Quantitative phosphoproteomic analysis of chicken DF-1 cells infected with Eimeria tenella, using tandem mass tag (TMT) and parallel reaction monitoring (PRM) mass spectrometry.

Eimeria tenella is an obligate intracellular parasite which causes great harm to the poultry breeding industry. Protein phosphorylation plays a vital role in host cell-E. tenella interactions. However, no comprehensive phosphoproteomic analyses of host cells at various phases of E. tenella infection have been published. In this study, quantitative phosphoproteomic analysis of chicken embryo DF-1 fibroblasts that were uninfected (UI) or infected with E. tenella for 6 h (PI6, the early invasion phase) or 36 h (PI36, the trophozoite development phase) was conducted. A total of 10,122 phosphopeptides matched to 3,398 host cell phosphoproteins were identified and 13,437 phosphorylation sites were identified. Of these, 491, 1,253, and 275 differentially expressed phosphorylated proteins were identified in the PI6/UI, PI36/UI, and PI36/PI6 comparisons, respectively. KEGG pathway enrichment analysis showed that E. tenella modulated host cell processes through phosphorylation, including focal adhesion, regulation of the actin cytoskeleton, and FoxO signaling to support its early invasion phase, and modulating adherens junctions and the ErbB signaling pathway to favor its trophozoite development. These results enrich the data on the interaction between E. tenella and host cells and facilitate a better understanding of the molecular mechanisms underlying host-parasite relationships.

Title: Analyse phosphoprotéomique quantitative de cellules DF-1 de poulet infectées par Eimeria tenella, par spectrométrie de masse avec marqueur de masse en tandem (TMT) et surveillance des réactions parallèles (PRM). Abstract: Eimeria tenella est un parasite intracellulaire obligatoire qui cause de graves dommages à l'industrie de l'élevage de volailles. La phosphorylation des protéines joue un rôle essentiel dans les interactions entre la cellule hôte et E. tenella. Cependant, aucune analyse phosphoprotéomique complète des cellules hôtes à différentes phases de l'infection par E. tenella n'a été publiée. Dans cette étude, une analyse phosphoprotéomique quantitative de fibroblastes DF-1 d'embryon de poulet non infectés (NI) ou infectés par E. tenella pendant 6 h (PI6, la phase d'invasion précoce) ou 36 h (PI36, la phase de développement des trophozoïtes) a été réalisée. Un total de 10 122 phosphopeptides correspondant à 3 398 phosphoprotéines de cellules hôtes ont été identifiés et 13 437 sites de phosphorylation ont été identifiés. Parmi celles-ci, 491, 1 253 et 275 protéines différentiellement phosphorylées exprimées ont été identifiées respectivement dans les comparaisons PI6/NI, PI36/NI et PI36/PI6. L'analyse d'enrichissement de la voie KEGG a montré qu'E. tenella modulait les processus de la cellule hôte par phosphorylation, y compris l'adhésion focale, la régulation du cytosquelette d'actine et la signalisation FoxO, pour aider sa phase d'invasion précoce, et la modulation des jonctions adhérentes et de la voie de signalisation ErbB pour favoriser le développement de son trophozoïte. Ces résultats enrichissent les données sur l'interaction entre E. tenella et les cellules hôtes et facilitent une meilleure compréhension des mécanismes moléculaires sous-jacents aux relations hôtes­parasites.

Graded levels of Eimeria challenge altered the microstructural architecture and reduced the cortical bone growth of femur of Hy-Line W-36 pullets at early stage of growth (0-6 wk of age).

