In vitro Development of First-Generation Schizonts of Eimeria canadensis (Bruce, 1921)*†

SYNOPSIS. In vitro development of Eimeria canadensis from cattle was studied in monolayer cultures of various bovine cell lines grown on coverslips in Leighton tubes. Excysted sporozoites were used for inoculation of the cell cultures. Sporozoites entered the host cells within a few minutes, but apart from a reduction in the number of refractile bodies, changed little in appearance during the first 9 days. Beginning at 91/2 days postinoculation, sporozoites developed into sporozoite-shaped schizonts or, less frequently, transformed into trophozoites. Sporozoite-shaped schizonts with as many as 8 nuclei were observed transforming into spheroid schizonts. At 111/2 days, intermediate schizonts had a characteristic single mass of refractile granules and 60–80 nuclei. Deep invaginations, which resulted in the formation of several blastophores, usually occurred when schizonts had about 100 nuclei. Merozoites were formed as a result of radial outgrowth from the surface of spheroid schizonts as well as of blastophores. Mature merozoites were seen 1st after 13 days.     

Development of Eimeria ninakohlyakimovae from Sheep In Cell Cultures*

SYNOPSIS. Monolayer cell line cultures of ovine trachea, thyroid, thymus, and kidney cells, as well as an established cell line (Madin-Darby) of bovine kidney cells, were inoculated with sporozoites of Eimeria ninakohlyakimovae and observed for a maximum of 24 days. Sporozoites were seen penetrating cells within 5 minutes after inoculation, as well as 2 and 3 days after inoculation, and leaving cells 3 days after inoculation. Transformation from sporozoites to trophozoites occurred by a widening or by a lateral outpocketing of the sporozoite body. Trophozoites and schizonts were first seen 3 days after inoculation in all ovine cell types. Large numbers of immature schizonts were observed, but only an estimated 0.4–4.3% of these became mature in the different kinds of cells. Usually, mature schizonts were first seen 10–11 days after inoculation in the ovine cells, but they sometimes occurred as early as 8 days. More mature schizonts were seen in the ovine kidney and trachea cells than in the others; the smallest number occurred in the bovine cells. The nucleoli of cells harboring large schizonts in each type of culture were enlarged and the chromatin clumps normally seen in the nuclei of non-infected cells were not visible. The cytoplasm of some infected cells was vacuolated. The formation of merozoites occurred by a budding process from blastophores, from the surface of schizonts, and/or from infoldings and invaginations of this surface. Merozoites were observed leaving host cells, but were not seen penetrating new cells. Intracellular first-generation merozoites were observed 13 and 15 days after inoculation in lamb trachea and kidney cells, respectively. No evidence of further development of such merozoites was found.     

Development of Eimeria alabamensis from Cattle in Mammalian Cell Cultures*

SYNOPSIS. Monolayer primary cultures of cells from bovine embryonic intestine (BEInt), kidney (BEK), spleen (BES), and thyroid (BETy) and cell line cultures of embryonic bovine trachea (EBTr) and synovium (BESy) as well as established cell line cultures of bovine kidney (Madin-Darby, MDBK), human intestine (Int 407) and Syrian hamster kidney (BHK) were inoculated with freshly excysted sporozoites of Eimeria alabamensis and observed for 4–5 days. Sporozoites penetrated all cell types; during the 1st 24 hr, intracellular sporozoites, trophozoites and binucleate schizonts were seen in all cell cultures. Mature schizonts were more numerous in BES and MDBK cells than in the others. Large schizonts, 14.2 (11–18.5) by 10.2 μ (8.5–11), with 6–14 short, stubby merozoites (each with 2 refractile bodies) occurred at 2 and 3 days in all cells except BESy, Int 407, and BHK. Small schizonts, 9.7 (5.5–13) by 6 μ (5–8.5), with 6–10 long, slender merozoites (each with 2 refractile bodies) were found 3 days after inoculation in all cell types. At 4 days, some intracytoplasmic merozoites and a few intranuclear 2nd generation trophozoites were found. After 4 days post-inoculation, intracellular parasites were rarely seen and these were apparently degenerate. Development within the host cell nucleus, the normal site of development in the host animal, was observed infrequently in cell cultures. Intranuclear sporozoites, found no earlier than 2 days after inoculation, developed similarly to those in the cytoplasm, and small intranuclear schizonts with 6–10 merozoites (each with 2 refractile bodies) occurred after 3 days in culture.     

