C. A. Speer

Oocyst-induced murine toxoplasmosis: life cycle, pathogenicity, and stage conversion in mice fed Toxoplasma gondii oocysts.

The development of sporozoites to tachyzoites and bradyzoites was studied in mice after feeding 1-7.5 x 10(7) Toxoplasma gondii oocysts. Within 2 hr after inoculation (HAI), sporozoites had excysted and penetrated the small intestinal epithelium. At 2 HAI, most sporozoites were in surface epithelial cells and in the lamina propria of the ileum, and by 8 HAI, T. gondii was also seen in mesenteric lymph nodes. At 12 HAI, sporozoites had divided into 2 tachyzoites in the lamina propria of the small intestine. By 48 HAI, there was a profuse growth of tachyzoites in the intestine and mesenteric lymph nodes of mice fed 7.5 x 10(7) oocysts. Parasites had disseminated via the blood and lymph to other organs by 4 days after inoculation (DAI). Toxoplasma gondii was first isolated from peripheral blood at 4 HAI. Tissue cysts were visible histologically in the brain at 8 DAI. By using immunohistochemical staining with anti-bradyzoite-specific (BAG-5 antigen) serum, BAG-5-positive organisms were first seen at 5 DAI in the intestine and at 8 DAI in the brain. Using the bioassay in cats, bradyzoites were first detected in mouse tissues between 6 and 7 DAI, and they were found in intestines before they were found in the brain. Cats fed murine tissues containing bradyzoites shed oocysts in their feces with a short (< 10 days) prepatent period, whereas cats fed tissues containing tachyzoites did not shed oocysts within 3 wk. Using a pepsin-digestion procedure and mouse bioassay, bradyzoites were first detected in brain tissue at 7 DAI and in many organs of mice at 51 and 151 DAI. Individual bradyzoites, small and large tissue cysts, and tachyzoites were seen in the brains of mice at 87 and 236 DAI.

Comparative ultrastructure of tachyzoites, bradyzoites, and tissue cysts of Neospora caninum and Toxoplasma gondii.

The ultrastructure of tachyzoites, bradyzoites and tissue cysts of the NC-1, NC-5 and NC-Liverpool strains of Neospora caninum are reviewed and compared with those of the VEG and ME-49 strains of Toxoplasma gondii. While each stage of N. caninum and T. gondii shared many ultrastructural characteristics, each parasite stage also had certain features or organelles that could be used to distinguish the two parasites. Some of the most prominent ultrastructural differences occurred in the number, appearance and location of rhoptries, looped-back rhoptries, micronemes, dense granules, small dense granules and micropores. The tissue cysts of both parasites were also basically similar, being surrounded by a cyst wall and not compartmentalised by septa. The cyst wall of N. caninum was irregular and substantially thicker, 0.5-4 microm, than those of T. gondii which were smooth and 0.5 microm thick.

Time lapse video microscopy and ultrastructure of penetrating sporozoites, types 1 and 2 parasitophorous vacuoles, and the transformation of sporozoites to tachyzoites of the veg strain of Toxoplasma gondii

Videomicroscopy and transmission electron microscopy were used to study the interaction of Toxoplasma gondii sporozoites with cultured cardiopulmonary artery endothelial, embryonic bovine tracheal and Madin-Darby bovine kidney cells. No moving junction or exocytosis of rhoptries, micronemes, and dense granules was detected during the initial penetration of sporozoites into cultured cells, whereas constriction of the sporozoite and partial exocytosis of rhoptries occurred during movement of the sporozoite from the first parasitophorous vacuole (PV1) into the second vacuole (PV2). The PV1 was unusually large, lacked a tubulovesicular membrane network (TMN), and had an indistinct parasitophorous vacuolar membrane (PVM). Comparatively, the PV2 was small, had a distinct PVM, contained a well-developed TMN, and was surrounded by numerous host cell mitochondria. Sporozoites that passed completely through cells carried with them an envelope of host cell membranes and cytoplasm. Cultured cells occasionally endocytosed sporozoites that were enveloped by host cell material. After formation of the PV2, sporozoites replicated by endodyogeny to form tachyzoites.

