Development of Eimeria callospermophili and E. bilamellata from the Uinta Ground Squirrel Spermophilus armatus in Cultured Cells*

SYNOPSIS. Cell lines or established cell lines of bovine, ovine or human origin and primary cells from whole embryos of groundsquirrels were used in a study of the in vitro development of Eimeria callospermophili and E. bilamellata from the Uinta ground squirrel, Spermophilus armatus. Monolayers in Leighton tube cultures were inoculated with sporozoites of either of these 2 species and examined with phase-contrast microscopy at various intervals. After such examination, coverslips were fixed in Schaudinn's or Zenker's fluid and variously stained. E. callospermophi sporozoites penetrated cells and underwent development to mature 1st generation schizonts in most cell types. At different times after inoculation, both species formed sporozoite-shaped schizonts, which later became spheroidal. Intracellular movements of sporo zoite-shaped schizonts of E. callospermophili were observed and such schizonts penetrated cells when freed by mechanical disintegration of the host cells. Merozoites were formed at the periphery of the schizont in both species. Mature 1st generation schizonts of E. callospermophili, with 6–14 merozoites, were first seen 15 hr after inoculation; the corresponding values for E. bilamellata were 12–27 merozoites and 4 days. Merozoites of both had anterior and posterior refractile bodies. Exposure to a trypsin-bile solution stimulated motility in merozoites of E. callospermophili. Second generation trophozoites and immature schizonts of E. callospermophili were seen in cultures of primary cells of whole ground-squirrel embryos 20–24 hr and 44–48 hr, respectively, after inoculation of sporozoites.     

Comparative Development of Eimeria tenella from Sporozoites to Oocysts in Primary Kidney Cell Cultures from Gallinaceous Birds

Eimeria tenella completed its endogenous life cycle in primary cultures of kidney cells from 2- to 3-week-old-chickens, guinea fowl, partridges, pheasants, quail, and turkeys. Similarity in percentage of infection at 4 hr suggested that sporozoites entered cells from all birds in equal numbers. Development was better, however, in chicken cells in that the percentage of survival and of developmental stages during the first 2 days were greater, developmental stages occurring after 2 days usually were found earlier, mature 2nd-generation schizonts and oocysts were larger, and oocyst production was far greater than in nonhost cells. Multinucleate macrogametes, which sometimes reached sizes 3–4 times greater than normal oocysts, are reported for the first time.     

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 meleagrimitis Tyzzer from Sporozoites and Merozoites in Turkey Kidney Cell Cultures*

SYNOPSIS. Sporozoites and 1st-, 2nd-, and 3rd-generation merozoites of Eimeria meleagrimitis were inoculated into primary cultures of turkey kidney cells. In vitro-excysted sporozoites developed into mature macrogamonts in 8 days; in vivo-excysted sporozoites developed into 2nd- or 3rd-generation schizonts within 5 to 7 days. First-generation merozoites obtained from infected turkeys produced mature 2nd-generation schizonts within 24 h. Second-generation merozoites from turkeys produced mature macrogamonts and oocysts within 72 h, whereas 3rd-generation merozoites produced these stages within 48 h. The oocysts that developed from 3rd-generation merozoites sporulated at 25 C and were infective for turkeys. The timing of the early stages and the intervals between schizogonic generations in cultures were comparable with those in turkeys. Morphologic parameters, however, indicated that some differences existed between in vitro and in vivo development. Second- and 3rd-generation schizonts and gamonts that developed after inoculation of cultures with merozoites were similar to stages in turkeys. Oocysts, however, were significantly smaller (P < 0.05) in cultures. All stages that developed after inoculation of cultures with sporozoites were smaller (P < 0.05) than their in vivo counter parts.     

