Clarence A. Speer

Factors Influencing Excystation in Cryptosporidium Oocysts from Cattle

Fisheries and Oceans, Biological Station, St. Andrews, New Brunswick, Canada EOG 2X0. We thank P. C. F. Hurley for organizing the collection of swordfish, Joyce Taylor for acquireries and Oceans, Bi logical Station, St. Ans, New Brunswick, C n da EOG 2X0. ing literature on pennellids, W. B. Scott for reviewing the manuscript, and Brenda Fullerton for typing the manuscript. pennel ids, . B. Scot for re-

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

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 Karnovskys 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.

Electron and light microscope studies of the oocyst walls, sporocysts, and excysting sporozoites of Eimeria callospermophili and E. larimerensis.

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 Karnovskys 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

Penetration of Eimeria larimerensis Sporozoites into Cultured Cells as Observed with the Light and Electron Microscopes

Cell cultures of Madin-Darby bovine kidney (MDBK) were used to study with the light and electron microscopes the process of penetration of cells by Eimeria larimerensis sporozoites. The fine structure of these sporozoites was also studied. Monolayers from Leighton tubes were covered with a concentrated suspension of sporozoites and immediately observed in double-coverslip preparations with phase-contrast microscopy. For electron microscope study, sporozoites were added to and mixed well with a suspension of MDBK cells, centrifuged for 4 min, fixed immediately, and prepared for study with the electron microscope. Frequently, the Golgi complex of the sporozoites was located in an indentation of the nucleus and partially surrounded by a fold of the nuclear envelope. The inner layer of the pellicle consisted of 2 unit membranes. Wavelike elevations of the pellicle were seen; these may be involved in locomotion. An intranuclear inclusion, consisting of microtubulelike fibrils, was observed. Vacuoles with particulate matter similar to that of the central vacuoles of the Golgi complex and with extensions running anteriorly into the conoid area were seen in extracellular sporozoites and in sporozoites entering cells. The bodies of sporozoites were usually constricted as they entered and left host cells and host cell nuclei; this was also observed in sporozoites moving through the cytoplasm of the host cell. During penetration, the host cell membrane was either interrupted at the initial site of entry or was interrupted after becoming invaginated for a short distance. Escape of host cell cytoplasm occurred frequently after sporozoites left host cells, but only seldom after entrance. Some intracellular sporozoites fixed 4 min or less after inoculation were surrounded by a host cell membrane; others were not. Some sporozoites which were fixed in the process of leaving host cells had a thin layer of host cell cytoplasm covering the portion of the body which was outside of the host cell, and some extracellular sporozoites with such a covering were seen. The process of host cell penetration by sporozoites of several Eimeria species has been studied in vitro with the light microscope. E. larimerensis is especially favorable for such a study because of the relatively large size of its sporozoites and because they enter cultured cells readily (Speer and Hammond, 1970). A detailed study with the light and electron microscopes of sporozoite penetration into cultured cells, as well as the fine structure of extraand intracellular sporozoites of E. larimerensis, is reported herein. MATERIALS AND METHODS Oocysts of Eimeria larimerensis were collected from experimentally infected ground squirrels (Spermophilus armatus), cleaned, sporulated, and sterilized as described previously (Speer, Hammond, and Anderson, 1970). They were then Received for publication 15 September 1970. * Supported in part by research grant AI-07488 from the NIAID, U. S. Public Health Service, and by Public Health Service Fellowship 1-F01GM44456-01 from the Institute of General Medical Sciences. Published as Journal Paper No. 1080, Utah Agricultural Experiment Station. incubated for 15 min in an excysting medium consisting of 0.25%o trypsin and 0.75% sodium taurocholate in saline A. The free sporozoites were washed and resuspended in minimum essential medium (MEM). For studying penetration, 2day-old cultures of Madin-Darby bovine kidney (MDBK) cells in Leighton tubes or 8-oz Brockway culture flasks were used. In the light microscope study, cover slips with monolayers were removed from Leighton tubes, covered with a few drops of suspended sporozoites (0.8 to 1 million sporozoites/ml), and immediately observed in doublecoverslip preparations (Parker, 1962). In the electron microscope study, the cell monolayer in an 8-oz Brockway culture flask was harvested with a trypsin-versene solution. The suspension was centrifuged and the pellet was resuspended in 3 ml of MEM. Five million sporozoites in 3 ml of MEM we added to the tube containing suspended MDBK cells. These were mixed well and immedi tely centrifuged at 750 g for 4 min. The fixative was added immediately after removing the supernatant. Some pellets were fixed according to the method described by Karnovsky (1965), using cacodylate buffer. Other pellets were fixed with 2.67% glutaraldehyde in cacodylate buffer for 1 hr, washed with buffer for 1/2 hr, and postfixed in 2.5% osmium tetroxide in cacodylate buffer for 1 hr. The fixed cells were dehydrated in 35 and 50% ethanol for 10 min each and stained with 1% uranyl acetate and 1% phosphotungstic

