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.     

Life Cycle of Eimeria ferrisi Levine & Ivens, 1965 in the Mouse, Mus musculus

SYNOPSIS. The life cycle of Eimeria ferrisi is described from experimentally infected Mus musculus. The prepatent period was 3 days and the patent period was 3–4 days. The endogenous stages were found only in the cecum and colon. Three generations of schizonts were found. Mature 1st-generation schizonts first seen 24 hr postinoculation (PI) measured 10.9 (7–14) × 10.2 (6–13) μm and had 9.6 (7–14) merozoites. Some 2nd-generation schizonts had uninucleate merozoites and others had multinucleate merozoites. The former were first seen in small numbers 36 hr PI and were most abundant 48 hr PI. They measured 9.6 (5–13) × 7.9 (6–12) μm and had 18 (6–25) merozoites. Schizonts with multinucleate merozoites were seen 72 hr PI. Mature 3rd-generation schizonts were seen 72 hr PI. They measured 14.0 (12–18) × 11-0 (9–13) μm and had 12.5 (5–16) merozoites. Macrogamonts were first seen in 72 hr sections. Each young macrogamont had a large nucleus with a prominent nucleolus. Only one type of cytoplasmic granule appeared to be involved in the formation of the oocyst wall. Mature macrogamonts were 11.0 (5–14) × 10.0 (6–13) μm. Crescent-shaped bodies were observed in the parasitophorous vacuole of trophozoites and young macrogamonts. Early microgamonts were first recognized at 96 hr by the presence of darkly stained and irregularly shaped nuclei. Usually, mature microgametes were arranged in long, narrow whorls at the periphery of the microgamont or in whorls at the surface of 2–5 compartments.     

Comparative Excystation of Four Species of Poultry Coccidia

SYNOPSIS. Examination of the crop, gizzard, and intestinal contents of chickens fed suspensions of either Eimeria acervulina or E. tenella oocysts and turkeys fed either E. meleagrimitis or E. gallopavonis oocysts indicated that, in all 4 species, (1) oocysts apparently remained unchanged while in the crop, (2) sporocysts were liberated from oocysts while the latter were passing through the gizzard, (3) sporozoites were activated and escaped from liberated sporocysts after they had reached the small intestine, and (4) sporozoites within intact oocysts in the crop, gizzard, and intestines were not activated.
In vitro , trypsin 1–300 alone caused a small percentage of sporozoites to excyst from mechanically liberated sporocysts. The percentage of excystation increased greatly when trypsin was added to sodium taurocholate and increased even more when it was combined with chicken or turkey bile.
The two duodenal species ( E. acervulina and E. meleagrimitis ) differed both in vivo and in vitro from the two cecal species ( E. tenella and E. gallopavonis ). The duodenal species excysted in less time and farther anteriorly in the small intestine than did the cecal species. In addition, sporozoites of the two cecal species survived much longer in media containing trypsin plus bile or sodium taurccholate than did those of the two duodenal species.     

Electrophoretic and immunologic characterization of proteins of merozoites of Eimeria acervulina, E. maxima, E. necatrix, and E. tenella.

Merozoites of Eimeria acervulina, Eimeria maxima, Eimeria necatrix, and Eimeria tenella were compared by gel electrophoresis, western-blotting with chicken antiserum, indirect fluorescent antibody reactions, and antiserum neutralization. Merozoites from the 4 species had dissimilar patterns of proteins and antigens in soluble and membrane fractions. Coomassie blue staining of SDS-PAGE gels revealed 16-22 protein bands depending on the species of merozoite but only 3 bands per species in the membrane fractions. Homologous and heterologous antisera recognized 5-12 soluble fraction bands and 3-7 membrane fraction bands on immunoperoxidase-stained western blots, depending on the species. When antisera from infected chickens were used in an indirect fluorescent antibody reaction, the merozoites of E. tenella and E. necatrix had a strong reaction with homologous and heterologous antisera. Merozoites of E. acervulina and E. maxima reacted with homologous antisera but had a weak or no reaction with heterologous antisera. Chicken antiserum against E. tenella had no effect on the viability of E. tenella merozoites when they were inoculated into chicken embryos.     

Schizonts and Microgametocytes of Eimeria auburnensis Christensen and Porter, 1939, in Calves

SYNOPSIS. Schizonts were found in the middle and lower third of the small intestine of two calves killed 12 and 14 days after they had been inoculated with pure cultures of oocysts of Eimeria auburnensis. The schizonts ranged from 78 to 250 μ long by 78 to 150 μ wide (mean 92 by 139.9 μ). They were usually located deep in the lamina propria near the muscularis mucosae instead of in the villi where most schizonts of Eimeria bovis are found. The schizonts of E. auburnensis resembled the previously described large microgametocytes of this species but were distinguishable morphologically and by histochemical stains. The microgametocytes were much larger than previously reported; one measured 91 by 287.5 μ.     

