Second-generation Merozoites Research Articles

Molecular characterization and functional analysis of Eimeria tenella ankyrin repeat-containing protein

Chicken coccidiosis causes disastrous losses to the poultry industry all over the world. Eimeria tenella is the most prevalent of these disease-causing species. Our former RNA-seq indicated that E. tenella ankyrin repeat-containing protein (EtANK) was expressed differently between drug-sensitive (DS) and drug-resistant strains. In this study, we cloned EtANK and analyzed its translational and transcriptional levels using quantitative real-time PCR (qPCR) and western blotting. The data showed that EtANK was significantly upregulated in diclazuril-resistant (DZR) strain and maduramicin-resistant (MRR) strain compared with the drug-sensitive (DS) strain. In addition, the transcription levels in the DZR strains isolated from the field were higher than in the DS strain. The translation levels of EtANK were higher in unsporulated oocysts (UO) than in sporozoites (SZ), sporulated oocysts (SO), or second-generation merozoites (SM), and the protein levels in SM were significantly higher than in UO, SO, and SZ. The results of the indirect immunofluorescence localization showed that the protein was distributed mainly at the anterior region of SZ and on the surface and in the cytoplasm of SM. The fluorescence intensity increased further with its development in vitro. An anti-rEtANK polyclonal antibody inhibited the invasive ability of E. tenella in DF-1 cells. These results showed that EtANK may be related to host cell invasion, required for the parasite’s growth in the host, and may be involved in the development of E. tenella resistance to some drugs.

Global profiling of protein S-palmitoylation in the second-generation merozoites of Eimeria tenella.

The intracellular protozoan Eimeria tenella is responsible for avian coccidiosis which is characterized by host intestinal damage. During developmental cycle, E. tenella undergoes versatile transitional stages such as oocyst, sporozoites, merozoites, and gametocytes. These developmental transitions involve changes in cell shape and cell size requiring cytoskeletal remodeling and changes in membrane proteins, which may require transcriptional and translational regulations as well as post-translational modification of proteins. Palmitoylation is a post-translational modification (PTM) of protein that orchestrates protein targeting, folding, stability, regulated enzymatic activity and even epigenetic regulation of gene expression. Previous research revealed that protein palmitoylation play essential role in Toxoplasma gondii, Trypanosoma cruzi, Trichomonas vaginalis, and several Plasmodium parasites. Until now, there is little information on the enzymes related to palmitoylation and role of protein acylation or palmitoylation in E. tenella. Therefore, palmitome of the second-generation merozoite of E. tenella was investigated. We identified a total of 2569 palmitoyl-sites that were assigned to 2145 palmitoyl-peptides belonging to 1561 protein-groups that participated in biological processes including parasite morphology, motility and host cell invasion. In addition, RNA biosynthesis, protein biosynthesis, folding, proteasome-ubiquitin degradation, and enzymes involved in PTMs, carbohydrate metabolism, glycan biosynthesis, and mitochondrial respiratory chain as well as vesicle trafficking were identified. The study allowed us to decipher the broad influence of palmitoylation in E. tenella biology, and its potential roles in the pathobiology of E. tenella infection. Raw data are publicly available at iProX with the dataset identifier PXD045061.

Comparative proteomic analysis across the developmental stages of the Eimeria tenella

