Complete Development of Caryospora bigenetica (Apicomplexa: Eimeriidae) In Vitro1

The development of Caryospora bigenetica in vitro is described by light microscopy. Sporozoites from snake-derived oocysts were purified and inoculated onto cultures of primary testicle cells of the cotton rat, cotton rat kidney cells, and human fetal lung cells. Intracellular sporozoites were observed one and two days postinoculation (DPI). Motile, extracellular first-generation merozoites were present 3 DPI, and second-generation merozoites were present 5 DPI. Mature gamonts were observed 9 DPI and developed into unsporulated oocysts by 10 DPI. Oocysts sporulated in vitro, and excystation was observed. Cells that were penetrated by in vitro-produced sporozoites formed caryocysts by 16 DPI. To test infectivity of in vitro-derived stages, merozoites were removed from cultured cells 5 DPI and inoculated intraperitoneally into a mouse; infection resulted. Sporulated oocysts removed from cell cultures 12 DPI produced facial swelling in an orally inoculated cotton rat.     

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.     

Direct Infection of Hepatocytes by Sporozoites of Plasmodium berghei1

ABSTRACT. To identify the unknown liver cell type initially invaded by sporozoiles of mammalian malaria, young rats were inoculated intravenously with large numbers of Plasmodium berghei sporozoites obtained from infected Anopheles stephensi mosquitoes. Fine structural studies of liver specimens obtained from the rats within 2 min after inoculation demonstrated the presence of morphologically unaltered sporozoites in the cytoplasm of hepatocytes. Many sporozoites were also observed undergoing cytolysis within the lysophagosomes of Kupffer cells, as well as other phagocytic cells. These observations strongly suggest direct infection of the hepatocyte by the sporozoite.     

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.     

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.     

Complete development of Caryospora bigenetica (Apicomplexa: Eimeriidae) in vitro

The development of Caryospora bigenetica in vitro is described by light microscopy. Sporozoites from snake-derived oocysts were purified and inoculated onto cultures of primary testicle cells of the cotton rat, cotton rat kidney cells, and human fetal lung cells. Intracellular sporozoites were observed one and two days postinoculation (DPI). Motile, extracellular first-generation merozoites were present 3 DPI, and second-generation merozoites were present 5 DPI. Mature gamonts were observed 9 DPI and developed into unsporulated oocysts by 10 DPI. Oocystes sporulated in vitro, and excystation was observed. Cells that were penetrated by in vitro-produced sporozoites formed caryocysts by 16 DPI. To test infectivity of in vitro-derived stages, merozoites were removed from cultured cells 5 DPI and inoculated intraperitoneally into a mouse; infection resulted. Sporulated oocysts removed from cell cultures 12 DPI produced facial swelling in an orally inoculated cotton rat.     

Direct infection of hepatocytes by sporozoites of Plasmodium berghei

To identify the unknown liver cell type initially invaded by sporozoites of mammalian malaria, young rats were inoculated intravenously with large numbers of Plasmodium berghei sporozoites obtained from infected Anopheles stephensi mosquitoes. Fine structural studies of liver specimens obtained from the rats within 2 min after inoculation demonstrated the presence of morphologically unaltered sporozoites in the cytoplasm of hepatocytes. Many sporozoites were also observed undergoing cytolysis within the lysophagosomes of Kupffer cells, as well as other phagocytic cells. These observations strongly suggest direct infection of the hepatocyte by the sporozoite.     

