Oocyst Wall Formation Research Articles

Comparative proteomic analysis of wall-forming bodies and oocyst wall reveals the molecular basis underlying oocyst wall formation in Eimeria necatrix

BackgroundThe durable oocyst wall formed from the contents of wall-forming bodies (WFBs) protects Eimeria parasites from harsh conditions and enhances parasite transmission. Comprehending the contents of WFBs and proteins involved in oocyst wall formation is pivotal to understanding the mechanism of the oocyst wall formation and the search for novel targets to disrupt parasite transmission.MethodsTotal proteins extracted from WFBs and the oocyst wall of Eimeria necatrix were subjected to comparative proteomic analysis using tandem mass tag in conjunction with liquid chromatography tandem-mass spectrometry techniques. After functional clustering analysis of the identified proteins, three proteins, including E. necatrix disulfide isomerase (EnPDI), thioredoxin (EnTrx) and phosphoglycerate kinase (EnPGK), were selected for further study to confirm their potential roles in oocyst wall formation.ResultsA total of 3009 and 2973 proteins were identified from WFBs and the oocyst wall of E. necatrix, respectively. Among these proteins, 1102 were identified as differentially expressed proteins, of which 506 were upregulated and 596 downregulated in the oocyst wall compared to the WFBs. A total of 108 proteins, including compositional proteins of the oocyst wall, proteases, oxidoreductases, proteins involved in glycosylation, proteins involved in synthesis of the acid-fast lipid layer and proteins related to transport, were proposed to be involved in oocyst wall formation. The approximate molecular sizes of native EnPDI, EnTrx and EnPGK proteins were 55, 50 and 45 kDa, respectively. EnPDI was present in both type 1 and type 2 WFBs, EnTrx was present only in type 2 WFB2 and EnPGK was present only in type 1 WFBs, whereas all of them were localized to the outer layer of the oocyst wall, indicating that all of them participate in the formation of the oocyst wall.ConclusionsTo the best of our knowledge, this is the first report on the proteomes of WFBs and the oocyst wall of E. necatrix. The data obtained from this study form a basis for deciphering the molecular mechanisms underlying oocyst wall formation of Eimeria parasites. They also provide valuable resources for future studies on the development of novel therapeutic agents and vaccines aimed at combating coccidian transmission.Graphical

Proteomic Analysis of Fractionated Eimeria tenella Sporulated Oocysts Reveals Involvement in Oocyst Wall Formation.

Eimeria tenella is the most pathogenic intracellular protozoan parasite of the Eimeria species. Eimeria oocyst wall biogenesis appears to play a central role in oocyst transmission. Proteome profiling offers insights into the mechanisms governing the molecular basis of oocyst wall formation and identifies targets for blocking parasite transmission. Tandem mass tags (TMT)-labeled quantitative proteomics was used to analyze the oocyst wall and sporocysts of E. tenella. A combined total of 2865 E. tenella proteins were identified in the oocyst wall and sporocyst fractions; among these, 401 DEPs were identified, of which 211 were upregulated and 190 were downregulated. The 211 up-regulated DEPs were involved in various biological processes, including DNA replication, fatty acid metabolism and biosynthesis, glutathione metabolism, and propanoate metabolism. Among these proteins, several are of interest for their likely role in oocyst wall formation, including two tyrosine-rich gametocyte proteins (EtGAM56, EtSWP1) and two cysteine-rich proteins (EtOWP2, EtOWP6). Concurrently, 96 uncharacterized proteins may also participate in oocyst wall formation. The present study significantly expands our knowledge of the proteome of the oocyst wall of E. tenella, thereby providing a theoretical basis for further understanding of the biosynthesis and resilience of the E. tenella oocyst wall.