Development of Eimeria crandallis Honess from Sheep in Cultured Cells*

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

Sporogonic development of Hepatozoon americanum (Apicomplexa) in its definitive host, Amblyomma maculatum (Acarina)

Light microscopic observations of the sporogonic development of Hepatozoon americanum are described in its acarine host, Amblyomma maculatum. Laboratory-reared nymphal ticks were fed on 2 dogs infected with H. americanum. Nymphal ticks were sampled daily, starting 3 days after being placed on a parasitemic dog, until 18 days after infestation (PI), and then every 3 or 4 days until replete nymphs molted. Ticks were examined as unstained wet mounts and hematoxylin-eosin-stained paraffin sections. Gametes were found within the gut cells of nymphs 4 and 6 days PI. Although differentiation of gamonts into gametes was not detected, syngamy and sporogony were observed. Sporogony appears to occur wholly within tick gut cells, followed by release of mature oocysts into the hemocoel. The earliest evidence of sporoblast formation was observed 23 days PI and of sporozoite formation, 10 days later. Mature oocysts were first found 42 days PI in newly molted adult ticks. Most adult ticks (>98%) that were dissected contained mature oocysts. Oocysts were multisporocystic, and sporocysts contained a variable number of sporozoites. Oocysts in various stages of development were often seen within the same tick, and the number of mature oocysts ranged from 4 to 573.     

Characterization in vitro and in vivo of resistance to ionophores in a strain of Eimeria tenella.

A field isolate of Eimeria tenella (FS139) was propagated several times in chickens medicated with 200 ppm of dietary monensin. In a laboratory test with 2-wk-old-chickens, the strain was resistant to monensin, salinomycin, and lasalocid given at double use level and was resistant to narasin and maduramicin at the normal use level. In comparison, a laboratory strain (WIS) was controlled by the normal use level of each product. When free WIS sporozoites were treated in vitro with 1.0 microgram/ml of monensin for 0.5 or 4.0 hr at 41 C and inoculated into primary cultures of chicken kidney cells the invasion was reduced by 35.6% or 96.3%, but invasion of FS139 sporozoites was increased by 18.5% by 0.5 hr treatment and was about the same as controls after 2 hr of treatment. Few sporozoites from the WIS strain developed into schizonts, but numerous sporozoites from the FS139 strain developed into normal first and second generation schizonts. The structure of free WIS sporozoites was distorted after 3 hr of treatment with 2.5 micrograms/ml of monensin at 41 C, as observed by light and scanning electron microscopy, whereas there was no change in structure of most treated FS139 sporozoites.     

Hepatozoon ursi n. sp. (Apicomplexa: Hepatozoidae) in Japanese black bear (Ursus thibetanus japonicus)

Morphological and genetic features of a new Hepatozoon species, Hepatozoon ursi n. sp., in Japanese black bear (Ursus thibetanus japonicus) were studied. Schizogonic developmental stages were observed in the lungs of Japanese black bears. The schizonts were sub-spherical in shape and 45.7+/-4.6 x 42.7+/-4.5 microm in size. Each mature schizont contained approximately 80-130 merozoites and 0-5 residual bodies. The merozoites were 7.0+/-0.7 x 1.8+/-0.3 microm in size. Intraleukocytic gametocytes were slightly curved, cigar-like in shape and had a beak-like protrusion at one end. The size of the gametocytes was 10.9+/-0.3 x 3.3+/-0.2 microm. The analyses of the18S rRNA gene sequences supported the hypothesis that H. ursi n. sp. is different from other Hepatozoon species. Mature Hepatozoon oocysts were detected in two species of ticks (Haemaphysalis japonica and Haemaphysalis flava) collected on the bears infected with H. ursi n. sp. Two measured oocysts were 263.2 x 234.0 microm and 331.8 x 231.7 microm, respectively. The oocysts contained approximately 40 and 50 sporocysts, respectively. The sporocysts were sub-spherical in shape and 31.2+/-2.5 x 27.0+/-2.9 microm in size. Each sporocyst contained at least 8-16 sporozoites, with the sporozoites being 12.2+/-1.4 x 3.5+/-0.5 microm in size. H. ursi n. sp. is the first Hepatozoon species recorded from the family Ursidae.     

Endogenous development of Eimeria intestinalis in rabbits (Oryctolagus cuniculus).

