Datus M. Hammond

Fine structure of macrogametes and oocysts of Coccidia and related organisms.

SummaryThe fine structure of the macrogametes of coccidia was investigated, described and diagrammatically depicted. A comparative analysis was made of representative species belonging to various genera. The following species were investigated: Eucoccidium dinophili, Aggregata eberthi, Eimeria perforans, E. stiedae, E. falciformis, E. bovis, E. auburnensis, E. tenella, E. maxima, Toxoplasma gondii, and Klossia helicina. The macrogametes of most of these Eimeria species are relatively uniform in their fine structure, but E. falciformis shows some differences and the macrogametes of T. gondii are also different in some respects. In the macrogametes of E. dinophili, A. eberthi, and K. helicina are found a number of special structures not seen in the others.ZusammenfassungDie Feinstruktur der Makrogameten von Coccidien und verwandten Gruppen wurde untersucht, beschrieben und schematisch dargestellt. Charakteristische Organelle folgender Arten: Eucoccidium dinophili, Aggregata eberthi, Eimeria perforans, Eimeria stiedae, E. falciformis, E. bovis, E. auburnensis, E. tenella, E. maxima, Toxoplasma gondii und Klossia helicina dienten als Ausgangsbasis zu einer vergleichenden Strukturanalyse. Die Makrogameten der Eimeria-Arten erwiesen sich in ihrem Feinbau als relativ einheitlich. Einige bedeutsame Unterschiede ergaben sich allerdings bei den weiblichen Gameten von E. falciformis und T. gondii im Vergleich zu den Verhältnissen bei den untersuchten Eimeria-Arten. Die Makrogameten von E. dinophili, A. eberthi und K. helicina enthielten spezielle Einschlüsse, die bei den Eimeria-Arten nicht in Erscheinung traten. Trotz aller Unterschiede liegt jedoch den hier untersuchten Makrogameten ein gemeinsamer Bauplan zu Grunde.

Electron microscope studies of microgametogenesis in coccidia and related groups

SummaryThe fine structure of the microgamonts and microgametes of coccidia and related groups was investigated and is herein described and diagrammatically depicted. Particular attention is given to the developmental processes during the differentiation of the microgametes. The species included in the study are: Eimeria perforans, E. maxima, E. tenella, E. auburnensis, E. falciformis, and Toxoplasma gondii. A comparative analysis of the developmental processes as well as the fine structure of the microgametes of these species and those of other Sporozoa is presented.

Fine structure of first-generation merozoites of Eimeria bovis.

