A monoclonal antibody reacts species-specifically with amylopectin granules of Eimeria bovis merozoites

An IgG1 monoclonal antibody (mAb 35B9) developed against first-generation merozoites of Eimeria bovis was shown by immunoelectron microscopy to react selectively with antigens localized in amylopectin granules. Amylopectin does not contribute to the epitope, as enzymatic degradation of carbohydrates in the parasite did not alter the binding pattern of mAb 35B9. When tested by immunoblotting, despite its organelle specificity the mAb recognized a variety of E. bovis merozoite I components with predominant molecules of 135 and 200 kDa. The epitope was not affected by treatment with endoglycosidase H; thus, N-linked sugar residues should not be involved in it. Alkaline cleavage (β-elimination), however, destroyed the epitope; thus, the involvement of O-linked carbohydrates cannot be excluded. Treatment of E. bovis merozoite extract with phospholipase C changed the binding pattern of mAb 35B9 in a way that suggests the presence of phosphorylcholine molecules on several antigens recognized by the mAb, albeit not belonging to the epitope but rather masking it. The epitope was not found in free sporozoites of E. bovis or young intracellular parasites up to day 4 after invasion of cells in vitro, whereas 5-day-old trophozoites were found to contain it. It seems to be species-specific, as it could not be shown in sporozoites or merozoites of E. tenella or in stages of several other Coccidia. Received: 24 November 1998 / Accepted: 10 December 1998     

Cross-reactivity of anti-Eimeria tenella antibody fragments on merozoites and sporozoites of different chicken Eimeria species

Eimeria tenella-specific antibodies were examined for the cross-reactivity on the sporozoites and merozoites of E. tenella, Eimeria maxima, Eimeria acervulina and Eimeria brunetti in an indirect fluorescence antibody test. Two of nine antibodies showed cross-reactivity with sporozoites of E. maxima, E. acervulina and E. brunetti; however, the localization of specific fluorescence differed between species. No antibody binding was observed on merozoites. The suitability of these antibodies to alter the infectivity of Eimeria sporozoites and/or merozoites must be verified in cell culture models and in vivo experimental infections.     

Dynamic development of parasitophorous vacuole of <Emphasis Type="Italic">Eimeria tenella</Emphasis> transfected with the yellow fluorescent protein gene fused to different signal sequences from apicomplexan parasites

Intracellular stages of Eimeria tenella reside within a membrane-bound parasitophorous vacuole (PV). PVs of apicomplexan parasites like E. tenella play important roles in nutrient acquisition, multiplication, and evasion of host immune responses. Different signal sequences from apicomplexan parasites were investigated in the transfected E. tenella for their functions in targeting yellow fluorescent protein (YFP) to subcompartments and the dynamic development of the PV of E. tenella was studied. Two 5′ terminal signal sequences derived from Toxoplasma gondii GRA8 protein and Plasmodium falciparum repetitive interspersed family protein, respectively, were confirmed to target YFP to the PVs of the transfected E. tenella, suggesting that signal sequences are functionally conserved among Apicomplexa. Three structurally different types of PVs were observed during the endogenous development of the transfected E. tenella in vitro. In addition, three subcompartments in the PV, namely, membranous extensions into the host cell cytosol, membranous extensions into the vacuolar lumen, and particle-like bodies, were detected during schizogony of the parasite.     

Production and characterization of monoclonal antibodies directed againstEimeria falciformis and cross-reactive with sporozoites from two species of avian coccidia

Monoclonal antibodies (mAbs) were obtained against the surface antigens of theEimeria falciformis sporozoite by immunizing mice with whole homogenized sporozoite. The hybridomas were selected by their reactivities against oocyst extracts, then against glutaraldehyde-treated sporozoites. Three mAbs recognized both the surface ofE. falciformis, E. tenella, andE. acervulina and their refractile bodies, whereas a fourth mAb recognized only one epitope on the refractile bodies. All mAbs bound to the same immunoaffinity-purified antigens in Western-blot analysis (P27 forE. falciformis and P25 forE. tenella andE. acervulina). Thus, the mAbs define at least two shared epitopes between sporozoite antigens from different eimerian species. Two of these mAbs are involved in the in vitro phagocytosis ofE. falciformis sporozoites by macrophages and also in their lysis by neutrophils. Altogether, these properties showed that the four mAbs came from different activated B-cells. The P27 antigen recognized by our mAbs represents a major target of the in vitro destructive immune response.     