Examination of gametocyte protein 22 localization and oocyst wall formation in Eimeria necatrix using laser confocal microscopy and scanning electron microscopy

BackgroundEimeria parasite infection occurs via ingestion of oocysts. The robust, bilayer oocyst wall is formed from the contents of wall-forming bodies (WFBs), WFB1 and WFB2, located exclusively in macrogametocytes. Eimeria necatrix gametocyte proteins 22 and 59 (EnGAM22 and EnGAM59) have been found to localize to WFBs and the oocyst wall. However, the exact localization of these two proteins is not clear.MethodsWFBs of E. necatrix were extracted from purified gametocytes using a cutoff filter and the extracts of purified WFBs and gametocytes were analyzed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Then, the localization of EnGAM22 and EnGAM59 proteins was determined using an indirect immunofluorescence assay. Finally, the development of macrogametocytes and the oocyst wall of E. necatrix was analyzed using laser confocal microscopy and scanning electron microscopy.ResultsPurified WFBs had the same shape and size as those observed in macrogametocytes. EnGAM22 protein localized to WFB1, whereas EnGAM59 protein localized to WFB2. Both EnGAM22 and EnGAM59 native proteins were detected in the extracts of WFBs and gametocytes. The outer layer of the oocyst wall was formed by the release of the contents of WFB1 at the surface of the macrogametocyte to form an anti-EnGAM22 positive layer. WFB2 then appeared to give rise to the inner layer, which was anti-EnGAM59 positive.ConclusionsEnGAM22 and EnGAM59 proteins localized to WFB1 and WFB2 and were involved in the formation of the outer and inner layers of the oocyst wall of E. necatrix, respectively. The processes of macrogametogenesis and oocyst wall formation of E. necatrix are similar to other Eimeria parasites. The anti-EnGAM22 antibody could be used as a tool to track the transport and secretion of proteins in WFB1 during oocyst development.Graphical

Recombinant GMA56 and ROP17 of Eimeria magna conferred protection against infection by homologous species.

One of the most common rabbits coccidia species, Eimeria magna is mainly parasitic in the ileal and jejunal epithelial cells. E. magna infection can affect the growth performance of rabbits or cause other secondary diseases. Traditional methods of anticoccidial treatment typically result in drug resistance and drug residue. Therefore, vaccination is a promising alternative. Gametocyte antigen 56 (GAM56) and rhoptry kinase family proteins (ROPs) are involved in oocyst wall formation and parasite invasion, respectively. A virulence factor, ROP17 contains a serine/threonine kinase catalytic domain. In this study, recombinant E. magna GAM56 (rEmGAM56) and ROP17 (rEmROP17) proteins were obtained from a prokaryotic expression system and their reactogenicity was investigated with immunoblotting. To assess the potential of rEmGAM56 and rEmROP17 as coccidiosis vaccines, New Zealand White rabbits were subcutaneously immunized with 100 μg rEmGAM56 (rGC group) or rEmROP17 (rRC group) twice at 2-week intervals followed by homologous oocyst challenge. The rabbit serum was collected weekly to detect the specific antibody levels. The cytokine levels of pre-challenge serum were measured by enzyme-linked immunosorbent assay and the rabbits were observed and recorded post-challenge for the onset of clinical symptoms. The weight gain, oocyst output, and feed conversion ratio were calculated at the end of the experiment. The results showed that both rEmGAM56 and rEmROP17 had good reactogenicity. The rEmGAM56- or rEmROP17-immunized rabbits had milder clinical symptoms and feed conversion ratios of 3.27:1 and 3.37:1, respectively. The rEmGAM56-immunized rabbits had 81.35% body weight gain and 63.85% oocyst output reduction; the rEmROP17-immunized rabbits had 79.03% body weight gain and 80.10% oocyst output reduction. The ACI of rGC and rRC groups were 162.35 and 171.03, respectively. The specific antibody levels increased rapidly after immunization. Significantly increased interleukin (IL)-2, interferon (IFN)-γ, and IL-17 levels were evident in the rGC and rRC groups (p < 0.05). The rEmGAM56 and rEmROP17 elicited humoral and cellular responses, which protected against E. magna infection in rabbits. Thus, rEmGAM56 and rEmROP17 are potential vaccine candidates against E. magna, and rEmROP17 performed better than rEmGAM56.