The fusiform merozoite is enclosed by a cell membrane. Another membrane underlying the cell membrane encloses the cytoplasm except at the anterior end of the merozoite where this inner membrane terminates forming the polar ring. Approximately 22 subpellicular fibrils extend posteriorly from the polar ring. A conoid, consisting of one or more fibrils wound in a tight helix, is situated within the polar ring. A paired organelle extends posteriorly through the conoid from the anterior end. Each member of the paired organelle is club-shaped having a narrow neck within the conoid region and a wider posterior portion. A median rod parallels the necks of the paired organelle. The region of the merozoite between the conoid and the ovoid glycogen bodies is tightly packed with many tortuous structures having indistinct borders. Numerous ribosomes as well as one or two mitochondria are scattered among these structures. A dense, membrane-enclosed body, possibly a lysosome, is occasionally seen near the mitochondria. The Golgi apparatus lies next to the flattened anterior edge of the nucleus. In some specimens a punctate invagination of the cell surface was seen near the level of the Golgi apparatus. Several cisternae of rough-surfaced endoplasmic reticulum are found both anterior and posterior to the nucleus. The merozoites lie free in a vacuole of the host cell. Blebbing of the host cells vacuolar membrane releases vesicles into the vacuole. The outer surface of the host cell has numerous microvilli. A fine, fibrous layer exists in the host cell cytoplasm surrounding the vacuole. The cytological characteristics of the firstgeneration merozoites of Eimeria bovis, as revealed by light microscopy, were reported by Hammond, Ernst, and Goldman (1965). The large number of merozoites (about 120,000) contained within the macroscopic host cell and the ease of collection of these host cells provided a simple means for studying the fine structure of this stage of the coccidial life cycle. Electron microscope studies of the merozoites of E. intestinalis (Mossevitch and Cheissin, 1961), E. perforans, and E. stiedae (Scholtyseck and Piekarski, 1965) have been reported. These species differ from E. bovis, however, in the size of the schizont and number of merozoites produced. This study was initiated to provide a detailed description of the ultrastructure of mature first-generation merozoites of E. bovis and the host cell in which they are located. The morphological characters of this stage of E. bovis will be compared with those of the corresponding stages of other species of Eimeria and of other Sporozoa. Received for publication 16 February 1966. * U. S. Department of Health, Education, and Welfare, Public Health Service, NIAID, Laboratory of Parasitic Diseases, Bethesda, Maryland 20014. t Department of Zoology, Agricultural Experiment Station, Utah State University, Logan, Utah. MATERIALS AND METHODS First-generation merozoites of E. bovis were obtained from infected calves as described by Hammond et al. (1965). Immediately after collection, by scraping the intestinal mucosa, the material was placed in a large volume of fixative without washing. Fixation was accomplished by one of three methods. Three per cent glutaraldehyde in Sorensens phosphate buffer (Sabatine, Bensch, and Barrnett, 1963) was used for some specimens. The material was fixed for 2 hr and then rinsed in buffer with sucrose. This was followed by postfixation in veronal-acetate-buffered Os04 for 2 hr. Some specimens were fixed for 2 hr in veronalacetate-buffered OsO (Caulfield, 1957); others were fixed for a similar period in Daltons chromeosmium fixative (Dalton, 1955). All fixatives were buffered at pH 7.4 and used at 0 to 4 C. Since it was necessary to mail specimens after fixation from one laboratory to another, they were left for several days in 70% alcohol, or, in the case of glutaraldehyde-fixed material, in the rinsing buffer. All specimens were dehydrated in a graded series of ethyl alcohol. They were then embedded in Epon (Sporn, Wanko, and Dingman, 1962) after being passed through propylene oxide to remove the alcohol. Polymerization was carried out for 16 to 18 hr at 60 C. Sectioning was done with an LKB Ultrotome using a diamond knife. Sections were mounted on bare, 400-mesh grids and stained with lead (Karnovsky, 1961). An RCA EMU-3G electron icroscope operating at 50 kv was used to view and photograph the specimens.

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

The fine structure of the developmental stages of the microsporidian Nosema apis Zander

Schizonts, sporonts and sporoblasts of Nosema apis from honey bees collected in the summer and winter were studied with the electron microscope. The nuclei usually had a diplokaryon arrangement. Intranuclear spindles with polar vesicles were associated with division. Schizonts had a single limiting unit membrane, whereas sporonts had a two-layered wall. Sporonts from summer bees had only a thin single limiting membrane in some areas and evidence of endocytosis was sometimes seen in these. Sporonts from winter bees had branched tubular outpocketings from the wall. In sporoblasts, the development of the polar filament was closely associated with a network of dense structures interwoven with a system of tubules evidently of ER derivation; the Golgi complex was associated with this network.

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

Electron microscope observations on the development of first-generation merozoites of Eimeria bovis.