Development of Eimeria callospermophili and E. bilamellata from the Uinta Ground Squirrel Spermophilus armatus in Cultured Cells*

SYNOPSIS. Cell lines or established cell lines of bovine, ovine or human origin and primary cells from whole embryos of groundsquirrels were used in a study of the in vitro development of Eimeria callospermophili and E. bilamellata from the Uinta ground squirrel, Spermophilus armatus. Monolayers in Leighton tube cultures were inoculated with sporozoites of either of these 2 species and examined with phase-contrast microscopy at various intervals. After such examination, coverslips were fixed in Schaudinn's or Zenker's fluid and variously stained. E. callospermophi sporozoites penetrated cells and underwent development to mature 1st generation schizonts in most cell types. At different times after inoculation, both species formed sporozoite-shaped schizonts, which later became spheroidal. Intracellular movements of sporo zoite-shaped schizonts of E. callospermophili were observed and such schizonts penetrated cells when freed by mechanical disintegration of the host cells. Merozoites were formed at the periphery of the schizont in both species. Mature 1st generation schizonts of E. callospermophili, with 6–14 merozoites, were first seen 15 hr after inoculation; the corresponding values for E. bilamellata were 12–27 merozoites and 4 days. Merozoites of both had anterior and posterior refractile bodies. Exposure to a trypsin-bile solution stimulated motility in merozoites of E. callospermophili. Second generation trophozoites and immature schizonts of E. callospermophili were seen in cultures of primary cells of whole ground-squirrel embryos 20–24 hr and 44–48 hr, respectively, after inoculation of sporozoites.     

Development of Eimeria auburnensis in Cell Cultures

SYNOPSIS. Monolayer established cell line cultures of bovine kidney (Madin-Darby) and human intestine (Intestine 407), as well as embryonic bovine tracheal and embryonic spleen cell line cultures were inoculated with E. auburnensis sporozoites and observed for a maximum of 22 days. Mature 1st generation schizonts developed in the kidney, tracheal and spleen cells. In the intestine cells, trophozoites were seen in 3 of 4 experiments, but schizonts were not found. Sporozoites penetrated cells, beginning within a few minutes after inoculation. Penetration was usually accomplished within 10 seconds, and the body of the sporozoite underwent a slight constriction as it passed thru the host cell membrane. Some sporozoites left cells. Numerous intracellular sporozoites were observed in kidney, tracheal and spleen cultures. Crescent bodies were seen in the parasitophorous vacuole as early as 1 day after inoculation. At this time, the nuclei of most intracellular sporozoites had changed from vesicular to compact. Beginning 4 days after inoculation, enlarged sporozoites and parasites having a sporozoite shape, but with 2-5 nuclei, were frequently seen. These enlarged sporozoites and sporozoite-shaped schizonts evidently transformed into trophozoites and spheroidal schizonts by means of lateral outpocketings. Few trophozoites were seen. More immature schizonts developed in kidney cells than in the other cell types. The numbers of mature schizonts observed in kidney and tracheal cells were similar, but development occurred less consistently in the latter. Few immature and mature schizonts developed in spleen cells. Mature schizonts, first seen 9 days after inoculation, were considerably smaller than those reported from calves. Some motile merozoites were seen; evidently no development beyond these occurred. The nucleus and nucleolus of host cells were enlarged; this enlargement was not as pronounced as in infections in calves. Multiple host cell nuclei were frequently observed. Degenerative changes in the cultured cells and in the parasites usually occurred, beginning 9-17 days after inoculation; these were more pronounced in the spleen cells than in the others.     

