Caryocyst-Like Host Cell Formation by Caryospora duszynskii (Apicomplexa: Eimeriidae) in Human Fetal Lung Cell Cultures1

ABSTRACT. Sporozoites of the coccidium, Caryospora duszynskii, penetrated human fetal lung cell cultures but did not undergo asexual or sexual multiplication during a 29-day observation period. Beginning three days postinoculation (PI), infected host cells lost their normal elongated fibroblast-like shape and became ellipsoidal in appearance and resembled caryocysts. These caryocyst-like infected cells were observed from 3 through 29 days PI. Sporozoites remained viable throughout the study as evidenced by motility of extracellular sporozoites in infected human fetal lung cell cultures. Results of this in vitro study suggest that some species of Caryospora may form caryocysts in secondary hosts without undergoing asexual or sexual multiplication in these hosts.     

Complete development of Caryospora bigenetica (Apicomplexa: Eimeriidae) in vitro

The development of Caryospora bigenetica in vitro is described by light microscopy. Sporozoites from snake-derived oocysts were purified and inoculated onto cultures of primary testicle cells of the cotton rat, cotton rat kidney cells, and human fetal lung cells. Intracellular sporozoites were observed one and two days postinoculation (DPI). Motile, extracellular first-generation merozoites were present 3 DPI, and second-generation merozoites were present 5 DPI. Mature gamonts were observed 9 DPI and developed into unsporulated oocysts by 10 DPI. Oocystes sporulated in vitro, and excystation was observed. Cells that were penetrated by in vitro-produced sporozoites formed caryocysts by 16 DPI. To test infectivity of in vitro-derived stages, merozoites were removed from cultured cells 5 DPI and inoculated intraperitoneally into a mouse; infection resulted. Sporulated oocysts removed from cell cultures 12 DPI produced facial swelling in an orally inoculated cotton rat.     

Complete Development of Caryospora bigenetica (Apicomplexa: Eimeriidae) In Vitro1

The development of Caryospora bigenetica in vitro is described by light microscopy. Sporozoites from snake-derived oocysts were purified and inoculated onto cultures of primary testicle cells of the cotton rat, cotton rat kidney cells, and human fetal lung cells. Intracellular sporozoites were observed one and two days postinoculation (DPI). Motile, extracellular first-generation merozoites were present 3 DPI, and second-generation merozoites were present 5 DPI. Mature gamonts were observed 9 DPI and developed into unsporulated oocysts by 10 DPI. Oocysts sporulated in vitro, and excystation was observed. Cells that were penetrated by in vitro-produced sporozoites formed caryocysts by 16 DPI. To test infectivity of in vitro-derived stages, merozoites were removed from cultured cells 5 DPI and inoculated intraperitoneally into a mouse; infection resulted. Sporulated oocysts removed from cell cultures 12 DPI produced facial swelling in an orally inoculated cotton rat.     

Serial transmission of Caryospora bigenetica Wacha and Christiansen, 1982 (Apicomplexa: Eimeriidae) between different species of rodents

Transmission of Caryospora bigenetica by cannibalism between cotton rats, between cotton rats and mice, and between mice was demonstrated. All experimental animals developed swollen muzzles and scrota 8 days after ingestion of infected tissues. Infections were confirmed by light microscopy of fresh tissue smears. Tissues of cannibalized animals contained caryocysts that, after ingestion by the next host, released sporozoites that underwent merogony, gamogony, sporogony, and caryocyst formation in dermal tissues. This study demonstrates that C. bigenetica can be transmitted by predation between species of rodents and that, in the recipient host, asexual and sexual reproduction occur before caryocysts appear.     

Development of the swine coccidium Eimeria debliecki Douwes, 1921 in mammalian cell cultures

Sporozoites of Eimeria debliecki entered human fetal lung and porcine kidney cells grown in cultures and underwent one merogenous cycle, terminating in the production of second-generation trophozoites. Sporozoites were intracellular 1 h post-inoculation (PI) and developed into sporozoite-shaped meronts at 40 h PI. These meronts, one of which was motile, had from two to ten nuclei. Sporozoite-shaped meronts then developed into elongate or spheroidal meronts with 10 to 24 nuclei by two days PI. Ten to 26 first-generation merozoites were formed by budding from the meront surface. Mature first-generation merozoites were most numerous three days PI. Most meronts had ruptured and released nonmotile merozoites into the culture medium by four days PI. Merozoites that were not released became rounded and developed into second-generation trophozoites. Refractile bodies were present in all developmental stages. No further development was observed five through eight days PI.     