Enrichment and proteomic identification of Cryptosporidium parvum oocyst wall

BackgroundCryptosporidium parvum is a zoonotic parasitic protozoan that can infect a variety of animals and humans and is transmitted between hosts via oocysts. The oocyst wall provides strong protection against hostile environmental factors; however, research is limited concerning the oocyst wall at the proteomic level.MethodsA comprehensive analysis of the proteome of oocyst wall of C. parvum was performed using label-free qualitative high-performance liquid chromatography (HPLC) fractionation and mass spectrometry-based qualitative proteomics technologies. Among the identified proteins, a surface protein (CpSP1) encoded by the C. parvum cgd7_5140 (Cpcgd7_5140) gene was predicted to be located on the surface of the oocyst wall. We preliminarily characterized the sequence and subcellular localization of CpSP1.ResultsA total of 798 proteins were identified, accounting for about 20% of the CryptoDB proteome. By using bioinformatic analysis, functional annotation and subcellular localization of the identified proteins were examined for better understanding of the characteristics of the oocyst wall. To verify the localization of CpSP1, an indirect immunofluorescent antibody assay demonstrated that the protein was localized on the surface of the oocyst wall, illustrating the potential usage as a marker for C. parvum detection in vitro.ConclusionThe results provide a global framework about the proteomic composition of the Cryptosporidium oocyst wall, thereby providing a theoretical basis for further study of Cryptosporidium oocyst wall formation as well as the selection of targets for Cryptosporidium detection.Graphical

Recent achievements and doors opened for coccidian parasite research and development through transcriptomics of enteric sexual stages

The Coccidia is the largest group of parasites within the Apicomplexa, a phylum of unicellular, obligate parasites characterized by the possession of an apical complex of organelles and structures in the asexual stages of their life cycles, as well as by a sexual reproductive phase that occurs enterically in host animals. Coccidian sexual reproduction involves morphologically distinct microgametes and macrogametes that combine to form a diploid zygote and, ultimately, following meiosis and mitosis, haploid, infectious sporozoites, inside sporocysts within an oocyst. Recent transcriptomic analyses have identified genes involved in coccidian sexual stage development and reproduction, including genes encoding for microgamete- and macrogamete-specific proteins with roles in gamete motility, fusion and fertilization, and in the formation of the resilient oocyst wall that allows coccidians to persist for long periods in the environment. Transcriptomics has also provided important clues about the regulation of gene expression in the transformation of parasites from one developmental stage to the next, a complex sequence of events that may involve transcription factors such as the apicomplexan Apetala2 (ApiAP2) family, alternative splicing, regulatory RNAs and MORC (a microrchida homologue and regulator of sexual stage development in Toxoplasma gondii). The molecular dissection of coccidian sexual development and reproduction by transcriptomic analyses may lead to the development of novel transmission-blocking strategies.

Insulinase-like Protease 1 Contributes to Macrogamont Formation in Cryptosporidium parvum.