An experiment was carried out to evaluate the impact of mixed Eimeria challenge on skeletal health of Hy-Line W-36 pullets. A total of 540, 16-day-old pullets were randomly allocated into 5 treatment groups, including a nonchallenged control. A mixed Eimeria species solution containing 50,000 E. maxima, 50,000 E. tenella, and 250,000 E. acervulina oocysts per mL was prepared and challenged to 1 group as a high-dose treatment. The 2-fold serial dilution was done to prepare the medium-high (25,000 E. maxima; 25,000 E. tenella; 125,000 E. acervulina), the medium-low (12,500 E. maxima; 12,500 E. tenella; 62,500 E. acervulina), and the low (6,250 E. maxima; 6,250 E. tenella; 31,250 E. acervulina) dose treatments which were challenged to 3 corresponding groups, respectively. The mineral apposition rate (MAR) was measured from 0 to 14 d post inoculation (DPI) and 14 to 28 DPI using calcein injection. The microstructural architecture of the femur was analyzed using the Skyscan X-ray microtomography (microCT) on 6, 14, and 28 DPI. The results showed that the MAR decreased linearly with an increase in the challenged dose (P < 0.05) during 0 to 14 DPI. The results of microCT revealed that cortical and total BMD, BMC, bone volume (BV), and bone volume as a fraction of tissue volume (BV/TV) of femur decreased both linearly (P < 0.05). Conversely, the total number of pores increased linearly with an increase in challenge dosages on 6 and 14 DPI. Trabecular BMD, BV, BV/TV, trabecular number, and trabecular thickness decreased linearly with an increase in the challenge dosages (P < 0.05) on 6 DPI. Furthermore, Eimeria infection significantly increased the number of osteoclasts and osteoclastic activity (P = 0.001). The result of this study suggests that the mixed Eimeria challenge negatively impacts the quality of skeletal health in a linear or quadratic manner with an increase in the concentration of Eimeria oocysts. The negative impact on long bone development might be due to malabsorption, nutrient deficiency during the infection, along with oxidative stress/inflammation disrupting the balance of osteoblastic and osteoclastic cells and their functions.

Investigating the effects of essential oils and pure botanical compounds against Eimeria tenella in vitro.

Essential oils (EO) and natural bioactive compounds are well-known antibacterial and anti-inflammatory factors; however, little is known about their anticoccidial activity and mode of action. EO deriving from basil (BEO), garlic (GAR), oregano (OEO), thyme (TEO), and their main bioactive compounds were investigated for their anticoccidial proprieties and compared to salinomycin (SAL) and amprolium (AMP) in vitro. The invasion of Eimeria tenella sporozoites was studied on 2 cell models: Madin-Darby Bovine Kidney (MDBK) cells and primary chicken epithelial cells (cIEC). Invasion efficiency was evaluated at 2 and 24 h postinfection (hpi) with counts of extracellular sporozoites and by detection of intracellular E. tenella DNA by PCR. Results show that at both timepoints, the EO were most effective in preventing the invasion of E. tenella with an average reduction of invasion at 24 hpi by 36% in cIEC and 55% in MDBK. The study also examined cytokine gene expression in cIEC at 24 hpi and found that AMP, BEO, OEO, TEO, carvacrol (CAR), and thymol (THY) significantly reduced interleukin (IL)8 expression, with CAR also reducing expression of IL1ß and IL6 compared to the infected control. In addition, this work investigated the morphology of E. tenella sporozoites treated with anticoccidial drugs and EO using a scanning electron microscope. All the treatments induced morphological anomalies, characterized by a reduction of area, perimeter and length of sporozoites. SAL had a significant impact on altering sporozoite shape only at 24 h, whereas CAR and THY significantly compromised the morphology already at 2 hpi, compared to the untreated control. OEO and GAR showed the most significant alterations among all the treatments. The findings of this study highlight the potential of EO as an alternative to traditional anticoccidial drugs in controlling E. tenella invasion and in modulating primary immune response.

Refractile bodies of Eimeria tenella are proteinaceous membrane-less organelles that undergo dynamic changes during infection.

Introduction: Refractile bodies (RB) are large membrane-less organelles (MLO) of unknown function found as a prominent mismatched pair within the sporozoite stages of all species of Eimeria, parasitic coccidian protozoa. Methods: High resolution imaging methods including time-lapse live confocal microscopy and serial block face-scanning electron microscopy (SBF-SEM) were used to investigate the morphology of RB and other intracellular organelles before and after sporozoite invasion of host cells. Results: Live cell imaging of MDBK cells infected with E. tenella sporozoites confirmed previous reports that RB reduce from two to one post-infection and showed that reduction in RB number occurs via merger of the anterior RB with the posterior RB, a process that lasts 20-40 seconds and takes place between 2- and 5-hours post-infection. Ultrastructural studies using SBF-SEM on whole individual sporozoites, both pre- and post-host cell invasion, confirmed the live cell imaging observations and showed also that changes to the overall sporozoite cell shape accompanied RB merger. Furthermore, the single RB post-merger was found to be larger in volume than the two RB pre-merger. Actin inhibitors were used to investigate a potential role for actin in RB merger, Cytochalasin D significantly inhibited both RB merger and the accompanying changes in sporozoite cell shape. Discussion: MLOs in eukaryotic organisms are characterised by their lack of a membrane and ability to undergo liquid-liquid phase separation (LLPS) and fusion, usually in an actin-mediated fashion. Based on the changes in sporozoite cell shape observed at the time of RB merger together with a potential role for actin in this process, we propose that RB are classed as an MLO and recognised as one of the largest MLOs so far characterised.