Development of First-Generation Schizonts of Eimeria bovis in Cultured Bovine Cells

SYNOPSIS. Monolayer primary and secondary cultures of embryonic bovine kidney, spleen, intestinal and testicle cells, and secondary cultures of embryonic bovine thymus, maintained in lactalbumin hydrolysate, Earle's balanced salt solution and ovine serum were observed for a maximum of 21 days after inoculation of E. bovis sporozoites. The sporozoites entered the cells in all of these cultures but underwent development only in primary cultures of kidney and intestinal cells and in secondary cultures of kidney, spleen, thymus, intestinal, and testicle cells. In acellular media, the sporozoites retained motility no longer than 21 hr. In the cell cultures, free motile sporozoites were seen for as long as 18 days after inoculation. Sporozoites entered cells anterior end first; the process of penetration required a few seconds to about a minute. Sporozoites were also observed leaving host cells. Intracellular sporozoites were first seen 3 min after inoculation; they were observed at various intervals up to 18 days after inoculation. In transformation of sporozoites into trophozoites a marked change in size and appearance of the nucleus took place before the change in shape of the body occurred. Trophozoites were first found 7 days after inoculation, multinucleate schizonts after 8 days, and schizonts with merozoites after 14 days. Schizonts containing merozoites were seen only in kidney, spleen, and thymus cells. The mature schizonts were smaller and represented a much lower proportion of the total number than in comparable stages of infections in calves. Schizonts with many nuclei occurred in intestinal cells; the most advanced stage seen in testicle cells was the binucleate schizont. Nuclear and cytoplasmic changes were observed in the infected cells.     

Ultrastructure of cytoplasmic and nuclear changes in Eimeria tenella during first-generation schizogony in cell culture.

Eimeria tenella sporozoites were inoculated into primary cultures of chick kidney cells. Cells fixed from 1 1/2 to 54 hr later were examined with the electron microscope. At 1 1/2 and 24 hr, most intracellular sporozoites were fusiform and retained organelles typical of extracellular sporozoites. However, at 35 hr, rounded trophozoites were present without these structures; only a refractile body, nucleus, mitochondria, and endoplasmic reticulum remained. Binucleate parasites were also present at that time, but at 48 hr many multinucleate schizonts were present. Nuclei, with adjacent conoids, were at the periphery of these schizonts. Partly developed merozoites, each containing a conoid and a nucleus, protruded into the parasitophorous vacuole. At 54 hr, fully developed merozoites were separated from the residual body. Merozoites resembled sporozoites but lacked the large refractile bodies seen in sporozoites. Linear inclusions were present near the merozoite nucleus and in the residual body. Round vacuoles and ribosomes were also found in the residuum. Nucleoli were first seen in sporozoite nuclei at 1 1/2 hr. They were also present in merozoites but were more prominent in trophozoites and schizonts. Peripheral and scattered nuclear heterochromatins were prominent in intracellular sporozoites and diminished in trophozoites, but increased after several nuclear divisions and were again prominent in the merozoite. Small, distinct interchromatin granules were found in all stages. Intranuclear spindles, centrocones, and centrioles were found in connection with nuclear divisions. Ultrastructure of first-generation schizogony in cell culture was similar to that described for second-generation E. tenella in the chicken and to schizogony of other species of Eimeria.     

Development of Eimeria meleagrimitis Tyzzer from Sporozoites and Merozoites in Turkey Kidney Cell Cultures*

SYNOPSIS. Sporozoites and 1st-, 2nd-, and 3rd-generation merozoites of Eimeria meleagrimitis were inoculated into primary cultures of turkey kidney cells. In vitro-excysted sporozoites developed into mature macrogamonts in 8 days; in vivo-excysted sporozoites developed into 2nd- or 3rd-generation schizonts within 5 to 7 days. First-generation merozoites obtained from infected turkeys produced mature 2nd-generation schizonts within 24 h. Second-generation merozoites from turkeys produced mature macrogamonts and oocysts within 72 h, whereas 3rd-generation merozoites produced these stages within 48 h. The oocysts that developed from 3rd-generation merozoites sporulated at 25 C and were infective for turkeys. The timing of the early stages and the intervals between schizogonic generations in cultures were comparable with those in turkeys. Morphologic parameters, however, indicated that some differences existed between in vitro and in vivo development. Second- and 3rd-generation schizonts and gamonts that developed after inoculation of cultures with merozoites were similar to stages in turkeys. Oocysts, however, were significantly smaller (P < 0.05) in cultures. All stages that developed after inoculation of cultures with sporozoites were smaller (P < 0.05) than their in vivo counter parts.     