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 Caryospora simplex (Apicomplexa: Eimeriidae) from Sporozoites to Oocysts in Human Embryonic Lung Cell Culture1

Leighton tubes containing monolayers of human embryonic lung cells were inoculated with 70,000 or 30,000 sporozoites of the viperid coccidium Caryospora simplex and examined at 1, 2, 4, 6, 8, 10, 12, 14, 16, and 18 days post-inoculation (PI). By day 1 PI, sporozoites had penetrated cells and were within parasitophorous vacuoles. Most sporozoites became spherical and then underwent karyokinesis several times between days 2 and 6 PI. Mature Type I meronts were found on days 6–16 PI and contained 8 to 22 short, stout merozoites. Mature Type II meronts were present on days 10–18 PI and contained 8 to 22 long, slender merozoites. Developing gamonts (undifferentiated sexual stages) were observed on days 14 and 16 PI. Mature micro- and macrogametes and thin-walled unsporulated oocysts were present on days 16 and 18 PI. Attempts to sporulate oocysts in tissue culture medium or in a 2.5% (w/v) aqueous solution of K₂Cr₂O₇ at 25/°C and 37°C were unsuccessful; only a few oocysts developed to the contracted sporont stage. Four Swiss-Webster mice injected intraperitoneally with merozoites obtained from Leighton tubes on day 10 PI did not acquire infections. This is the second coccidium reported to complete its entire development, from sporozoite to oocyst, in cell culture.     

Ultrastructural differentiation of Toxoplasma gondii schizonts (types B to E) and gamonts in the intestines of cats fed bradyzoites

The ultrastructural characterisitics of four types of Toxoplasma gondii schizonts (types B, C, D and E) and their merozoites, microgamonts and macrogamonts were compared in cats killed at days 1, 2, 4 and 6 after feeding tissues cysts from the brains of mice. Schizonts, merozoites and gamonts contained most of the ultrastructural features characteristic of the phylum Apicomplexa. All four types of schizonts developed within enterocytes or intraepithelial lymphocytes. Occasionally, type B and C schizonts developed within enterocytes that were displaced beneath the epithelium into the lamina propria. Type D and E schizonts and gamonts developed exclusively in the epithelium. Tachyzoites occurred exclusively within the lamina propria. Type B schizonts formed merozoites by endodyogeny, whereas types C to E developed by endopolygeny. The parasitophorous vacuoles surrounding type B and C schizonts consisted of a single membrane, whereas those surrounding types D and E schizonts were comprised of two to four electron-dense membranes. The parasitophorous vacuole of type B schizonts had an extensive tubulovesicular membrane network (TMN); the TMN was reduced or absent in type C schizonts and completely absent in types D and E schizonts and gamonts. Type B merozoites were ultrastructurally similar to tachyzoites, except that they were slightly larger. Type C merozoites exhibited a positive periodic acid-Schiff reaction by light microscopy and ultrastructurally contained amylopectin granules. Rhoptries were labyrinthine in type B merozoites but were electron-dense in types C-E. The development of microgamonts, macrogamont and oocysts is also described.     

Gametogenesis and Sporogony of Hepatozoon Mocassini (Apicomplexa: Adeleina: Hepatozoidae) In an Experimental Mosquito Host, Aedes Aegypti

ABSTRACT. The sexual life cycle of the hemogregarine Hepatozoon mocassini was studied in Aedes aegypti , an experimental mosquito host, using transmission electron microscopy. Gamonts were observed leaving the host snake erythrocyte as early as 30 min after mosquitoes ingested infected blood, and some gamonts had penetrated the gut epithelial cells by this time. Six hours post-feeding, gamonts were identified within cells of the abdominal fat body. Twenty-four hours post-feeding, gamonts were often entrapped within the peritrophic membrane, but were no longer observed within the gut wall. Parasites pairing up in syzygy and undergoing sexual differentiatioe were observed within fat cells at this time, and by 48 hours post-feeding, well-developed macro- and microgametocytes as well as microgametes were discernible. Developing zygotes observed 3 days post-feeding were enclosed within a panoitophorous vacuole. By day 6, multinucleate oocysts with crystalloid bodies in the cytoplasm were seen. Sporazoites developing within sporocysts appeared by day 12. Seventeen days post-feeding, mature oocysts with sporocysts containing approximately 16 sporozoites were observed upon dissection of mosquitoes. Large crystalloid bodies no longer bound by rough endopbsmic reticulum were located anterior and posterior to the sporozoite nucleus. Free sporozoites were not observed.     