In vivo evaluation of anticoccidial effect of antibody fragments expressed in pea (Pasum sativum) on Eimeria tenella sporozoites

Coccidiosis in chicken causes great economic losses. The increasing resistance of Eimeria species to anticoccidials has induced the search for alternative methods of control. In vivo antibody neutralization assay was conducted to study the inhibitory effect of nine antibody fragments (Ab1–Ab9) on Eimeria tenella sporozoites. These anti-E. tenella antibody fragments were expressed in pea plant (Pasum sativum). To assess the inhibitory effect on parasite reproduction, the in vivo antibody neutralization assay was done by retrograde infection of chicken with sporozoites previously incubated with antibody fragments. The pre-incubated sporozoites with the examined antibody fragments displayed a reduced ability to reproduce following intracloacal application to chicken (especially Ab1, Ab3, Ab5, and Ab9). Other antibody fragments (Ab2, Ab4, Ab6, Ab7, and Ab8) were less capable to reduce sporozoite infectivity and reproduction.     

Localization and immunogenicity of a low molecular weight antigen of Eimeria tenella

A low molecular weight (LMW) antigen recognized by a murine monoclonal antibody (C₃4F₁) was localized within endogenous stages of Eimeria tenella (USDA strain 80). Using indirect fluorescent antibody assay and immunoelectron microscopy, the LMW antigen was found in: sporozoites, first, second and third generation meronts, gamonts, unsporulated oocysts, and sporocysts. The antigen was observed in the cytoplasm and pellicle of the parasite, and in the parasitophorous vacuole, sporocyst walls and cytoplasm of infected host cells. The immunogenicity of this LMW antigen was assessed by antigen-specific serum antibody responses in chickens orally inoculated with live oocysts or injected intramuscularly with dead sporozoites. LMW antigen-specific serum antibodies were detected using Western blots of E. tenella sporozoites as early as 4 days after sporozoite injection and 6 days after oocyst inoculation. Unusually, the monoclonal antibody C₃4F₁ reduced the binding of immune chicken serum to the antigen in a competitive antibody binding assay, but not the reverse, suggesting that there is a single, immunodominant epitope on this antigen.     

Identification and characterization of cDNA clones encoding antigens of Eimeria tenella

An Eimeria tenella cDNA library was constructed in the expression vector λgt11 from poly (A⁺) RNA extracted from sporulating oocysts. The library was screened with rabbit antiserum raised against antigens extracted from fully sporulated oocysts. All of the antigen-expressing plaque-purified clones were initially characterized by cross screening with antisera raised against different stages of the E. tenella life cycle, as well as with antiserum raised against sporozoites of a related species, namely E. acervulina. A selected number of clones were further characterized by antibody selection coupled with immunoblotting and DNA cross hybridization. Three different E. tenella antigens were identified. All three appear to be constitutively expressed at the protein level during sporogony.     

The ultrastructure of first generation development of Eimeria tenella (railliet and lucet, 1891) fantham, 1909 in cell cultures

Summary Primary cultures of embryonic chick kidney cells, inoculated with freshly excysted Eimeria tenella sporozoites, were grown in Leighton tubes containing Melinex coverslips. After penetration of the host cell, several micropores appear in the pellicle of the sporozoite. Most intracellular sporozoites possess only one paranuclear body. The cytoplasm of an early segmenting schizont contains many ribosomes, a highly developed endoplasmic reticulum, and large spherical electron-pale vesicles. The nuclei of the first generation merozoites, lying in cytoplasmic protrusions of the schizont, later separate from the mother cell, thus forming a new generation. Essentially, the ultrastructure of E. tenella asexual stages cultivated in vitro appears similar to that reported from in vivo material.Published with the approval of the Director of the New Hampshire Agricultural Experiment Station as Scientific Contribution Nr. 512. Supported in part by Research Grant AI 05745-05 from the National Institute of Allergy and Infectious Diseases, U.S. Public Health Service, and the Deutsche Forschungsgemeinschaft.Dedicated to Prof. Dr. G. Piekarski on the occasion of his 60th birthday.     

