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SYNOPSIS. Eimeria larimerensis was found in 5 species of ground squirrels and the white-tailed prairie dog. The hosts included Spermophilus armatus from Utah and Montana, S. variegatus from Utah, S. tridecemlineatus from Wyoming, S. lateralis from Utah, S. beecheyi from California and Cynomys leucurus from Wyoming. Oocysts were not present in fecal samples of S. richardconi from Montana, S. lateralis from California or S. columbianus from Washington. This coccidium could not be experimentally transmitted to S. richardsoni; however, patent infections were established in S. armatus, S. lateralis, and S. variegatus. No infections were found after inoculation of least chipmunks (Eutamius minimus), Mongolian gerbils (Meriones unguiculatus), or laboratory rats even tho excystation occurred in these animals. Resistance to infection did not develop during repeated experimental infections of S. armatus, S. lateralis, or S. variegatus. No outward signs of coccidiosis were seen in any of the experimentally infected animals. In experimentally infected S. armatus, the prepatent period was 5 days, and the patent period lasted 3–7 (mean 6.5) days. The endogenous stages were located in the epithelial cells of the jejunum and ileum. Mature 1st-generation schizonts, 1st seen 2.5 days after inoculation, contained 16–32 merozoites. Mature 2nd generation schizonts were present 3.5 days after inoculation and contained 22–46 merozoites of a larger size than those of the 1st generation. Gametocytes were 1st seen 3.5 days after inoculation and developing oocysts were present 4 days after inoculation. Macrogametes contained eosinophilic granules which coalesced to form the oocyst wall. Formation of the macrogametes took place around cytoplasmic masses within the microgametocytes.

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.

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.

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.

SYNOPSIS. A Tritrichomonas foetus-likt flagellate was found in the stomach, small intestine, and cecum as well as in the nasal cavity of pigs. Xo appreciable differences in morphology or response to cultivation could be found among the trichomonads from the different sites; therefore, it is considered that they.belong to a single species, Tritrichomonas suis (Gruby & Delafond). a description of which is given. This organism could be grown indefinitely in various media, and, after a short period of cultivation, it was the only species surviving in cultures that originated from cecal samples containing 2 or 3 species. T. suis was found in the nasal cavity in 55 of 100 pigs, in the stomach in 41 (8.0%) of 512, in the small intestine in 3 of 100. and in the cecum in 215 (43.370) of 496. A T. batrachorum-type trichomonad, herein described as Tritrichomonas rotunda n. sp., was found in the cecum in 52 (10.5%) of 496 pigs. This species is typically broadly pyriform or rotund (average length 8.95 ± 0.83 ii, range, 6.83-11.4), with 3 equal or subequal anterior flagella slightly longer than the body, a relatively low undulating membrane extending y2 to 2/1 of the length of the body, and a posterior free flagellum usually a little shorter than the body. The narrow axostyle, expanded anteriorly into a curved capitulum closely associated with the large, spherical, anteriorly located nucleus, projects from the posterior surface of the flagellate for a distance which equals about 2/3 half of the body length. The parabasal apparatus is biramus. This species could be maintained only temporarily in media not containing extracts of cecal contents. A Trichomonas pronazeki-typt flagellate, found in the cecum in 126 (25.4%) of 496 pigs and in the small intestine in 1 of 100. is described as Trichomonas buttreyi n. sp. This organism is relatively small (average length 5.92 ± 0.79 μ, range 4.55-7.49). ellipsoidal in shape, with 5 to 4 flagella up to twice as long as the body, a relatively high undulating membrane of body length, a narrow axostyle with an inconspicuous capitulum closely associated with the usually oval nucleus, a projecting part of the axostyle that equals about j; of the body length, and a disc-shaped parabasal body lying dorso-lateral to the nucleus. In media without cecal extract this species could not be subcultured.

SYNOPSIS. In the nearly mature macrogametes of Eimeria auburnensis, the cell membrane is a unit membrane, with underlying and overlying osmiophilic layers usually present. Cup-shaped micropores were occasionally seen. Smaller, V-shaped invaginations were also found in considerable numbers at the surface. At the deepest point, these invaginations were bounded only by a unit membrane. Immediately adjacent to this point, vesicles with homogenous electron-pale contents bounded by a similar unit membrane, were frequently seen. Pinocytosis evidently occurs at the site of these invaginations. Numerous folds of the host cell membrane bordering the vacuole in which the parasite lay extended about 0.1–0.7 μ into the vacuole. These “intravacuolar folds” varied in depth and number in different specimens. In some, the majority of folds had apparently become disconnected from the host cell membrane. A highly developed smooth endoplasmic reticulum occurred in the adjacent host cell cytoplasm. The intravacuolar folds may assist in transfer of nutrients, including membrane material, from the host cell to the parasite. The evidence indicates that in this species of Eimeria nutrients are taken into the parasite primarily as fluids by pinocytosis and possibly other processes.