The endogenous life cycle of a pure strain of Eimeria intestinalis was studied by light and electron microscopy in coccidia-free rabbits. Four schizont generations could be observed: the first one, not previously described, was seen between 36 and 144 hr postinoculation (PI), the second one between 64 and 168 hr PI, the third one between 96 and 192 hr PI, and the fourth one between 168 and 240 hr PI. Gamogony apparently started as early as 144 hr PI. Thus, it was possible for oocysts to develop from third generation merozoites, later oocysts developing after the fourth schizont generation. Electron microscopic observation suggested that oocysts were derived mainly from merozoites of the fourth schizont generation. During the first stage of the life cycle, sporozoites were seen in intraepithelial lymphocytes. All asexual generations, except the fourth, were characterized by 2 schizont types: the first, regarded as female, contained mononuclear merozoites and the second, regarded as male, contained polynuclear merozoites.     

The Life Cycle of Eimeria bateri (Protozoa, Eimeriidae) in the Japanese Quail Coturnix coturnix japonicum

SYNOPSIS. Eimeria bateri, a parasite of the Indian quail, was described from laboratory infections in Japanese quail. First generation schizonts developed in the glands of the duodenum and upper intestine. Second, 3rd and 4th generation schizonts and gametocytes occurred in the villous epithelium. There was a gradual spread along the small intestine until the whole organ was affected. The prepatent period was 4 days and the patent period 6 days. Graded doses from 5,000 to 1,280,000 oocysts produced very little pathogenic effect in young quail. E. bateri did not infect young pheasants or chicks.     

Eimeria maxima (Apicomplexa): a comparison of sporozoite transport in naive and immune chickens

This study compared the early stages of infection in naive and immune chickens infected with Eimeria maxima. An immunoperoxidase stain was developed and used to detect sporozoites and early schizonts in tissue sections of intestinal epithelium. A significantly higher proportion of sporozoites was present in the crypts of naive chickens, 48 hr postinoculation of oocysts, compared to immune chickens. Sporozoites in immune birds tended to remain in the lamina propria rather than migrate to the crypts. Sporozoites were found within intraepithelial lymphocytes (IEL's) in the epithelium, the lamina propria, and the crypts of both naive and immune chickens. Parasites in IEL's of immune birds at the ultrastructural level and there were no apparent morphological abnormalities. Livers and spleens, of both immune and naive chickens that had been inoculated with Eimeria maxima, produced patent infections when fed to susceptible chickens. Infections could be transferred up to 72 hr post-inoculation of the donor birds. Peak oocyst production in the recipient birds occurred 7-8 days after the transfers. This time period approximates the prepatent period in a natural infection and thus implies that the extraintestinal stage was a sporozoite.     

Endogenous stages of Eimeria tuskegeensis (Protozoa: Eimeriidae) in the cotton rat, Sigmodon hispidus

Endogenous stages of Eimeria tuskegeensis were studied in experimentally infected cotton rats, Sigmodon hispidus. Almost all parasites were located on the basilar side of the nucleus in epithelial cells on the sides and tips of villi of the small intestine. The endogenous cycle consisted of three generations of schizogony followed by gametogony. First-, second-, third-generation schizonts could be distinguished by time of appearance, size and shape of the schizont, and number, size, shape, and arrangement of merozoites. Immature gametogonous stages appeared to 84 hr postinoculation (PI) and developed into mature microgametocytes and macrogametes by 96 hr PI. Microgametocytes had a mono-centric type of development. Intermediate macrogametes had small, basophilic wall-forming bodies and mature macrogametes had large, eosinophilic wall-forming bodies. It was not possible to determine whether these were two distinct types of wall-forming bodies or whether they were different stages of a single type. Two nuclei were seen in the host's epithelial cells parasitized by schizonts, microgematocytes, macrogametes, and oocysts. This binucleate condition was apparently parasite-induced.     

