Development of Eimeria crandallis Honess from Sheep in Cultured Cells*

SYNOPSIS. Cell lines of embryonic lamb trachea (LETr), lamb thyroid (LETh), and bovine liver (BEL) as well as an established cell line of Madin-Darby bovine kidney (MDBK) were used in a study of the in vitro development of Eimeria crandallis from sheep. Excysted sporozoites were inoculated into Leighton tubes containing coverslips with monolayers of the different cell types. Coverslips were examined with phase-contrast and interference-contrast at various intervals up to 20 days after inoculation; thereafter the monolayers were fixed and stained in various ways. Freshly excysted sporozoites, with 2–10 spheroidal refractile bodies, entered all of the cell types in relatively small numbers; intracellular sporozoites were first seen 2 min after inoculation. After 24 hr, most intracellular sporozoites had only 1 or 2 refractile bodies. Before and during transformation of sporozoites, the nucleus and peripheral nucleolus increased markedly in size. Transformation resulted in usually spheroid but sometimes ellipsoid trophozoites. Trophozoites were seen first 3–4 days, and binucleate schizonts at 4–5 days after inoculation. Immature schizonts increased considerably in size and eventually had large numbers of nuclei. Some of the parasites became lobulated and the lobules often separated to form individual schizonts. In BEL, LETr and LETh cells, mature schizonts, up to 150 μm in diameter, were seen first 11–14 days after inoculation. The BEL cells were the most favorable for development. Merozoites were formed by a budding process from the surface of the schizonts as well as from blastophores. Some merozoites were seen leaving mature schizonts, but no further development was observed. Merozoites frequently were motile and had a sharply bent posterior end. Marked nuclear and cytoplasmic changes were observed in parasitized cells.     

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 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 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.     

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 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 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.     

Invasion of different cell types by sporozoites of Eimeria species and effects of monoclonal antibody 1209-C2 on invasion of cells by sporozoites of several apicomplexan parasites

Sporozoites of avian Eimeria species differed markedly in their ability to invade cells in vitro. Invasion by E. tenella and E. adenoeides was significantly greater in baby hamster kidney (BHK) and chicken cecal cell (CC) cultures than in primary chicken (PCK) or turkey kidney (PTK) cell cultures. Moreover, invasion of BHK cell cultures by E. adenoeides was significantly greater than that of other Eimeria species, and invasion by E. acervulina sporozoites was significantly lower. Monoclonal antibody 1209-C2 (MAb 1209-C2) reacted by immunofluorescent labeling (IFA) with refractile bodies of sporozoites of 5 species of Eimeria and Caryospora bigenetica, but not with sporozoites of Toxoplasma gondii, Hammondia hammondi, or Cryptosporidium parvum, which have no refractile bodies. The MAb also cross-reacted with formalin-fixed BHK, CC, turkey cecal (TC) cells, and PTK. Pretreatment of BHK cells with MAb 1209-C2 significantly reduced invasion of the cells by sporozoites of E. tenella, E. acervulina, E. meleagrimitis, and C. bigenetica, but did not alter invasion by T. gondii, C. parvum, or H. hammondia. Apparently, reactivity of MAB 1209-C2 with the sporozoites was required for inhibition of invasion despite the fact that the inhibition resulted from pre-treatment of the host cell. Conversely, although MAb 1209-C2 also reacted moderately with PTK and TC cells, pre-treatment of these cell cultures with the MAb did not inhibit invasion by either MAB 1209-C2-reactive or -nonreactive parasites. Collectively, the data indicated that refractile body antigens of sporozoites of Eimeria and Caryospora, which are recognized by MAb 1209-C2, may function in cellular invasion, but also suggest that cellular invasion is probably not mediated by interactions between the conserved epitopes in sporozoites and cultured host cells that are recognized by the MAb.     