Transmission Electron Microscopy of Intracellular Sporozoites of Eimeria vermiformis (Apicomplexa,Eucoccidiida) in the Mouse1

ABSTRACT. Sporozoites of Eimeria vermiformis from the mouse were first seen in the epithelial cells of villus tips and the crypts of Lieberkühn four hours after inoculation (HAI). They were always within a parasitophorous vacuole. By 12 HAI, most were in crypt epithelial cells between the basement membrane and host cell nucleus. The sporozoites in the villus tips had 26 subpellicular microtubules, two polar rings, two preconoidal rings, two refractile bodies surrounded by amylopectin-like granules, a lamellar Golgi apparatus, numerous micronemes, and rhoptries. The sporozoites in the crypt cells had fewer amylopectin-like granules, micronemes, and rhoptries. A nucleolus was visible, as were pieces broken off from the posterior refractile body. Later, the sporozoites folded over to become U-shaped; the infolded membranes fused; and then the inner membranes disappeared so that spherical meronts were formed. Folding sporozoites were first seen 16 HAI and persisted until 52 HAI.     

The Development of First Generation Schizonts of Eimeria bovis*

SYNOPSIS. In young first generation schizonts of E. bovis, the nuclei appeared to have a random distribution. In calves killed 8 days after inoculation some of the schizonts had the nuclei arranged in a single layer at the periphery, with a few infoldings of this layer into the interior. In further development, such ingrowths of the nuclear layer resulted in the formation of compartments of varying size. In schizonts of calves killed 12 days after inoculation spherical or ellipsoidal bodies (blastophores), about 5–20 μ in diameter with a single peripheral layer of nuclei were formed. Merozoites developed as radial outgrowths from the blastophores, leaving residual bodies of variable size, which later disappeared. The response of the host cell to the presence of the schizont was characterized by marked growth of both the nucleus and cytoplasm. The nucleolus became greatly enlarged, and the chromatin was distributed in relatively fine granules. In the host cell cytoplasm, 2 concentric layers were observed; the inner was more dense than the outer. After growth of the schizont was completed its host cell was stretched into a thin covering layer about 1 μ thick. In some schizonts, the host cell disintegrated, and the schizont was then invaded by eosinophils, macrophages and other cells, which eventually destroyed the merozoites.     

Transmission electron microscopy of intracellular sporozoites of Eimeria vermiformis (Apicomplexa, Eucoccidiida) in the mouse

Sporozoites of Eimeria vermiformis from the mouse were first seen in the epithelial cells of villus tips and the crypts of Lieberkühn four hours after inoculation (HAI). They were always within a parasitophorous vacuole. By 12 HAI, most were in crypt epithelial cells between the basement membrane and host cell nucleus. The sporozoites in the villus tips had 26 subpellicular microtubules, two polar rings, two preconoidal rings, two refractile bodies surrounded by amylopectin-like granules, a lamellar Golgi apparatus, numerous micronemes, and rhoptries. The sporozoites in the crypt cells had fewer amylopectin-like granules, micronemes, and rhoptries. A nucleolus was visible, as were pieces broken off from the posterior refractile body. Later, the sporozoites folded over to become U-shaped; the infolded membranes fused; and then the inner membranes disappeared so that spherical meronts were formed. Folding sporozoites were first seen 16 HAI and persisted until 52 HAI.     

Development of Hepatozoon griseisciuri in Cultured Squirrel Cells*

Sporocysts of Hepatozoon griseisciuri obtained from laboratory-reared spiny rat mites (Echinolaelaps echidninus) and laboratory-reared squirrel mites (Haemogamasus reidi) were made bacteria-free and incubated in trypsin-bile for 30 min at 37 C to release sporozoites. Hepatozoon griseisciuri sporozoites were inoculated into monolayer cultures of primary adult squirrel kidney (PSK) cells and cell line cultures of neonatal squirrel kidney (SK), heart (SH), and spleen (SS) cells. Extracellular sporozoites underwent flexing, gliding, and pivoting movements similar to other coccidian sporozoites. Sporozoites entered cells in all the cultures used and were found intracellularly as early as 1 hr and as late as 10 days after inoculation. In SK, SH, and SS cells, development proceeded only to the trophozoite stage. In PSK cells, immature schizonts and mature schizonts containing 12–40 merozoites were present from 5 through 10 days after inoculation. The finding of pairs of intracellular organisms within a single parasitophorous vacuole in PSK cells suggested that endodyogeny or limited schizogony had occurred.     