CAPPING OF IMMUNE COMPLEXES BY SPOROZOITES OF EIMERIA TENELLA

Sporozoites of Eimeria tenella were incubated for 10, 20, or 30 min with parasite-specific monoclonal IgG antibody 3D3II from mice and then rinsed in a Tris-buffered glucose saline solution (TBGS). Some sporozoites were then incubated for 10, 20, or 30 min with ferritin- or colloidal gold-conjugated goat anti-mouse IgG antibody and then fixed in 2.5% glutaraldehyde and prepared for transmission (TEM) or scanning (SEM) electron microscopy. Other sporozoites that had been previously exposed to monoclonal antibody were prefixed with 0.25% glutaraldehyde, incubated with ferritin- or colloidal gold-conjugated anti-mouse IgG antibody and then fixed and prepared for TEM or SEM. Control preparations consisted of sporozoites exposed only to TBGS, monoclonal antibody 3D3II or to ferritin- or colloidal gold-conjugated anti-mouse IgG antibody. Capping of immune complexes occurred only on the surface of those sporozoites exposed to monoclonal antibody 3D3II followed by ferritin- or gold-conjugated antibody. Immune complexes moved laterally and posteriorly on the outer surface of the parasite plasma membrane to form a cap at the posterior end of the sporozoite. Capping did not occur in TBGS controls nor in sporozoites treated with monoclonal antibody 3D3II and prefixed in 0.25% glutaraldehyde before exposure to ferritin- or gold-conjugated antibody. Thus, capping of surface antigens did not occur in the presence of monoclonal 3D3II antibody only, whereas specimens exposed to both monoclonal and ferritin- or colloidal gold-conjugated antibodies were able to cap immune complexes.

Ultrastructure of the Sporocyst Wall during Excystation of Isospora endocallimici

Sporocysts of Isospora endocallimici, a parasite of marmosets, were exposed to minimal essentials medium (MEM) or a trypsin-bile salt solution (TBS) and then fixed and prepared for transmission electron microscopy. Excystation occurred in TBS but not MEM. The sporocyst wall has 2 layers, a thin outer layer (15 to 110 nm thick) and a thick inner layer (65 to 180 nm thick), which is composed of 4 separate curved plates. The outer layer consists of 1 to 3 membranes interspersed with lipid droplets. In the inner layer, a thin layer of material connects the peripheral margins of 2 apposing plates. Immediately beneath this layer, a thin strip of material is interposed between the 2 apposing plates. Ultrastructural changes preparatory to excystation occur primarily in the inner layer of the sporocyst wall. The TBS acts upon the site of apposition between 2 plates causing the interposed strip to swell and separate from the margin of each plate which leads to collapse of the sporocyst. As the sporocyst collapses, the margins of each curved plate curl inward toward the center of the sporocyst.

Effects of nylon wool purification on infectivity and antigenicity of Eimeria falciformis sporozoites and merozoites.