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.     

Storage Polysaccharide in Coccidial Sporozites after Excystation and Penetration of Cells

SYNOPSIS. Eimeria acervulina, E. necatrix , and E. meleagrimitis sporozoites were examined for carbohydrates by cytochemical methods during dormancy, after excystation, and after penetration of cells. The only carbohydrate found was amylopectin, a homogeneous polymer of glucose. It was distributed in 3 regions: (a) in front of the anterior refractile globule, (b) around the nucleus, and (c) behind the posterior refractile globule. The relative amounts decreased after excystation and penetration of cells until only small amounts remained around the nucleus. The quantity of amylopectin decreased following excystation from 30.0-36.7 to 9.4-13.3 μg glucose/10⁶ oocysts. Over a 6 yr period of storage at 4 C, there was a decrease in the quantity of amylopectin in dormant sporozoites of E. acervulina from 33.3 μg glucose/10⁶ oocysts at 3 mos to 1.5 μg at 6 years. Coincidentally, 3 month- and 1 year-old oocysts of E. acervulina produced patent infections in chicks with a dosage of 5 × 10⁴ oocysts, but only a few of the oocysts that had been stored for 2 years were infective; a dosage of 2 × 10⁶ oocysts was necessary to produce a patent infection. Oocysts which had been stored 6 years did not produce a patent infection.
It was concluded that amylopectin is the energy source for excystation and subsequent penetration of cells. Small amounts of amylopectin are used during dormancy and, when the content in the sporozoite falls below a certain level, the sporozoites lack sufficient energy to infect cells.     

Oxygen Consumption of Coccidial Sporozoites During in Vitro Excystation

SYNOPSIS. Oxygen consumption of sporozoites of Eimeria acervulina, E. necatrix , and E. meleagrimitis was determined during in vitro excystation. Increase in O₂ consumption occurred only when the permeability of the oocyst membrane was altered by either grinding or incubation in cystine buffer with CO₂ when excystation fluid was present. Addition of 10⁻³ M KCN completely inhibited respiration. The O₂ consumption reached a peak during the time the sporozoites were excysting, and then decreased to a lower level where it remained steady while the sporozoites were awaiting stimulus to penetrate host cells.     

Eimeria dispersa and Eimeria gallopavonis: Infectivity, Survival, and Development in Primary Chicken and Turkey Kidney Cell Cultures

SYNOPSIS. Eimeria dispersa (turkey strain) and Eimeria gallopavonis sporozoites were inoculated into primary cultures of chicken kidney (CK) and turkey kidney (TK) cells. Eimeria dispersa sporozoites were more infective in either cell type than those of E. gallopavonis : at 4 hr, the percentage of infection was 67-98 for E. dispersa but only 23-56 for E. gallopavonis . E. dispersa also survived better in culture: at 2 days, losses of E. dispersa in both cell types were only 4-19%, whereas losses of E. gallopavonis were 35-47% in TK cells and 60–95% in CK cells. However, E. gallopavonis developed further than E. dispersa . Location and increase in numbers of intracellular stages at 4 days indicated that E. dispersa proceeded through 2 schizogonic generations before development stopped.     

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.     

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.     

Endogenous Stages of the Life Cycle of Isospora rivolta in the Dog

SYNOPSIS. Coccidia-free beagle puppies were experimentally infected with a cloned culture of Isospora rivolta oocysts. The endogenous stages were found in the posterior 1/2 of the small intestine, and rarely in the cecum and colon. Maximum numbers of all stages occurred just anterior to the ileocecal valve. Endogenous stages were found in the distal third of the villi, predominantly parasitizing subepithelial cells of the lamina propria; however, stages were occasionally present in epithelial cells. The number of asexual generations could not be determined from their structure, but evidence based on oocyst production suggested that there were at least 2 asexual generations. The schizonts were 17–24 by 12–25 μ and contained 4–24 merozoites, the most common number being 4 or 8. Schizonts with mature merozoites were found as early as 72 hr, but were present in maximum numbers at 96 hr. Merozoites had slender curved bodies and were 10.5–13.4 by 2.3–3.0 μ. Mature gamonts were found by 144 hr. Mature microgametocytes were 13.4 by 8.7 μ and contained 50–70 microgametes. Microgametes had slightly curved tapering bodies (5.8–6.4 by 0.6 μ) with 2 posteriorly directed flagella 11–14 μ long. Mature macrogametes had reticular cytoplasm and a uniformly large nucleus and nucleolus.
The prepatent period was 142–146 hr. The patent period was 13–23 days with an average of 19 days.     