At the earliest stage observed in the development of the first-generation merozoites of Eimeria bovis the schizont cytoplasm was subdivided into many lobes or spheroidal blastophores; their peripheries being lined by the many nuclei resulting from repeated divisions. The beginning of merozoite production is characterized by the formation of a complex of structures which later comprises the anterior end of the merozoite. A thickened layer forms under the plasma membrane and eventually becomes the inner membrane of the merozoite. Adjacent to a central opening in this layer lies a conoid. Subpellicular fibrils, which radiate from the opening, are closely applied to the inner membrane. As development proceeds, the blastophore membrane is elevated into a cone-shaped projection which later elongates into a fingerlike bud. This bud, the developing merozoite, contains the primordia of the paired organelle, a nucleus with adjacent Golgi apparatus, and other cytoplasmic constituents derived from the blastophore. With further growth of the merozoite, the outer and inner membranes become extended posteriorly; after the early stages, this is associated with an infolding of the membranes into the blastophore. In a late stage of development, the merozoites are completely formed except for an attachment of their posterior ends to the remains of the blastophore. Finally, the attachment is broken, resulting in free merozoites and residual bodies. Eimeria bovis is one of a group of Eimeria species which has unusually large schizonts. Each first-generation schizont of this species produces approximately 120,000 merozoites (Hammond et al., 1946). The literature pertaining to the development of such schizonts was reviewed by Hammond, Ernst, and Miner (1966), in connection with a study of the development of first-generation schizonts of E. bovis with the light microscope. These authors found that in early schizonts the nuclei became arranged in a peripheral layer. This layer then grew inward at a number of places, forming compartments of various sizes. These compartments later gave rise to spherical or ellipsoidal bodies called blastophores, having a single peripheral layer of nuclei. Radial outgrowths of the blastophores formed merozoites, with some cytoplasm remaining as residual bodies. No studies with the electron microscope of schizogony in Eimeria species with large schizonts have been reported. Since the fine structure of the first-generation merozoites of E. bovis is known (Sheffield and Hammond, 1966) the present study was initiated to provide more detailed information concerning the Received for publication 1 March 1967. * Laboratory of Parasitic Diseases, NIAID, NIH, Public Health Service, U. S. Department of Health, Education and Welfare, Bethesda, Maryland 20014. t Department of Zoology, Agricultural Experiment Station, Utah State University, Logan, Utah. cytological events occurring during the development of these merozoites. MATERIALS AND METHODS First-generation schizonts of E. bovis were obtained from calves 12 to 15.5 days after inoculation as described by Hammond, Ernst, and Goldman (1965), except that mucosal scrapings containing schizonts were fixed immediately without prior washing, in order to obtain optimal fixation. The material was fixed in either 3% glutaraldehyde in Sorensens phosphate buffer (Sabatine, Bensch, and Barrnett, 1963) followed by postfixation in veronal acetate-buffered OsO4, or in Daltons chrome-osmium fixative (Dalton, 1955). All fixatives were buffered at pH 7.4 and used at 0 to 4 C. Specimens were fixed in Logan then sent to Bethesda in rinsing buffer in the case of glutaraldehyde-fixed materials, or in 70% ethyl alcohol when OsO4 fixative was used. Before the material was shipped the schizonts were concentrated by rotation in petri dishes as previously described (Hammond et al., 1965). With the aid of a dissecting microscope a rough separation of the immature schizonts from the completely mature ones was made by observing degree of opacity and size (immature schizonts were less opaque). All material was dehydrated in a graded series of ethyl alcohol, passed through propylene oxide, and then embedded in Epon according to the procedure of Sporn, Wanko, and Dingman (1962). Polymerization was carried out for 16 to 18 hr at 60 C. Sections were cut with a diamond knife on an LKB Ultrotome. They were mounted on bare, 400mesh grids and stained with lead (Karnovsky, 1961). Photographs were taken with an RCA EMU-3G electron microscope operating at 50 kv.

Fine structural study of the microgametogenesis of Eimeria auburnensis.