Transfected Eimeria tenella could complete its endogenous development in vitro

Live attenuated coccidiosis vaccines could be used as powerful carriers, expressing exogenous viral and bacterial antigens, to induce protective immunity against pathogenic organisms. We investigated the ability of Eimeria tenella to express an exogenous gene in vitro. Eimeria tenella sporozoites were transfected with the plasmid pH4-2EYFP-Actin3 containing the yellow fluorescent protein gene (yfp) and inoculated into primary chicken kidney cells (PCKCs), followed by incubation at 41 C in a 5% CO2 chamber. Fluorescent sporozoites were observed as early as 15-20 hr post-inoculation (PI). Fluorescence displayed by the expressed YFP protein was visible throughout the schizogony and gametogony stages of the tranfected E. tenella. Fluorescent oocysts were found between 200-327 hr PI. Higher fluorescence intensity was observed in the nucleus than in other compartments of the transfectants, while little or no fluorescence was seen in the refractile globule. The diversity of schizonts, particularly of the first generation, was presented by fluorescent nuclei arranged in different patterns. Our results demonstrated the ability of E. tenella to express an exogenous gene throughout the endogenous development in vitro. Completion of the endogenous development of transfected E. tenella in cell cultures will facilitate the study of foreign antigen expression in Eimeria spp., paving the way for the development of an Eimeria spp. vector vaccine that also carries and delivers other vaccines by oral administration.     

Development of Eimeria auburnensis in Cell Cultures

SYNOPSIS. Monolayer established cell line cultures of bovine kidney (Madin-Darby) and human intestine (Intestine 407), as well as embryonic bovine tracheal and embryonic spleen cell line cultures were inoculated with E. auburnensis sporozoites and observed for a maximum of 22 days. Mature 1st generation schizonts developed in the kidney, tracheal and spleen cells. In the intestine cells, trophozoites were seen in 3 of 4 experiments, but schizonts were not found. Sporozoites penetrated cells, beginning within a few minutes after inoculation. Penetration was usually accomplished within 10 seconds, and the body of the sporozoite underwent a slight constriction as it passed thru the host cell membrane. Some sporozoites left cells. Numerous intracellular sporozoites were observed in kidney, tracheal and spleen cultures. Crescent bodies were seen in the parasitophorous vacuole as early as 1 day after inoculation. At this time, the nuclei of most intracellular sporozoites had changed from vesicular to compact. Beginning 4 days after inoculation, enlarged sporozoites and parasites having a sporozoite shape, but with 2-5 nuclei, were frequently seen. These enlarged sporozoites and sporozoite-shaped schizonts evidently transformed into trophozoites and spheroidal schizonts by means of lateral outpocketings. Few trophozoites were seen. More immature schizonts developed in kidney cells than in the other cell types. The numbers of mature schizonts observed in kidney and tracheal cells were similar, but development occurred less consistently in the latter. Few immature and mature schizonts developed in spleen cells. Mature schizonts, first seen 9 days after inoculation, were considerably smaller than those reported from calves. Some motile merozoites were seen; evidently no development beyond these occurred. The nucleus and nucleolus of host cells were enlarged; this enlargement was not as pronounced as in infections in calves. Multiple host cell nuclei were frequently observed. Degenerative changes in the cultured cells and in the parasites usually occurred, beginning 9-17 days after inoculation; these were more pronounced in the spleen cells than in the others.     

Production of quail (Coturnix japonica) germline chimeras by transfer of gonadal primordial germ cells into recipient embryos

The possibility of producing quail germline chimeras by the transfer of gonadal primordial germ cells (gPGCs) into recipient embryos was investigated. Japanese quail of the black (D: homozygous for the autosomal incomplete dominant gene D) and wild-type plumage (WP: d+/d+) strains were used as donors and recipients, respectively. Gonadal cells were retrieved from the gonads of 5-day-old D embryos, and gPGCs were enriched by magnetism-activated cell sorting. Fresh (noncultured) gPGCs or those isolated after culture for 3 days with gonadal stromal cells present in the mixed cell population were introduced into the dorsal aorta of 2-day-old recipient WP embryos. Hatchability of the recipient embryos was 23.7% (31/131) and 34.4% (31/90) for those transfused with cultured or noncultured gPGCs, respectively. Of the hatched quail, 28 acquired sexual maturity; among these animals, 7.1% (1/14) and 21.4% (3/14) of those that received cultured or noncultured gPGCs, respectively, were proved to be germline chimeras. The percentage of germline transmission to the donor-derived gametes in the chimeras that received cultured and noncultured gPGCs were 1.9 and 2.2-4.7%, respectively. In conclusion, quail gPGCs retrieved from 5-day-old embryos were thus transmitted in the germline after their transfer to quail embryos of a different strain. This property of the gPGCs was not adversely affected by culture for up to 3 days.     