Extraintestinal Development of Caryospora simplex (Apicomplexa: Eimeriidae) in Experimentally Infected Mice,Mus musculus1

Developmental stages of Caryospora simplex were found in connective tissue of the cheek, tongue, and nose of Swiss-Webster and C57 BL/6 mice (Mus musculus) from 8 through 70 days after oral inoculation with 50,000 or 250,000 oocysts, or 60,000 free sporocysts of the same species obtained from an Ottoman viper, Vipera xanthina xanthina. The earliest developmental stages were seen on day 8 post-inoculation (PI) and consisted of two types of meronts and gamonts (undifferentiated sexual stages). Gamonts, microgametocytes, macrogametes, and unsporulated oocysts were found on days 10 and 12 PI. Fully sporulated, thin-walled oocysts containing eight sporozoites surrounded by a thin sporocyst membrane were first seen 12 days PI. Monozoic cysts (caryocysts) were first seen 12 days PI and appeared fully viable throughout the duration of the study, 70 days PI. Four mice injected intra-peritoneally with 150,000 free sporozoites and killed 12 days PI contained unsporulated and sporulated oocysts in connective tissues of the cheek, tongue, and nose, suggesting that sporozoites may be carried to the site of infection via the lymphatic/circulatory system. Four cotton rats, Sigmodon hispidus, inoculated orally with 250,000 oocysts all had unsporulated and sporulated oocysts of C. simplex in connective tissue of the cheek, tongue, and nose when killed on day 12 PI, indicating extraintestinal development in the secondary host is not species specific. This is the first report of a heteroxenous coccidium with both asexual and sexual development in the primary (predator) and secondary (prey) hosts.     

Extraintestinal development of Caryospora simplex (Apicomplexa: Eimeriidae) in experimentally infected mice, Mus musculus

Developmental stages of Caryospora simplex were found in connective tissue of the cheek, tongue, and nose of Swiss-Webster and C57 BL/6 mice (Mus musculus) from 8 through 70 days after oral inoculation with 50,000 or 250,000 oocysts, or 60,000 free sporocysts of the same species obtained from an Ottoman viper, Vipera xanthina xanthina. The earliest developmental stages were seen on day 8 post-inoculation (PI) and consisted of two types of meronts and gamonts (undifferentiated sexual stages). Gamonts, microgametocytes, macrogametes, and unsporulated oocysts were found on days 10 and 12 PI. Fully sporulated, thin-walled oocysts containing eight sporozoites surrounded by a thin sporocyst membrane were first seen 12 days PI. Monozoic cysts (caryocysts) were first seen 12 days PI and appeared fully viable throughout the duration of the study, 70 days PI. Four mice injected intra-peritoneally with 150,000 free sporozoites and killed 12 days PI contained unsporulated and sporulated oocysts in connective tissues of the cheek, tongue, and nose, suggesting that sporozoites may be carried to the site of infection via the lymphatic/circulatory system. Four cotton rats, Sigmodon hispidus, inoculated orally with 250,000 oocysts all had unsporulated and sporulated oocysts of C. simplex in connective tissue of the cheek, tongue, and nose when killed on day 12 PI, indicating extraintestinal development in the secondary host is not species specific. This is the first report of a heteroxenous coccidium with both asexual and sexual development in the primary (predator) and secondary (prey) hosts.     

