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.     

Trypanorhyncha cestodes of hygienic-sanitary importance infecting flounders Paralichthys patagonicus Jordan, 1889 and Xystreurys rasile (Jordan, 1891) of the Neotropical region, Brazil

Serine protease inhibitors (serpins) mediate many biological processes, including immune responses to pathogenic infection. In this study, a member of the serpin superfamily was identified from the common poultry parasite Eimeria tenella by expressed sequence tag analysis and the rapid amplification of cDNA ends technique. The full-length cDNA was 1,918 bp and had an open reading frame of 1,248 bp encoding a polypeptide of 415 amino acids with the theoretical isoelectric point of 5.26 and predicted molecular weight of 45.5 kDa. Real-time quantitative PCR analysis revealed that the serpin gene was expressed at higher levels in sporozoites than in the other developmental stages (unsporulated oocysts, sporulated oocysts, and second-generation merozoites). The sequence encoding the mature protein was amplified by PCR, cloned into the pET28(a) vector, and expressed in Escherichia coli. Specific antiserum generated against the recombinant protein was prepared and used to determine invasion inhibition capacity and localization; the results suggested that the serpin may play an important role in invasion and survival of the sporoziotes in the host.     

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.     

Development ofEimeria dispersa Tyzzer, 1929, from bobwhite quail (Colinis virginianus), in bovine kidney cell cultures

Summary The development ofEimeria dispersa Tyzzer, a parasite of bobwhite quail, in Madin-Darby bovine kidney cell cultures was investigated. Excysted sporozoites were inoculated into Leighton tubes containing cell monolayers on glass coverglasses and maintained in minimum essential medium supplemented with heat-inactivated fetal calf serum. Sporozoites became intracellular within 2 h. Sporozoite-shaped schizonts, schizonts with developing merozoites, and mature first-generation schizonts were seen 24 h postinoculation. Intracellular first-generation merozoites, second-generation trophozoites, and early second-generation schizonts containing two nuclei were first observed 72 h postinoculation. Second-generation schizonts containing developing merozoites as well as mature second-generation schizonts were first seen 96h postinoculation. Gametogony was not observed.DM developing merozoite - HN host nucleus - IM intracellular merozoite - M merozoite - N nucleus - R refractile body - RB residual body - V parasitophorous vacuole     

A merozoite-specific 22-kDa rhoptry protein of the coccidium Eimeria nieschulzi (Sporozoa,Coccidia) is exocytosed in the parasitophorous vacuole upon host cell invasion

A monoclonal antibody (Mab D12A4) was used to follow the genesis and fate of rhoptries from early first-generation merogony through second-generation merozoites of the rat coccidium Eimeria nieschulzi. The epitope recognized by Mab D12A4 belongs to a 22-kDa protein which can be localized first in developing meronts 25 h post-infection. Early rhoptries appear as distinct granules in the cytoplasm of developing meronts and elongate into mature organelles before merozoite release. The 22-kDa protein is found in the parasitophorous vacuole after host cell invasion. Western blotting and immunofluorescence showed that the 22-kDa rhoptry protein is expressed in schizonts and merozoites but not in sporozoites. Received: 9 August 1997 / Accepted: 14 October 1997     

Some aspects ofLeucocytozoon caulleryi reinfection in chickens

The role of first- and second-generation schizonts in acquired immunity toLeucocytozoon caulleryi in chickens was studied. The chickens, which had recovered from a primary infection with various doses of sporozoites at 22–95 days of age, were challenged with sporozoites. First-generation merozoites were found in all of the challenged chickens, but no second-generation merozoites and gametocytes were seen in 30 of 32 chickens challenged with sporozoites. Almost all of the chickens that had recovered from a primary infection with sporozoites showed complete resistance to reinfection, and those that had recovered from a primary infection with first-generation merozoites showed resistance to reinfection with sporozoites. These results indicate that the second-generation schizont ofL. caulleryi appears to be more immunogenic than the first-generation schizont and that some immune factors acquired by the chickens in the second generation of schizogony may inhibit the development of second-generation schizonts.Presented as paper during the 6th Japanese-German Cooperative Symposium on Protozoan Diseases, München, September 21–25, 1987     