Ultrastructural observations of development ofEimeria tenella in a novel established avian-derived cell line

Transmission electron microscopy was used to study the in vitro development ofEimeria tenella in a novel established avian-derived cell line (designated CEV-1/F7) used for antigen production in chicken immunization studies. Sporozoites ofE. tenella were inoculated onto cell monolayers and the cells were fixed at 24-h intervals. Large numbers of intracellular sporozoites were seen at 24 h postinoculation (p.i.), and trophozoites were identified at 24–48 h p.i. Immature schizonts, some with budding merozoites, were seen by 48 h p.i. At 72–96 h p.i., immature and mature schizonts and extracellular merozoites were observed. No merozoite invasion occurred, but immature second-generation schizogony was seen in parasitophorous vacuoles of first-generation schizonts. No further development occurred and degeneration of most schizonts was seen by 120–144 h p.i. The results confirmed synchronous development ofE. tenella until 48 h p.i., followed by asynchronous development and ultrastructural degeneration with increased incubation time.     

Binding of a Monoclonal Antibody to Sporozoites of Sarcocystis singaporensis Enhances Escape from the Parasitophorous Vacuole, Which Is Necessary for Intracellular Development

Early intracellular development in vitro of the cyst-forming protozoon Sarcocystis singaporensis and the influence of a monoclonal antibody on invasion, intracellular localization, and development of sporozoites were studied. As revealed by immunofluorescence using parasite-specific antibodies which labeled the parasitophorous vacuole membrane (PVM) and by ultrastructural analysis, sporozoites invaded pneumonocytes of the rat via formation of a parasitophorous vacuole (PV). About half of the sporozoites left this compartment within the first 8 h postinfection to enter the host cell cytosol. By semiquantitative analysis of acetyl-histone H4 expression of sporozoites, a marker linked to early gene expression of eukaryotic cells, we show (supported by ultrastructural analysis) that escape from the PV appears to be necessary for early intracellular development. More than 90% of sporozoites located in the cytosol expressed high levels of acetylated histone H4 in the nucleus, whereas only a quarter of the intravacuolar sporozoites exhibited a similar signal. As revealed by ultrastructural analysis, young schizonts all resided in the cytosol. Specific binding of a monoclonal antibody (11D5/H3) to sporozoites before invasion significantly enhanced their escape from the PV, whereas cell invasion itself remained unaffected. The antibody actually increased proliferation of the parasites in vitro, providing a further link between residence in the cytosol and successful intracellular development. Monoclonal antibody 11D5/H3 precipitated a major 58-kDa antigen from oocyst-sporocyst extracts and reacted with the cytoplasm and the surface of sporozoites in immunofluorescence assays. Collectively, the observed antibody-parasite interaction suggests the existence of a signaling event that influences intracellular development of Sarcocystis.     

Immunity to Plasmodium berghei exoerythrocytic forms derived from irradiated sporozoites

