Monosaccharide transport by Eimeria tenella sporozoites

Eimeria tenella sporozoites were incubated in the presence of 3 different [14C]-labeled sugars; D-glucose, 2-deoxy-D-glucose and 3-O-methyl-D-glucose. The initial velocity, Vi, of uptake of D-glucose and 2-deoxy-D-glucose was similar, 41 micrograms/10(10) sporozoites/min and 46 micrograms/10(10) sporozoites/min, respectively; whereas that for 3-O-methyl-D-glucose was significantly lower, 17 micrograms/10(10) sporozoites/min. Initial velocity studies also revealed that glucose uptake was a saturable event, with an apparent KT of 20 mM and an apparent Vmax of 312 micrograms/10(10) sporozoites/min. Uptake was unaffected by exogenous sodium levels or the presence of ouabain. However, 0.1 mM phloretin significantly inhibited glucose uptake. Thus, it would appear that E. tenella sporozoites possess a Na-independent, phloretin-sensitive, carrier-mediated monosaccharide-transport system.     

Excystation of Eimeria tenella sporozoites impaired by antibody recognizing gametocyte/oocyst antigens GAM22 and GAM56

Eimeria tenella is the causative agent of coccidiosis in poultry. Infection of the chicken intestine begins with ingestion of sporulated oocysts releasing sporocysts, which in turn release invasive sporozoites. The monoclonal antibody E2E5 recognizes wall-forming body type II (WFBII) in gametocytes and the WFBII-derived inner wall of oocysts. Here we describe that this antibody also binds to the stieda body of sporocysts and significantly impairs in vitro excystation of sporozoites. Using affinity chromatography and protein sequence analysis, E2E5 is shown to recognize EtGAM56, the E. tenella ortholog of the Eimeria maxima gametocyte-specific GAM56 protein. In addition, this antibody was used to screen a genomic phage display library presenting E. tenella antigens as fusion proteins with the gene VIII product on the surfaces of phagemid particles and identified the novel 22-kDa histidine- and proline-rich protein EtGAM22. The Etgam22 mRNA is expressed predominantly at the gametocyte stage, as detected by Northern blotting. Southern blot analysis in combination with data from the E. tenella genome project revealed that Etgam22 is an intronless multicopy gene, with approximately 12 to 22 copies in head-to-tail arrangement. Conspicuously, Etgam56 is also intronless and is localized adjacent to another gam56-like gene, Etgam59. Our data suggest that amplification is common for genes encoding oocyst wall proteins.     

Eimeria tenella: immunogenicity of arrested sporozoites in chickens

Groups of chickens were medicated with the anticoccidial drug, decoquinate, and starting 1 day after this medication they were given daily inoculations of either 1 X 10(4) (Experiment 1) or 1 X 10(5) (Experiment 2) oocysts of a decoquinate-sensitive strain of Eimeria tenella. This assured the presence of large numbers of drug-inhibited sporozoites in the cecal tissues. The immunity arising from the presence of these inhibited sporozoites was assessed by challenging the medicated chickens with a 2.5 X 10(5) oocysts of a decoquinate-resistant strain of E. tenella. The response to challenge was assessed by weight gain, the severity of cecal lesions, hematocrits, and cecal oocyst numbers. The inhibited sporozoites promoted little (if any) immunity judged by clinical signs of disease. However, judged by body weight changes after challenge, the presence of inhibited sporozoites provided substantial protection against the body-weight-depressing effects of the challenge dose. These findings emphasize the importance of stage-specific antigen expression in Eimeria spp. infections and support the notion that immunogenicity is associated with tropic stages of the parasite.     

Flotillin-1 localization on sporozoites oF Eimeria tenella

In an attempt to identify parasite surface components involved in the interaction with the host cell, the present research focuses on the rafts of Eimeria tenella that might be involved in the host cell invasion process. To that end, this study was undertaken to investigate the expression of flotillin-1, which is an important component and marker of lipid rafts at the plasma membrane of sporozoites of E. tenella. The expression of this plasma membrane protein was identified by an antibody that specifically reacts with flotillin- and was studied by electron microscopy. Flotillin-1 was found to occur in patches on the surface of E. tenella sporozoites. Immunoblot analysis of the total proteins of the sporozoites showed only 1 band of approximately 48 kDa. This indicates that the antibody exclusively recognized the molecules of flotillin-1 expressed on the surface of E. tenella sporozoites. The presence of flotillin-1 on the cellular membrane of sporozoites predominantly at the apical tip suggests that flotillin-1 belongs to the invasion machinery of E. tenella.     