The apicomplexan parasite Cryptosporidium parvum contains an expanded family of 22 insulinase-like proteases (INS), a feature that contrasts with their otherwise streamlined genome. Here, we examined the function of INS1, which is most similar to the human insulinase protease that cleaves a variety of small peptide substrates. INS1 is an M16A clan member and contains a signal peptide, an N-terminal domain with the HXXEH active site, followed by three inactive domains. Unlike previously studied C. parvum INS proteins that are expressed in sporozoites and during merogony, INS1 was expressed exclusively in macrogamonts, where it was localized in small cytoplasmic vesicles. Although INS1 did not colocalize with the oocyst wall protein recognized by the antibody OW50, immune-electron microscopy indicated that INS1 resides in small vesicles in the secretory system. Notably, these small INS1-positive vesicles were often in close proximity to large OW50-positive vacuoles resembling wall-forming bodies, which contain precursors for oocyst wall formation. Genetic deletion of INS1, or replacement with an active-site mutant, resulted in lower formation of macrogamonts in vitro and reduced oocyst shedding in vivo Our findings reveal that INS1 functions in the formation or maturation of macrogamonts and that its loss results in attenuated virulence in immunocompromised mice.IMPORTANCE Cryptosporidiosis is a debilitating diarrheal disease in young children in developing countries. The absence of effective treatments or vaccines makes this infection very difficult to manage in susceptible populations. Although the oral dose of oocysts needed to cause infection is low, infected individuals shed very high numbers of oocysts, readily contaminating the environment. Our studies demonstrate that the protease INS1 is important for formation of female sexual stages and that in its absence, parasites produce fewer oocysts and are attenuated in immunocompromised mice. These findings suggest that mutants lacking INS1, or related proteases, are useful for further characterizing the pathway that leads to macrogamont maturation and oocyst wall formation.

Polyphyletic origin, intracellular invasion, and meiotic genes in the putatively asexual agamococcidians (Apicomplexa incertae sedis)

Agamococcidians are enigmatic and poorly studied parasites of marine invertebrates with unexplored diversity and unclear relationships to other sporozoans such as the human pathogens Plasmodium and Toxoplasma. It is believed that agamococcidians are not capable of sexual reproduction, which is essential for life cycle completion in all well studied parasitic apicomplexans. Here, we describe three new species of agamococcidians belonging to the genus Rhytidocystis. We examined their cell morphology and ultrastructure, resolved their phylogenetic position by using near-complete rRNA operon sequences, and searched for genes associated with meiosis and oocyst wall formation in two rhytidocystid transcriptomes. Phylogenetic analyses consistently recovered rhytidocystids as basal coccidiomorphs and away from the corallicolids, demonstrating that the order Agamococcidiorida Levine, 1979 is polyphyletic. Light and transmission electron microscopy revealed that the development of rhytidocystids begins inside the gut epithelial cells, a characteristic which links them specifically with other coccidiomorphs to the exclusion of gregarines and suggests that intracellular invasion evolved early in the coccidiomorphs. We propose a new superorder Eococcidia for early coccidiomorphs. Transcriptomic analysis demonstrated that both the meiotic machinery and oocyst wall proteins are preserved in rhytidocystids. The conservation of meiotic genes and ultrastructural similarity of rhytidocystid trophozoites to macrogamonts of true coccidians point to an undescribed, cryptic sexual process in the group.

Correlative light and electron microscopy of wall formation in Eimeria nieschulzi

Coccidian parasites possess complex life cycles involving asexual proliferation followed by sexual development leading to the production of oocysts. Coccidian oocysts are persistent stages which are secreted by the feces and transmitted from host to host guaranteeing life cycle progression and disease transmission. The robust bilayered oocyst wall is formed from the contents of two organelles, the wall-forming bodies type I and II (WFBI, WFBII), located exclusively in the macrogametocyte. Eimeria nieschulzi has been used as a model parasite to study and follow gametocyte and oocyst development. In this study, the gametocyte and oocyst wall formation of E. nieschulzi was analyzed by electron microscopy and immuno-histology. A monoclonal antibody raised against the macrogametocytes of E. nieschulzi identified a tyrosine-rich glycoprotein (EnGAM82) located in WFBII. Correlative light and electron microscopy was used to examine the vesicle-specific localization and spatial distribution of GAM82-proteins during macrogametocyte maturation by this monoclonal antibody. In early and mid-stages, the GAM82-protein is ubiquitously distributed in WFBII. Few hours later, the protein is arranged in subvesicular structures. It was possible to show that the substructure of WFBII and the spatial distribution of GAM82-proteins probably represent pre-synthesized cross-linked materials prior to the inner oocyst wall formation. Dityrosine-cross-linked gametocyte proteins can also be confirmed and visualized by fluorescence microscopy (UV light, autofluorescence of WFBII).