"Mango in all her majesty"-the potential of mangiferin and its analogues in the inhibition of Eimeria tenella hexokinase-a computational approach.

The potential of natural products in mitigating infections and diseases are being considered lately. Herein, via in silico methods, we report the possible molecular mechanism of mangiferin (isolated from the fruit, peel, bark and leaves of mango tree) and its derivatives in inhibiting Eimeria tenella hexokinase. We evaluated the binding affinity of these inhibitors to the glucose binding site of EtHK and thereafter proceeded to molecular dynamics simulation. The Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) reveals that three of the derivatives (CPAMM, MxPAMM and NAMM) had better total binding free energy than mangiferin. The ADMET and physicochemical properties assessed shows that inhibitors also hold a potential to be drug-likely. Finally, in mediating their inhibitory potentials, the ligands stabilize both the global and local structures of the protein. This study provides a theoretical premise on which the anti-coccidial propensities of mangiferin most especially its derivatives can be investigate in vitro and in vivo.Communicated by Ramaswamy H. Sarma.

In Vitro Study to Evaluate the Efficacy of Ultrasonicated Ethanolic Extract of Rosmarinus officinalis and its Chitosan-Based Nanoparticles Against Eimeria tenella Oocysts of Chickens.

In this study, chitosan nanoparticles (CsNPs) were used as nanocarrier for ultrasonicated ethanolic extract of Rosmarinus officinalis (UEERO) as a new nanoformulation against Eimeria tenella. Herein, CsNPs have been synthesized by ionic gelation method at pH 3 (CsNPs3) and pH 5 (CsNPs5), followed by characterization of morphology, size, polydispersity index (PDI), surface charge, and loading efficiency of UEERO. An in vitro sporulation inhibition assay (10, 5, 2.5, 1.25, 0.62, 0.31, 0.15, 0.07, 0.04, 0.02, and 0.01 mg/ml normal saline solution) against E. tenella was conducted. Results showed that free CsNPs and UEERO-CsNPs3/5 were cubic- and spherical-shaped with positive charge and average size of ~ 150.8 nm (314.4 nm) and 151.7 nm (321.1 nm), respectively. The total loading efficiency using UV-vis spectrophotometer, was 80.05 at pH 5 and 64.39% at pH 3. The in vitro sporulation inhibition assay revealed that UEERO, CsNPs3/5, and UEERO-CsNPs3/5 showed a potential inhibitory effect on sporulation (%), distortion in wall (%), and sporocyst abnormality (%) in a dose-dependent manner. Accordingly, the concentration (10 mg/ml) showed the best efficacy after 24 h in UEERO, free CsNPs, and UEERO-CsNPs. Moreover, UEERO-CsNPs3 and UEERO-CsNPs5 had stopped the sporulation (%) after 72 h. Taken all together, UEERO-CsNPs3 and UEERO-CsNPs5 are best effective against E. tenella in a dose-dependent manner in terms of sporulation (%), distortion in wall (%), and sporocysts abnormality.

Role of EtMIC4 EGF-like in regulating the apoptosis of Eimeria tenella host cells via the EGFR pathway.

This study aimed to explore the role and key point of EtMIC4 EGF-like recombinant protein in regulating the apoptosis of Eimeria tenella host cells via the epidermal growth factor receptor (EGFR) pathway. The cells were treated with EtMIC4 EGF-like protein, EGFR-specific siRNA, or both. Infection and apoptosis rates as well as dynamic changes in the key genes and proteins of the EGFR signaling pathway in the host cells were determined. Results showed that the E. tenella and EtMIC4 EGF-like group had the highest infection rate (P < 0.01). In cells treated with EtMIC4 EGF-like for 4 to 24 h, the apoptosis rate was significantly decreased (P < 0.01) and the relative mRNA expression and protein phosphorylation levels of EGFR, protein kinase B (AKT), and extracellular regulated protein kinases (ERK) were significantly increased (P < 0.01). In E. tenella sporozoites infected for 4 to 96 h, the rate of host cell apoptosis induced by E. tenella infection was significantly (P < 0.01) reduced by EtMIC4 EGF-like. The relative mRNA expression and protein phosphorylation levels of EGFR, AKT, and ERK in the host cells of E. tenella + EtMIC4 EGF-like group were significantly increased (P < 0.01). These results indicated that E. tenella could activate the EGFR pathway through EtMIC4 EGF-like and regulate the expression of key genes in the AKT and ERK signaling pathways, thereby inhibiting cell apoptosis.