which was always found in large numbers in each of the study sites occupied by infected L. variegatus. Since both urchins are commonly found in close proximity to each other in beds of Thalassia, and apparently feed on the same materials, there may be physiological differences which account for the host-parasite specificity. Unforwhic was always found in arge numbers in each tunately, nothing is known about the life cycles of species of Syndesmis. Although it would appear the parasites and/or their eggs could pass from the host intestine with the feces, it is not known how they could pass from the coelomic cavity of the host to the exterior. Future comparative studies of the physical, chemical and biological features of the back-reef areas may provide information helpful to our understanding of the life cycle of this parasite. Also, it would be of interest to examine urchins from other bays which have similar characteristics to those of Discovery Bay to determine if the parasite exhibits a restricted distribution in these areas. We wish to thank Dr. Donald W. Duszynski of the Department of Biology at the University of New Mexico, Albuquerque, New Mexico, for reading the manuscript and providing helpful comme ts. We thank the following individuals who helped with the collections: C. Ennesser, C. Reich, A. Mucerino, M. Macuga, K. Lamminen, S. Cowie, B. Merrill, and W. Way. Contribution number 325 from the Discovery Bay Marine Laboratory, University of the West Indies.

Stimulation of motility in merozoites of five Eimeria species by bile salts.

First-generation schizonts of Eimeria callospermophili, E. larimerensis, and E. bilamellata from the Uinta ground squirrel, E. nieschulzi from the rat, and E. ninakohlyakimovae from the sheep were grown in cell lines of embryonic bovine intestine, kidney, and liver. Extracellular merozoites and mature first-generation schizonts with merozoites in monolayers were observed with phase-contrast microscopy for degree of motility. Various agents in minimal essential medium (MEM) were then added to the preparation; these included 0.2% trypsin, 4% bovine bile, 0.5% sodium taurocholate, 0.5% sodium glycotaurocholate, and 0.2% trypsin combined with 4% bovine bile or 0.5% sodium taurocholate; also, MEM with no agents added was used as a control. Little or no motility was observed in preparations to which MEM or trypsin was added. In all preparations to which bile, a bile salt, or one of these in combination with trypsin was added, the extracellular merozoites were markedly motile, and the merozoites still within schizonts except for those of E. ninakohlyakimovae became active and left the host cells. In such preparations, the merozoites flexed and glided more frequently and moved for longer distances than normal; in E. callospermophili, merozoites were observed penetrating new host cells and sporozoite-shaped schizonts were seen leaving host cells. Speer, Hammond, and Anderson (1970) reported that Eimeria callospermophili merozoites which developed in cell cultures underwent a marked increase in motility when treated with a trypsin-bovine bile solution. We report herein additional information concerning stimulation of motility in merozoites of E. callospermophili, E. larimerensis, and E. bilamellata from the Uinta ground squirrel, E. nieschulzi from the rat, and E. ninakohlyakimovae from sheep. All of the above species of coccidia were grown in cell cultures. MATERIALS AND METHODS Cell lines of embryonic bovine intestine, kidney, and liver were used in this investigation. The methods for maintaining and examining the cell monolayers were similar to those described by Fayer and Hammond (1967). Oocysts were collected and handled as in previous work (Speer, Hammond, and Anderson, 1970; Kelley and Hammond, 1970). Monolayers from Leighton tube cultures were examined in double-coverslip preparations (Parker, 1961) with phase-contrast microscopy at the appropriate interval after inoculation of sporozoites of each Eimeria species at which mature schizonts usually occur (24 hr after inoculation for E. callospermophili, 36 hr for E. nieschulzi, 48 hr for E. larimerensis, 4 days for E. bilamellata, and 10 days for E. ninakohlyakimovae). Extracellular merozoites or mature first-generation Received for publication 17 February 1970. Supported in part by research grant AI-07488 from the NIAID, U. S. Public Health Service. Published as Journal Paper No. 1005, Utah Agricultural Experiment Station. schizonts with merozoites were located and the degree of motility was observed before applying various solutions of trypsin and/or bile salts or bovine bile in minimal essential medium (MEM, Nutritional Biochemical) to the edge of the double cover slip. The solutions included 0.2% trypsin (1-300, Nutritional Biochemical) and 4% bovine bile, 0.2% trypsin and 0.5% sodium taurocholate (bile salt), 0.2% trypsin, 0.5% sodium taurocholate, 0.5% sodium glycotaurocholate, 4% bovine bile, and fresh MEM as a control. The pH of all solutions was adjusted to approximately 7.4 with a 7.5% sodium bicarbonate solution. During observation, the monolayers were maintained at 37 C with the aid of a warm stage and additional examinations in each experiment were conducted at room temperature (about 22 C).

Nuclear divisions and refractile-body changes in sporozoites and schizonts of Eimeria callospermophili in cultured cells.

