Search

Four Eimeria species have been reported from American bison (Bison bison): E. auburnensis, E. bovis, E. brasiliensis, and E. canadensis. We report on finding two additional species: E. ellipsoidalis and E. zuernii. In 1993, the prevalence of these six species in 31 yearling bison in a recently-established herd on private ranchland in Montana (USA) was: E. bovis, 97%; E. canadensis, 90%; E. zuernii, 32%; E. ellipsoidalis, 32%; E. auburnensis, 9.7%; and E. brasiliensis, 3.2%. Eimerian oocysts also were recovered from five (14%) of 36 bison in 1993 from West Yellowstone; the species involved were: E. canadensis, E. bovis, E. zuernii and E. auburnensis.

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.

Nuclear division and microgametogony of Eimeria nieschulzi were studied by transmission electron microscopy. All nuclear divisions occurred in close proximity to the gamont surface with four centrioles situated between the nuclear envelope and the gamont plasmalemma. During early nuclear divisions, each nucleus had a rod or hourglass-shaped nucleolus, indicating that the nucleolus probably pinched in two during nuclear division. Nuclear divisions occurred by nearly centrally located intranuclear spindles. Two centrioles were associated with each centrocone. Spindle microtubules, which originated from each centrocone, either traversed to the other centrocone or terminated at kinetochores. Four to 6 chromosome-like structures were present in each dividing nucleus. The formation of 50 to 100 biflagellate microgametes occurred at the margin of the microgamont by a process similar to that described for other coccidian species. Microgametes were limited by a single membrane with a prominent glycocalyx, 13 nm thick.

Immunoelectron microscopy was used to study the localization of monoclonal IgG (13.9 and 15.84) and IgM (10.84) antibodies generated against Eimeria tenella sporozoites on sporozoites, sporocysts, and oocysts of Eimeria acervulina and E. tenella. A uniform layer of ferritin was present on sporozoites of E. tenella fixed chemically before the addition of 10.84, 13.90, or 15.84 (called prefixed), whereas postfixed (fixed chemically after exposure to monoclonal antibody) sporozoites lacked ferritin, indicating that the latter had capped immune complexes. Patches of ferritin were present on prefixed and postfixed sporozoites of E. acervulina exposed to 15.84, indicating that immune complexes containing 15.84 were not capped. Sporocysts of E. tenella exposed to 10.84 had a uniform layer of ferritin on their outer surface; ferritin was localized in patches on those exposed to 13.90 or 15.84. In E. acervulina sporocysts exposed to 15.84, ferritin was widely scattered on the outer surface but formed a uniform layer on the inner surface of the sporocyst wall. Patches of ferritin occurred on the inner layer of the oocyst walls of E. tenella and E. acervulina exposed to 10.84, 13.90, or 15.84. These findings indicate the shared antigen detected by 15.84 differed in relative amount, spatial distribution, and structural location in sporozoites and sporocysts of E. acervulina and E. tenella.

Oocysts of Eimeria nieschulzi from the laboratory rat, Rattus norvegicus, were studied by scanning and transmission electron microscopy. Oocysts had a rough outer wall with apparent random depressions. The oocyst wall is composed of 2 layers: an osmiophilic outer layer consisting of a rough external and smooth internal surface, and a relatively thick, electron-lucent inner layer. The outer layer is composed of a dense, coarsely granular matrix. The inner layer consists of homogenous fine granular material interspersed with coarse osmiophilic granules and contains one closely applied membrane on the outermost surface. Several raised lenticular areas are seen on the coarse outer surface of the inner layer. These layers are 102 (75-128) and 176 (135-204) nm thick, respectively. The sporocyst wall is thin, consisting of 3 to 4 unit membranes, and measures (18-34) nm thick.