The role of Ca2+ in the injury of host cells during the schizogenic stage of E. tenella.

Cecal epithelial cell damage is a key factor in host injure during the development of E. tenella. The intracellular free Ca2+ of the host cell is closely related to the invasion, development and proliferation of intracellular parasites, and cell damage. To determine the relationship between Ca2+ and host cell damage in the schizogenic stage of E. tenella, we established a chick embryo cecal epithelial cells model of E. tenella infection. Fluorescence staining, flow cytometry, transmission electron microscopy, inhibition and blocking experiments were used to detect the damage effect and mechanism of host cells during the schizogenic stage of E. tenella. The results showed that the host cells cytoskeletal remodeling, cell and organelle structure was destroyed, and apoptosis and necrosis were increased during the schizont stage of E. tenella. Furthermore, the above-mentioned effects of the schizogenic stage of E. tenella on cells can be alleviated by reducing the intracellular Ca2+ concentration in the host cells. These observations indicate that the effect of host cell injury was closely related to Ca2+ during schizont stage of E. tenella.

Sanguinarine induces apoptosis in Eimeria tenella sporozoites via the generation of reactive oxygen species.

Eimeria tenella (E. tenella) is the most pathogenic genus in Eimeria and can lead to a huge number of deaths of chickens, causing significant economic losses in the poultry industry worldwide. As a natural alkaloid, sanguinarine has many medicinal effects; to a certain extent, it can replace antibiotics and has good application prospects in veterinary medicine. To evaluate the effect of sanguinarine on sporozoites of E.tenella, we used flow cytometry and immunofluorescence staining to detect reactive oxygen species (ROS), mitochondrial membrane potential (MMP), calcium ion (Ca2+), and caspase-3 activation in E.tenella sporozoites treated with different concentrations of sanguinarine. The results of flow cytometry showed that sanguinarine could inhibit the invasion of sporozoites of E.tenella in vitro (P < 0.05) and increase the reactive oxygen species and calcium ions in the sporozoites (P < 0.05). The results of immunofluorescence staining showed that sanguinarine could decrease the mitochondrial membrane potential of sporozoites. Our analysis suggests that sanguinarine can induce apoptosis of E. tenella sporozoites through reactive oxygen species-mediated reduction of the mitochondrial membrane potential and an increase in calcium ion concentration. It follows that sanguinarine is likely to be a novel type of anticoccidiosis drug with good research and clinical application prospects.

Effects of host vimentin on Eimeria tenella sporozoite invasion.

BACKGROUND: Chicken coccidiosis is a parasitic disease caused by Eimeria of Apicomplexa, which has caused great economic loss to the poultry breeding industry. Host vimentin is a key protein in the process of infection of many pathogens. In an earlier phosphorylation proteomics study, we found that the phosphorylation level of host vimentin was significantly regulated after Eimeria tenella sporozoite infection. Therefore, we explored the role of host vimentin in the invasion of host cells by sporozoites. METHODS: Chicken vimentin protein was cloned and expressed. We used qPCR, western blotting, and indirect immunofluorescence to detect levels of mRNA transcription, translation, and phosphorylation, and changes in the distribution of vimentin after E. tenella sporozoite infection. The sporozoite invasion rate in DF-1 cells treated with vimentin polyclonal antibody or with small interfering RNA (siRNA), which downregulated vimentin expression, was assessed by an in vitro invasion test. RESULTS: The results showed that vimentin transcription and translation levels increased continually at 6-72 h after E. tenella sporozoite infection, and the total phosphorylation levels of vimentin also changed. About 24 h after sporozoite infection, vimentin accumulated around sporozoites in DF-1 cells. Treating DF-1 cells with vimentin polyclonal antibody or downregulating vimentin expression by siRNA significantly improved the invasion efficiency of sporozoites. CONCLUSION: In this study, we showed that vimentin played an inhibitory role during the invasion of sporozoites. These data provided a foundation for clarifying the relationship between Eimeria and the host.