Characterization of the antigen SO7 during development of Eimeria tenella

The developmental expression of the antigen SO7, which has been previously shown to protect chickens against infection by several Eimeria species, was investigated. Using RT-PCR, mRNA for SO7 was found to be restricted primarily to unsporulated oocysts (0 hr). Western blot (WB) analysis with an antibody to recombinant SO7 (rbSO7) revealed expression of the protein from 6 to 72 hr (fully sporulated) of sporulation and in sporozoites (SZ). SO7 was absent in host-derived second-stage merozoites (MZ) and was present in culture-derived first-stage MZ but at a level of only 25% of that exhibited by SZ. During invasion of Madin-Darby bovine kidney (MDBK) cells by SZ in vitro, the level of SO7 within cells, as determined by WB analysis, remained relatively constant until 48 hr of development and then decreased by about 40% at the next time point (72 hr). The SO7 secreted into the culture media during in vitro development increased to a relative maximum at 48 hr and then decreased to about 20% of maximum at 72 hr. Immunostaining with anti-rbSO7 indicates that SO7 is highly concentrated in both refractile bodies (RB) of SZ, with some limited distribution in the apical complex. Anti-rbSO7 intensively stained the intracellular parasites and the first-stage schizonts during in vitro development of E. tenella in MDBK cells. Upon release from the schizonts, the first-stage merozoites stained with 1 or 2 bright spots typically at each end. The results suggest that SO7 is closely associated with the SZ RB and is developmentally regulated but may not play a direct role in cellular invasion.     

Survival and Development of Eimeria meleagrimitis Tyzzer, 1929 in Bovine Kidney and Turkey Intestine Cell Cultures

SYNOPSIS. Monolayer cell cultures of embryonic turkey intestine (primary) and bovine kidney (cell line, 20th passage), maintained at 40.6 and 43 C for alternating intervals of approximately 12 hours in Basal Medium Eagle and fetal calf serum at pH 7.0–7.4, were observed for 144 hours after inoculation of Eimeria meleagrimitis sporozoites.
In turkey intestine cultures, which consisted of fibroblast-like cells and patches of epitheliul-like cells, there were decreases of 80 and 81% in the numbers of parasites between 5 and 48 hrs; in bovine cultures, 21–41% decreases. Decreases in the turkey cultures, however, were due to the nonsurvival of sporozoites in fibroblast-like cells; in epitheliul-like cells there was a 42% dcrease between 5 and 48 hrs and only 27% between 48 and 144 hours.
Trophozoites were present in bovine cells at 5 hrs. Small, mature schizonts containing only 12-28 merozoites were present in the bovine cultures and in the epitheliul-like cells within turkey intestine cultures from 48-144 hrs. Larger schizonts (50-115 by 20-70 μ) were present in bovine but not in turkey cultures from 72–144 hrs. Many of these schizonts contained far more merozoites than schizonts of any of the 3 generations described from the host.
In bovine cultures, there was an abundance of liberated merozoites at 50, 52, 74, and 76 hrs; many had reinvaded cells, sometimes as many as 50–60 per cell. In turkey cultures, liberated merozoites were found once at 144 hrs and none were intracellular. At 120 and 144 hrs in bovine cultures, abnormally developed and degenerate forms appeared; in turkey cultures, all were normal.     

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.     

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.     

Further studies on the development of gamonts and oocysts of Eimeria magna in cultured cells

Monolayer, cell-line cultures of embryonic bovine trachea, Madin-Darby bovine kidney (MDBK), and monolayers (RK-1) or aggregates of primary rabbit kidney cells were inoculated with merozoites obtained from rabbits that had been inoculated 3 to 5 1/2 days earlier with Eimeria magna. Merozoites obtained from from rabbits 3 days entered cells and underwent only merogony, whereas 3 1/2-5 1/2-day-old merozoites formed gamonts as well as meronts. Merozoites arising from the first or second meront generation in culture formed another meront generation or gamonts. Third-generation merozoites formed only gamonts. Most merozoites remained within the parasitophorous vacuole of the original host cell and transformed into macro- or microgamonts or meronts. Some such macro- and microgamonts then fused with each other to form larger multinucleated bodies. Such microgamonts formed microgametes, but multinucleate macrogamonts did not form oocysts. Mature microgamonts were 34 microns in diameter, and contained several hundred biflagellate microgametes. Mature macrogamonts measured 29.1 x 21.5 microns, unsporulated oocysts were 31.2 x 22 microns, and sporulated oocysts were 32 x 23.1 microns. Oocysts obtained from cell cultures were sporulated and then inoculated by gavage into rabbits, which passed E. magna oocysts 6--10 days later. Sporozoites, obtained from oocysts produced in culture or from rabbits that had been inoculated with the vitro-produced oocysts, developed to first- and second-generation meronts in MDBK or RK-1 cultures.     

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.     