Macrophage (M phi)-activating lymphokines present in concanavalin A-stimulated bovine T-lymphocyte cultures (ConAS) were studied by assessing their effects on Eimeria bovis and Toxoplasma gondii growth in cultured bovine monocytes (BM) and mouse M phi. The in vitro development of both parasites was assessed by incorporation of [3H]uracil and by microscopic examination of parallel cultures. Incorporation of [3H]uracil into infected cultures was an accurate indicator of growth of both E. bovis and T. gondii in BM and mouse M phi. Sporozoites of E. bovis underwent merogony in untreated BM but not in mouse M phi, whereas T. gondii developed in both cell types. Inhibition of T. gondii growth was greatest in ConAS-treated BM, whereas preincubation of mouse M phi with ConAS resulted in about 80% growth inhibition. There was no significant difference between the inhibition of either T. gondii sporozoite- or tachyzoite-induced growth in ConAS-treated cells, showing that activation pathways are equally effective against both stages. Treatment of ConAS with glycine-hydrochloride buffer (pH 2) resulted in a total loss of antiviral activity mediated by gamma interferon (IFN-gamma). When pH 2 dialyzed ConAS was used to activate BM, inhibition of T. gondii growth was only partially affected. Because bovine IFN-gamma does not activate mouse M phi and due to the partial effects of pH 2 on ConAS-induced growth inhibition, the major component(s) of ConAS responsible for T. gondii growth inhibition is distinct from IFN-gamma. Furthermore, IFN-gamma may act synergistically rather than being part of a priming sequence for M phi responsiveness to other lymphokines. Murine recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) was tested for any microbistatic activity against T. gondii sporozoites and tachyzoites. There was no significant difference in either colony formation or [3H]uracil incorporation between rGM-CSF-treated and control cultures, regardless of host cell type. Thus, rGM-CSF does not induce adequate M phi activation to kill T. gondii and is not a major microbistatic component of ConAS. rGM-CSF also had no effect on T. gondii infection in vivo.

Oocysts of Eimeria crotalviridis sp. n. are described from praire rattlesnakes, Crotalus viridis viridis in New Mexico on the basis of light and electron microscopy and in vitro excystation of sporozoites. Sporulated oocysts of E. crotalviridis are elliptical, 26.4 X 22.3 (23-29 X 20-24) micrometer with ovoid sporocysts 11.7 X 8.1 (11-13 X 7-9) micromiter. A micropyle, micropyle cap and polar bodies are absent, but oocyst and sporocyst residua and Stieda and substieda bodies are present. Excysted sporozoites are 12.4 X 2.8 (11-13 X 2-3) micromiter and have 1 large posterior refractile body and a nucleus with a prominent nucleolus. Ultrastructurally, the oocyst wall has 2 layers, a thick, electron-dense, highly sculptured outer layer composed of a fine granular matrix and a thin, granular, osmiophilic inner layer, separated from the outer layer by at least one unit membrane. These layers are 441 (353-510) and 21.6 (19-29) nm thick, respectively. Within 15 min after exposure to a trypsin-sodium taurocholate fluid, sporozoites of E. crotalviridis excysted from 5-month-old sporocysts.

Sporozoites of Eimeria tenella were incubated for 10, 20, or 30 min with parasite-specific monoclonal IgG antibody 3D3II from mice and then rinsed in a Tris-buffered glucose saline solution (TBGS). Some sporozoites were then incubated for 10, 20, or 30 min with ferritin- or colloidal gold-conjugated goat anti-mouse IgG antibody and then fixed in 2.5% glutaraldehyde and prepared for transmission (TEM) or scanning (SEM) electron microscopy. Other sporozoites that had been previously exposed to monoclonal antibody were prefixed with 0.25% glutaraldehyde, incubated with ferritin- or colloidal gold-conjugated anti-mouse IgG antibody and then fixed and prepared for TEM or SEM. Control preparations consisted of sporozoites exposed only to TBGS, monoclonal antibody 3D3II or to ferritin- or colloidal gold-conjugated anti-mouse IgG antibody. Capping of immune complexes occurred only on the surface of those sporozoites exposed to monoclonal antibody 3D3II followed by ferritin- or gold-conjugated antibody. Immune complexes moved laterally and posteriorly on the outer surface of the parasite plasma membrane to form a cap at the posterior end of the sporozoite. Capping did not occur in TBGS controls nor in sporozoites treated with monoclonal antibody 3D3II and prefixed in 0.25% glutaraldehyde before exposure to ferritin- or gold-conjugated antibody. Thus, capping of surface antigens did not occur in the presence of monoclonal 3D3II antibody only, whereas specimens exposed to both monoclonal and ferritin- or colloidal gold-conjugated antibodies were able to cap immune complexes.