Development of the Swine Coccidium Eimeria debliecki Douwes, 1921 in Mammalian Cell Cultures1

Sporozoites of Eimeria debliecki entered human fetal lung and porcine kidney cells grown in cultures and underwent one merogenous cycle, terminating in the production of second-generation trophozoites. Sporozoites were intracellular 1 h post-inoculation (PI) and developed into sporozoite-shaped meronts at 40 h PI. These meronts, one of which was motile, had from two to ten nuclei. Sporozoite-shaped meronts then developed into elongate or spheroidal meronts with 10 to 24 nuclei by two days PI. Ten to 26 first-generation merozoites were formed by budding from the meront surface. Mature first-generation merozoites were most numerous three days PI. Most meronts had ruptured and released nonmotile merozoites into the culture medium by four days PI. Merozoites that were not released became rounded and developed into second-generation trophozoites. Refractile bodies were present in all developmental stages. No further development was observed five through eight days PI.     

Ultrastructure of In Vivo-Produced Caryocysts Containing the Coccidian Caryospora bigenetica (Apicomplexa: Eimerüdae)

A cotton rat was inoculated orally with oocysts of Caryospora bigenetica from the feces of a rattlesnake. Sixteen days later the rat was euthanized, and portions of the scrotum, foot pad and muzzle were processed for histological sections and transmission electron microscopy. Sporozoites within caryocysts had typical coccidian features such as an anterior and posterior refractile body, centrally located nucleus, micronemes, rhoptries, a conoid, a micropore near the anterior refractile body, a posterior pore, amylopectin granules, lipid bodies, a Golgi-like body, a mitochondrion and subpellicular microtubules. The infected host cell was spherical and surrounded by a fibrous wall-like covering, 0.35–1.00 μm thick. This outer covering, when viewed in stained histological sections, was periodic acid-Schiff (PAS)-positive.     

Ultrastructure of in vivo-produced caryocysts containing the coccidian Caryospora bigenetica (Apicomplexa: Eimeriidae)

A cotton rat was inoculated orally with oocysts of Caryospora bigenetica from the feces of a rattlesnake. Sixteen days later the rat was euthanized, and portions of the scrotum, foot pad and muzzle were processed for histological sections and transmission electron microscopy. Sporozoites within caryocysts had typical coccidian features such as an anterior and posterior refractile body, centrally located nucleus, micronemes, rhoptries, a conoid, a micropore near the anterior refractile body, a posterior pore, amylopectin granules, lipid bodies, a Golgi-like body, a mitochondrion and subpellicular microtubules. The infected host cell was spherical and surrounded by a fibrous wall-like covering, 0.35-1.00 microns thick. This outer covering, when viewed in stained histological sections, was periodic acid-Schiff (PAS)-positive.     

Development of Hammondia heydorni in cultured bovine and ovine cells

Sporozoites were excysted from oocysts of Hammondia heydorni obtained from a naturally-infected dog and inoculated into monolayer cultures of bovine pulmonary artery endothelial cells (CPA), Madin-Darby bovine kidney (MDBK) cells, bovine monocytes (M617), or ovine monocytes (WOMO). Sporozoites penetrated all four cell lines and underwent asexual reproduction by endodyogeny (as determined by electron microscopy) to form cyst-like structures at four to nine days after sporozoite inoculation (DAI). At 4-10 DAI, considerably more zoites were harvested from M617 cultures (80.1 x 10(6) zoites) than from CPA (17.4 x 10(6], MDBK (47.3 x 10(6], and WOMO (53.5 x 10(6]. Little or no parasite multiplication occurred at 10-16 DAI. Zoites harvested at 7 DAI and transferred to freshly prepared cultures did not penetrate cells nor develop further.     

Examination of tissue cyst formation by Toxoplasma gondii in cell cultures using bradyzoites, tachyzoites, and sporozoites

Tissue cyst formation by a goat isolate (GT-1) of Toxoplasma gondii was examined in bovine monocyte, human fetal lung, and Madin-Darby bovine kidney cell cultures. Transmission electron microscopy (TEM) and cat feeding studies indicated that tissue cysts were present in all 3 cell lines examined. Tissue cysts were first seen 3 days postinoculation (PI) using TEM. Standard cell culture procedures were used and no additional condition was needed to induce tissue cyst formation. Cats fed cell cultures excreted T. gondii oocysts in their feces 5-7 days PI. These oocysts caused lethal infections in mice. Tissue cysts were produced in cell cultures regardless if the initiating inoculum consisted of bradyzoites, sporozoites, or a mixture of bradyzoites and tachyzoites. Tissue cyst formation has been followed through 40 subpassages of infected cells. By TEM tissue cysts still were present after 40 passages, but when 40th-passaged cultures were fed to cats, oocytsts were not excreted. This indicates that the parasite had become oocystless after repeated passage in vitro.     