 The nature of immunity generated by Plasmodium berghei exoerythrocytic (EE) stages developing from irradiated sporozoites was studied using in vivo parameters of host protection on immunization with irradiated sporozoites and in vitro parameters of inhibition of sporozoite invasion and EE form development by serum antibodies from immunized mice. On in vivo challenge of immunized mice by sporozoites, protection was observed in an irradiation-dose-dependent manner. This finding stresses that protection is dependent on the irradiation dose of sporozoites that allows sporozoite penetration yet controls EE form development within the liver. Using the human hepatoma line Hep G2 as host cells in vitro, we observed that serum antibodies raised in mice immunized with irradiated sporozoites reacted with sporozoite- and hepatic-stage parasites in an immunofluorescent antibody test (IFAT). No reactivity was observed with blood-stage parasites. Serum antibodies from mice immunized with 6- to 18-krad-irradiated sporozoites inhibited sporozoite invasion and caused severe inhibition of EE form development in hepatoma cells, pointing to the antigenic content of EE forms developing from irradiated sporozoites (irra EE forms) as critical immunogens. Moreover, in an enzyme-linked immunosorbent assay (ELISA), serum antibodies raised to 12-krad-irradiated sporozoites showed reactivity to synthetic peptides representing the conserved Region II sequences of the P. falciparum circumsporozoite (CS) protein as well as the P. falciparum liver-stage-specific antigen (LSA-1)-based repeat sequences, thus implicating an important role for both the sporozoite and the hepatic stage in protection. Received: 21 June 1995 / Accepted: 27 Oktober 1995     

Partial characterization of a non-proteinaceous, low molecular weight antigen of Eimeria tenella

A low molecular weight (LMW) antigen of Eimeria tenella, initially identified using a murine monoclonal antibody (mAb C₃4F₁) raised against E. tenella sporozoites, was partially characterized using enzymatic degradation, solvent extraction, and immunization into various inbred lines of mice. The LMW antigen could be isolated using Folch extraction (methanol/chloroform/water) and the epitope recognized by mAb C₃4F₁ was resistant to degradation by α-amylase, pronase, and proteinase K, but was sensitive to sodium m-periodate treatment or digestion using mixed glycosidases (from Turbo cornutus). These observations suggest that the antigenic epitope recognized by mAb C₃4F₁ is carbohydrate-dependent and, based on our ability to isolate the LMW antigen by Folch extraction, the epitope probably resides on a polar glycolipid. The inability of sporozoite-immunized nude mice to elicit a serum antibody response to this molecule indicates that it acts as a T-dependent antigen. Furthermore, sporozoite-immunized male CBA/N mice (with an X-linked immunodeficiency) also failed to elicit a serum antibody response to this molecule, which is consistent with a carbohydrate antigenic epitope. We propose that this antigenic molecule be designated ET-GL1 to reflect its origin and probable structure (E. tenella glycolipid 1). Received: 30 June 1999 / Accepted: 2 December 1999     

Improved techniques for the in vitro cultivation ofEimeria tenella in primary chick kidney cells

Primary kidney cells of 1- to 4-week-old chickens (PCKC) grown in Flexiperm chambers or culture flasks were infected with ultrapure sporozoites of twoEimeria tenella strains. For the 24-h parasite-free adaptation phase of the PCKC culture, Williams E medium plus 10% foetal calf serum (FCS) was used, and for the subsequent parasite-containing 168-h maintenance phase, we used medium 199 plus 2.5% FCS. Monolayers established during that time enabled the routine development of all schizont generations as well as, in general, young oocysts. The parasite stages propagated in FLEX were rendered visible by modified PAS-AO staining. Sporulated oocysts differed in length and width from those recovered after their passage through chickens. These results show thatE. tenella can reliably be reproduced from sporozoites to oocysts in PCKC cultures. However, the yield of oocysts was generally low, indicating that mass production of oocysts is achieved only by passaging sporulated oocysts through chickens.     

Studies on the excystation of different species ofEimeria in vitro

Summary The role of enzymes in the excystation of an embryo-adapted strain ofE. tenella (TA) was investigated; 0.1% crude trypsin or pure trypsin in combination with 0.5% bile salt resulted in 75% and 58% excystation respectively. Both enzymes were more effective than chymotrypsin. Addition of 0.01% chymotrypsin to 0.1% purified trypsin resulted in 76% excystation suggesting that both trypsin and chymotrypsin may be important for excystation in vitro. Pre-incubation of sporocysts in 2% bile salt prior to the addition of enzyme resulted in lower excystation. Sporozoites released at various enzyme concentrations did not differ in infectivity when inoculated into chicken embryos.Excystation of the Houghton strains ofE. acervulina, E. praecox andE. maxima occurred more rapidly at lower concentrations of crude trypsin plus 0.5% bile salt than the Houghton strains ofE. brunetti orE. tenella. Sixty per cent of sporozoites ofE. acervulina had excysted after 25 min incubation with 0.001% crude trypsin plus 0.5% bile salt. At the same concentrations 72%E. praecox had excysted after 25 min, 43%E. maxima after 60 min, 20%E. brunetti after 105 min and 1%E. tenella after 105 min. Maximum numbers of sporozoites ofE. brunetti andE. tenella were found after 105 min incubation with 0.1% crude trypsin.Whereas incubation ofE. acervulina, E. praecox andE. maxima for 105 min in 0.01% crude trypsin resulted in sporozoite lysis, no lysis was found withE. brunetti orE. tenella after incubation in crude trypsin.     