Asexual stages of Eimeria falciformis var. pragensis in Swiss-Webster mice were studied by scanning electron microscopy. Sporozoites were present in the cecum and colon 2 h post-inoculation (PI) and measured 11.3 X 2.1 micrometer (9-13.9 X 2-2.2 micrometer). Sporozoites penetrated epithelial cells with an extended anterior end and were constricted at the site of entry. Asexual generations were found in the cecum and colon epithelial cells. In meronts found at days 3-9 PI, merozoites matured synchronously, were oriented in the same direction, and were arranged in a helical pattern. Such meronts measured 11.3 X 6.4 micrometer (8-13.7 X 5-7.4 micrometer) and contained 8-12 merozoites, which measured 11.9 X 1.5 micrometer (7.4-15.7 X 1.3-1.8 micrometer). Meronts which were present at day 7 PI measured 9.5 X 8.2 micrometer (9-10.5 X 7-9.5 micrometer) and contained 20-50 small merozoites which budded asynchronously from a central residuum. At days 3-7 PI, parasitized epithelial cells had shorter and fewer or no microvilli. The lumenal plasmalemma of the host cell was often disrupted or absent in cells containing mature meronts and escaping merozoites. At day 6 PI, phagocytic cells appeared on the epithelial surface, some of which were in contact with merozoites. Small foci of exposed basal lamina were present at day 7 PI in areas where cells had sloughed from the epithelium.

Sporozoites of Eimeria bovis penetrated and developed normally to first-generation meronts in bovine monocytes (BM) and Madin-Darby bovine kidney (MDBK) cells that had been pretreated with culture medium (CM) or supernatant (NS) from nonstimulated bovine T cells. At 240 h after sporozoite inoculation (ASI), the mean percent development (meronts/[sporozoites + meronts]) in CM- and NS-pretreated BM was 52 and 28%, respectively; values for MDBK cells were 36 and 35%, respectively. Pretreatment of BM and MDBK cells with supernatant (ConAS) from concanavalin A-stimulated bovine T cells had no effect on the ability of sporozoites to penetrate cells; however, at 240 h ASI, only 1% of the sporozoites in ConAS-pretreated BM cultures had developed to meronts. In contrast, ConAS had no adverse effect on the ability of E. bovis sporozoites to develop to first-generation meronts in MDBK cells. At 240 h ASI, E. bovis meronts in ConAS-pretreated BM were abnormal in appearance and retarded in development, whereas sporozoites appeared structurally normal by light microscopy. Pretreatment of BM with ConAS had no effect on the ability of sporozoites of Eimeria papillata (Apicomplexa) to penetrate cells. Sporozoites of E. papillata did not develop to meronts in ConAS-pretreated BM and, in contrast to E. bovis, most sporozoites were destroyed intracellularly.

Heat shock proteins (hsps) are a group of highly conserved polypeptides found in a wide variety of organisms. Polypeptides of sporozoites and merozoites of Eimeria bovis, blotted onto nitrocellulose, were probed with antibodies to artificially constructed peptides representing portions of the cDNA-generated fragment of pf75, the 75K hsp of merozoites of Plasmodium falciparum. Polypeptide antigens of sporozoites and merozoites of E. bovis with molecular weights of 46K, 71-72K, and 75K reacted with antibodies against pf75, indicating that they are hsp70 (the 70K family of hsps) or hsp70 cognates (noninducible proteins homologous to hsps). Radiolabeling with 125I and treatment with antibodies against pf75 detected a 71K antigen on the merozoite surface. Hsps in sporozoites of E. bovis are either constitutive or evoked by treatment at 37 C for in vitro excystation. If hsp70 is mandatory for parasite survival, it may prove to be an appropriate antigen for a vaccine against bovine coccidiosis.