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 oocysts did not produce as much immunity as 50,000 oocysts. 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 oocysts, but inoculation with 100,000 oocysts, resulted in substantially longer and more severe illness than 50,000 oocysts. 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 oocysts 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.

SYNOPSIS. Stages of merogony of Eimeria magna were observed with the electron microscope in schizonts in ultrathin intestinal sections from white rabbits killed 4 days after inoculation. Some of the mature schizonts observed had uninucleate merozoites, whereas others had multinucleate ones. In each of the latter were rough endoplasmic reticulum, mitochondria, micronemes, refractile bodies, and occasionally, lipid droplets; some nuclei had a nucleolus. In the interior of some multinucleate merozoites, anlagen of daughter merozoites were observed. Each anlage was associated closely with a nucleus. In some of these nuclei, a cone-shaped pole of a spindle was directed toward the anlage. Each early anlage consisted of an inner membrane complex with a rhoptry anlage. A Golgi complex frequently was seen at the base of the anlage. One multinucleate merozoite, still attached to the residual body, had a merozoite anlage. In later stages of merogony, the anlagen were longer and each had a conoid. In one such merozoite, 2 merozoite anlagen were observed in close association with an eccentric intranuclear spindle, and 1 anlage had a Golgi adjunct. Another merozoite had an eccentric spindle and associated centrioles, but no visible anlagen. The finding of stages of merogony in multinucleate merozoites of E. magna indicates that these might represent a further schizogonic generation occurring in the original parasitophorous vacuole.

First-generation merozoites of Eimeria bovis, obtained from calves which had been inoculated 14, 14 1/2 and 15 days earlier, were inoculated into monolayers of Madin-Darby bovine kidney (MDBK) and primary embryonic bovine kidney (BEK-1), as well as kidney cell aggregates (BEK-2). Cultures were examined at intervals of 1–12 hr for 168 hr with phase-contrast microscopy and then fixed, stained and re-examined with bright-field microscopy. Fourteen-day merozoites entered all cell types, whereas 14 1/2- and 15-day merozoites did not enter any. Development beyond the first-generation merozoite stage occurred in BEK-1 and BEK-2, but not MDBK. Intermediate and mature second-generation schizonts with 24–36 merozoites were seen at 48–108 hr. Merozoites were 4.5 by 1.4 μm, with a posteriorly located nucleus. Young, intermediate, and mature microgamonts were seen at 72–96 hr, 72–120 hr, and 96–120 hr, respectively. Young, intermediate, and mature macrogamonts were seen at 48–96 hr, 72–120 hr, and 96–120 hr, respectively. Mature microgamonts and macrogamonts were 14.7 μm in diameter and 19.5 by 15 μm, respectively. At 120–168 hr, zygotes in various stages of oocyst wall formation and oocysts (24.4 by 15 μm) were seen. A crescent body was present in the parasitophorous vacuole of some schizonts and gamonts.

SYNOPSIS. Eimeria bovis merozoites occurred in tissue culture medium removed from Leighton tube cultures of embryonic bovine tracheal cells beginning 12-14 days after inoculation with 270,000-369,000 sporozoites per tube. The number of merozoites produced in these cultures increased daily until a peak was reached 18-21 days after inoculation. In 3 experiments an average of 2.0–15.6 million merozoites per tube was produced during the 20-day observation period. When such merozoites were frozen in liquid nitrogen and stored 26–42 days, some were motile upon thawing. These merozoites as well as others freshly obtained from cell cultures and from calves were inoculated into 11 different types of cultured mammalian cells including primary, cell line and established cell line cultures. Some merozoites were exposed to substances normally found in the lumen of the gut, before or at the time of inoculation. Altho small numbers of intracellular merozoites were found, no further development was observed. Gametocytes were observed in the cecum of a calf 4 days after merozoites from cell cultures were introduced into a ligated cecum of the calf.