Molecular cloning and characterization of lactate dehydrogenase gene from Eimeria tenella

Lactate dehydrogenase (LDH) is a key enzyme in the glycolytic pathway and is crucial for parasite survival. In this study, we cloned and expressed the LDH of Eimeria tenella (EtLDH). Real-time polymerase chain reaction and Western blot analysis revealed that the expression of EtLDH was developmentally regulated at the messenger RNA (mRNA) and protein levels. EtLDH mRNA levels were higher in second-generation merozoites than in other developmental stages (unsporulated oocysts, sporulated oocysts, and sporozoites). EtLDH protein expression levels were most prominent in second-generation merozoites, moderately expressed in unsporulated oocysts and sporulated oocysts, and weakly detected in sporozoites. Immunostaining with anti-recombinant EtLDH (rEtLDH) antibody indicated that EtLDH was mainly located in the anterior region in free sporozoites and became concentrated in the anterior region of intracellular sporozoites except for the apex after invasion into DF-1 cells. Specific staining of EtLDH protein was more intense in trophozoites and immature first-generation schizonts, but decreased in mature first-generation schizonts. Inhibition of EtLDH function using specific antibodies cannot efficiently reduce the ability of E. tenella sporozoites to invade host cells. These results suggest that EtLDH may be involved in glycolysis during the first-generation merogony stage in E. tenella and has little role in host invasion.     

Ultrastructural studies of the effects of the ionophore lasalocid onEimeria tenella in chickens

The ultrastructural development ofEimeria tenella was studied in experimentally infected chicks fed 90 ppm lasalocid, an ionophorous anticoccidial antibiotic. Drug treatment was timed to target-specific endogenous stages. At 6 h after infection, many sporozoites within the epithelium showed degradation as a result of drug action. Only a few intact sporozoites were seen. The drug caused outer-membrane blistering, large surface swellings and enlarged mitochondria in both first-and second-generation merozoites. No effect on the gamonts was discerned.Abbreviations AM Abnormal merozoites - B bleb - C cytoplasm - EM enlarged mitochondrion - HCN host cell nucleus - I intraepithelial lymphocyte - ID incomplete division - IM inner membrane of merozoite - MA macrogamete - ME merozoite - MI microgamont - N nucleus - OM outer membrane of merozoite - PG polysaccharide granules - R refractile body - RC residual cytoplasm - RH rhoptry - S sporozoite - SR swollen region of merozoite     

Identification and characterization of an Eimeria-conserved protein in Eimeria tenella