Ultrastructure of cryptosporidial life cycle in chicken host cells

Ultrastructural studies were conducted on cryptosporidia in various stages of the life cycle, attached to the epithelial cell surface of the bursa of Fabricius, caecum and trachea in six 5 and 6-week-old chickens fed faeces containing cryptosporidial oocysts. The stages observed were sporozoites or merozoites penetrating the host cells, trophozoites, schizonts, macrogametocytes, microgametocytes and oocysts. The cryptosporidia in the chickens were morphologically and developmen-tally similar to those reported previously in other animals and humans. All the life cycle stages were recognised in each organ examined and sporulating oocysts were found adherent to the host cells. Trophozoites, schizonts and macrogametocytes were common in each organ examined, while oocysts were found only occasionally and microgametocytes and sporozoites or merozoites were uncommon.     

Development of Eimeria larimerensis from the Uinta ground-squirrel in cell cultures

Summary Cell cultures of embryonic bovine liver, lamb kidney, whole ground-squirrel embryos and adult Madin-Darby bovine kidney were used to study the in vitro development of Eimeria larimerensis from the Uinta ground-squirrel, Spermophilus armatus. Leighton tubes were inoculated with sporozoites and the monolayers were examined with phase-contrast microscopy at various intervals; fixed and stained material also was studied. In all cell types, sporozoites penetrated and developed to trinucleate second-generation schizonts. The intracellular sporozoites increased in size and underwent nuclear divisions. The sporozoite-shaped schizonts had motility similar to that of the sporozoites. After the formation of 4–10 nuclei, transformation from the sporozoite-shaped schizont to a spheroidal schizont occurred by a lateral outpocketing in the anterior region or a progressive increase in width of the body. In 10 specimens, completion of the transformation process at 37 C required an average of 57 min. The greatest numbers of spheroidal schizonts were seen at 48 hr. Merozoites were apparently formed by a radial budding process, which required an average of 72 min at 37 C. The largest numbers of mature first-generation schizonts were seen at 60 hr after inoculation; these had 4–36 merozoites. Each merozoite had anterior and posterior refractile bodies; some also had small posterior refractile granules. The refractile bodies of the merozoite were derived from the posterior refractile body of the sporozoite. Second-generation trophozoites were seen at 60 hr, and second-generation bi- and trinucleate schizonts were seen at 60 and 72 hr after inoculation.Supported in part by research grant AI-07488 from the NIAID U.S. Public Health Service. Published as Journal Paper No. 1044, Utah Agricultural Experiment Station.     

Protective immunity induced by a DNA vaccine encoding Eimeria tenella rhomboid against homologous challenge

Rhomboid protein in Apicomplexa was associated with the process of host cell invasion. To evaluate the potential of the protein in eliciting protective immunity against challenge, a DNA vaccine pVAX1-Rho encoding Eimeria tenella rhomboid was constructed. Recombinant protein was expressed in Hela cells and verified by indirect immunofluorescence and western blotting analysis. In vivo experiments, 1-week-old chickens were randomly divided into three groups. Experimental group of chickens were immunized with DNA vaccines while control group of chickens were injected with pVAX1 plasmid alone or sterile water. Two weeks following the booster dose, all chickens were inoculated orally with 5?×?10⁴ sporulated oocysts of E. tenella. The host immunity and protective efficacy of this vaccine against E. tenella challenge in broilers were evaluated. Results showed that specific antibody, the levels of interleukin-2 (IL-2), interferon-γ (IFN-γ), and the percentages of CD4⁺ and CD8⁺ T lymphocyte cells were significantly increased in the pVAX1-Rho group. Challenge experiments demonstrated that pVAX1-Rho vaccination could reduce oocyst excretion, decrease cecal lesion, increase bodyweight gains and provide chickens with oocysts decrease ratio around 75.8 %. These results suggest that the pVAX1-Rho was able to induce humoral and cellular responses and generate protective immunity against E. tenella infection.     