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

1. The neuromotor system of Euplotes patella Ehrenberg was studied in different stages of the life-cycle, including the adult, and asexual and sexual reproduction. 2. In the adult the basal granules of all the cirri are arranged in straight primary and secondary rows. The fibrils from the bases of the cirri are in general parallel with either the primary or secondary rows of basal granules of the cirri to which they are attached. This is interpreted as an indication of the evolution of the organism from an ancestor with a simple neuromotor system in which the cilia were uniformly distributed over the surface of the body in longitudinal, slightly spiral rows. 3. The basal apparatus of a bristle consists of a basal granule at the bottom of a depression in the ectoplasm which is surrounded by a group of rodlets and ends at the surface in a ring connected with the polygonal system. The polygonal or silverline system is pellicular in position. 4. During asexual reproduction the entire set of cirri is resorbed and replaced by two new sets of cirri, one for each daughter. One frontal cirrus for the anterior daughter develops in the old peristomial field of the parent, while the corresponding cirrus for the posterior daughter develops in the new peristome. The right marginal cirri arise on the dorsal surface along the rows of bristles and later move to the ventral surface. The fibrils develop outward from the bases of the cirri. 5. During binary fission a new peristome arises in a depression in the ectoplasm independently of the old peristome. The old peristome remains in the anterior daughter without at the time undergoing any visible change in structure. 6. The multiplication of the bristles is limited to a zone on either side of the future plane of constriction. Multiplication occurs by a series of fissions of the basal granules in this area. Dedifferentiation of bristles was not seen. 7. The polygonal system is replaced over the entire surface of the body with the exception of the old peristomial field. The new system originates in many separate loci in connexion with the bristles on the dorsal surface, and with the rudiments (Anlagen) of the cirri and peristome on the ventral surface. 8. During conjugation there are two successive reorganizations of the neuromotor system, that of the ‘gamete’ (conjugant) and that of the zygote. 9. The first reorganization, that of the gamete, begins during the maturation divisions preparatory to fertilization. During this reorganization the posterior portion of the peristome is resorbed and the remaining anterior portion is later replaced. The eighteen cirri are replaced by a set containing only seventeen cirri. 10. The second reorganization, that of the zygote, occurs during the differentiation of the nuclei in the exconjugants. It involves the completion of the peristome by differentiation of the missing posterior portion, and the replacement of the set of seventeen cirri by a complete set of eighteen cirri. 11. These protoplasmic reorganizations, invariably associated with both asexual and sexual reproduction, involve a structural and presumably a physiological rejuvenation resulting in the possibility of an indefinitely continued existence of the individual in the Protozoa. It may be that sexual reproduction is not necessary to bring about this result, but further work on the relationship of endomixis to the problem is needed.

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.

SYNOPSIS. Mechanically released sporocyts of Eimeria bovis, E. ellipsoidalis and E. auburnensis readily excysted when incubated in a mixture of trypsin, steapsin, and bovine bile. Excystation of intact E. bovis, E. ellipsoidalis and E. auburnensis oocysts occurred after pretreatment at 37°C in aqueous 0.02 M cysteine hydrochloride under an atmosphere of CO2-air or CO2-N2, followed by incubation in an enzyme-bile mixture. Nine % of E. bovis oocysts were activated after 6 hr of incubation at 37 C in a mixture of 0.5% (w/v) trypsin, 0.5% (w/v) steapsin, and 5.0% (v/v) bile at pH 7.5 with 10 hr pretreatment under air and 20% CO2; with 30% CO2, 80% activation occurred; with 50% CO2, 91% of the oocysts were activated. At CO2 levels from 10 to 40%, pretreatment with CO2 and N2 yielded less activation than CO2 and air. Pretreatment with CO2-N2 (50–50) for 2 hr resulted in 37% activation; for 6 hr, 60%; for 10 hr, 94%; and for 14 hr, 99%. After pretreatment under 50% CO2 for 10 hr, 95% excystation was obtained by incubation for 6 hr in 0.5% trypsin and 5.0% bile. Only 76 or 69% excystation occurred when 0.25% trypsin or 1.0% bile, respectively, was substituted. Addition of steapsin to this mixture caused little change in the results. In oocysts pretreated 14 to 21 hr with steapsin, 2% or less excystation occurred after 6 hr in a trysinbile or bile mixture. E. ellipsoidalis oocysts, pretreated for 10 hr under CO2-N2 (50–50), underwent 61 and 80% excystation when incubated for 2 and 6 hr, respectively, in trypsin, steapsin, and bile. E. bovis oocysts enclosed in a dialysis bag inside an intestinal fistula became thinned or flattened at the micropyle after 24 hr, and excystation occurred when these oocysts were mixed with trypsin, steapsin, and bile and returned to the fistula for 4 hr. No excystation occurred when oocysts without added enzymes were left 48 hr in the fistula. Sporozoites escaped first from sporocysts and then from oocysts.

Sporozoites, trophozoites, and early schizonts of Eimeria ninakohlyakimovae grown in cultured cells were examined with the electron microscope. Intracellular sporozoites had ultrastructural characteristics similar to those of extracellular sporozoites, except that apparently functional micropores were observed. During transformation of sporozoites to trophozoites, the conoid, annuli and subpellicular tubules, as well as some portions of the inner membrane complex of the pellicle, disappeared. Trophozoites and binucleate schizonts had a reduced number of micronemes and rhoptries. In schizonts with 3 nuclei, only a few remnants of the micronemes and rhoptries were seen and the inner membrane complex had almost completely disappeared. One such schizont had one or more centrioles associated with each nucleus; in one, an eccentric spindle with a dense, coneshaped structure at each pole was observed. A moderately dense, granular crescent body, probably derived from host cell material, was seen within the parasitophorous vacuole in some specimens.