SummaryThe microgametogenesis of Eimeria auburnensis from experimentally infected calves killed 16 to 18 days after inoculation was investigated with the electron microscope. Early microgametocytes had nuclei with compact nucleoli and relatively few electron-dense masses. In this stage, deep invaginations from the surface evidently served for the intake of nutrients from the parasitophorous vacuole. In older stages, the nuclei had no compact nucleoli and had more elctrondense material. An intranuclear fiber apparatus was present in some nuclei, apparently in an early stage of division. Nuclear division appeared in electron micrographs to occur by a kind of fission, in which the intranuclear fiber apparatus may participate. Numerous fissures appeared in the interior of the microgametocyte and the nuclei were arranged in irregular rows in association with these. Centrioles, either single or double, were observed between the nuclei and the membrane lining the fissures. Such nuclei often had an intranuclear fiber apparatus; one of the osmiophilic poles of this apparatus protruded outward from the nucleus in the vicinity of the centriole. Directly over this pole, electron-dense material, probably representing the anlage of the perforatorium, occurred immediately beneath the surface membrane of the fissure. As many as 9 closely arranged micropores were observed in this membrane in some specimens. In the nearly mature microgamete, the basal bodies of the flagella lay at the anterior end; the mitochondrion, with numerous, regularly arranged tubules began slightly posterior to this. The strongly osmiophilic, condensed nucleus was a little farther posterior; this remained connected by a narrow stalk with the uncondensed portion of the nucleus in the residual cytoplasm until the microgametes were almost completely mature. Usually, 2 flagella were present. Rarely, 3 flagella were observed, and evidence of a rudimentary middle flagellum was found in a number of specimens.ZusammenfassungDie Mikrogamogonie des Rindercoccids Eimeria auburnensis wurde mit Hilfe des EM untersucht. Junge Mikrogamonten haben Nuklei mit kompaktem Nukleolus und relativ wenig elektronendichten, unregelmäßig im Kernplasma verteilten Partikeln. Schon in jungen Mikrogamonten zeigen sich von der Oberfläche her tiefe, kanalartige Einstülpungen, die tief bis in das Zentrum des Gamonten führen und mit amorphem Material angefüllt sind, wie es in der parasitophoren Vakuole enthalten ist. In älteren Stadien ist der kompakte Nukleolus nicht mehr vorhanden. Dafür sind große Anteile des Kernplasmas stark elektronendicht. Innerhalb der Kerne werden intranukleäre Fibrillen beobachtet. Die Kernteilung erscheint im elektronenoptischen Bild als Zweiteilung, an der sich vermutlich die intranukleären Fibrillen beteiligen. Im Cytoplasma der Gamonten entstehen zahlreiche Spalträume, um die sich die Kerne anordnen. Centriolen in Ein- oder Zweizahl sind zwischen den einzelnen Kernen und den Membranen der Spalträume sichtbar. In diesem Stadium zeigen sich oft ebenfalls intranukleäre Fibrillen, die aus einem osmiophilen Pol in der Nähe der Centriolen entspringen. Oberhalb dieses spindelartigen Pols entwickelt sich eine osmiophile Platte unter der Membran des Spaltraumes. In dieser Membran können sich bis zu 9 Mikroporen dicht nebeneinander befinden. In der Spitze des langgestreckten Mikrogameten liegen die Basalkörper der Geißeln, weiter unterhalb ein besonders strukturiertes Mitochondrium und schließlich der langgestreckte, stark osmiophile Kern. Meistens werden an den Mikrogameten 2 Geißeln beobachtet, in wenigen Fällen sind aber 3 vorhanden und manchmal ergeben sich nur Hinweise auf eine dritte rudimentäre Geißel.