Development of Caryospora simplex (Apicomplexa: Eimeriidae) from sporozoites to oocysts in human embryonic lung cell culture

Leighton tubes containing monolayers of human embryonic lung cells were inoculated with 70,000 or 30,000 sporozoites of the viperid coccidium Caryospora simplex and examined at 1, 2, 4, 6, 8, 10, 12, 14, 16, and 18 days post-inoculation (PI). By day 1 PI, sporozoites had penetrated cells and were within parasitophorous vacuoles. Most sporozoites became spherical and then underwent karyokinesis several times between days 2 and 6 PI. Mature Type I meronts were found on days 6-16 PI and contained 8 to 22 short, stout merozoites. Mature Type II meronts were present on days 10-18 PI and contained 8 to 22 long, slender merozoites. Developing gamonts (undifferentiated sexual stages) were observed on days 14 and 16 PI. Mature micro- and macrogametes and thin-walled unsporulated oocysts were present on days 16 and 18 PI. Attempts to sporulate oocysts in tissue culture medium or in a 2.5% (w/v) aqueous solution of K2Cr2O7 at 25 degrees C and 37 degrees C were unsuccessful; only a few oocysts developed to the contracted sporont stage. Four Swiss-Webster mice injected intraperitoneally with merozoites obtained from Leighton tubes on day 10 PI did not acquire infections. This is the second coccidium reported to complete its entire development, from sporozoite to oocyst, in cell culture.     

Development of Hammondia heydorni in Cultured Bovine and Ovine Cells1

Sporozoites were excysted from oocysts of Hammondia heydorni obtained from a naturally-infected dog and inoculated into monolayer cultures of bovine pulmonary artery endothelial cells (CPA), Madin-Darby bovine kidney (MDBK) cells, bovine monocytes (M617), or ovine monocytes (WOMO). Sporozoites penetrated all four cell lines and underwent asexual reproduction by endodyogeny (as determined by electron microscopy) to form cyst-like structures at four to nine days after sporozoite inoculation (DAI). At 4–10 DAI, considerably more zoites were harvested from M617 cultures (80.1 times 10⁶ zoites) than from CPA (17.4 times 10⁶), MDBK. (47.3 times 10⁶), and WOMO (53.5 times 10⁶). Little or no parasite multiplication occurred at 10–16 DAI. Zoites harvested at 7 DAI and transferred to freshly prepared cultures did not penetrate cells nor develop further.     

Development of Caryospora bigenetica n. sp. (Apicomplexa,Eimeriidae) in Rattlesnakes and Laboratory Mice1

During a survey of the coccidian parasites of reptiles from Iowa, three specimens of Crotalus horridus L., the Timber Rattlesnake, and one of Sistrurus catenatus (Rafinesque), the Massasauga Rattlesnake, were found to be passing oocysts of a Caryospora, here described as C. bigenetica n. sp. Since these snakes (family Crotalidae) are known to subsist mainly on small mammals, oocysts from one of the Timber Rattlesnakes were fed to laboratory white mice (Mus musculus L.) to determine if mammals might be involved as alternate hosts in the life cycle. At necropsy, tissues of the tongue and dermis of the mice revealed a sequence of stages which included mature male and female gamonts, fully sporulated sporocysts, “excysted” sporozoites, and “resting” sporozoites that lay individually in solitary, cyst-like host cells termed “caryocysts.” A coccidia-free Massasauga that was fed an infected mouse, at a time when caryocysts in the mouse would have been present, later passed oocysts similar to those of the original inoculum. These results, along with the discovery of endogenous stages (asexual and sexual) in the intestine of the Timber Rattlesnake and the experimentally infected Massasauga, suggest that this parasite has a heteroxenous life cycle pattern, with sexual stages occurring both in the ophidian and the mammalian hosts.     

