned, placed on 200-mesh grids, stained with lead citrate, and examined with a Zeiss EM 9S electron microscope.