Re-evaluation of endogenous development of Eimeria bareillyi Gill, Chhabra and Lall, 1963 in water buffalo (Bubalus bubalis).

Water buffalo (Bubalus bubalis) is important for the economy of Asia, South America and parts of Europe. Coccidiosis is an important cause of neonatal mortality in livestock, including buffalo. Of more than 12 species of Eimeria in buffalo, Eimeria bareillyi is the most pathogenic. There are uncertainties concerning its asexual and sexual development. During a previously reported outbreak of fatal enteritis associated with E. bareillyi in buffaloes in the Netherlands, sections of small intestine were re-evaluated histologically and by transmission electron microscopy (TEM) to seek details of endogenous development. Profuse asexual multiplication occurred in the jejunum and ileum. Light microscopic examination revealed that parasites divided in two (probably endodyogeny) or more organisms. There were two or more generations of morphologically different merozoites; some of these observations were confirmed by TEM. Details of gametogonic development, including oocyst wall formation are provided. Schizogonic and gametogonic development described in the present study can serve as a guide for differential diagnosis of Eimeria species in histological sections of intestines of buffaloes.

Identification and analysis of Eimeria nieschulzi gametocyte genes reveal splicing events of gam genes and conserved motifs in the wall-forming proteins within the genus Eimeria (Coccidia, Apicomplexa).

The genus Eimeria (Apicomplexa, Coccidia) provides a wide range of different species with different hosts to study common and variable features within the genus and its species. A common characteristic of all known Eimeria species is the oocyst, the infectious stage where its life cycle starts and ends. In our study, we utilized Eimeria nieschulzi as a model organism. This rat-specific parasite has complex oocyst morphology and can be transfected and even cultivated in vitro up to the oocyst stage. We wanted to elucidate how the known oocyst wall-forming proteins are preserved in this rodent Eimeria species compared to other Eimeria. In newly obtained genomics data, we were able to identify different gametocyte genes that are orthologous to already known gam genes involved in the oocyst wall formation of avian Eimeria species. These genes appeared putatively as single exon genes, but cDNA analysis showed alternative splicing events in the transcripts. The analysis of the translated sequence revealed different conserved motifs but also dissimilar regions in GAM proteins, as well as polymorphic regions. The occurrence of an underrepresented gam56 gene version suggests the existence of a second distinct E. nieschulzi genotype within the E. nieschulzi Landers isolate that we maintain.

Use of fluorescent nanoparticles to investigate nutrient acquisition by developing Eimeria maxima macrogametocytes.

The enteric disease coccidiosis, caused by the unicellular parasite Eimeria, is a major and reoccurring problem for the poultry industry. While the molecular machinery driving host cell invasion and oocyst wall formation has been well documented in Eimeria, relatively little is known about the host cell modifications which lead to acquisition of nutrients and parasite growth. In order to understand the mechanism(s) by which nutrients are acquired by developing intracellular gametocytes and oocysts, we have performed uptake experiments using polystyrene nanoparticles (NPs) of 40 nm and 100 nm in size, as model NPs typical of organic macromolecules. Cytochalasin D and nocodazole were used to inhibit, respectively, the polymerization of the actin and microtubules. The results indicated that NPs entered the parasite at all stages of macrogametocyte development and early oocyst maturation via an active energy dependent process. Interestingly, the smaller NPs were found throughout the parasite cytoplasm, while the larger NPs were mainly localised to the lumen of large type 1 wall forming body organelles. NP uptake was reduced after microfilament disruption and treatment with nocodazole. These observations suggest that E. maxima parasites utilize at least 2 or more uptake pathways to internalize exogenous material during the sexual stages of development.

Ultrastructural effects of acetamizuril on endogenous phases of Eimeria tenella.