Life Cycles of Leucocytozoon dubreuili Mathis and Leger, 1911 and L. fringillinarum Woodcock, 1910 (Haemosporidia: Leucocytozoidae)*

SYNOPSIS The development of Leucocytozoon dubreuili and L. fringillinarum was studied on successive days in simuliid and avian hosts. Sporogony of both parasites is completed in at least 5 species of sylvatic Simuliidae in a minimum of 4-5 days at 21 C. The pattern of development of the 2 species is similar but the size of the oocysts and the number of sporozoites differ. Sporozoites of L. dubreuili and L. fringillinarum were injected into uninfected robins (Turdus m. migratorius) and grackles (Quiscalus quiscula versicolor), respectively. Hepatic biopsies were performed on some of the injected birds. These and others were killed at intervals following inoculation and their tissues examined to detect stages of schizogony. Blood and macerated tissues from birds injected with sporozoites were transferred to uninfected birds to determine whether asexual stages would develop in the latter as a result of the inoculations. The 1st asexual cycle of L. dubreuili is completed in hepatic parenchymal cells in a minimum of 84 hr. Merozoites produced by the hepatic schizonts apparently follow one of 3 courses: invade hepatic parenchymal cells to initiate another cycle; penetrate blood cells and become gametocytes; penetrate tubular cells of the kidneys and grow into renal schizonts. The minimum prepatent period in infections with L. fringillinarum is 76 hr. The 1st asexual cycle occurs in hepatic parenchymal cells and in tubular cells of the kidney. A schizogonic cycle is completed in a minimum of 72 hr in the former and 96 hr in the kidney. Merozoites from the primary hepatic schizonts apparently give rise to (a) gametocytes; (b) secondary hepatic schizonts; (c) renal schizonts. Thus the schizogonic cycles of L. dubreuili and L. fringillinarum differ from each other and from those of L. simondi in ducks.     

The Life Cycle of Isospora felis Wenyon, 1923, a Coccidium of the Cat*

SYNOPSIS. A pure strain of Isospora felis derived from a single oocyst was used to study the endogenous cycle. One and a half to two-month-old laboratory-reared, coccidia-free kittens were used thruout the study. The endogenous stages occurred in the epithelial cells of the distal parts of the villi in the ileum and occasionally duodenum and jejunum. All stages lay above the host cell nucleus. There were 3 asexual generations. The 1st generation schizonts were 11–30 by 10–23 μ when mature and contained 16–17 banana-shaped merozoites 11–15 by 3–5 μ. They became mature in 96 or sometimes in 120 hours. The 1st generation merozoites entered new host cells, rounded up and formed 2nd generation schizonts. These formed within themselves 2–10 or more spindle-shaped bodies resembling 1st generation merozoites in shape and size. These were 2nd generation merozoites. They were uninucleate 120 hours after inoculation, but by 144 hours they became larger, multinucleate and some lost their elongate shape and became ovoid. They were then 3rd generation schizonts. They were 12–16 by 4–5 μ. Each formed up to 6 or more banana-shaped merozoites 6–8 by 1–2 μ. The 3rd generation schizonts and merozoites developed within the same host cell and parasitophorous vacuole as the 2nd generation schizonts and merozoites. Mature schizonts containing only 3rd generation merozoites appeared 144 hours after inoculation, were most abundant 168 hours after inoculation, and might be present as late as 216 hours after inoculation. They were 14–36 by 13–22 μ and contained 36 to more than 70 merozoites. The 3rd generation merozoites entered the sexual cycle. The mature microgametocytes were 24–72 by 18–32 μ and contained a central residuum and a large number of microgametes 5–7 by 0.8 μ with 2 posteriorly-directed flagella. The mature macrogametes were 16–22 by 8–13 μ. Gametogony occurred 144–216 hours after inoculation. The prepatent period was 168–192 hours and the patent period 10–11 days. Peak oocyst production occurred on the 6th day of the patent period.     