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.

SYNOPSIS. The sporulated oocysts of Eimeria utahensis sp. n. from 2 species of kangaroo rats in northwestern Utah are described. The oocysts averaged 42.0 by 39.3 μ, and had rough, yellow-brown walls, about 3 μ thick. In each sporocyst, a substiedal body was seen beneath the Stieda body; this is the 1st report of such a structure in any Eimeria species. E. utahensis was found in 3.6% of 196 Dipodomys ordii and 19.4% of 36 D. microps. The incidence was higher in young adult than in adult Ord kangaroo rats and lowest in juveniles; in D. microps only adults were infected. The mean prepatent period was 9.8 days. The patent period could not be determined because of apparent reinfections; there was no evidence of immunity.

Amoeboid movement of spheroid or ovoid immature or mature macrogamonts within cultured Madin-Darby bovine cells usually began with the formation of a pseudopodium-like protrusion at the margin of the gamont. The protrusion enlarged as the gamont cytoplasm and nucleus moved into the protruded area. During movement, macrogamonts had an elongate shape. The body of the gamont was constricted as it moved through the cell cytoplasm or from one cell to an adjacent one. After movement had ceased, the gamont resumed its ovoid or spheroidal shape.

SYNOPSIS. Monolayers of bovine kidney cells were overlaid with Eimeria magna sporozoites and observed with phase-contrast optics until penetration of the cells by the parasites had begun. Cells and penetrating parasites were fixed with glutaraldehyde and OsO4-containing ruthenium red, dehydrated, and embedded in situ. Cells being penetrated were selected for study in the electron microscope. The lack of intracellular staining with ruthenium red and intact plasmalemmas of cells being penetrated, was accepted as evidence that the sporozoites did not disrupt the plasma membranes. The sporozoite caused invagination of the host cell plasmalemma until the parasite was entirely within the cell, after which the invagination was sealed off by short pseudopodia enclosing the sporozoite within a membrane-lined vacuole inside the cell. Often myelin-forms, apparently of host cell origin, were seen in the space between the sporozoite and the cell.

SYNOPSIS. Oocysts of Eimeria bovis were found to undergo excystation when subjected at 39 C to a pretreatment consisting of exposure for 24 hr to CO2 and air (50–50), and a treatment for 7 hr with a mixture of bile and trypsin. At pH's of 6.0 thru 10.0 with tris-maleate buffer, excystation occurred over the entire range of pH tested, with the highest levels at pH 7.5-8.5. No adverse or inhibitive effect on excystation or the viability of the sporozoites was observed. Disintegration of sporozoites occurred within the sporocysts of intact oocysts at each of the pH levels studied when boric acid-borax, ammediol, and glycine-sodium hydroxide buffers were used in the treatment medium. Phosphate buffer inhibited excystation when used in the excysting medium. Excystation occurred at levels above 90% in all dilutions of taurocholic, glycocholic, glycotaurocholic, and cholic acids included in the study (0.5-10.0%) except for the 10% and 5% dilutions of cholic acid and the 10% dilution of glycotaurocholic acid. In the latter 3 dilutions, sporozoites within the sporocysts of intact oocysts disintegrated. Excystation levels above 90% were observed in the 50% and 10% dilutions of fresh bovine bile, and in the 5% dilution of lyophilized bovine bile. Lower levels of excystation occurred in greater dilutions of both kinds of bile. No excystation occurred when any of the bile acids, fresh bovine bile or lyophilized bile were used without trypsin, except for fresh bile that contained a heavy suspension of bacteria and fungi. In a medium containing trypsin and heat-treated bile, heat-treated bile acids, or no bile, 2.5–8% of the oocysts excysted. The findings indicate that satisfactory excystation can be obtained with a treatment medium containing tris-maleate at pH 7.5–8.5, 0.25% trypsin, and 1% of one of the bile acids.