Analysis of transcripts from intracellular stages of Eimeria acervulina using expressed sequence tags

Coccidiosis in chickens is caused by 7 species of Eimeria. Even though coccidiosis is a complex disease that can be caused by any combination of these species, most of the molecular research concerning chicken coccidiosis has been limited to Eimeria tenella. The present study describes the first large-scale analysis of expressed sequence tags (ESTs) generated primarily from second-stage merozoites (and schizonts) of E. acervulina. In total, 1,847 ESTs were sequenced; these represent 1,026 unique sequences. Approximately half of the ESTs encode proteins of unknown function, or hypothetical proteins. Twenty-nine percent of the E. acervulina ESTs share significant sequence identity with sequences in the E. tenella genome. Additionally, EST hits seem to be much different compared with those of E. tenella. One of the differences is the very low number of ESTs that encode putative microneme proteins. This study underlines the potential differences in the molecular aspects of 2 Eimeria species that in the past were thought to be highly similar in nature.     

Scanning Electron Microscopy of Schizogony in Eimeria tenella

SYNOPSIS. Developing 2nd- and 3rd-generation schizonts of Eimeria tenella were found in the ceca of chicks infected orally with sporulated oocysts. Several free 2nd-generation schizonts, which varied in diameter from 11 to 21.6 μm, were found on the epithelial surface of the cecum. Some schizonts appeared to have lost merozoites. Other schizonts were intact, one of which was surrounded by an unbroken membrane that followed the contours of the merozoites. Third-generation schizonts, much smaller than 2nd-generation schizonts and with fewer merozoites, were found only on cut or fractured surfaces of the cecal tissue. Third-generation merozoites appeared shorter and thicker than those of the 2nd-generation and were attached to the schizont residuum. A form with conical protuberances and another with 4 triangular segments were found; they were believed to be developing stages 3rd-generation schizonts.     

The Life Cycle of Eimeria dispersa Tyzzer, 1929 in Turkeys

SYNOPSIS. The life cycle of a turkey strain of Eimeria dispersa Tyzzer was studied in Beltsville Small White turkeys. There were 4 asexual generations. Mature schizonts of the first generation were present 30 h postinoculation (PI); those of the 2nd, 3rd, and 4th generations were present 48, 72, and 96 h PI, respectively. Average size of schizonts and number and size of merozoites for each generation were as follows: first , 14.3 × 13.0 μm with 19.2 merozoites, each 4.5 × 1.2 μm; second , 8.0 × 7.2 μm with 13.5 merozoites, each 4.5 × 1.1 μm; third , 8.9 × 8.9 μm with 15.1 merozoites, each 5.6 × 2.1 μm; fourth , 11.6 × 10.5 μm with 6.7 merozoites, each 8.2 × 2.0 μm. Sporozoites and developmental stages of the first generation were in close association with an epithelial cell nucleus and located between the brush border and the "row" of epithelial cell nuclei; developmental stages of the other 3 generations were not associated with a nucleus and were located just under the brush border. Early macrogametes and microgametocytes were present 96 h PI. Development was confined to the epithelial cells of the villus and extended from the tip of the villus to ∼ 1/2 the distance down the sides in all areas of the intestine except the cecum. The prepatent period was between 114 and 120 h. Percentage of sporulation was 15, 57, and 90, at 24, 36, and 48 h, respectively. Sporulated oocysts averaged 24.5 × 20.2 μm.     

In vitro cultivation of Sarcocystis neurona from the spinal cord of a horse with equine protozoal myelitis

Asexual stages of Sarcocystis neurona were seen in cultured bovine monocytes (M617) inoculated with tissue homogenates from the spinal cord of a horse with naturally acquired protozoal myelitis. Organisms first were observed as intracytoplasmic schizonts and later as motile extracellular zoites capable of infecting surrounding M617 cells. Parasites most often occurred as clusters of merozoites dispersed throughout the host cell cytoplasm; however, schizonts also contained merozoites arranged in a radial fashion surrounding a prominent residual body. Schizonts divided by endopolygeny. The parasite has been maintained beyond 280 days in the laboratory by serial passage of infected M617 cells.     

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 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.     

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.     

Equine protozoal myelitis in Panamanian horses and isolation of Sarcocystis neurona.

Schizonts of Sarcocystis neurona were identified microscopically in hematoxylin-eosin-stained spinal cord sections from 2 native Panamanian horses that exhibited clinical signs of equine protozoal myelitis (EPM). Spinal cord homogenate from a third Panamanian horse with EPM was inoculated onto monolayers of cultured bovine monocytes (M617). Intracytoplasmic schizonts containing merozoites arranged in rosette forms surrounding a central residual body first were observed 13 wk postinoculation. Parasites divided by endopolygeny and lacked rhoptries. Schizonts from each horse reacted with Sarcocystis cruzi antiserum in an immunohistochemical test.