Eimeria bovis and Toxoplasma gondii differ in their susceptibility to macrophages activated by lymphokines. Interferon-gamma can activate macrophages to totally inhibit E. bovis sporozoite development, whereas growth of T. gondii tachyzoites in macrophages is not totally affected. The susceptibility of these parasites to oxygen intermediates and their ability to evade the oxidative burst by macrophages were investigated in cell-free systems. Using a logistic model to assess growth inhibition, T. gondii growth was impaired by 50% at 10(-4.25) M (56 microM) H2O2, with 30 min as the optimum time for measuring inhibition. Preliminary results indicate that T. gondii follows mode-one and mode-two killing with relation to time after exposure to H2O2, implying a role for OH. and the induction of a DNA repair mechanism. The same model was used to assess inhibition of E. bovis growth that was more susceptible, being inhibited to 50% by 10(-5) M (10 microM) H2O2. Both parasites were susceptible to the effects of xanthine-xanthine oxidase that releases a full complement of oxygen intermediates (H2O2, OH., (1)O2, and O2-). Adding quenchers or scavengers to the system confirmed that T. gondii was susceptible to products of the interaction of O2- and H2O2 (OH. and (1)O2), and that E. bovis sporozoites were at least partially susceptible to H2O2 and O2-, but extremely susceptible to OH.. These data were supported by studies on scavenging enzymes present in the parasites. Toxoplasma gondii was rich in superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPO), and E. bovis had less catalase and SOD.(ABSTRACT TRUNCATED AT 250 WORDS)

P20 is an immunodominant surface antigen of Eimeria bovis sporozoites. As parasites underwent merogony within cultured bovine monocytes and Madin-Darby bovine kidney (MDBK) cells, P20 appeared to be shed gradually by meronts and was absent in type 1 and 2 first-generation merozoites. Meronts of E. bovis appeared to shed P20 into the parasitophorous vacuole of bovine monocytes, whereas MDBK cells evidently released P20 into the culture medium or destroyed its antigenic determinant.

The interaction of Eimeria falciformis sporozoites with the intestinal epithelium and with the intestinal contents from the cecum and colon of normal and specifically immunized mice was studied by light (LM) and scanning electron (SEM) microscopy. Fecal (FM) and enterocyte-associated (EAM) mucus were removed from the cecum and colon of normal mice and mice that had been immunized 1, 6, 12, or 20 days earlier with a series of oral inoculations of E. falciformis oocysts. Sporozoite-specific IgA, but neither IgM nor IgG, was detected by the immunofluorescent antibody test in FM and EAM from immunized mice. No sporozoite-specific immunoglobulin was detected in normal mice. When examined by LM, sporozoites exposed to all FM and EAM preparations exhibited greater motility and excystation from sporocysts. At 4 h after incubation in FM or EAM from normal or immune mice, about 10% of the sporozoites appeared damaged, being non-motile and non-refractile. Immune FM and EAM caused agglutination of sporozoites and sporocysts and oocyst walls of E. falciformis, but did not agglutinate those of E. ferrisi. Scanning electron microscopy of in vitro interactions between E. falciformis sporozoites and intestinal contents revealed that sporozoites exposed to immune EAM were coated with particulate material whereas those exposed to normal EAM were relatively clean. Sporozoites exposed to immune FM were usually embedded within the mucus whereas those exposed to normal FM were situated on top of the mucus. No significant differences occurred between the length/width (L/W) ratios of sporozoites incubated in normal and EAM or in PBS.(ABSTRACT TRUNCATED AT 250 WORDS)

Five monoclonal antibodies (MAbs) were partially characterized and tested for their ability to inhibit penetration of Madin-Darby bovine kidney (MDBK) cells by sporozoites of Eimeria bovis. By indirect fluorescent-antibody assays, all MAbs reacted with acetone-fixed sporozoites, but only two MAbs, EbS9 (immunoglobulin G1) and EbS11 (immunoglobulin G2a), localized specifically on the plasmalemma of live sporozoites. Two of the five MAbs also reacted with acetone-fixed first-generation merozoites of E. bovis; however, none of the MAbs reacted with live merozoites. Treatment of live sporozoites with EbS9 or EbS11 resulted in 79 and 73% decreases, respectively, in sporozoite penetration of MDBK cells. No significant differences in cell penetration occurred in MDBK cells inoculated with sporozoites that had been treated with the other three MAbs. Both EbS9 and EbS11 reacted in Western blots (immunoblots) of sporozoites with the same 20,000-relative-molecular-weight protein. The antigens against which these neutralizing MAbs react might be useful in immunizing against bovine coccidiosis.

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 Freund's 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.