Excystation of the Sporozoites of Eimeria tenella in Apparently Unbroken Oocysts in the Chicken

SYNOPSIS. Chickens experimentally fed sporulated oocysts of Eimeria tenella were necropsied 5–300 minutes later to study excystation, especially in apparently unbroken oocysts. In birds killed 75–125 minutes after inoculation, there was evidence of excystation in some oocysts recovered from the small and large intestines. Altho not visibly broken, the shells of about 10% of the ones studied were wrinkled or otherwise deformed; many contained active sporozoites, some inside and some outside the sporocysts. During examinations, numerous sporozoites emerged from the sporocysts. Altho evidence was not obtained that excystation from unbroken oocysts was occurring inside the birds, the fact that it occurred in some oocysts during examinations may indicate that it could be taking place. Evidence of excystation was not seen in oocysts with shells that were not visibly altered.     

Capping of immune complexes by sporozoites of Eimeria tenella

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.     

A Cold Storage Technic for Studying Excystation of Eimeria tenella*

SYNOPSIS. Activities of sheep coccidia in stages of in vitro excystation, and of Eimeria tenella removed in stages of excystation from the chicken were arrested by lowering the temperature of the parasites; in a cooled condition, the parasites remained apparently unchanged for at least 1-2 days. When transferred later to the warming stage of the microscope, they quickly resumed excystation. A cold storage technic for E. tenella was developed. The technic might also help in studying other gastrointestinal parasites and might also provide material for classroom work.     

Carbon Dioxide as the Initial Stimulus for Excystation of Eimeria tenella Oocysts*

SYNOPSIS. The stimulus necessary to initiate in vitro excystation of the chicken coccidium Eimeria tenella was provided by exposure of intact sporulated oocysts to an atmosphere of carbon dioxide. This stimulus produced a thinning and indentation at the micropylar region and oocysts became permeable to trypsin and bile. Sporozoites became active and began to escape from sporocysts into the oocyst cavity and then to the outside thru the altered micropyle after incubation in the enzyme-bile mixture. Activation of sporozoites when CO₂-pretreated oocysts were incubated in trypsin and bile, was used as the criterion to determine the number of oocysts responding to the initial stimulus. Thus, activation of sporozoites within intact oocysts was an indirect measurement of the number of oocysts stimulated during CO₂-pretreatment. Approximately 90% of the oocysts contained active sporozoites after 18 hr of pretreatment with carbon dioxide and 8 hr incubation in trypsin and bile at 38 or 41 C, respectively. Pretreatment of oocysts with air, N₂, O₂, or He resulted in 8% or less activation during incubation in trypsin and bile. Approximately 83% of the oocysts responded to the stimulus during 8 hr CO₂-pretreatment at 41 C, whereas at 38 C, 16 hr of pretreatment were required for a similar response. The stimulus did not elicit a response from oocysts held at 23 C during the pretreatment gasphase. No significant difference occurred in number of oocysts containing active sporozoites after sufficient CO₂-pretreatment for maximum stimulation of oocysts and incubation in trypsin and bile at 38 or 41 C.     

Eimeria tenella: Incomplete Excystation in the Presence of EDTA in a Taurodeoxycholate-Based Medium

ABSTRACT. Normally, sporozoites of Eimeria tenella are efficiently excysted in vitro with trypsin and bile salts. However, a one hour treatment at °40C with a chelator-supplemented excystation medium (purified trypsin and chymotrypsin, taurodeoxycholate and ethylenediaminetetraacetic acid in buffered saline) produced incomplete excystation. The treatment removed the sporocyst plug and left an opened sporocyst containing motile sporozoites, but the release of sporozoites was greatly reduced (<12% release). Some of the sporozoites extended a portion of their anterior end through the sporocyst opening then retracted it into the sporocyst. Sporozoites were released when magnesium was added to the chelator-supplemented medium. Manganese was less effective and calcium was ineffective in producing release. Also, sporozoites were released when the incompletely excysted sporocysts were transferred to buffered saline with albumin and this became the basis for a new assay. The assay demonstrated that ethylenediaminetetraacetic acid reduced release in the presence of taurodeoxycholate but not in its absence. Hydrophobic and hydrophilic chelators were tested in the assay. Ethylene-dioxy diethylene-dinitrilotetraacetic acid and 8-hydroxyquinoline were inactive. The chelator 1,10-phenanthroline did not require bile salt to reduce release. The inhibitory effects by phenanthroline were eliminated in the presence of magnesium or manganese, while calcium had no effect. Thus, although certain chelators can inhibit release, a consistent correlation between chelation and inhibition of release has not been established. The application of ethylenediaminetetraacetic acid with taurodeoxycholate as a reversible inhibitor of release is discussed.