Eimeria tenella: use of a monoclonal antibody in determining the intracellular fate of the refractile body organelles and the effect on in vitro development

A monoclonal antibody, which recognizes the refractile body of Eimeria sporozoites, was used to study the developmental fate of this organelle during asexual development of E. tenella and to determine the effect of this monoclonal antibody on in vitro development of the parasite. Through use of immunofluorescent antibody and gold-labeling techniques at the light and electron microscopy level, the refractile body at 48 to 96 hr postinoculation was found to separate into 6 to 10 small globules, then diffuse throughout the schizont cytoplasm, and eventually reconcentrate as a small dot of material in each of the mature first-generation merozoites. The schizont did not develop to maturity if diffusion of the refractile body did not occur. The refractile body material was quickly lost as the merozoite left the schizont and invaded new cells and was not detected in any later developmental stages. The in vitro development of first- and second-generation schizonts of E. tenella was greatly inhibited (up to 100%) with exposure to the monoclonal antibody. There was an increase in the number of schizonts with nondispersed refractile body in the monoclonal antibody-treated cells when compared to the untreated controls, and the few mature schizonts seen had up to a 50-fold decrease in the number of merozoites. Immunofluorescent antibody labeling of the refractile body of intracellular sporozoites and schizonts treated in vitro with the monoclonal antibody for 24-96 hr postinoculation indicated that the antibody had crossed the host cell and parasite plasma membrane during incubation.     

Schizogony in Haemoproteus columbae Kruse*

SYNOPSIS To fill in some of the gaps in our knowledge of Schizogony of Haemoproteus columbae Kruse, transmission experiments involving inoculation into pigeons (Columba livia Gmelin) of sporozoites from salivary glands of the hippoboscid fly Pseudolynchia canariensis (Macquart) were carried out. We were unable to detect prepatent schizonts or to observe schizogonic development when infection became chronic. Schizonts were mainly confined to lung tissue. Observations of parapatent schizonts were made in smears and tissue sections. A variety of forms was found. Cytomeres were rarely encountered. Two types of morphologically distinct merozoites were seen. One type was twice as large as the other and was thought to repeat the process of schizogony several times before invading erythrocytes. Schizonts with cytoplasmic clefts were not common in our material due to the fixatives used (Bouin's and Carnoy's). Merozoites were occasionally observed inside monocytes, probably being phagocytosed.     

Life Cycle and Host Specificity of Eimeria callospermophili Henry, 1932 From the Uinta Ground Squirrel Spermophilus armatus*

SYNOPSIS. Eimeria callospermophili was found in 6 species of ground squirrels and the white-tailed prairie dog. The hosts included Spermophilus armatus from Utah and Montana, S. richardsoni from Montana and Wyoming, S. beecheyi from California, S. lateralis and S. variegatus from Utah, and S. tridecemlineatus and Cynomys leucurus from Wyoming. Infections were generally transmissible from each species of ground squirrel to S. armatus and S. richardsoni. Oocysts from C. leucurus caused infections in S. armatus and S. richardsoni. No infections were found after inoculation of E. callospermophili oocysts into least chipmunks (Eutamius minimus), Mongolian gerbils (Meriones unguiculatus), or laboratory rats; however, excystation occurred in these animals. Resistance to infection did not develop in S. armatus, S. richardsoni, or S. variegatus, but did occur after 5 or more infections in S. lateralis. Eimeria callospermophili had little or no effect on the host in S. armatus, S. lateralis, or S. variegatus, but caused bloody diarrhea in severely infected individuals of S. richardsoni. The oocysts had an oocyst residuum consisting of several distinct bodies, which later coalesced to form a large homogeneous body. Each sporozoite had an unusually large refractile body. In experimentally infected specimens of S. armatus the prepatent period and patent period lasted for 5 and 9 days, respectively. Mature 1st-generation schizonts, first seen 2 days after inoculation, had 8–12 merozoites. Mature 2nd-generation schizonts, first seen 3 days after inoculation, had an average of 18 merozoites which were smaller than those of the 1st generation. Mature gametes were 1st seen 4 days after inoculation. Mature microgametocytes were only slightly larger than mature macrogametes.     

