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.     

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.     

Fine Structure of Schizonts and Merozoites of Eimeria nieschulzi*

SYNOPSIS. Schizonts of E. nieschulzi lie in a vacuole within the host cell. After nuclear division the cell membrane invaginates forming merozoites. Differentiation of the pellicle and other organelles occurs while merozoites are still attached to the schizont cytoplasm. Merozoites have a pellicle thickened at the anterior end to form a polar ring. Radiating posteriorly from the ring, directly beneath the pellicle, are about 25 microtubules. Within the polar ring is a dense conoid. Extending posteriorly from within the conoid is a paired organelle. The paired organelle varies in size and shape in each generation of merozoites. Numerous toxonemes occupy the anterior half of the merozoites. Two paranuclear bodies are present in 1st generation merozoites. One or 2 granular bodies were seen in the anterior end of 2nd generation merozoites. In 3rd generation merozoites 6 or more granular bodies were seen anterior to the nucleus. Each merozoite has a single nucleus containing diffuse chromatin material. Elongate mitochondria and glycogen granules are present. The vacuole surrounding mature merozoites contains residual cytoplasm of the schizont and some granular material. Microvilli project into the vacuole from the host cell membrane.     

Studies on the Storage Polysaccharides of the Coccidia Eimeria bovis and Eimeria stiedai

Polysaccharides have been isolated from Eimeria bovis and E. stiedai by the use of alkali. The purified polysaccharides stain purple with iodine and have been shown by chemical and enzymic means to be amylopectin. The storage amylopectins of these coccidia differ slightly from typical plant amylopectins in their fine structure.     

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.     

Ultrastructural Study of Schizogony of Eimeria bovis in Cell Cultures*

SYNOPSIS. First-generation schizogony of Eimeria bovis in bovine cell culture was studied by electron microscopy. The intracellular sporozoite retained its structure for at least 6 days at which time it rounded up and lost its apical complex. Although the refractile body underwent certain morphologic changes, it was retained throughout the parasite's growth. The beginning of mitosis was marked by the formation of a cytoplasmic funnel which traversed the nucleus opening on each side toward a pair of centrioles. Subsequently, there developed an intranuclear spindle. Separation of the daughter nuclei was preceded by the formation of typical centrocones. Differentiation of merozoites was accomplished by exogenesis during the last mitotic division. A dense fiber, interpreted as a link connecting the merozoite anlage with its nucleus, extended from the developing apical complex to the nearest division pole. In the anlage, the inner membrane complex was at first composed of patches associated with pairs of subpellicular microtubules. Rhoptries appeared early in merogenesis, whereas micronemes formed at the time the merozoites detached from the residuum. The level of amylopectin, low in schizonts, rose at the beginning of merozoite formation.     

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.     

Resistance of Calves to Reinfection with Eimeria bovis

SYNOPSIS. An immunity to reinfection with E. bovis was demonstrated in 3 experiments involving 60 calves. This immunity develops rapidly, as indicated by resistance to a challenge given 14 days after the immunizing inoculation. In 3 groups of 3 to 6 young calves each, immunity was still present to a moderate degree 2 to 3 months after inoculation; in one group of 5 animals about a year old there was apparently a high degree of immunity about 7 months after the last inoculation. In one experiment an immunizing inoculum of 10,000 oöcysts did not produce as much immunity as 50,000 oöcysts. In 2 experiments there appeared to be little difference in the immunity produced by a single inoculation of 50,000 as compared with 100,000 oöcysts, but inoculation with 100,000 oöcysts, resulted in substantially longer and more severe illness than 50,000 oöcysts. There appeared to be no appreciable difference in clinical symptoms or development of immunity between calves given a single immunizing inoculum and those given the same number of oöcysts in 5 equal inocula on successive days. Treatment with sulfamethazine and sulfamerazine (Merameth) 13 to 15 days after inoculation alleviated the clinical symptoms of coccidiosis without interfering appreciably with the development of immunity. In one experiment with 7 calves, no beneficial effect was noted from 1 or 2 transfusions of 500 ml. of plasma and leucocytes from immune calves into 4 calves 1 and 12 days or 11 days after a challenge inoculation.     

Alternative mechanism of Eimeria bovis sporozoites to invade cells in vitro by breaching the plasma membrane

In vitro Eimeria bovis sporozoites invade a wide range of cell types, and in the case of bovine cells, they may develop to first-generation schizonts. Often, however, they subsequently leave their host cell to invade a new one, which seems contrary to the classical way of infecting a cell by forming a parasitophorous vacuole. Using a standard, "cell wound assay," we show that E. bovis can invade bovine endothelial cells by breaching the plasma membrane and may again leave the surviving cell. Eimeria bovis sporozoites also infected VERO and HT29 cells but obviously without damaging the plasma membrane. The same held true when bovine endothelial cells were exposed to tachyzoites of Toxoplasma gondii and Neospora caninum. According to a literature report dealing with Plasmodium yoelii sporozoites, breaching the membrane of certain host cells may be a common phenomenon for coccidian sporozoites but may not be for merozoites.     