In vitro infection of Madin-Darby bovine kidney (MDBK) cells with Eimeria acervulina sporozoites: quantitative analysis of parasite cellular invasion and replication using real-time polymerase chain reaction (PCR).

Poultry coccidiosis causes considerable economical losses to the livestock industry. Eimeria parasites are responsible for this disease. On a global scale, E. acervulina and E. tenella are amongst the most common Eimeria spp. infecting broilers. E. tenella is commonly used as infection model in in vivo and in vitro studies. On the other hand, E. acervulina has barely been studied under in vitro conditions. A well established and widely used in vitro model for E. tenella infection is the Madin-Darby bovine kidney cell line (MDBK); however, little is known regarding suitability of MDBK cells as host cells for E. acervulina. We infected MDBK monolayers with two different doses, 5 × 104 and 2 × 105, of E. acervulina sporozoites and evaluated cultures at 24 and 96 h post infection (hpi). For comparison, we ran an identical infection assay using E. tenella sporozoites. To assess parasite reproduction, the number of DNA copies of E. acervulina SCAR marker and E. tenella ITS-1 gene was quantified using real-time quantitative PCR. We found that the number of E. acervulina copies increased significantly at 24 hpi in comparison to E. tenella (p < 0.05). After 96 hpi, E. acervulina gene copies were considerably reduced while E. tenella continued to multiply (p < 0.05). Our results show that MDBK monolayers could be used for in vitro research aimed to study E. acervulina sporozoite cell invasion. Nevertheless, modifications of in vitro cultivation appear necessary to allow qualitative and quantitative studies over longer periods of parasite reproduction.

Transcriptome Analysis Revealed Ameliorative Effects of Bacillus Based Probiotic on Immunity, Gut Barrier System, and Metabolism of Chicken under an Experimentally Induced Eimeria tenella Infection.

In this study, we performed transcriptome analysis in the cecum tissues of negative control untreated non-challenged (NC), positive control untreated challenged (PC), and Bacillus subtilis (B. subtilis) fed challenged chickens (BS + ET) in order to examine the underlying potential therapeutic mechanisms of Bacillus based probiotic feeding under an experimental Eimeria tenella (E. tenella) infection. Our results for clinical parameters showed that birds in probiotic diet decreased the bloody diarrhea scores, oocyst shedding, and lesion scores compared to positive control birds. RNA-sequencing (RNA-seq) analysis revealed that in total, 2509 up-regulated and 2465 down-regulated differentially expressed genes (DEGs) were detected in the PC group versus NC group comparison. In the comparison of BS + ET group versus PC group, a total of 784 up-regulated and 493 down-regulated DEGs were found. Among them, several DEGs encoding proteins involved in immunity, gut barrier integrity, homeostasis, and metabolism were up-regulated by the treatment of probiotic. Functional analysis of DEGs also revealed that some gene ontology (GO) terms related with immunity, metabolism and cellular development were significantly affected by the exposure of probiotic. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis showed that the DEGs in the cecum of B. subtilis-fed challenged group were mainly participated in the pathways related with immunity and gut barrier integrity, included mitogen-activated protein kinase (MAPK) signaling pathway, toll-like receptor (TLR) signaling pathway, extracellular matrix (ECM)-receptor interaction, tight junction, and so on. Taken together, these results suggest that Bacillus based probiotic modulate the immunity, maintain gut homeostasis as well as barrier system and improve chicken metabolism during E. tenella infection.

Dual RNA-Seq transcriptome analysis of chicken macrophage-like cells (HD11) infected in vitro with Eimeria tenella.

The study aimed to monitor parasite and host gene expression during the early stages of Eimeria tenella infection of chicken cells using dual RNA-Seq analysis. For this, we used chicken macrophage-like cell line HD11 cultures infected in vitro with purified E. tenella sporozoites. Cultures were harvested between 2 and 72 h post-infection and mRNA was extracted and sequenced. Dual RNA-Seq analysis showed clear patterns of altered expression for both parasite and host genes during infection. For example, genes in the chicken immune system showed upregulation early (2­4 h), a strong downregulation of genes across the immune system at 24 h and a repetition of early patterns at 72 h, indicating that invasion by a second generation of parasites was occurring. The observed downregulation may be due to immune self-regulation or to immune evasive mechanisms exerted by E. tenella. Results also suggested pathogen recognition receptors involved in E. tenella innate recognition, MRC2, TLR15 and NLRC5 and showed distinct chemokine and cytokine induction patterns. Moreover, the expression of several functional categories of Eimeria genes, such as rhoptry kinase genes and microneme genes, were also examined, showing distinctive differences which were expressed in sporozoites and merozoites.