Development of Eimeria tenella in MDBK cell culture with a note on enhancing effect of preincubation with chicken spleen cells

Eimeria tenella, an intracellular protozoan parasite infecting the epithelial cells of the ceca of chickens, causes severe diarrhea and bleeding that can lead its host to death. It is of interest that E. tenella first penetrate into the mucosal intraepithelial lymphocytes (IEL) before they parasitize crypt or villous epithelial cells. This in vitro study was undertaken to know whether the penetration of E. tenella into such a lymphoid cell is a beneficial step for the parasite survival and development. Three sequential experiments were performed. First, the in vitro established bovine kidney cell line, MDBK cells, were evaluated for use as host cells for E. tenella, through morphological observation. Second, the degree of parasite development and multiplication in MDBK cells was quantitatively assayed using radioisotope-labelled uracil (3H-uracil). Third, the E. tenella sporozoites viability was assayed after preincubation of them with chicken spleen cells. E. tenella o?cysts obtained from the ceca of the infected chickens were used for the source of the sporozoites. Spleen cells (E) obtained from normal chickens (FP strain) were preincubated with the sporozoites (T) at the E:T ratio of 100:1, 50:1 or 25:1 for 4 or 12 hours, and then the mixture was inoculated into the MDBK cell monolayer. Morphologically the infected MDBK cells revealed active schizogonic cycle of E. tenella in 3-4 days, which was characterized by the appearance of trophozoites, and immature and mature schizonts containing merozoites. The 3H-uracil uptake by E. tenella increased gradually in the MDBK cells, which made a plateau after 48-60 hours, and decreased thereafter. The uptake amount of 3H-uracil depended not only upon the inoculum size of the sporozoites but also on the degree of time delay (preincubation; sporozoites only) from excystation to inoculation into MDBK cells. The 3H-uracil uptake became lower as the preincubation time was prolonged. In comparison, after preincubation of sporozoites with spleen cells for 4 or 12 hours, the 3H-uracil uptake was significantly increased compared with that of control group. From the results, it was inferred that, although the penetration of E. tenella sporozoites into the lymphoid cells such as IEL is not an essential step, it should be at least a beneficial one for the survival and development of sporozoites in the chicken intestine.     

Ultrastructural Study of Schizogony in Eimeria callospermophili*

SYNOPSIS The development of 1st generation schizonts of Eimeria callospermophili was studied with cell cultures and with experimentally infected host animals, Spermophilus armatus. Sporozoite-shaped schizonts each had 5-10 nuclei and all of the organelles of the sporozoite; each nucleus had a nucleolus and an associated Golgi apparatus. In stages immediately preceding merozoite formation, an intranuclear spindle apparatus with conical polar areas were observed near the outer margin of each nucleus. Two centrioles, each having 9 single peripheral tubules and one central tubule, were observed near each pole in some specimens. Merozoite formation began internally, with anlagen of 2 merozoites developing near each nucleus. The inner membrane of the merozoites first appeared as 2 dense thickenings adjacent to the polar cones and centrioles; subpellicular microtubules appeared simultaneously. Two anterior annuli and the conoid formed between the 2 thickenings. Vesicles, possibly of Golgi origin, were located next to the forming inner membrane. As the forming merozoites underwent elongation, a rhoptries anlage, a Golgi apparatus, refractile bodies, and mitochondria were incorporated into each. Sporozoite-shaped schizonts with merozoite anlagen transformed into spheroid or ovoid schizonts; at this time the conoid, rhoptries, micronemes, and the inner membrane of the pellicle gradually disappeared; several small refractile bodies were formed from the larger one. When development was about 1/3 complete, the immature merozoites began to grow outward from the surface of the schizont. In this phase of development, the single surface membrane of the schizont became the outer membrane of the merozoite's pellicle, and additional organelles, including the nucleus, were incorporated. Finally, the merozoites became pinched off, leaving a residual body. Development in cell cultures and host tissues was similar. This type of schizogony, previously undescribed in Eimeria, is compared with corresponding stages of development in other species of Eimeria and Sporozoa.     