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.     

Intestinal coccidiosis in a spinner dolphin (Stenella longirostris)

Intestinal coccidiosis was diagnosed histologically in the small intestine of a spinner dolphin (Stenella longirostris). Numerous intralesional coccidia were present in mucosal epithelial cells. Schizonts, gamonts, and unsporulated oocysts were seen. Schizonts were up to 30 x 20 microm and contained up to 16 merozoites, which measured 10-12 x 2 microm. Unsporulated oocysts were about 9-12 x 8-10 microm. This is the first report of intestinal coccidiosis in a spinner dolphin.     

Development of Caryospora simplex (Apicomplexa: Eimeriidae) from sporozoites to oocysts in human embryonic lung cell culture

Leighton tubes containing monolayers of human embryonic lung cells were inoculated with 70,000 or 30,000 sporozoites of the viperid coccidium Caryospora simplex and examined at 1, 2, 4, 6, 8, 10, 12, 14, 16, and 18 days post-inoculation (PI). By day 1 PI, sporozoites had penetrated cells and were within parasitophorous vacuoles. Most sporozoites became spherical and then underwent karyokinesis several times between days 2 and 6 PI. Mature Type I meronts were found on days 6-16 PI and contained 8 to 22 short, stout merozoites. Mature Type II meronts were present on days 10-18 PI and contained 8 to 22 long, slender merozoites. Developing gamonts (undifferentiated sexual stages) were observed on days 14 and 16 PI. Mature micro- and macrogametes and thin-walled unsporulated oocysts were present on days 16 and 18 PI. Attempts to sporulate oocysts in tissue culture medium or in a 2.5% (w/v) aqueous solution of K2Cr2O7 at 25 degrees C and 37 degrees C were unsuccessful; only a few oocysts developed to the contracted sporont stage. Four Swiss-Webster mice injected intraperitoneally with merozoites obtained from Leighton tubes on day 10 PI did not acquire infections. This is the second coccidium reported to complete its entire development, from sporozoite to oocyst, in cell culture.     

Tyzzeria chalcides N. Sp. from the Ocellated Skink,Chalcides ocellatus1

ABSTRACT A new species of coccidium, Tyzzeria chalcides, is described from the ocellated skink, Chalcides ocellatus, from Egypt. Meronts occur in the epithelial cells of the bile ducts and gamonts within the epithelial cells of the gall bladder. Fully sporulated oocysts are cylindrical (L/W ratio 1.88) without a micropyle, oocyst residuum, or polar granule and contain eight spindle-shaped sporozoites and no sporocysts. Sporulation is completed within the gall bladder lumen. Comparison with other species of the genus found in reptiles indicates that it is a new species.     

Developmental stages of Hepatozoon hemprichii sp. nov. infecting the skink Scincus hemprichii and the tick Hyalomma impeltatum from Saudi Arabia

The life cycle of Hepatozoon hemprichi n. sp. is described; the vertebrate host is Scincus hemprichii and it is vectored by Hyalomma impeltatum. Erythrocytic stages of 18 ± 1.8 × 4 ± 0.8 μm developed in the hemocoel of ticks to sporozoites within 16-18 days. Schizogony occurred in the liver parenchyma and the endothelial cells of blood capillaries in lung and spleen. Mature schizonts measuring 27 ± 3.11 × 20.13 ± 3.0 μm produced 28 merozoites (on average). The merozoites were 13 ± 1.21 × 1.21 ± 0.72 μm with nuclei 5 ± 0.65 × 2.1 ± 0.51 μm. Syzygy and differentiation of gamonts took place in tick's hemocoel up to the third day post-infection (PI). The microgamont (16 ± 0.31 × 18 ± 0.42 μm) produced 4, uniflagellated microgametes at 4-5 days PI. The microgamete measured 15.2 ± 0.31 μm while the flagellum was always at least 26 μm. The macrogamete was very large in size (31 ± 3.11 μm) with a central nucleus. After fertilization, (5-6 days PI) zygotes developed into oocysts (55 ± 3.41 × 52 ± 4.11 μm) in which repeated mitotic divisions with centripetal invaginations occurred; each contained 18 banana-shaped sporozoites, 13.61 ± 0.8 × 1.2 ± 0.31 μm in size. Experimental transmission was successfully carried out by oral administration or by intra-peritoneal inoculation of the infective stages (sporozoites) to uninfected skinks and led to the appearance of blood stages after 5 wk and 4 wk, respectively.     