Actin-depolymerizing factor of second-generation merozoite in Eimeria tenella: clone, prokaryotic expression, and diclazuril-induced mRNA expression

Actin depolymerizing factor (ADF) is an essential actin-binding protein that plays a key role in the control of actin dynamics and actin-based motility processes in intracellular parasites. To determine the effects of diclazuril on ADF gene of second-generation merozoites (mz-ADF) mRNA expression in Eimeria tenella, mz-ADF gene was cloned by RT-PCR from extracted RNA in second-generation merozoite of E. tenella and successfully expressed by pET-28a vector in Escherichia coli BL21(DE3). Results showed that the full length of the cloned cDNA sequence of the mz-ADF gene is 476 bp including an ORF of 375 bp. The sequence has 100% homology with a published sequence of sporozoite stage E. tenella ADF mRNA (GenBank EF195234.1). The recombinant protein was induced to be expressed by 1 mM isopropyl β-d-1-thiogalactopyranoside in vitro. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis showed that 16.99 kDa fusion protein existed in solvable form. Compared with the infected/control group, mz-ADF mRNA expression level was downregulated by 63.86% in the infected/treatment group with the treatment of diclazuril. In conclusion, the data presented here indicate that mz-ADF gene participates in an important role in the invasion host of E. tenella. Downregulation of mz-ADF mRNA expression enrich the mechanism study of diclazuril on E. tenella.     

Development in cell cultures of Eimeria vermiformis Ernst,Chobotar and Hammond, 1971

Summary Development of Eimeria vermiformis from sporozoite to mature first-generation schizonts in cultured bovine kidney cells, Madin-Darby bovine kidney cells, and primary cultures of whole mouse embryos is described. Intracellular sporozoites were seen at 5 min, and for as long as 120 h after inoculation. Sporozoites were observed penetrating cells, with uninucleate trophozoites and immature schizonts with 2–6 nuclei first appearing 24 h after inoculation. Schizonts with 6 or more nuclei, as well as mature schizonts containing first-generation merozoites, were first seen between 36 and 48 h after inoculation of all 3 cell types used. The first indication of merozoite formation was determined by the appearance of small protuberances of cytoplasm at the periphery of schizonts. Merozoites began development at the periphery of schizonts and were later observed radiating from a central body of cytoplasm, 14–20 merozoites being formed. Some mature schizonts retained a small spherical residual body after merozoite formation was completed. After the rupture of schizonts, intracellular merozoites, which contained anterior and posterior refractile granules, were seen at 48, 72 and 96 h postinoculation. Merozoites were not seen entering or leaving cells. No further development was observed.     

Effects of Acquired Resistance on Infection with Eimeria falciformis var. pragensis in Mice

Mice immunized with infections of 500, 5,000, or 20,000 oocysts of E. falciformis var. pragensis were reinfected with 20,000 and 100,000 oocysts at 20 and 38 days, respectively, after the initial infection. After the first challenge infection, none of the immunized mice showed clinical signs of coccidiosis; a few mice passed very low numbers of oocysts, and oocyst discharge seemed to correlate negatively with immunizing dose. None of the mice immunized twice passed oocysts after challenge. Mice immunized with three infections were completely immune to challenge for 4 months. The effect of the immune response on the life cycle of the coccidium was determined by histological examination of the intestines of immune and nonimmune mice infected with the parasite. In both the immune and nonimmune groups, sporozoites penetrated absorptive epithelial cells and migrated to crypt epithelial cells during the first 6 to 24 h postinfection. At 48 to 72 h postinfection, the sporozoites developed into mature first-generation schizonts in the nonimmune mice, whereas the developing first-generation schizonts degenerated within the crypt epithelial cells of the immune mice. In nonimmune mice, third-generation merozoites, inoculated intracecally, developed into mature fourth-generation schizonts, whereas in immune mice the developing fourth-generation schizonts degenerated before maturing. The possibility that a cellmediated immune mechanism is responsible for the arrest in schizogony is discussed.     