Fine Structure of the Endogenous Stages of Eimeria labbeana. 5. Schizonts and Merogony,with Special Reference to the Rhoptry-Microneme System*

SYNOPSIS The fine structure of the 3 generations of meronts, merogony, and merozoites of Eimeria labbeana Pinto from the ileal mucosa of experimentally infected pigeons, Columba livia Linnaeus, was described and compared to that of similar stages in other species of Eimeria. Sporozoite-trophozoite transition stages, trophozoites (5.8 × 4.2 μm), young meronts (10.1 × 8.4 μm), and mature meronts with free merozoites of the first generation, were observed at 20, 28, 36, and 48 hr post-infection, respectively. The 2nd and 3rd generation merogony were completed at 96 and 144 hr. Merogony was essentially of the ectomerogonous type without cytomere formation, as in most species. The average number of merozoites per meront in the 3 generations was 10 (5–15), 14 (8–19), and 7.5 (6–16); and the average size was 4.4 × 2.1 (4.1–5.9 × 1.8–2.2) μm, 4.2 × 1.8 (4.0–4.8 × 1.5–2.0) μm, and 5.4 × 1.8 (5.2–7.8 × 1.6–2.0) μm, respectively. Aggregation and subsequent degeneration of micronemes within membrane-bounded vesicles in the sporozoite-trophozoite stage, was observed as a possible mode of eliminating certain organelles present in the motile stages. Centrioles with (9 + 1) microtubular composition, and centrocones, were frequently seen in early meronts. Anlagen of micronemes, without any apparent association with the Golgi complex and the merozoite bud, were seen to develop in the cytoplasm of the meront. A single, median structure, probably representing the anlage of the rhoptry-microneme system was observed within the conoid of an early merozoite bud. Connections between the micronemes and the bulbous portion of the rhoptries, and a branched (interconnected ?) structure of the rhoptries observed in the present study, substantiate the present contention that the micronemes and rhoptries are functional forms of the same complex of organelles, the rhoptry-microneme system.     

Further Studies on Antigenic Changes in Trypanosoma lewisi*

SYNOPSIS. An investigation of day-to-day changes in adult form antigens of T. lewisi in the rat shows that adult antigens, 2 of which are present in trace amounts in young forms, suddenly appear in large amounts in 9-day-old trypanosomes. An additional antigen is formed on the 13th day. In addition, 2-day-old trypanosomes contain some antigens not present in 6-day-old forms. The combined results of the present paper and a previous one indicate that there are antigenically 3 distinct stages of T. lewisi in the rat: 1- to 3-day-old, 4- to 8-day-old, and 9- to 14-day-old forms. From 9- to 11-day-old trypanosomes, there is a form containing both young and adult antigens.     

Ultrastructural and Cytologic Studies of the Sporozoites of Four Eimeria Species*

SYNOPSIS. Studies were made with the light microscope of live sporozoites of E. ninakohlyakimovae and E. ellipsoidalis as well as sporozoites fixed with Schaudinn's, Stieve's and Zenker's fluids, methanol and ethanol saturated with picric acid. Sporozoites were stained with Giemsa, bromphenol blue, modified PAS-AO, Feulgen, Harris’hematoxylin and eosin Y, and iron hematoxylin. Sporozoites of the above species as well as those of E. auburnensis and E. bovis were also fixed with glutaraldehyde and osmium tetroxide or negatively stained for study with the electron microscope. Living sporozoites had gliding, pivoting, flexing, and probing movements. Each sporozoite of each species was covered by a pellicle consisting of an outer limiting unit membrane that was continuous around the sporozoite and an inner membrane that terminated at the polar ring. Twenty-four subpellicular microtubules were longitudinally arranged just beneath the inner membrane. At the anterior end of the sporozoites was a protruded or retracted conoid composed of spirally-arranged fibrillar structures, 2 rings anterior to the conoid, and the polar ring, a thickening at the anterior termination of the microtubules and inner membrane. Other organelles observed with the electron microscope were a nucleus with or without a net-like nucleolus, club-shaped organelles, refractile bodies, micronemes, endoplasmic reticulum, Golgi apparatus, mitochondria with tubular cristae, micropores, lipoid-like bodies, oval polysaccharide bodies and ribosomes. The fine structure of these sporozoites is compared to that of related Sporozoa.     

The Influence of Dexamethasone on Attempts to Transmit Eimeria meleagrimitis to Chickens and E. tenella to Turkeys

SYNOPSIS. Eimeria meleagrimitis completed development in chickens given daily intramuscular injections of dexamethasone, but not in chickens fed mash containing dexamethasone, or in unmedicated chickens.
Eimeria tenella did not develop in dexamethasone-medicated turkeys, or in unmedicated turkeys.