Electron and Light Microscope Studies of the Oocyst Walls, Sporocysts, and Excysting Sporozoites of Eimeria callospermophili and E. larimerensis

Abstract

Oocysts of E. callospermophili and E. larimerensis were ground to release the sporocysts, which were studied by bright-field and phase-contrast microscopy. Sporocysts and broken oocyst walls were fixed with Karnovsky's fixative for study with the electron microscope. The oocyst walls of each species had 2 layers. The outer layer of E. callospermophili consisted of hexagonal columnar projections arranged in a honeycomb pattern, whereas in E. larimerensis, the outer layer was electron-dense and homogeneous, with knoblike projections and heterogeneous material at its surface. Each sporocyst of each species had an outer membrane covering the wall, as well as a Stieda body and substiedal body at the anterior end. Inside were 2 sporozoites, fluid which coagulated during fixation, and a membranebound residual body with vacuoles, lipoid granules, and amylopectin. The sporocysts of E. callospermophili had an additional wall layer that resembled the coagulated internal fluid. The additional wall layer and internal fluid were no longer present in sporocysts with excysting sporozoites. The Stieda body of each species was bipartite; one portion filled a gap in the sporocyst wall and the other formed a cap over the anterior end. The homogeneous substiedal body was immediately posterior to the Stieda body. In sporocysts of each species exposed to a mixture of 0.75% sodium taurocholate (bile salt) and 0.25% trypsin, the Stieda body became less dense and the substiedal body began to evaginate. The Stieda body then disappeared and the substiedal body popped out and disintegrated, after which the sporozoites excysted. In sporocysts exposed to 0.25% trypsin alone, excystation occurred less rapidly and at a lower percentage than when trypsin and the bile salt were used; the substiedal body disintegrated in situ. In sporocysts of each species exposed to saline A for 1 hr, 4 to 5% had motile sporozoites, and 34 to 58% of sporocysts exposed for 1 hr to 0.75% sodium taurocholate solution had motile sporozoites; no excystation was seen in any of these sporocysts. Evidently, the bile salt stimulates motility of the sporozoites. Little is known as to the morphological changes associated with excystation in Eimeria species. Roberts and Hammond (1970) described the fine structure of a sporozoite of E. bovis from cattle in the process of escaping from the sporocyst. Hammond, Ernst, and Chobotar (1970) reported light microscope observations on the excystation of sporozoites from sporocysts in E. utahensis from the kangaroo rat, Dipodomys ordii. We describe herein our findings with the electron and light microscopes as to the oocyst walls, sporocysts, and excysting sporozoites of E. callospermophili and E. larimerensis. Both of these species have substiedal bodies and are from the Uinta ground squirrel, Spermophilus armatus. Received for publication 5 March 1970. Supported in part by NSF research grant GB8252, research grant AI-07488 from the NIAID, U. S. Public Health Service. Published as Journal paper No. 1014, Utah Agricultural Experiment Station. MATERIALS AND METHODS Oocysts of E. callospermophili and E. larimerensis were obtained from experimentally infected ground squirrels. They were cleaned of fecal debris and sporulated by methods described by Nyberg and Hammond (1964). Some of the oocysts later used for study of oocyst walls were exposed to 5.25% sodium hypochlorite solution (Clorox) for 10 or 30 min. Free sporocysts were obtained by mechanically breaking oocysts in a round-bottomed pyrex grinding vessel with a motor-driven tefloncoated pestle. Sporocysts for light-microscope study were placed in saline A and concentrated by centrifugation. One drop of the resulting suspension of sporocysts was placed on each of 4 slides, and to this drop was added a drop of 0.25% trypsin (1-300, Nutritional Biochemical) in saline A, 0.75% sodium taurocholate (bile salt) in saline A, 0.25% trypsin and 0.75% sodium taurocholate in saline A, and saline A, respectively. The slides were then examined at room temperature (about 22 C) with phase and bright-field microscopy for observation of the excystation process. Sporocysts for electron-microscope studies were placed in a saline A solution containing 0.75% sodium taurocholate and 0.25% trypsin for 5 min