SYNOPSIS. Oocysts of Eimeria bilamellata were found in Spermophilus armatus, Utah and Wyoming; S. beecheyi, California; and S variegatus, Utah. Oocysts were not found in S. lateralis, S. richardsoni, S. columbianus, S. tridecemlineatus, or the white-tailed prairie dog, Cynomys leucurus. Experimental infections were established in S. armatus, S. variegatus, and S. lateralis, but not in S. richardsoni, least chipmunks Eutamius minimus, laboratory rats Rattus norvegicus, or Mongolian gerbils Meriones unguiculatus. After one experimental infection S. armatus and S. variegatus were immune to further infections. Spermophilus lateralis could be infected 3 or 4 times before the animals were immune. However, individuals of S. armatus in a natural population had more than one infection with E. bilamellata; probably infections must be of a certain level before immunity develops. When S. armatus were inoculated with about 100,000 oocysts, the animals usually died on the 7th day after incoculation. Oocysts were 33-37 by 25-31 μ (mean 34.5 by 28.2 μ). The oocyst wall was brown and composed of 2 layers. A distinct micropyle was present. Sporocysts were 18-23 by 9-12 μ (mean 19.9 by 10.3 μ). In experimental infections, the prepatent period was 10 days and the patent period 5–21 (mean 9) days. Schizonts were 1st seen 7 days after inoculation. They were located above the host cell nuclei of epithelial cells at the tips of the villi of the jejunum and ileum. One or more earlier generations of schizonts were thought to occur, but these were not observed. Gametogony took place in epithelial cells of the jejunum and ileum. Shortly after the merozoites entered the cells, the cells became enlarged and were displaced into the lamina propria. The microgametocytes were considerably larger than the macrogametes and contained a central residual body. Macrogametes had a peripheral eosinophilic layer as well as cytoplasmic granules; both apparently participated in formation of the oocyst wall.

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.

Cell cultures of embryonic bovine liver, lamb kidney, whole ground-squirrel embryos and adult Madin-Darby bovine kidney were used to study the in vitro development of Eimeria larimerensis from the Uinta ground-squirrel, Spermophilus armatus. Leighton tubes were inoculated with sporozoites and the monolayers were examined with phase-contrast microscopy at various intervals; fixed and stained material also was studied. In all cell types, sporozoites penetrated and developed to trinucleate second-generation schizonts. The intracellular sporozoites increased in size and underwent nuclear divisions. The sporozoite-shaped schizonts had motility similar to that of the sporozoites. After the formation of 4–10 nuclei, transformation from the sporozoite-shaped schizont to a spheroidal schizont occurred by a lateral outpocketing in the anterior region or a progressive increase in width of the body. In 10 specimens, completion of the transformation process at 37 C required an average of 57 min. The greatest numbers of spheroidal schizonts were seen at 48 hr. Merozoites were apparently formed by a radial budding process, which required an average of 72 min at 37 C. The largest numbers of mature first-generation schizonts were seen at 60 hr after inoculation; these had 4–36 merozoites. Each merozoite had anterior and posterior refractile bodies; some also had small posterior refractile granules. The refractile bodies of the merozoite were derived from the posterior refractile body of the sporozoite. Second-generation trophozoites were seen at 60 hr, and second-generation bi- and trinucleate schizonts were seen at 60 and 72 hr after inoculation.

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.

Two experiments were conducted to investigate the possible presence of humoral antibodies in young Holstein-Friesian calves experimentally infected with Eimeria bovis. A total of 10 calves received 25,000 oocysts and 1 million oocysts 26 or 27 days later, and 8 calves were inoculated once with 1 million oocysts. Blood samples were taken periodically and the sera collected were tested against first-generation merozoites by the indirect method of fluorescent staining and the slide agglutination test, and against an oocyst extract by the precipitin ring test. In vitro observations were also made on the effect of immune serum upon the first-generation merozoites. Fluorescence of the merozoites was first observed in serum collected 10–22 days after inoculation. The calves which received only one large inoculum attained on the average higher titers in less time (mean = 1:462 in 22.1 days) than did the calves inoculated twice (mean = 1:250 in 28.4 days). Agglutinating antibody was first detected 7–17 days after inoculation. However, in this case calves which received a second inoculum developed higher titers (mean = 1:464 in 35 days) than did those inoculated once (mean = 1:150 in 26 days). The results of the precipitin ring tests paralleled closely those of the slide agglutination tests, and suggest that the asexual and sexual stages of E. bovis share certain antigenic properties. The antibody level declined gradually in each calf, but titers detectable by all 3 methods were still present in all calves several months after the last inoculation. First-generation merozoites incubated for 1 hour in whole immune serum or immune serum diluted 1:20 underwent lysis or became immobilized, swollen, and granulated; non-immune whole serum had a similar effect on merozoites, but little or no effect when diluted 1:20.

The fine structure of the macrogametes of coccidia was investigated, described and diagrammatically depicted. A comparative analysis was made of representative species belonging to various genera. The following species were investigated: Eucoccidium dinophili, Aggregata eberthi, Eimeria perforans, E. stiedae, E. falciformis, E. bovis, E. auburnensis, E. tenella, E. maxima, Toxoplasma gondii, and Klossia helicina. The macrogametes of most of these Eimeria species are relatively uniform in their fine structure, but E. falciformis shows some differences and the macrogametes of T. gondii are also different in some respects. In the macrogametes of E. dinophili, A. eberthi, and K. helicina are found a number of special structures not seen in the others.