To explore the primary stage or site of action of acetamizuril (AZL), a novel triazine anticoccidial compound, the ultrastructural development of Eimeria tenella at different endogenous stages was studied in experimentally infected chickens treated with a single oral dose of 15 mg/kg AZL. As a result of drug action, the differentiations of second-generation schizonts and microgamonts were largely inhibited and merozoites became irregular in shape. Meanwhile, the outer membrane blistering and perinuclear space enlargement were obvious in the second-generation schizonts and microgamonts, which were never observed in the classic triazine anticoccidiosis drug diclazuril-treated E. tenella. The chromatin aggregation, anachromasis, and marginalization were visible in merozoites and microgamonts. Furthermore, the abnormal evagination of microgametes finally resulted in the degeneration of microgamonts and the failure of subsequent fertilization. The most marked micromorphological alteration occurring in the macrogamonts was the WFB2. Even if the fertilization occurred, the formation of oocyst wall became malformed and the zygote proceeded to the obvious degeneration. In addition, mitochondria swelling and cytoplasm vacuolization were discerned in respective intracellular stages, while endoplasmic reticulum and Golgi body swelling was less seen. These alterations may be the causes leading to the final death of E. tenella and also provide some information for further exploring the mechanism of action of AZL at the molecular level.

Two COWP-like cysteine rich proteins from Eimeria nieschulzi (coccidia, apicomplexa) are expressed during sporulation and involved in the sporocyst wall formation.

BackgroundThe family of cysteine rich proteins of the oocyst wall (COWPs) originally described in Cryptosporidium can also be found in Toxoplasma gondii (TgOWPs) localised to the oocyst wall as well. Genome sequence analysis of Eimeria suggests that these proteins may also exist in this genus and led us to the assumption that these proteins may also play a role in oocyst wall formation.MethodsIn this study, COWP-like encoding sequences had been identified in Eimeria nieschulzi. The predicted gene sequences were subsequently utilized in reporter gene assays to observe time of expression and localisation of the reporter protein in vivo.ResultsBoth investigated proteins, EnOWP2 and EnOWP6, were expressed during sporulation. The EnOWP2-promoter driven mCherry was found in the cytoplasm and the EnOWP2, respectively EnOWP6, fused to mCherry was initially observed in the extracytoplasmatic space between sporoblast and oocyst wall. This, so far unnamed compartment was designated as circumplasm. Later, the mCherry reporter co-localised with the sporocyst wall of the sporulated oocysts. This observation had been confirmed by confocal microscopy, excystation experiments and IFA. Transcript analysis revealed the intron-exon structure of these genes and confirmed the expression of EnOWP2 and EnOWP6 during sporogony.ConclusionsOur results allow us to assume a role, of both investigated EnOWP proteins, in the sporocyst wall formation of E. nieschulzi. Data mining and sequence comparisons to T. gondii and other Eimeria species allow us to hypothesise a conserved process within the coccidia. A role in oocyst wall formation had not been observed in E. nieschulzi.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-0982-3) contains supplementary material, which is available to authorized users.

F-actin distribution and function during sexual development in Eimeria maxima.

To determine the involvement of the actin cytoskeleton in macrogametocyte growth and oocyst wall formation, freshly purified macrogametocytes and oocysts were stained with Oregon Green 514 conjugated phalloidin to visualize F-actin microfilaments, while Evans blue staining was used to detect type 1 wall forming bodies (WFB1s) and the outer oocyst wall. The double-labelled parasites were then analysed at various stages of sexual development using three-dimensional confocal microscopy. The results showed F-actin filaments were distributed throughout the entire cytoplasm of mature Eimeria maxima macrogametocytes forming a web-like meshwork of actin filaments linking the type 1 WFBs together into structures resembling 'beads on a string'. At the early stages of oocyst wall formation, F-actin localization changed in alignment with the egg-shaped morphology of the forming oocysts with F-actin microfilaments making direct contact with the WFB1s. In tissue oocysts, the labelled actin cytoskeleton was situated underneath the forming outer layer of the oocyst wall. Treatment of macrogametocytes in vitro with the actin depolymerizing agents, Cytochalasin D and Latrunculin, led to a reduction in the numbers of mature WFB1s in the cytoplasm of the developing macrogametocytes, indicating that the actin plays an important role in WFB1 transport and oocyst wall formation in E. maxima.