Comparative Cultivation of Poultry Coccidia in Mammalian Kidney Cell Cultures

SYNOPSIS. Excysted sporozoites of Eimeria meleagrimitis, E. necatrix, E. acervulina , and E. gallopavonis were inoculated into monolayer cell cultures of bovine, ovine, porcine, and human kidney. E. meleagrimitis developed only in bovine embryonic kidney. Mature schizonts were found in the 11th, 16th, and 20th serial passages, but only immature schizonts were in the 4th and 6th passages. E. necatrix developed to mature schizonts in the 3rd, 4th, 6th, 11th, 16th, and 20th passages of bovine kidney and also to immature schizonts in the 175th and 189th passages of PK-15 (cell line porcine kidney). Schizonts, however, did not develop in the 140th and 145th passages of CCI-33 (cloned PK-15). Neither E. meleagrimitis nor E. necatrix developed in the primary, 1st or 2nd passages of bovine embryonic kidney, primary porcine kidney, 45th and 52nd passages of a human embryonic kidney cell line, or in the primary, 5th and 18th passages of ovine kidney. Eimeria acervulina and E. gallopavonis did not develop in any of the cultures.
E. meleagrimitis and E. necatrix probably completed only one asexual generation in culture. The structure of mature schizonts of both species differed greatly from those in the natural host. Schizonts of E. meleagrimitis present at 48 hours were small (13–18 by 12–14 μ) and contained only 12–28 merozoites that were 3.2–3.8 μ long. At 48 hours, E. necatrix schizonts were 15–18 μ in diameter or less and contained only 15–20 merozoites (2.0–3.5 μ long); at 96 hours they were 50–70 by 10–35μ and contained either hundreds of small merozoites (2.0–3.5 μ long) or a lesser number of larger merozoites (9–11 μ).     

Cystoisospora belli: in vitro multiplication in mammalian cells

Intracellular development of Cystoisospora belli was demonstrated in 4 different mammalian cell lines. Human ileocecal adenocarcinoma (HCT-8), epithelial carcinoma of lung (A549), Madin-Darby bovine kidney (MDBK), and African green monkey kidney (VERO) were exposed in vitro to C. belli sporozoites, which had been isolated from the feces of HIV-AIDS patients. Parasites invaded all the cellular types between 4 and 12h after exposure and multiplication was demonstrated after 24 h. Grater number of merozoites formed in VERO cells, followed by HCT-8. In the MDBK and HCT-8 cells, the parasitophorous vacuole was less evident and immobile merozoites were observed in the cytoplasm. In VERO cells, one or several parasitophorous vacuoles contained up to 16 mobile sporozoites. No oocysts were found in any of the cell types used. VERO cells may be suitable for studies of the interaction between parasite and host cells.     

Schizogonic Development of Leucocytozoon smithi

ABSTRACT The schizogonic development of Leucocytozoon smithi in the liver of experimentally infected turkey poults was examined by electron microscopy. Following intraperitoneal injection, sporozoites migrated to the liver and entered hepatic cells to become intracellular trophozoites. Three to four days post inoculation (PI), trophozoites underwent asexual multiple fission known as merogony or schizogony. Two generations of schizonts were observed. The primary or first generation schizonts, abundant on day 4 PI, appeared as interconnected cytoplasmic masses (pseudocytomeres). Each pseudocytomere was enclosed by a membranous vacuole and contained varying numbers of nuclei. As nuclear division and growth of the schizonts continued, larger discrete cytoplasmic masses or cytomeres were formed with rhoptries and multiple nuclei in various stages of division. Synchronous multiple cytoplasmic cleavage of the schizont resulted in the formation of numerous uninucleate merozoites. Second generation schizonts, which developed from hepatic merozoites released from primary schizonts, were abundant in hepatocytes on day 6 PI. Although tissue samples from liver, lung, spleen, kidney, intestine, brain, blood vessels and lymph nodes were examined, schizogonous forms were observed in liver only. No megaloschizonts were detected in any host tissue examined. Schizogonic development was completed by day 7 PI as merozoites developed into gametocytes within mononuclear phagocytes.     

Schizogonic development of Leucocytozoon smithi.

The schizogonic development of Leucocytozoon smithi in the liver of experimentally infected turkey poults was examined by electron microscopy. Following intraperitoneal injection, sporozoites migrated to the liver and entered hepatic cells to become intracellular trophozoites. Three to four days post inoculation (PI), trophozoites underwent asexual multiple fission known as merogony or schizogony. Two generations of schizonts were observed. The primary or first generation schizonts, abundant on day 4 PI, appeared as interconnected cytoplasmic masses (pseudocytomeres). Each pseudocytomere was enclosed by a membranous vacuole and contained varying numbers of nuclei. As nuclear division and growth of the schizonts continued, larger discrete cytoplasmic masses or cytomeres were formed with rhoptries and multiple nuclei in various stages of division. Synchronous multiple cytoplasmic cleavage of the schizont resulted in the formation of numerous uninucleate merozoites. Second generation schizonts, which developed from hepatic merozoites released from primary schizonts, were abundant in hepatocytes on day 6 PI. Although tissue samples from liver, lung, spleen, kidney, intestine, brain, blood vessels and lymph nodes were examined, schizogonous forms were observed in liver only. No megaloschizonts were detected in any host tissue examined. Schizogonic development was completed by day 7 PI as merozoites developed into gametocytes within mononuclear phagocytes.