The fine-structural aspects of development of microgamonts of Eimeria magna were studied in kidney cell cultures and in experimentally infected rabbits. Spheroidal masses of gamont-like cytoplasm containing ribosomes, polyribosomes, and amylopectin granules were found within the parasitophorus vacuole; these bodies were apparently pinched off the surface of the gamont. Nucleoli were present in the early stages of nuclear division but disappeared as development proceeded.

Eimeria bovis has two types of first-generation merozoites with distinct ultrastructural characteristics. Type I merozoites were relatively large (means = 13.2 x 1.5 micron) and crescent-shaped, contained numerous micronemes and amylopectin granules, had a posteriorly located nucleus and a conical-shaped posterior tip, and were highly motile and capable of penetrating cultured cells. Type II merozoites were small (means = 5.9 x 0.9 micron) and spindle-shaped, had a centrally-located nucleus, few micronemes, few or no amylopectin granules, a dome-shaped posterior tip and little motility, and appeared to be incapable of penetrating cultured cells. It is possible that these two types of merozoites have considerably different roles in the life cycle of E. bovis.

Autoradiography identified six 125I-labeled proteins, ranging in molecular weight (Mr) from 20,000 to approximately 110,000, on the plasmalemma of Eimeria bovis sporozoites. Immunoblotting with bovine antiserum generated by intravenous inoculations of sporozoites and with immune serum generated by per os inoculations of oocysts revealed that protein 20 (i.e., 20,000 Mr) was an immunodominant antigen on the surface of E. bovis sporozoites.

Monolayer, cell-line cultures of embryonic bovine trachea, Madin-Darby bovine kidney (MDBK), and monolayers (RK-1) or aggregates of primary rabbit kidney cells were inoculated with merozoites obtained from rabbits that had been inoculated 3 to 5 1/2 days earlier with Eimeria magna. Merozoites obtained from from rabbits 3 days entered cells and underwent only merogony, whereas 3 1/2-5 1/2-day-old merozoites formed gamonts as well as meronts. Merozoites arising from the first or second meront generation in culture formed another meront generation or gamonts. Third-generation merozoites formed only gamonts. Most merozoites remained within the parasitophorous vacuole of the original host cell and transformed into macro- or microgamonts or meronts. Some such macro- and microgamonts then fused with each other to form larger multinucleated bodies. Such microgamonts formed microgametes, but multinucleate macrogamonts did not form oocysts. Mature microgamonts were 34 microns in diameter, and contained several hundred biflagellate microgametes. Mature macrogamonts measured 29.1 x 21.5 microns, unsporulated oocysts were 31.2 x 22 microns, and sporulated oocysts were 32 x 23.1 microns. Oocysts obtained from cell cultures were sporulated and then inoculated by gavage into rabbits, which passed E. magna oocysts 6--10 days later. Sporozoites, obtained from oocysts produced in culture or from rabbits that had been inoculated with the vitro-produced oocysts, developed to first- and second-generation meronts in MDBK or RK-1 cultures.

Monoclonal IgG antibodies against sporozoites of Eimeria tenella were obtained from the ascites fluid of BALB/c mice. Oocysts, sporocysts, and sporozoites were exposed to medium 199, normal ascites fluid, or monoclonal antibodies 1A, 9D, 3D3II, or 2G8f. Specimens were then incubated with ferritin-conjugated goat anti-mouse IgG antibody. Ferritin was uniformly distributed over the surface of sporozoites exposed to 1A, 9D, or 3D3II; ferritin was localized in patches on sporozoites exposed to 2G8f. A uniform layer of ferritin was present on the inner layer of oocyst walls and on the Stieda body, but not on the sporocyst wall. No ferritin was found on specimens exposed to medium 199 or normal ascites fluid. Monoclonal antibodies 1A, 9D, and 3D3II, but not 2G8F, caused complement-mediated lysis of sporozoites. These findings indicate that oocysts, sporocysts, and sporozoites of E. tenella contain common antigens specific for each monoclonal antibody tested.

Oocysts of Eimeria procyonis, from the American raccoon (procyon lotor), were broken, added to a cell suspension, fixed in Karnovsky's fluid, and studied with the electron microscope. The oocyst wall has three layers: a thin electron-dense inner layer (8-15 nm), an electron-lucent middle layer (25-35 nm), and a thick outer layer (120-140 nm). The outer layer has an electron-dense inner portion and an electron-lucent outer portion that contains membrane-bound vesicles. When exposed to a trypsin-sodium taurocholate fluid, sporozoites excysted from most sporocysts which were 35-43 months old, but not from sporocysts that looked normal and were 106 months old. Excysted sporozoites measured 13-16 x 3-4 (mean 14.3 x 3.2) micrometer, usually had two refractile bodies, and had a nucleus with a prominent nucleolus.