The microgametogenesis of Eimeria auburnensis from experimentally infected calves killed 16 to 18 days after inoculation was investigated with the electron microscope. Early microgametocytes had nuclei with compact nucleoli and relatively few electron-dense masses. In this stage, deep invaginations from the surface evidently served for the intake of nutrients from the parasitophorous vacuole. In older stages, the nuclei had no compact nucleoli and had more elctrondense material. An intranuclear fiber apparatus was present in some nuclei, apparently in an early stage of division. Nuclear division appeared in electron micrographs to occur by a kind of fission, in which the intranuclear fiber apparatus may participate. Numerous fissures appeared in the interior of the microgametocyte and the nuclei were arranged in irregular rows in association with these. Centrioles, either single or double, were observed between the nuclei and the membrane lining the fissures. Such nuclei often had an intranuclear fiber apparatus; one of the osmiophilic poles of this apparatus protruded outward from the nucleus in the vicinity of the centriole. Directly over this pole, electron-dense material, probably representing the anlage of the perforatorium, occurred immediately beneath the surface membrane of the fissure. As many as 9 closely arranged micropores were observed in this membrane in some specimens. In the nearly mature microgamete, the basal bodies of the flagella lay at the anterior end; the mitochondrion, with numerous, regularly arranged tubules began slightly posterior to this. The strongly osmiophilic, condensed nucleus was a little farther posterior; this remained connected by a narrow stalk with the uncondensed portion of the nucleus in the residual cytoplasm until the microgametes were almost completely mature. Usually, 2 flagella were present. Rarely, 3 flagella were observed, and evidence of a rudimentary middle flagellum was found in a number of specimens.

SYNOPSIS. Macrogamonts in tissues from rabbits killed 5 1/2 days after inoculation with Eimeria magna oocysts were studied with the electron microscope. In young macrogamonts, parts of cytoplasm, sometimes including micronemes, were pinched off into the parasitophorous vacuole. In all stages of development, small segments of the inner membrane complex were present beneath the limiting membrane. Micropores also were seen in all stages, and some apparently functional ones were present in mature macrogametes. Wall-forming bodies of Type I and Type II were observed in relatively early stages. The former were less numerous than the latter, which had a more compact appearance than in other species. Usually, several Golgi complexes were present and several Golgi adjuncts occurred in the vicinity of the nucleus in all stages of development. Microgametes were observed in the cytoplasm of host cells harboring immature macrogametes.

Schizonts, sporonts and sporoblasts of Nosema apis from honey bees collected in the summer and winter were studied with the electron microscope. The nuclei usually had a diplokaryon arrangement. Intranuclear spindles with polar vesicles were associated with division. Schizonts had a single limiting unit membrane, whereas sporonts had a two-layered wall. Sporonts from summer bees had only a thin single limiting membrane in some areas and evidence of endocytosis was sometimes seen in these. Sporonts from winter bees had branched tubular outpocketings from the wall. In sporoblasts, the development of the polar filament was closely associated with a network of dense structures interwoven with a system of tubules evidently of ER derivation; the Golgi complex was associated with this network.

SYNOPSIS. Eimeria vermiformis sp. n. and E. papillata sp. n. are described from the mouse Mus musculus. The sporulated oocysts of E. vermiformis are 18–26 by 15–21 μ (mean 23.1 by 18.4 μ); its sporocysts are 11–14 by 6–10 μ (mean 12.8 by 7.9 p). The sporulated oocysts of E. papillata are 18–26 by 16–24 μ (mean 22.4 by 19.2 μ); its sporocysts are 10–13 by 6–9 μ (mean 11.2 by 8.0 μ). A substiedal body is present in E. papillata sporocysts. Patent infections were produced in white laboratory mice with both species. Fourteen species of Eimeria have now been described from the genus Mus.

Summary Three experiments were conducted to determine whether schizonts of E. bovis are appreciably influenced by the immune reaction in calves. Each experiment involved two groups each of four to six calves. Each calf in one group was given an immunizing inoculation of 60,000 to 100,000 oocysts, and after 30 to 35 days each calf in both groups was given a challenging inoculation of 800,000 to 5 million oocysts. One calf in each group was killed on successive days beginning 15 to 17 days after the second inoculation. Numbers of schizonts in the small intestine were estimated by examination with a dissecting microscope and frequency of sexual stages in the cecum and colon was observed in sections. No consistent difference was found in concentration, estimated total number, or size of schizonts in the two groups, although non-immunized calves had a much higher frequency of sexual stages. The results indicate that the schizonts and preceding stages are not appreciably influenced by the immune reaction, so that immunity must chiefly affect the merozoites or sexual stages.