Sporozoites of Toxoplasma gondii lack dense-granule protein GRA3 and form a unique parasitophorous vacuole

The invasion of host cells by sporozoites of Toxoplasma gondii leads to the formation of parasitophorous vacuoles that are distinctly different from those surrounding tachyzoites. In sporozoite-infected cells, the fluid-filled space surrounding the sporozoite is many times larger in volume than the sporozoite, essentially lacks granular or tubular structures, and has no detectable continuous parasitophorous vacuolar membrane when prepared by conventional electron microscopic methods. Consistent with the ultrastructural differences, dense-granule protein GRA3, which associates with the parasitophorous vacuolar membrane of tachyzoites, was not detected by indirect immunofluorescence in sporozoite-infected cells 2-12 h post-inoculation or by Western blot analysis of sporozoite extracts. Western blots incubated with the alpha ROP/DG antiserum, which recognizes tachyzoite rhoptry and dense-granule proteins, revealed numerous other antigenic differences between sporozoites and tachyzoites. Cell cultures inoculated with sporozoites were monitored at various intervals for the expression of GRA3 and the developmentally-regulated tachyzoite surface protein SAG1. Expression of SAG1 and GRA3 was first observed in 30% of the sporozoite-infected cells at 12 and 15 h post-inoculation, respectively, and in all intracellular parasites at 24 h. Parasite replication was only observed in sporozoite-infected cells that were positive for GRA3 and SAG1. Thus, these data indicate that sporozoites and their interaction with host cells differ substantially from tachyzoites and the expression of tachyzoite-specific proteins is likely required for parasite replication.

Redescription of the Sarcocysts of Sarcocystis rileyi (Apicomplexa: Sarcocystidae)

Abstract The intermediate hosts for Sarcocystis rileyi (Stiles 1893) Minchin 1913 are ducks (Anas spp.), and the striped skunk (Mephitis mephitis) is its definitive host. The structure of sarcocysts from an experimentally infected shoveler duck (Anas cylpeata) fed sporocysts from an experimentally-infected M. mephitis was studied and compared with type specimens from a naturally infected duck. The experimentally infected duck was killed 154 d after feeding sporocysts. By light microscopy the sarcocyst wall was 3–5 μm thick with indistinct villar protrusions. Ultrastructurally, the sarcocyst wall was a type-23 cyst wall with anastomosing villar protrusions that were up to 7.5 μm long. The villar projections contained filamentous structures. The bradyzoites were 12–14 μm long. Structurally, the sarcocyst from the naturally infected and experimentally infected ducks appeared similar.

Developmental gene expression in Eimeria bovis

By differential screening of stage-specific cDNA libraries of Eimeria bovis, we have identified and isolated a large set of genes that are regulated during development of the sporozoites and merozoites. Duplicate lifts of cDNA libraries constructed from partially sporulated oocysts and merozoites were probed with radioactively labeled first-strand cDNA prepared from partially sporulated oocyst and merozoite mRNA. Out of 60,000 plaques screened in each case, over 250 plaques from the partially sporulated oocyst library preferentially hybridized with the oocyst cDNA probe and 67 plaques from the merozoite library preferentially hybridized with the merozoite cDNA probe. Three of the oocyst phage and 7 of the merozoite phage were selected for further characterization. Northern analysis revealed a common pattern of mRNA expression for the oocyst cDNA clones. Consistent with the results of the differential screen, no hybridization to merozoite RNA was detected with any of these 3 oocyst cDNA clones. The expression of the merozoite cDNA clones was more complex, with 3 different classes of merozoite genes being identified based on their pattern of developmental regulation. Although each of the merozoite clones was expressed to some extent during sporulation, in all cases, expression was higher in merozoites than in partially sporulated oocysts, consistent with the restriction of expression defined by the differential screen. Sequence analysis revealed that 2 of the merozoite cDNA clones encode elongation factor 1 alpha and the ubiquitin/ribosomal protein fusion, and 1 of the sporozoite cDNAs displays a significant identity to insulin-degrading enzyme. The developmental expression of E. bovis genes involved in protein synthesis and degradation provides additional evidence for the importance of regulation of protein metabolism during parasite development.