Ultrastructural study of developmental stages of Mattesia dispora (Neogregarinorida: Lipotrophidae), a parasite of the flour moth Ephestia kuehniella (Lepidoptera)

The ultrastructure of merozoites, gamonts and oocysts of the neogregarine Mattesia dispora and their development in larvae of the flour moth Ephestia kuehniella were studied by electron microscopy. The apical complex of free macronuclear merozoites was very distinct in micrographs of sections, the polar rings being especially prominent. Two gamonts associated in head-to-head syzygy and the apical complexes served as the contact point during pairing. At this stage the rhoptries became reduced and the conoid widened. The gamonts had a foam-like appearance in the light microscope. Paired gamonts formed an envelope and developed into a gametocyst, within which the gamonts were separated by a distinct border. Four gametes and two residual cells developed inside the gametocyst. The gametes were covered with a single membrane. The gametes fused in pairs to form two spherical zygotes, each covered by two membranes and with one large nucleus. The external layer appeared more undulated than the inner one. A single membrane covered each residual cell. Walls were formed around both zygotes to produce two oocysts. Each mature oocyst was lemon-shaped with polar plugs and eight peripheral sporozoites, which had a pellicle similar to that of the merozoites, lay beneath the thick oocyst wall.     

Complete Development of Isospora suis of Swine in Chicken Embryos1

Development of the swine coccidium, Isospora suis, in embryonated chicken eggs is described. The allantoic cavities of eight-to-ten-day-old white Leghorn embryos were inoculated with either 100,000 or 200,000 sporozoites. Developmental stages morphologically similar to those found in the intestines of piglets were present in the endodermal layer of the chorioallantoic membrane (CAM), beginning three days post inoculation (PI). No stages were found in the mesodermal or ectodermal layers of the CAM and none were observed in heart, lung, liver, or spleen. Type I meronts and merozoites were found on days 3 through 10 PI. Type II meronts and merozoites were found days 4 through 10 PI. Mature microgamonts, macrogamonts, and oocysts were found on days 7 through 10 PI. Oocysts appeared to be retained in the endodermal cells and in ovo sporulation did not occur. Attempts to sporulate CAM-derived oocysts were not successful. Isospora suis was not pathogenic for embryos under the conditions of this study. This study represents the first fully documented report of complete development of a mammalian coccidium in chicken embryos.     

Cultivation of Eimeria tenella in Japanese quail embryos (Coturnix coturnix japonica).

Complete development of Eimeria tenella in Japanese quail embryos was observed. Sporozoites were inoculated into the allantoic cavity of 7-day-old Japanese quail embryos (Coturnix coturnix japonica), after which the infected embryos were incubated at 41 C. In the chorioallantoic membrane mature first generation schizonts, mature second generation schizonts, and gametes were detected at 48 hr postinoculation of sporozoites (PI), 84 hr PI, and 126 hr PI, respectively. Mature gametes and zygotes were found at 132 hr PI, and oocysts were detected at 138 hr PI. Mortality of embryos increased with increment of inoculum size of sporozoites. LD50 was 1.7 x 10(2) sporozoites. Oocyst production was also dependent on inoculum size. Oocysts harvested from embryos sporulated. The oocysts were inoculated into 13-day-old chickens, and oocysts, capable of sporulating normally, were recovered from ceca 7 days after inoculation.     

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.