Exogenous nitric oxide stimulates early egress of Eimeria tenella sporozoites from primary chicken kidney cells in vitro.

Egress plays a vital role in the life cycle of apicomplexan parasites including Eimeria tenella, which has been attracting attention from various research groups. Many recent studies have focused on early egress induced by immune molecules to develop a new method of apicomplexan parasite elimination. In this study, we investigated whether nitric oxide (NO), an immune molecule produced by different types of cells in response to cytokine stimulation, could induce early egress of eimerian sporozoites in vitro. Eimeria tenella sporozoites were extracted and cultured in primary chicken kidney cells. The number of sporozoites egressed from infected cells was analyzed by flow cytometry after treatment with NO released by sodium nitroferricyanide (II) dihydrate. The results showed that exogenous NO stimulated the rapid egress of E. tenella sporozoites from primary chicken kidney cells before replication of the parasite. We also found that egress was dependent on intra-parasitic calcium ion (Ca2+) levels and no damage occurred to host cells after egress. The virulence of egressed sporozoites was significantly lower than that of fresh sporozoites. The results of this study contribute to a novel field examining the interactions between apicomplexan parasites and their host cells, as well as that of the clearance of intracellular pathogens by the host immune system.

TITLE: L'oxyde nitrique exogène stimule in vitro la sortie précoce des sporozoïtes d'Eimeria tenella des cellules primaires de rein de poulet. ABSTRACT: La sortie des cellules joue un rôle vital dans le cycle de vie des parasites Apicomplexa, y compris Eimeria tenella, ce qui a attiré l'attention de plusieurs groupes de recherche. De nombreuses études récentes se sont concentrées sur la sortie précoce induite par des molécules immunitaires, pour développer une nouvelle méthode d'élimination des parasites Apicomplexa. Dans cette étude, nous avons examiné si l'oxyde nitrique (NO), une molécule immunitaire produite par différents types de cellules en réponse à la stimulation des cytokines, pouvait induire in vitro une sortie précoce des sporozoïtes des Eimeria. Les sporozoïtes d'E. tenella ont été extraits et cultivés dans des cellules primaires de rein de poulet. Le nombre de sporozoïtes sortant des cellules infectées a été analysé par cytométrie en flux après traitement avec du NO libéré par le nitroferricyanure de sodium (II) dihydraté. Les résultats ont montré que le NO exogène stimulait la sortie rapide des sporozoïtes d'E. tenella des cellules primaires de rein de poulet avant la réplication du parasite. Nous avons également constaté que la sortie dépendait des niveaux intra-parasitaires d'ions calcium (Ca2+) et qu'aucun dommage n'est survenu aux cellules hôtes après la sortie. La virulence des sporozoïtes sortis était significativement inférieure à celle des sporozoïtes frais. Les résultats de cette étude contribuent à un nouveau domaine d'étude des interactions entre les parasites Apicomplexa et leurs cellules hôtes, ainsi qu'à celui relatif à l'élimination des pathogènes intracellulaires par le système immunitaire de l'hôte.

Activation of ChTLR15/ChNF-κB-ChNLRP3/ChIL-1ß signaling transduction pathway mediated inflammatory responses to E. tenella infection.

Avian coccidiosis caused by Eimeria leads to severe economic losses in the global poultry industry. Although chicken Toll-like receptor 15 (ChTLR15) was reported to be involved in Eimeria infection, the detailed mechanism underlying its role in the inflammatory response remains to be discovered. The present study demonstrated that the mRNA expression levels of ChTLR15, ChMyD88, ChNF-κB, ChNLRP3, ChCaspase-1, ChIL-18 and ChIL-1ß and the protein levels of ChTLR15 and ChNLRP3 in cecal tissues of Eimeria-infected chickens were significantly elevated at 4, 12, and 24 h compared with those in noninfected control chickens (p < 0.01). Moreover, the mRNA levels of molecules in the ChTLR15/ChNF-κB and ChNLRP3/ChIL-1ß pathways and the protein levels of ChTLR15 and ChNLRP3 in chicken embryo fibroblast cells (DF-1) stimulated by E. tenella sporozoites were consistent with those in Eimeria-infected chickens. Furthermore, overexpression of ChTLR15 in DF1 cells augmented activation of the ChTLR15/ChNF-κB and ChNLRP3/ChIL-1ß pathways when stimulated with E. tenella sporozoites, while knockdown of ChTLR15 in DF1 cells showed inverse effects. Taken together, the present study provides evidence that E. tenella sporozoites specifically activate ChTLR15 and then trigger activation of the ChNLRP3/ChIL-1ß pathway, which partially mediates inflammatory responses to Eimeria infection.