In vitro development of first- and second-generation schizonts of Eimeria contorta haberkorn, 1971 (coccidia,sporozoa)

Summary Sporozoites of Eimeria contorta inoculated into monolayer cultures of bovine embryonic kidney cells and Madin-Darby bovine kidney cells developed to mature second-generation schizonts, which were first seen 72 hr after inoculation. In cultures of bovine embryonic intestine cells, only mature first-generation schizonts developed. Intracellular sporozoites became U-shaped before transforming into trophozoites with a prominent refractile body. Sporozoite-shaped schizonts were not seen. At 30 hr after inoculation, merozoites began to form as conical protrusions of the surface of schizonts with 25–35 nuclei. Mature first-generation schizonts (25 by 20), with 25–35 merozoites each having two refractile bodies, were first observed at 46 hr. The merozoites averaged 9.5 by 2.0 . After enough sodium taurocholate to make a concentration of 0.01% was added to the cultures, merozoites left their host cells, entered others and transformed into trophozoites within 4 hr. Merozoite formation frequently began with the appearance of conical protrusions at opposite poles of second-generation schizonts. During the late stages of their development, the 8–15 merozoites were spirally arranged around the central residual body. Mature schizonts and merozoites averaged 19 by 8.5 and 22.5 by 1.4 , respectively. In some cultures, larger schizonts with 30–40 merozoites, arranged rectilinearly with respect to the residual body, were observed. On the basis of a comparison with the corresponding stages of E. nieschulzi, it is concluded that E. contorta is a valid species.This investigation was supported by PHS Research Grant No. AI-07488 from the National Institute of Allergy and Infectious Diseases and by a fellowship awarded to B. E. G. Müller by the German National Fellowship Foundation. Published as Journal Paper No. 1339, Utah Agricultural Experiment Station.     

Immunohistochemical identification of the cells parasitized by second-generation schizonts of Eimeria tenella

Conflicting reports exist in the literature concerning the type of cells within the lamina propria of the ceca that harbor second-generation schizonts of Eimeria tenella. Most of the previous studies concerning these cells have been performed using routine light or electron microscopy. Consequently, difficulties are evident in precise definition of the type of these cells using normal morphological criteria, since growth of the schizonts of E. tenella alters the morphology of the parasitized cell, making it difficult to recognize the cell type. This has led us to investigate the possibility of precisely identifying the subepithelial cells that are parasitized by mature schizonts. For this purpose we used cytoskeletal markers, namely, keratin and vimentin intermediate filaments, which allow the discrimination between epithelial and mesenchymal cells. Localization of keratin and vimentin on frozen cecal sections was studied immunohistochemically using specific monoclonal antibodies. Sites of antigenicity were detected by the avidin-biotin complex (ABC) immunoperoxidase technique and visualized by the deposition of diaminobenzidine. The identity of the cells was confirmed by the immunodetection of keratin intermediate filaments in the cytoplasm of the cells. Immunoreactivity for vimentin was absent in the parasitized cells. Therefore, we conclude that the development of second-generation schizonts of E. tenella takes place in epithelial cells within the lamina propria, which are presumably crypt epithelial cells that leave the crypts and enter the lamina propria after infection by first-generation merozoites. Received: 15 April 1997 / Accepted: 16 July 1997     

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.     

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.     

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.     

Transformation of oocysts from several coccidian species by heat treatment

The transformation of sporocysts in oocysts ofEimeria caviae, E. intestinalis, E. nieschulzi, E. separata, E. tenella, Isospora canis, I. heydorni, andToxoplasma gondii by heat treatment was examined. Fresh unsporulated oocysts from these species were heated at 50° or 55°C for less than 5 min and cultivated at 25°C for 1 week. No transformation of sporocysts was observed in the sporulated oocysts following heating at 55°C, but sporulated oocysts ofTyzzeria andCaryospora types were found in someI. canis andI. heydorni oocysts after heating at 50°C followed by cultivation. A few sporulated oocysts of theIsospora type were observed inE. intestinalis oocysts after cultivation. All transformed sporulated oocysts contained eight sporozoites. In contrast, the number of sporocysts varied from two to 0. These results indicate that sporocyst formation in oocysts was affected by heat treatment. The relationship between the total numbers of sporozoites and sporocysts in the transformed oocysts was consistent with that of the Octozoic group classified by Hoare.