Eimeria arloingi: Further studies on the development of some endogenous stages

The present study investigated the ultrastructural characteristics of gamogony and oocyst wall formation of Eimeria arloingi in experimentally infected kids. The 18 newborn animals allocated to 3 equal groups. Two of groups, A, B were inoculated with a single dose of 1×103 and 1×105 sporulated oocysts of E. arloingi, respectively. At 7, 14, 21, 28, 35, and 42days postinoculation (DPI), 1 kid from each group was necropsied for ultrastructural studies. Transmission electron microscopy was used to screen for the presence of developmental stages of the parasite. All stages of microgametocyte and macrogametocyte developments and also oocyst wall formation were observed from 7 to 42DPI. Different stages of schizigony accompanied by marked proliferation of endoplasmic reticulum, mitochondria and several granular dividing nuclei were diagnosed in the affected epithelial cells. Young microgamonts were recognizable by an electron lucent parasitophorous vacuole and several dividing nuclei with prominent nucleolar and peripheral chromatin in the cytoplasm. At a later stage, the nuclei began to elongate and a single mitochondrion and two basal bodies were observed in close proximity nucleus. These bodies eventually protruded from the surface of the gametocyte and formed two flagellar structures. Up to 80–120 microgametes were produced per microgamont. Macrogamonts were recognized by the presence of wall-forming bodies of types 1 and 2. Electron lucent WFB2 appeared earlier than the electron denser WFB1 during the process of macrogametogenesis. The outer layer of the oocyst wall was formed by the release of the contents of WFB1 at the surface to form an electron dense layer. The WFB2 appeared, subsequently, to give rise to the electron lucent inner layer. WFB1 plays a major role in oocyst wall formation.

Cloning and characterization of an Eimeria necatrix gene encoding a gametocyte protein and associated with oocyst wall formation

BackgroundGametocyte proteins of Eimeria (E.) spp. are important components of the oocyst wall and some have been used to develop transmission-blocking vaccines against avian coccidiosis.MethodsTotal RNA isolated from E. necatrix gametocytes was utilized as templates for RT-PCR amplification and sequencing of cDNA encoding a gametocyte protein using gene-specific primers. The cDNA was cloned into the bacterial expression vector pET28a(+) and expressed in E. coli BL21 cells. The antigenicity of the recombinant gametocyte protein and its localization in different E. necatrix life-cycle stages were determined by western blot and indirect immunofluorescence analyses, respectively.ResultsA 731-nucleotide sequence of cDNA [GenBank: KF649255] of E. necatrix had 97.7% identity to that of Etgam22 of E. tenella. The cDNA ORF encoded a 186-amino acid protein containing a histidine-proline-rich region. The recombinant gametocyte protein (rEnGAM22) was predominately expressed in the insoluble inclusion body and recognized by antiserum from chickens immunized with oocysts of E. necatrix, E. maxima and E. tenella. A specific antibody to the rEnGAM22 protein recognized the wall-forming bodies in macrogametocytes and the walls of oocysts and sporocysts.ConclusionsThe gene cloned from E. necatrix gametocytes is an ortholog to Etgam22 of E. tenella and presents a potential target for future recombinant subunit vaccines against coccidiosis.

The spatial organization and extraction of the wall-forming bodies ofEimeria maxima

Eimeria maxima has been used as a model apicomplexan parasite to study sexual stage development and oocyst wall formation. A complete understanding of the wall's biochemical and biophysical properties is of great interest in research on all apicomplexan parasites. Purified gametocytes, zygotes and oocysts were analysed by three-dimensional confocal microscopy, and wide-field fluorescent microscopy was used to investigate the appearance and spatial organization of the 2 types of wall-forming bodies (WFBs). In addition, a variety of staining procedures and immunoassays were used to assess the biosynthesis, metabolic activity, intactness and molecular composition of the WFBs in situ. WFBs were extracted from gametocytes/zygotes and their composition was assessed by microscopy and SDS-PAGE analysis. It was concluded that isolated gametocytes are intact and metabolically active. Additionally, it was observed that the Type 1 WFBs are aligned at the periphery of the parasite and fuse together producing neutral lipid rich patches that appear to be inserted into the space between 2 parasite-specific membranes. Finally, it was shown that the WFBs extracted from purified gametocytes had the same shape, size and staining properties as those observed in situ, and contained the major glycoprotein antigens known to be present in these organelles.