Molecular characterization and functional analysis of Eimeria tenella citrate synthase.

Chicken coccidiosis, caused by an obligate intracellular protozoan parasite of the genus Eimeria, is a major parasitic disease in the intensively reared poultry industry. Due to the widespread use of anticoccidial drugs, resistance has become an inevitable problem. In our previous study, Eimeria tenella citrate synthase (EtCS) was found to be up-expressed in two drug-resistant strains (diclazuril-resistant and maduramycin-resistant strains) compared to drug-sensitive strain by RNA sequence. In this study, we cloned and expressed EtCS and obtain its polyclonal antibodies. Quantitative real-time polymerase chain (qPCR) reactions and Western blots were used to analyze the transcription and translation levels of EtCS in sensitive and three drug-resistant strains. Compared with the sensitive strain, the transcription of EtCS was both significantly upregulated in diclazuril-resistant and maduramycin-resistant strains, but was not significantly different in salinomycin-resistant strain. No significant difference was seen in translation level in the three drug-resistant strains. Indirect immunofluorescence indicated that EtCS was mainly located in the cytoplasm of sporozoites except for posterior refractile bodies and in the cytoplasm and surface of merozoites. Anti-rEtCS antibody has inhibitory effects on E. tenella sporozoite invasion of DF-1 cells and the inhibition rate is more than 83%. Binding of the protein to chicken macrophage (HD11) cells was confirmed by immunofluorescence assays. When macrophages were treated with rEtCS, secretion of nitric oxide and cell proliferation of the macrophages were substantially reduced. These results showed that EtCS may be related to host cell invasion of E. tenella and involve in the development of E.tenella resistance to some drugs.

Identification and Characterization of the ATG8, a Marker of Eimeria tenella Autophagy / Identificação e Caracterização do ATG8, um marcador de autofagia em Eimeria tenella

Abstract Autophagy plays an important role in maintaining cell homeostasis through degradation of denatured proteins and other biological macromolecules. In recent years, many researchers focus on mechanism of autophagy in apicomplexan parasites, but little was known about this process in avian coccidia. In our present study. The cloning, sequencing and characterization of autophagy-related gene (Etatg8) were investigated by quantitative real-time PCR (RT-qPCR), western blotting (WB), indirect immunofluorescence assays (IFAs) and transmission electron microscopy (TEM), respectively. The results have shown 375-bp ORF of Etatg8, encoding a protein of 124 amino acids in E. tenella, the protein structure and properties are similar to other apicomplexan parasites. RT-qPCR revealed Etatg8 gene expression during four developmental stages in E. tenella, but their transcriptional levels were significantly higher at the unsporulated oocysts stage. WB and IFA showed that EtATG8 was lipidated to bind the autophagosome membrane under starvation or rapamycin conditions, and aggregated in the cytoplasm of sporozoites and merozoites, however, the process of autophagosome membrane production can be inhibited by 3-methyladenine. In conclusion, we found that E. tenella has a conserved autophagy mechanism like other apicomplexan parasites, and EtATG8 can be used as a marker for future research on autophagy targeting avian coccidia.