Lymphoproliferative responses against a preparation of Eimeria bovis antigens (EBAg) were measured in E. bovis-immune and naive animals. Optimal lymphocyte responsiveness could be measured after 7 days of culture in the presence of antigen at a cell concentration of 2 X 10(5) cells per well. The specificity of the reaction was confirmed by limiting dilution analysis. Whereas immune peripheral blood mononuclear cells responded to EBAg (f = 1/18,824), naive cells did not (f = 0). The helper function of cells proliferating in response to EBAg was investigated by raising T-cell lines and a clonal population derived from a line. The T-cell line showed an enhanced reactivity to EBAg by limiting dilution analysis (f = 1/256) and was interleukin-2 dependent. Limiting dilution analyses indicated at least two populations of cells: one that was interleukin-2 restricted and antigen dependent and another that was antigen independent. Supernatants from T-cell lines and the clone were analyzed for the production of lymphokines after antigen stimulation. Minimal amounts of interleukin-2 were produced. The T-cell line produced both gamma interferon (IFN-gamma) (750 U) and IFN-alpha (1,250 U), whereas the clone produced IFN-gamma (1,250 U) only. Short-term (4-day) stimulation of immune cells by EBAg induced the production of IFN-gamma (600 U) and a non-IFN macrophage-activating lymphokine. We conclude that this macrophage-activating lymphokine is only produced after short-term culture and that further culture of T cells results in the proliferation of other clones producing other factors (such as IFN).

Mature macrogamonts were present in the small intestine of rats 5.5 to 7.5 days postinoculation with Eimeria nieschulzi oocysts; oocysts were present at 6 to 7.5 days. Types I and II wall-forming bodies in macrogamonts began to undergo ultrastructural changes within zygotes to form the outer and inner layers of the oocyst wall. Before and during oocyst wall formation a total of 5 membranes (M1-5) were formed at or near the surface of the zygote. The outer and inner oocyst wall layers formed between M2 and M3, and M4 and M5, respectively. The mature oocyst was loosely surrounded by M1 and M2, had an electron-dense outer layer, 100-275 nm thick, and an electron-lucent inner layer, 160-180 nm thick. It also contained an electron-lucent line consisting of M3 and M4 interposed between the outer and inner layers of the oocyst wall. The micropyle, measuring 935 x 47 nm, was located in the outer layer of the oocyst wall and consisted of 10-14 alternating layers of electron-dense and lucent material. The sporont of mature oocysts was covered by M5, immediately beneath which were M6 and M7. The sporont contained a nucleus and nucleolus, lipid and amylopectin bodies, mitochondria, ribosomes, as well as smooth and rough endoplasmic reticulum. Canaliculi, Golgi complexes, and types I and II wall-forming bodies were absent.

B-cell responses of 3 immunoglobulin isotypes (IgA, IgG, and IgM) were investigated in the large intestine and mesenteric lymph nodes (MLN) of naive or immune mice after inoculation of oocysts of Eimeria falciformis. Primary and anamnestic IgA and IgG lymphocyte responses to E. falciformis occurred in the large intestine of nonimmune and immune mice, respectively. IgA-containing lymphocytes (IgAc) were the largest population of responding B cells in the large intestine. In infected mice, IgAc accumulated in the apical portion of the lamina propria, whereas IgG-containing lymphocytes (IgGc) were more numerous at the base of the lamina propria. No significant increase in the number of IgM-containing lymphocytes (IgMc) was observed in the lamina propria of the large intestine. Primary but no anamnestic B-cell responses occurred in the MLN, and immune mice actually had reduced numbers of IgAc and IgGc in the MLN when compared with naive mice. IgGc were the largest population of responding B cells in the MLN. Thus, IgAc appear to accumulate preferentially at the site of parasite development, whereas IgGc are primarily localized deeper in the lamina propria of the large intestine and in the draining lymph nodes of mice infected with E. falciformis.

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.