The precocious lines of Eimeria spp. have unique phenotypes. However, the genetic basis of the precocious phenotype is still poorly understood. The identification of Eimeria genes controlling the precocious phenotype is of immense importance in the fight against coccidiosis. In the present study, a novel gene of Eimeria maxima was cloned using rapid amplification of cDNA ends (RACE) based on the expressed sequence tag (EST). Homologous genes were also found in Eimeria tenella and Eimeria acervulina. Alignment of the amino acid sequences from E. tenella, E. maxima, and E. acervulina showed 80–86 % identity, demonstrating a conserved protein in different Eimeria spp. This gene, designated Eimeria-conserved protein (ECP), contained 235 amino acids with a predicted molecular mass of 25.4 kDa and had 100 % identity with one annotated protein from E. maxima (Emax_0517). Real-time PCR and Western blot analysis revealed that the expression of ECP at mRNA and protein level in E. tenella is developmentally regulated. Messenger RNA levels from the ECP gene were higher in sporozoites than in other developmental stages (unsporulated oocysts, sporulated oocysts, and second-generation merozoites). Expression of ECP protein was detected in unsporulated oocysts, increased in abundance in sporulated oocysts, and was most prominent in sporozoites. Thereafter, the level of the ECP protein decreased, and no ECP-specific protein was detected in second-generation merozoites. Immunostaining with anti-rECP indicated that ECP is highly concentrated in both refractile bodies (RB) of free sporozoites, but is located at the apical end of the sporozoites after invasion of DF-1 cells. The specific staining of the ECP protein becomes more intense in trophozoites and immature first-generation schizonts, but decreases in mature first-generation schizonts. Inhibition of the function of ECP using specific antibodies reduced the ability of E. tenella sporozoites to invade host cells. Compared with the parent strain, both mRNA and protein expression levels in the sporulated oocyst were downregulated in the precocious line of E. tenella. These results suggest that ECP may be involved in invasion and development of the first-generation merogony stage of E. tenella. Findings of downregulation of ECP mRNA and protein expression in the precocious line enrich the study of the precocious phenotype of Eimeria.     

Interactions in vitro between sporozoites of Eimeria tenella and host peritoneal exudate cells: Electron microscopal observations

Summary The interactions of sporozoites of Eimeria tenella with peritoneal cells from normal and from immunized chickens were examined in vitro. Although the uptake of sporozoites by cells from immunized birds was greater than by cells from susceptible birds, no differences were apparent in their appearance in electron micrographs. Entry into both macrophages and heterophils (comparable to mammalian neutrophils) was by phagocytosis. The findings are discussed.     

Extracellular calcium deficiency and ryanodine inhibit Eimeria tenella sporozoite invasion in vitro

An in vitro assay system with Eimeria tenella sporozoites was used to study the effects of extracellular calcium and active agents affecting the invasion of parasites into host cells. At concentrations of 900M Ca²⁺ and less the invasion rates were distinctly decreased. Ryanodine, a herbal alkaloid known for binding to internal Ca²⁺ channels (ryanodine receptors), showed an inhibitory effect on E. tenella sporozoite invasion. Preincubation tests and staining with a fluorescent derivative of ryanodine assured that the compound bound specifically to the sporozoites and affected them rather than the host cells.All experiments described in this publication comply with the current laws of Germany     

Effects of curcumin (diferuloylmethane) on Eimeria tenella sporozoites in vitro

The negative effects of coccidiosis on poultry health and productivity and increasing problems related to drug resistance have stimulated the search for novel and alternative methods of control. The present study evaluates the anticoccidial activity of curcumin (diferuloylmethane), a natural polyphenolic compound abundant in the rhizome of the perennial herb turmeric (Curcuma longa) which is a spice and food colourant commonly used in curries and also used as medicinal herb. Its effects were evaluated on Eimeria tenella sporozoites, including morphological alterations, sporozoite viability and infectivity to Madin–Darby bovine kidney (MDBK) cells. Morphological alterations of the sporozoites were recorded as deformation due to swelling and cell membrane corrugations. Curcumin at concentrations of 25, 50, 100, 200 and 400 μM showed considerable effects on sporozoite morphology and viability in a dose-dependent manner after incubation over 3, 6, 18 and 24 h while lower curcumin concentrations (6.25 and 12.5 μM) were not effective. In comparison to the untreated control, sporozoite infectivity was reduced at curcumin concentrations of 100 and 200 μM by 41.6% and 72.8%, respectively. Negative effects of curcumin on MDBK cells were not seen at these concentrations; however, curcumin at concentrations of 1,800, 600 and 400 μM was toxic to MDBK cells and affected cell proliferation. In conclusion, curcumin exhibited a marked inhibitory effect in vitro on E. tenella sporozoites inducing morphological changes and reducing sporozoite viability and infectivity.