Resumo A autofagia desempenha um papel importante na manutenção da homeostase celular através da degradação de proteínas desnaturadas e outras macromoléculas biológicas. Nos últimos anos, muitos pesquisadores se concentraram no mecanismo da autofagia em parasitas apicomplexos, mas pouco se sabe sobre esse processo na coccidia aviária. No presente estudo, a clonagem, sequenciamento e caracterização de gene relacionado à autofagia Etatg8 foram investigados pela PCR quantitativa em tempo real (RT-qPCR), mancha ocidental (WB), ensaios indiretos de imunofluorescência (IFAs) e microscopia eletrônica de transmissão (TEM), respectivamente. Os resultados mostraram que o gene Etatg8 de E. tenella possui uma ORF de 375 bp, codificando uma proteína de 124 aminoácidos com estrutura e propriedades semelhantes à de outros apicomplexos. RT-qPCR revelou que Etatg8 é expresso durante os quatro estágios de desenvolvimento de E. tenella. Entretanto, seus níveis transcricionais foram significativamente mais elevados na fase de oocisto não esporulados. Os ensaios de manchas ocidental (WB) e de imunofluorescência (IFA) mostraram que a proteína EtATG8 foi lipidada para ligar-se à membrana do autofagossomo sob condições de deficiência nutritiva (em presença de rapamicina) e se agregar no citoplasma de esporozoítas e merozoítas. No entanto, o processo de produção de membrana do autofagossomo pode ser inibido por um inibidor de autofagia (3-meetiladeninatiladenina, 3-MA). Em conclusão, foi demonstrado que E. tenella tem um mecanismo de autofagia conservado, semelhante ao de outros parasitas apicomplexos, e que EtATG8 pode ser usado como um marcador para futuras pesquisas sobre autofagia direcionada à coccidiose aviária.

Apicomplexan co-infections impair with phagocytic activity in avian macrophages.

Mixed infections of Toxoplasma gondii and Eimeria tenella are likely to occur frequently due to the high prevalence of both pathogens in free-ranging chickens. In this study, we investigated the co-occurrence of the two parasites in the same immune-competent host cell towards altered patterns of parasite-host interactions. Chicken blood monocyte-derived macrophages were co-infected with T. gondii RH tachyzoites and E. tenella Houghton sporozoites in vitro for 24 h. Through monitoring the uptake of pH-sensitive pHrodo™ Zymosan BioParticles ("Zymosan") by macrophages, we created a three-dimensional model and to analyze quantitatively phagocytosis using confocal laser scanning microscopy. Assessments of parasite populations were performed by qPCR at 2, 6, 12, and 24 h post-infection (hpi). At 6 hpi, phagocytosis was inhibited in the E. tenella-infected cultures while no inhibition of phagocytosis was observed due to T. gondii. Phagocytosis activity revealed more complex interactions during co-infection. At 12 and 24 hpi, phagocytosis response to "Zymosan" was distinctly weaker in co-infected cells than in all other groups except for cells mono-infected with high doses of E. tenella at 24 hpi. By qPCR, significantly reduced numbers of both intracellular parasites were recorded (10-fold) in all infected groups at 2 hpi. At 12 hpi, the T. gondii population reached lowest values but dramatically increased by 24 hpi. Our data confirm that macrophage phagocytosis is involved in the control of invasion by apicomplexan parasites in chicken which particularly applies to E. tenella infection and it was able to be altered by the co-existing parasites.

EtMIC3 and its receptors BAG1 and ENDOUL are essential for site-specific invasion of Eimeria tenella in chickens.

Avian coccidian parasites exhibit a high degree of site specificity in different Eimeria species. Although the underlying mechanism is unclear, an increasing body of evidence suggests that site specificity is due to the interaction between microneme proteins (MICs) and their receptors on the surface of target host cells. In this study, the binding ability of E. tenella MICs (EtMICs) to different intestinal tissue was observed by immunofluorescence to identify the key surface molecule on the parasite responsible for the site specificity. Subsequently, we identified the corresponding host-cell receptors by yeast two-hybrid screening and glutathione-S-transferase pull-down experiments and the distribution of these receptors was observed by immunofluorescence in chicken intestinal tissues. Finally, we evaluated the efficacy of receptor antiserum against the infection of E. tenella in chickens. The results showed that EtMIC3 could only bind to the caecum while EtMIC1, EtMIC2, and EtAMA1 did not bind to any other intestinal tissues. Anti-serum to EtMIC3 was able to block the invasion of sporozoites with a blocking rate of 66.3%. The receptors for EtMIC3 were BCL2-associated athanogene 1 (BAG1) and Endonuclease polyU-specific-like (ENDOUL), which were mainly distributed in the caecum. BAG1 and ENDOUL receptor antiserum reduced weight loss and oocyst output following E. tenella infection, showing partial inhibition of E. tenella infection. These data elucidate the mechanism of site specificity for Eimeria infection and reveal a potential therapeutic avenue.