New observations on first-generation merogony of Eimeria tuskegeensis in Sigmodon hispidus

First-generation development of Eimeria tuskegeensis was evaluated using light microscopy. Sporozoite-shaped meronts containing a prominent refractile body were observed in small intestinal cells of an experimentally infected cotton rat at 24 h post inoculation (PI). Mature spherical or subspherical meronts containing crescent-shaped merozoites were observed at 36 h PI. Refractile bodies were observed in some of these merozoites. Sporozoite-shaped meronts that were isolated from host intestinal cells and inoculated onto human fetal lung cell cultures penetrated the cultured cells by 2 h PI. A mature, subspherical, first-generation meront containing seven merozoites was observed at 9 h PI in cell culture, indicating that sporozoite-shaped meronts isolated from the host retained their infectivity.     

New Observations on First-Generation Merogony of Eimeria tuskegeensis in Sigmodon hispidus1

First-generation development of Eimeria tuskegeensis was evaluated using light microscopy. Sporozoite-shaped meronts containing a prominent refractile body were observed in small intestinal cells of an experimentally infected cotton rat at 24 h post inoculation (PI). Mature spherical or subspherical meronts containing crescent-shaped merozoites were observed at 36 h PI. Refractile bodies were observed in some of these merozoites. Sporozoite-shaped meronts that were isolated from host intestinal cells and inoculated onto human fetal lung cell cultures penetrated the cultured cells by 2 h PI. A mature, subspherical, first-generation meront containing seven merozoites was observed at 9 h PI in cell culture, indicating that sporozoite-shaped meronts isolated from the host retained their infectivity.     

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

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

Induction of aryl hydrocarbon hydroxylase in human fetal liver cell and fibroblast cultures by polycyclic hydrocarbons

Cells originating from the human fetal liver and grown as a primary monolayer culture for 4 to 11 days contain an enzyme system that metabolizes benzo(α) pyrene. The basal level of the enzyme varied about three-fold. The activity was increased from 1.4- to 5.1-fold by the exposure of cells for 24 hours to benz(α) anthracene, the magnitude of increase depending on the amount of inducer, on the individual cell batch studied and on the stage of cell growth. Also 3-methylcholanthrene, but not benzo(α)pyrene, induced the enzyme activity in fetal liver cell cultures at concentrations used. Fibroblast cultures derived from the human fetal lung or skin exhibited less benzo(α)pyrene metabolism and the inducibility of the enzyme activity was less marked than in hepatic cell cultures.     

Theileria parva: Early events in the development of bovine lymphoblastoid cell lines persistently infected with macroschizonts

The cellular origin and development of bovine lymphoblastoid cell lines persistently infected with macroschizonts of Theileria parva was studied. Cultures of lymphoblastoid cells isolated from cattle with patent East Coast fever were compared with those obtained by infecting normal lymphocytes in vitro with sporozoites. The young lines were contrasted with a continuous line which had been isolated earlier. The mononuclear cells were separated from the blood and the inoculum enriched for lymphoblastoid cells and/or lymphocytes by removing the monocytes. The lines arose directly from lymphoblastoid cells transplanted into culture or from lymphocytes infected by sporozoites. In primary cultures of lymphoblastoid cells from the peripheral blood, there was an increase in the proportion of infected cells without the eclipse of the parasite, the macroschizonts were larger than those observed in the inoculum or the continuous line, and there was concurrent microschizont differentiation. In lymphocyte cultures challenged with sporozoites, small mononucleated trophozoites were observed after 2 days which differentiated into typical macroschizonts but microschizonts were rare. In all cultures, the infected cells had mitotic indices of 4 to 5%. As the young lines were passaged, the parasites came to resemble those of the continuous line. The macroschizont size in the continuous line was stable and most had six to eight nuclei but when cultured at high cell concentrations the number of parasite nuclei increased. Minicultures of lymphocytes were used to quantitate the infectivity of sporozoites obtained from organ cultures of Rhipicephalus appendiculatus savliary glands. Sporozoites from ticks fed on rabbits for 5 days were approximately six times more infective than those from glands of ticks fed for 2 days and then cultured at 32 °C for 3 days. Glands from unfed ticks cultured for 5 days failed to yield infective sporozoites.