Pre-Erythrocytic Development and Associated Host Responses to Haemoproteus meleagridis (Haemosporina: Haemoproteidae) in Experimentally Infected Domestic Turkeys12

ABSTRACT. Two generations of pre-erythrocytic schizogony occurred in skeletal and cardiac muscle of domestic turkeys infected with sporozoites of Haemoproteus meleagridis. First generation schizonts reached maturity approximately five days post-inoculation (DPI) and developed in capillary endothelial cells and myofibroblasts. The schizonts ranged from 12 to 20 μm in diameter and produced long (5–6 μm), slender merozoites. Early second generation schizonts were first detected in capillary endothelial cells between 5 and 8 DPI. They were cylindrical and ranged in size from 5 to 8 μm in diameter and up to 28 μm in length. Second generation schizonts which reached maturity by 17 DPI were surrounded by a thick, hyaline wall and were packed with numerous spherical merozoites less than 1 μm in diameter. Mature megaloschizonts were fusiform, ranged from 30 to 113 μm in diameter, and extended as much as 465 μm along the long axis of muscle fibers. Merozoites developed as buds from cytomeres that formed between 8 and 14 DPI. Infected turkeys developed a moderate to severe myositis within 5 DPI and were lame in one or both legs. The myositis was associated with the necrosis of scattered groups of muscle fibers. Muscle fibers surrounding mature megaloschizonts were swollen and hyaline. Megaloschizonts were surrounded occasionally by fibroblasts and infiltrates of mononuclear cells. The morphology and site of development of mature megaloschizonts of Haemoproteus meleagridis are contrasted with those of other avian haemosporidians.     

Ultrastructural development of first- to second-generation merozoites in Eimeria contorta Haberkorn, 1971.

The development of first-generation merozoites to second-generation schizonts and merozoites of Eimeria contorta in one of its natural hosts, the mouse, was investigated with the electron microscope. Merozoites inside a host cell show a marked U-shape and a degeneration of the inner-pellicular membrane complex prior to transformation into schizonts. These processes closely resemble those seen in transforming sporozoites. In young schizonts with about 3-5 nuclei, the Golgi-adjuncts (structures of unknown function) form a large interconnected network. Nuclear divisions in growing schizonts involve the formation of a centroc?ne, which develops in a pocket-like indentation of the nuclear envelope. At least one centriole is present immediately adjacent to this indentation. In a later stage, the centroc?ne forms a conical nuclear protrusion directed towards a merozoite-anlage. This developing merozoite contains anlagen of a conoid, of rhoptries, and of micronemes and a refractile body in addition to the nucleus, centrioles, and a Golgi-adjunct. The merozoite-anlage is limited by a triple unit membrane complex. Schizonts give rise to 8-15 second-generation merozoites. Interesting features of these merozoites are the high number of micronemes, the finding of one single large mitochondrion per merozoite, and the occurrence of 26 subpellicular microtubules, i.e. the same number as in sporozoites of E. contorta. At the end of their development, merozoites come into direct contact with the host cell cytoplasm as the parasitophorous vacuole breaks down.     

Development of Eimeria meleagrimitis Tyzzer from sporozoites and merozoites in turkey kidney cell cultures

Sporozoites and 1st-, 2nd-, and 3rd-generation merozoites of Eimeria meleagrimitis were inoculated into primary cultures of turkey kidney cells. In vitro-excysted sporozoites developed into mature macrogamonts in 8 days; in vivo-excysted sporozoites developed into 2nd- or 3rd-generation schizonts within 5 to 7 days. First-generation merozoites obtained from infected turkeys produced mature 2nd-generation schizonts within 24 h. Second-generation merozoites from turkeys produced mature macrogamonts and oocysts within 72 h, whereas 3rd-generation merozoites produced these stages within 48 h. The oocysts that developed from 3rd-generation merozoites sporulated at 25 C and were infective for turkeys. The timing of the early stages and the intervals between schizogonic generations in cultures were comparable with those in turkeys. Morphologic parameters, however, indicated that some differences existed between in vitro and in vivo development. Second- and 3rd-generation schizonts and gamonts that developed after inoculation of cultures with merozoites were similar to stages in turkeys. Oocysts, however, were significantly smaller (P less than 0.05) in cultures. All stages that developed after inoculation of cultures with sporozoites were smaller (P less than 0.05) than their in vivo counter parts.