The nuclear stage and the location and number of refractile bodies in Eimeria callospermophili sporozoites and schizonts were observed at 0.5 to 3-hr intervals from 3 to 20 hr after inoculation of sporozoites into monolayer cultures of Madin-Darby bovine kidney and embryonic bovine kidney and intestine cells. At about 6 hr, the nucleus and nucleolus had changed from a spheroidal to an ellipsoidal shape and had markedly increased in size. The first nuclear division usually occurred 8 to 10 hr after inoculation. Beginning at about 8 hr, the nucleolus became elongated and then assumed a dumbbell shape. At about 9 hr, the nucleolus divided into 2 separate nucleoli and 2 daughter nuclei were formed by infolding of the nuclear membrane between the 2 nucleoli. Between 11 and 15 hr, additional divisions occurred similarly, resulting in 4 to 6 nuclei. In schizonts with more than 6 nuclei, these were small and indistinct. Most freshly excysted sporozoites had a spherical anterior refractile body and a large posterior refractile body. At 6 to 10 hr, the anterior refractile body underwent a decrease in size until it finally disappeared. During this time, granules were formed at the periphery of the anterior refractile body, and these later assumed a random distribution. At 6 to 15 hr, the posterior refractile body decreased in size. The formation of granules at its surface was only rarely observed. After transformation of the sporozoite-shaped schizont to a spheroidal schizont, which occurred about 15 to 20 hr after inoculation, the posterior refractile body usually formed several smaller spherical bodies. In previous work we described the development in cell cultures of first-generation schizonts of Eimeria callospermophili and E. bilamellata from the Uinta ground squirrel (Speer, Hammond, and Anderson, 1970). In the course of this work we observed details of nuclear division and changes in refractile bodies in the former species, which are reported herein. MATERIALS AND METHODS Cell lines of embryonic bovine intestine and kidney as well as established cell lines of MadinDarby bovine kidney cells were used in this investigation. The cell monolayers were maintained as described previously (Fayer and Hammond, 1967). Oocysts were also handled as described before (Speer, Hammond, and Anderson, 1970). Three experiments were conducted with each cell type. Cover slips from Leighton tube cultures were examined in double-coverslip preparations (Parker, 1962) with phase-contrast microscopy at 0.5to 3-hr intervals from 3 to 20 hr after inoculation of sporozoites. The observations were made with a Zeiss photomicroscope, 100X neofluar phase-contrast objective, 8x Kpl oculars, and optovar set at 1.25. The appearance of the nucleus or nuclei and Received for publication 9 December 1969. * Supported in part by research grant AI-07488 from the NIAID U. S. Public Health Service. Published as Journal Paper No. 982, Utah Agricultural Experiment Station. the refractile bodies in 80 to 120 intracellular specimens was determined for each time interval in each experiment by examination of living specimens. Measurements were made with an ocular micrometer at a magnification of 1,600X. Each measurement in the following descriptions is in microns and, unless otherwise stated, represents the mean of 30 or more living specimens, with the range in parentheses.

Motility of Plasmodium berghei ookinetes in vitro.

Abstract Motility of Plasmodium berghei ookinetes, which developed in primary and established cell line cultures obtained from Anopheles stephensi mosquitoes, was studied by using still photomicrographs and normal speed cinephotomicrography. At 18–72 hr after inoculation of P. berghei infected blood from hamsters or mice, motile ookinetes were seen in both mosquito cell cultures; the most active specimens were observed at 24–30 hr. Ookinetes underwent a sporadic forward gliding movement, during which a variable degree of rotation of the body upon its longitudinal axis usually occurred. Some specimens rotated repeatedly upon their axes without any forward progression. The direction of the gliding movement always coincided with the curvature of the ookinete body. In those specimens in which no rotation of the body occurred, a circular course resulted. Ookinetes covered a distance of as much as 50 μm during a single gliding movement. A few ookinetes undergoing locomotion appeared to leave a path or trail on the substrate. Occasionally, an ookinete penetrated a red cell with its slender anterior projection, resulting in lysis of the cell. After red cells had been penetrated by ookinetes, the parasites already within these cells fused with each other to form larger spheroidal bodies. Penetration of cultured cells was not observed.