Mature macrogametes of Eimeria bovis in the mucosa of the colon of calves killed 17 days after inoculation had a characteristic fine structure. Numerous osmiophilic wall-forming bodies of type I were located peripherally in the macrogamete, whereas the wall-forming bodies of type II were relatively large and located more centrally. Numerous polysaccharide bodies and lipid inclusions were also present. The parasitophorous vacuole, when present, was narrow and had electron-pale contents. The host cell cytoplasm was vacuolated; in some specimens the host cell appeared to have disintegrated. A microgamete was observed within a vacuole in the cytoplasm of a macrogamete from a calf killed 19 days after inoculation. This microgamete had two flagella and a homogeneous osmiophilic nucleus. The wall-forming bodies of type I and type II were altered in this macrogamete as compared with those of mature macrogametes, indicating that oocyst wall formation had begun. Many microgametes were observed in the cytoplasm of the host cell as well as in the parasitophorous vacuole of the macrogamete.

The fine structure of the microgamonts and microgametes of coccidia and related groups was investigated and is herein described and diagrammatically depicted. Particular attention is given to the developmental processes during the differentiation of the microgametes. The species included in the study are: Eimeria perforans, E. maxima, E. tenella, E. auburnensis, E. falciformis, and Toxoplasma gondii. A comparative analysis of the developmental processes as well as the fine structure of the microgametes of these species and those of other Sporozoa is presented.

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.

SYNOPSIS. Eimeria scholtysecki n. sp. is described from Ord's kangaroo rat Dipodomys ordii. The sporulated oocysts are broadly ovoid to ellipsoid, averaging 24.6 by 19.6 μ. A polar granule is present. A micropyle and oocyst residuum are absent. The ovoid sporocysts average 12.1 by 8.0 μ, and have small, flattened Stieda bodies. The distinctive sporocyst residuum is composed of coarse granules. The mean prepatent period was 8.2 days. Five inoculated rats apparently became reinfected and discharged oocysts for 30 days or more.

Trichomonas ovis (Robertson, 1932) Morgan, 1946 was found in the ceca of 12 out of 16 domestic sheep in Illinois and Utah and was cultivated readily at 37 C in Diamond's medium, CPLM medium and bovine cecal extract medium. It was studied by phase-contrast microscopy and after fixation with osmium tetroxide or Bouin's or Hollande's fixatives and staining with Giemsa, Heidenhain's iron hematoxylin, or silver impregnation. It was piriform, 6 to 9 by 4 to 8 A with a mean of 7 by 6 ,u, with an anterior nucleus, with a prominent pelta at the anterior end, with a slender, hyaline axostyle which protruded a mean of 5 , beyond the body and gradually tapered to a point, without a chromatic ring at the point of exit of the axostyle, with 4 anterior flagella of unequal length averaging 14, 11, 9, and 7 u, respectively, with a prominent undulating membrane which extended from three-fourths to the full length of the body and usually had two to three undulations, with a posterior, free flagellum extending beyond the undulating membrane, with a prominent costa and several irregular rows of paracostal granules, with an ovoid or club-shaped parabasal body averaging 2 by 1 a and containing an intensely chromophilic body, and with a parabasal filament. This is the only species of enteric trichomonad of sheep or cattle which has been described in detail following study by modern techniques. Robertson (1932) described a trichomonad which he observed in the "gut" in one of 86 sheep in England. He said that this trichomonad possessed only two anterior flagella and therefore proposed the name Ditrichomonas ovis for it. Since that time there appears to have been no further investigation of this parasite or of any other trichomonad from sheep. In 1958, Dr. Krishna N. Mehra of the College of Veterinary Medicine, University of Illinois, Urbana, Illinois, isolated a strain of cecal trichomonad from a domestic sheep (Ovis aries). This strain, labeled "0," has been used since that time in connection with experiments on the survival of various trichomonad species during extended frozen storage. This strain possessed four anterior flagella and also differed in other morphological aspects, such as body size and prominence of the undulating memReceived for publication 15 March 1962. * This investigation was supported in part by a research grant (E790C) from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, and in part from a Utah State University Research Fellowship, Utah State University, Logan, Utah. t Present address: Department of Zoology, Utah State University, Logan, Utah. brane, from the trichomonad described previously by Robertson. Four other strains which were subsequently isolated from sheep during 1961 (one from a sheep raised in Illinois, one from a sheep shipped to Illinois from Texas, and two from sheep killed in Logan, Utah) all appeared identical with strain "0" when observed with the phase-contrast microscope. Because of the obvious differences from the trichomonad described by Robertson as Ditrichomonas ovis, it was decided to make a detailed study of the morphology of these cecal trichomonads. MATERIALS AND METHODS All observations reported in this paper were made on trichomonads of strain "O." Cultures were grown at 37 C in Diamond's (1957) medium without agar or phosphate buffer, in Johnson and Trussell's (1943) CPLM medium, and in bovine cecal extract medium (Jensen, 1961), which was a modification of the cecal extract medium used by Hibler et al. (1960) in their investigation of porcine trichomonads. A Zeiss photomicroscope was used for both phase-contrast and bright-field observations. Morphological studies were based upon observations of living trichomonads, of specimens fixed for one minute in the vapors of 2% osmium tetroxide solution, and of specimens stained with Giemsa's stain