Cultivation of Eimeria bovis in Three Established Cell Lines and in Bovine Tracheal Cell Line Cultures

Monolayer established cell line cultures of bovine kidney (Madin-Darby, 1958), human intestine (Intestine 407), and mouse fibroblasts (L cells), as well as bovine tracheal cell line cultures, were inoculated with E. bovis sporozoites and observed for a maximum of 21 days. Mature first-generation schizonts developed in each of the cell types, except for the L cells. In these, large numbers of sporozoites entered cells but only a few transformed into trophozoites; the most advanced stage seen was a trinucleate schizont. In the tracheal cells, relatively numerous schizonts developed and mature schizonts were first observed 8 days after inoculation. Thus, the rate of development of the schizont in the tracheal cells was unusually rapid, exceeding even that in calves. Some mature schizonts were relatively large; the maximum, 292 by 118 ,u, was observed in an 18-day culture. Intracellular merozoites and early schizonts of the second generation were found in 18and 19-day cultures of tracheal cells. Many schizonts occurred also in established kidney cell line cultures, but the rate of development was slower than that in tracheal cells, and the first mature schizonts were observed in a 14-day culture. The largest mature schizont seen (55 by 38 u) was in a 21-day culture. In the intestinal cells, relatively small numbers of schizonts developed; the rate of development was approximately the same as that in kidney cells. The largest mature schizont observed, 109 by 55 ,u, was seen in a 13day culture. Of the four cell types studied, the bovine tracheal cells appear to provide the best conditions for development of E. bovis. In earlier work (Fayer and Hammond, 1967), we found that sporozoites of E. bovis developed into mature first-generation schizonts in cell line cultures of bovine kidney, spleen, and thymus cells. Development only to binucleate and multinucleate schizonts occurred in cultures of bovine testicle and intestinal cells, respectively. Others (Patton, 1965; Strout et al., 1965; Doran and Vetterling, 1967a, b) have found that Eimeria species from chickens and turkeys undergo development in a variety of cells. These include established cell lines, as well as primary and cell line cultures, from species other than the normal host. In further work on the in vitro cultivation of E. bovis, we have attempted to obtain additional information about the variety of host cells in which sporozoites of this species will grow. In the present paper, we report the results of such work with bovine, human, and murine established cell lines, and with bovine tracheal cell line cultures. MATERIALS AND METHODS Three of the four cell types used in the experiments were obtained from the American Type CulReceived for publication 12 January 1968. Supported in part by research grant A1-07488 from the NIAID, U. S. Public Health Service, and by a traineeship from the National Science Foundation. Published as Journal Paper no. 744, Utah Agricultural Experiment Station. ture Collection Cell Repository in Rockville, Maryland. The fourth type, L cell (mouse fibroblast) cultures, was obtained from Dr. Rex Spendlove, Department of Bacteriology, Utah State University. Between serial passages, cultures were maintained in Brockway prescription bottles in a 37 C incubator. Madin-Darby bovine kidney (MDBK) cells and embryonic bovine trachea (EBTr) cells were cultured in Eagles minimal essential medium (MEM) with Earles salts, 1% sodium pyruvate, 1% nonessential amino acids, and 10% fetal bovine serum. Embryonic human intestinal (Int 407) cells (Henle and Deinhardt, 1957) were cultured in Eagles basal medium (BME) with Hanks salts and 15% fetal bovine serum. L cells (Sanford, Earle, and Likely, 1948) were cultured in MEM and 10% fetal bovine serum. The cells to be used in each experiment were transferred, at the rate of 150,000 cells/ml, to Leighton tubes containing 9by 35-mm or 10.5by 50-mm cover slips. These tubes were incubated at 37 C until the cells attached to the cover slip, at which time sporozoites were added. The terminology used for cell cultures is that recently proposed by the Tissue Culture Association (Fedoroff, 1967). By the use of methods previously described (Fayer and Hammond, 1967), oocysts were collected, cleaned, sterilized, and excysted. The total number of sporozoites in suspension was determined with the aid of a hemocytometer. Concentrations of 150,000 to 350,000 sporozoites per 1.7 ml aliquot were obtained by diluting the suspension with serum-free tissue culture medium appropriate for the cell type being investigated. A sterile Cornwall syringe was used to pipette 1.7 ml of suspended sporozoites into each of six to 20 Leighton tubes containing cell cultures for each experiment. In each experiment up to 20