Molecular characterisation of a novel family of cysteine-rich proteins of Toxoplasma gondii and ultrastructural evidence of oocyst wall localisation

Among apicomplexan parasites, the coccidia and Cryptosporidium spp. are important pathogens of livestock and humans, and the environmentally resistant stage (oocyst) is essential for their transmission. Little is known of the chemical and molecular composition of the oocyst wall. Currently, the only parasite molecules shown to be involved in oocyst wall formation are the tyrosine-rich proteins gam56, gam82 and gam230 of Eimeria spp. and the cysteine-rich proteins COWP1 and COWP8 of Cryptosporidium parvum. In the present study, we searched the ToxoDB database for the presence of putative Toxoplasma gondii oocyst wall proteins (OWPs) and identified seven candidates, herein named TgOWP1 through TgOWP7, showing homology to the Cryptosporidium COWPs. We analysed a cDNA library from partially sporulated oocysts of T. gondii and cloned the full-length cDNAs encoding TgOWP1, TgOWP2 and TgOWP3, which consist of 499, 462 and 640 amino acids, respectively. The three proteins share 24% sequence identity with each other and a markedly similar overall structure, based on the presence of an N-terminal leader peptide followed by tandem duplications of a six-cysteine amino acid motif closely related to the Type I repeat of COWPs. Using antisera to recombinant TgOWP1, TgOWP2 and TgOWP3, we showed by Western blot that these molecules are expressed in T. gondii oocysts but are not detectable in tachyzoites. The solubilisation of TgOWP1–3 strictly depended on the presence of reducing agents, consistent with a likely involvement of these proteins in multimeric complexes mediated by disulphide bridges. Immunofluorescence analysis allowed the localisation of TgOWP1, TgOWP2 and TgOWP3 to the oocyst wall. Additionally, using immunoelectron microscopy and the 1G12 monoclonal antibody, TgOWP3 was specifically detected in the outer layer of the oocyst wall, thus representing the first validated molecular marker of this structure in T. gondii.

Effect of artemisinin on oocyst wall formation and sporulation during Eimeria tenella infection

The anticoccidial effect of a product extracted from the natural herb Artemisia annua, artemisinin, which has a potential use as a dietary supplement, has been studied. Commercial artemisinin was administered at 10 and 17 ppm in food and tested against infection with Eimeria tenella. A battery trial to quantify the effect of artemisinin on the reproductive and infective capabilities of E. tenella was carried out. For that purpose flow cytometry was combined with electron microscopy and immunofluorescence techniques in order to study the effect of artemisinin on E. tenella gametogenesis. Significantly reduced oocyst output and lesion scores were found in chickens treated with artemisinin. In addition, evidence to support a lower oocyst sporulation rate was obtained. Though the ultrastructural studies showed normal development of gametogenesis in artemisinin-treated chickens, the oocyst wall formation was significantly altered. This resulted in both death of developing oocysts and reduced sporulation rate. Immunofluorescent studies provided evidence that treatment with artemisinin inhibited sarcoplasmic–endoplasmic reticulum calcium ATPase (SERCA) expression in macrogametes. According to these findings, artemisinin has a deleterious effect on fertilized macrogametes (early zygotes) by inhibiting SERCA. The altered secretion of the wall-forming bodies may be the result of Ca 2+-dependent ATPase impaired activity which, in turn, is the result of SERCA inhibition.