First-generation schizonts of Eimeria callospermophili, E. larimerensis, and E. bilamellata from the Uinta ground squirrel, E. nieschulzi from the rat, and E. ninakohlyakimovae from the sheep were grown in cell lines of embryonic bovine intestine, kidney, and liver. Extracellular merozoites and mature first-generation schizonts with merozoites in monolayers were observed with phase-contrast microscopy for degree of motility. Various agents in minimal essential medium (MEM) were then added to the preparation; these included 0.2% trypsin, 4% bovine bile, 0.5% sodium taurocholate, 0.5% sodium glycotaurocholate, and 0.2% trypsin combined with 4% bovine bile or 0.5% sodium taurocholate; also, MEM with no agents added was used as a control. Little or no motility was observed in preparations to which MEM or trypsin was added. In all preparations to which bile, a bile salt, or one of these in combination with trypsin was added, the extracellular merozoites were markedly motile, and the merozoites still within schizonts except for those of E. ninakohlyakimovae became active and left the host cells. In such preparations, the merozoites flexed and glided more frequently and moved for longer distances than normal; in E. callospermophili, merozoites were observed penetrating new host cells and sporozoite-shaped schizonts were seen leaving host cells. Speer, Hammond, and Anderson (1970) reported that Eimeria callospermophili merozoites which developed in cell cultures underwent a marked increase in motility when treated with a trypsin-bovine bile solution. We report herein additional information concerning stimulation of motility in merozoites of E. callospermophili, E. larimerensis, and E. bilamellata from the Uinta ground squirrel, E. nieschulzi from the rat, and E. ninakohlyakimovae from sheep. All of the above species of coccidia were grown in cell cultures. MATERIALS AND METHODS Cell lines of embryonic bovine intestine, kidney, and liver were used in this investigation. The methods for maintaining and examining the cell monolayers were similar to those described by Fayer and Hammond (1967). Oocysts were collected and handled as in previous work (Speer, Hammond, and Anderson, 1970; Kelley and Hammond, 1970). Monolayers from Leighton tube cultures were examined in double-coverslip preparations (Parker, 1961) with phase-contrast microscopy at the appropriate interval after inoculation of sporozoites of each Eimeria species at which mature schizonts usually occur (24 hr after inoculation for E. callospermophili, 36 hr for E. nieschulzi, 48 hr for E. larimerensis, 4 days for E. bilamellata, and 10 days for E. ninakohlyakimovae). Extracellular merozoites or mature first-generation Received for publication 17 February 1970. Supported in part by research grant AI-07488 from the NIAID, U. S. Public Health Service. Published as Journal Paper No. 1005, Utah Agricultural Experiment Station. schizonts with merozoites were located and the degree of motility was observed before applying various solutions of trypsin and/or bile salts or bovine bile in minimal essential medium (MEM, Nutritional Biochemical) to the edge of the double cover slip. The solutions included 0.2% trypsin (1-300, Nutritional Biochemical) and 4% bovine bile, 0.2% trypsin and 0.5% sodium taurocholate (bile salt), 0.2% trypsin, 0.5% sodium taurocholate, 0.5% sodium glycotaurocholate, 4% bovine bile, and fresh MEM as a control. The pH of all solutions was adjusted to approximately 7.4 with a 7.5% sodium bicarbonate solution. During observation, the monolayers were maintained at 37 C with the aid of a warm stage and additional examinations in each experiment were conducted at room temperature (about 22 C).

During the past several years an intensive study of bovine coccidia has been under way at this laboratory. Since Eimeria bovis is so widespread and causes such serious losses, it has received more attention than the other species. Christensen (1941) pointed out that E. smithi is a synonym of E. bovis, redescribed the unsporulated o6cyst, and determined the time required for sporulation under optimum conditions. Boughton (1942) reported that the cyst-like structures in the mucosa of the small intestines of cattle described under the names of Globidium and Gastrocystis are the schizonts of E. bovis. He (1943) found also that sulfaguanidine can be used as a preventive in experimentally inoculated calves. Hammond, Davis, and Bowman (1944) described the symptoms and pathology observed in calves inoculated with this species, and recorded a definite relation between the size of the inoculum and the symptoms produced. This paper reports the results of further studies of this parasite with emphasis on the endogenous phase of its life cycle.