IN VITRO CULTIVATION OF MERONTS OF SARCOCYSTIS CRUZI

Several established cell lines were tested for their ability to support in vitro development of meronts of Sarcocystis cruzi. Sporozoites penetrated bovine monocytes (BM), bovine pulmonary artery endothelial cells (CPA), Madin-Darby bovine kidney cells and mouse macrophages, but developed to meronts in BM and CPA only. Sporozoites developed to large meronts that contained approximately 180-350 merozoites, whereas merozoites formed small meronts with 50-100 merozoites. Mature large meronts were present at 18-86 days after inoculation (DAI) in BM and at 16-72 DAI in CPA. Small meronts were present at 23-115 and 23-91 DAI in BM and CPA. Considerably more merozoites developed in CPA than in BM. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that merozoites harvested at 36 and 48 DAI each had 1 unique protein as well as numerous common proteins.

PROTEINS AND ANTIGENS OF MEROZOITES AND SPOROZOITES OF EIMERIA BOVIS (APICOMPLEXA)

Proteins and antigens of first-generation merozoites and sporozoites of Eimeria bovis were examined using standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and lactoperoxidase iodination procedures. SDS-PAGE gels revealed both common and unique protein bands in merozoite and sporozoite extracts, ranging in molecular weight (Mr) from 15,000 to 215,000. Nitrocellulose immunoblots of separated proteins, when probed with sera obtained from immunized calves, revealed numerous IgG-binding antigens of Mr 18,000 to 180,000 in merozoites and Mr 28,000 to approximately 118,000 in sporozoites. Although merozoite and sporozoite preparations each contained antigens of different molecular weights, 4 antigens had the same migratory distance in both preparations (Mr 58,000, 70,000, 83,000, 98,000). Of 3 types of immune sera used to probe immunoblots, serum taken from a calf that had been inoculated with oocysts of E. bovis and boosted 10 wk later by subcutaneous injection with 2 X 10(7) live merozoites emulsified in Freunds complete adjuvant consistently identified and reacted more intensely with more antigens of merozoites and sporozoites than the other immune sera tested. Autoradiographic analysis of radioiodinated parasites revealed major surface proteins on merozoites of between 15,000 and 18,000 Mr and 3 surface proteins on sporozoites of Mr 28,000, 77,000, and 183,000. All but the 183,000 protein elicited an IgG antibody response in the host.

Comparative ultrastructure and expression of L-selectin on bovine alphabeta and gammadelta T cells.

Compared to most αβ T cells, bovine γδ T cells express two to five times the level of L‐selectin and have a much higher capacity to roll on monolayers of endothelial cells, platelets, and leukocytes in assays done under physiological flow. To gain additional insight into the basis for these differences, scanning (SEM) and transmission (TEM) electron microscopy were used to compare the cellular ultrastructure of bovine γδ and αβ T cells and to study the expression of L‐selectin on these cells. It is interesting that γδ T cells had more than twice as many microvilli and other surface projections on a per cell basis as αβ T cells. This was not due to the γδ T cell being larger; after adhesion and fixation procedures used for the EM studies, γδ T cells averaged 3.9 ± 0.01 μm in diameter, whereas αβ T cells were slightly larger (5.7 ± 0.01 μm in diameter). As previously shown for human neutrophils and lymphocytes, L‐selectin was preferentially localized to the tips of the microvilli. In contrast, WC1, a lineage‐specific antigen on γδ T cells, was localized on the plasmalemma between the microvilli. Our findings suggest that more effective rolling of γδ T cells in various in vitro flow assays may be due to the greater number of microvilli on γδ T cells leading to a higher number of contact sites during adhesion events. In addition, this physical parameter may explain the increased level of L‐selectin expression on γδ versus αβ T cells because L‐selectin is clustered at the tips of microvilli. J. Leukoc. Biol. 64: 104–107; 1998.

Biochemical and ultrastructural observations of coccidian parasite and host cell interactions.

In general, the interactions of protozoan or metazoan parasites and their animal hosts are less understood than animal—virus interactions. Consequently, there is little biochemical information about the effects of these parasites on their animal hosts. One critical aspect of this field that is only beginning to receive attention is the study of the biochemical mechanisms involved in the communication between intracellular protozoa and their host cells.