Complete development of Isopora suis of swine in chicken embryos

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

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.     

The life cycle and pathogenicity of coccicium Eimeria nocens (Kotlán, 1933) in domestic goslings

Twenty-four 10-day-old, artificially reared, coccidia-free goslings (Anser cygnoides var. domestica) were inoculated orally with 1.0x10(5)-1.0x10(6) sporulated oocysts of Eimeria nocens, and killed at intervals from 30 to 336 hr postinoculation (PI). Parts of the visceral organs, including intestines, kidney, liver, gallbladder, and spleen from inoculated goslings, were fixed and sectioned. The life cycle of E. nocens and histologic changes during infection were examined microscopically. The results showed that at least 3 generations of meronts developed in the endogenous stage of the life cycle of E. nocens. Two types of meronts were found. The first completed maturation at 54 to 78 hr PI. These meronts were the first generation, with each forming about 12 merozoites. The second completed maturation at 102 to 240 hr PI. These meronts were the second or third generations, with each meront forming about 24 merozoites. Development of gamonts began at about 198 hr after infection. The prepatent period was 9 days and discharge of oocysts continued for 4 days. Sporulation of oocysts occurred in 60-72 hr at 25 C. Eimeria nocens invaded the posterior jejunum, ileum, caecum, rectum, and cloaca. Developmental stages were localized within the epithelial cells of villi and crypts, and in lamina propria. Marked histological changes, including desquamation and necrosis of intestinal epithelium, submucosal edema, hemorrhages, infiltration of inflammatory cells, and villous atrophy, were seen during the periods of late merogony, gamogony, and oocyst shedding. They were most pronounced in the ileum and the regions nearby. The infected goslings showed severe diarrhea, bloody feces, anorexia, emaciation, and even death, suggesting that E. nocens is highly pathogenic for goslings.     

Ultrastructure of cytoplasmic and nuclear changes in Eimeria tenella during first-generation schizogony in cell culture.

Eimeria tenella sporozoites were inoculated into primary cultures of chick kidney cells. Cells fixed from 1 1/2 to 54 hr later were examined with the electron microscope. At 1 1/2 and 24 hr, most intracellular sporozoites were fusiform and retained organelles typical of extracellular sporozoites. However, at 35 hr, rounded trophozoites were present without these structures; only a refractile body, nucleus, mitochondria, and endoplasmic reticulum remained. Binucleate parasites were also present at that time, but at 48 hr many multinucleate schizonts were present. Nuclei, with adjacent conoids, were at the periphery of these schizonts. Partly developed merozoites, each containing a conoid and a nucleus, protruded into the parasitophorous vacuole. At 54 hr, fully developed merozoites were separated from the residual body. Merozoites resembled sporozoites but lacked the large refractile bodies seen in sporozoites. Linear inclusions were present near the merozoite nucleus and in the residual body. Round vacuoles and ribosomes were also found in the residuum. Nucleoli were first seen in sporozoite nuclei at 1 1/2 hr. They were also present in merozoites but were more prominent in trophozoites and schizonts. Peripheral and scattered nuclear heterochromatins were prominent in intracellular sporozoites and diminished in trophozoites, but increased after several nuclear divisions and were again prominent in the merozoite. Small, distinct interchromatin granules were found in all stages. Intranuclear spindles, centrocones, and centrioles were found in connection with nuclear divisions. Ultrastructure of first-generation schizogony in cell culture was similar to that described for second-generation E. tenella in the chicken and to schizogony of other species of Eimeria.