Eimeria tenella is the main pathogen responsible for coccidiosis in chickens. The life cycle of E. tenella is, arguably, the least complex of all Coccidia, with only one host. However, it presents different developmental stages, either in the environment or in the host and either intracellular or extracellular. Its signaling and metabolic pathways change with its different developmental stages. Until now, little is known about the developmental regulation and transformation mechanisms of its life cycle. In this study, protein profiles from the five developmental stages, including unsporulated oocysts (USO), partially sporulated (7 h) oocysts (SO7h), sporulated oocysts (SO), sporozoites (S) and second-generation merozoites (M2), were harvested using the label-free quantitative proteomics approach. Then the differentially expressed proteins (DEPs) for these stages were identified. A total of 314, 432, 689, and 665 DEPs were identified from the comparison of SO7h vs USO, SO vs SO7h, S vs SO, and M2 vs S, respectively. By conducting weighted gene coexpression network analysis (WGCNA), six modules were dissected. Proteins in blue and brown modules were calculated to be significantly positively correlated with the E. tenella developmental stages of sporozoites (S) and second-generation merozoites (M2), respectively. In addition, hub proteins with high intra-module degree were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway enrichment analyses revealed that hub proteins in blue modules were involved in electron transport chain and oxidative phosphorylation. Hub proteins in the brown module were involved in RNA splicing. These findings provide new clues and ideas to enhance our fundamental understanding of the molecular mechanisms underlying parasite development.

Anti-Eimeria tenella activity of Ethanamizuril in vitro and in vivo.

The prevalence of chicken coccidiosis in the poultry industry is a significant concern, further exacerbated by the emergence of drug-resistant coccidia resulting from the indiscriminate use of medications. Ethanamizuril, a novel triazine anti-coccidial compound, has been used to combat drug resistance. Currently, it is known that Ethanamizuril acts on the second-generation merozoites and early gametogenesis stages of Eimeria. Limited information exists regarding its impact on the early merozoites and exogenous stage of Eimeria. In the present study, the anti-coccidial properties of Ethanamizuril were evaluated both in vitro and in vivo. The in vitro experiments demonstrated that Ethanamizuril effectively inhibits the sporulation of E. tenella oocysts in a dose-dependent manner and significantly reduces the sporozoite excystation rate. Furthermore, in vivo tests revealed that treatment with 10 mg/L Ethanamizuril in drinking water significantly decreased the copy number of first-generation and secondary-generation merozoites in the chicken cecum, indicating that it can inhibit the development of whole schizonts development. Moreover, treatment with Ethanamizuril demonstrated excellent protective efficacy with an anti-coccidial index (ACI) of 180.2, which was manifested through higher body weight gains, lighter cecal lesion, lower fecal oocyst shedding score and reduced liver index. Collectively, this study suggests that Ethanamizuril effectively treats E. tenella infection by inhibiting both endogenous and exogenous stages development.

Ponazuril: Clinical efficacy, ultrastructure, and histopathology studies of in vivo anticoccidial action against Eimeria tenella

Ponazuril, a novel antiprotozoal drug in the class of triazine, has shown a promising application on apicomplexan infections in poultry and livestock. However, the effect and mechanism of action of ponazuril against Eimeria tenella (E. tenella) are unclear. The efficacy against E. tenella was initially studied by administering different doses of ponazuril in drinking water. The treated stage and site of ponazuril on E. tenella were observed through ultrastructural and histopathological analyses. Chicks were orally treated with a dose of 15 mg/kg body weight of ponazuril at different endogenous stages of E. tenella post-infection. According to the clinical study, the values of anticoccidial indices (ACI) were 157.0, 162.3, 196.9, 194.5, and 190.9, respectively, when the ponazuril was administered in drinking water at doses of 5, 10, 20, 40, and 50 mg/L for two consecutive days after infection. Among them, the 20 mg/L ponazuril group showed the best anticoccidial effect, which was superior to that of the toltrazuril treatment group, with an ACI value of 191.7. Histological analysis indicated that ponazuril effectively relieved cecal lesions, and decreased the number of merozoites. Transmission electron micrographs (TEM) observed that merozoites became irregular in shape, and some apparent protrusions of the outer membrane were presented especially the second-generation merozoites. Additionally, abnormalities in the development of WFBI and WFBII in the macrogametocyte were observed, which may affect the formation of the ovule wall. Moreover, merozoites in the treated group showed uneven and marginalized chromatin and mitochondrial swelling. These results suggested ponazuril is a potential anticoccidial drug, providing information on the mechanism of anticoccidial effects.

Expression of a SAG protein with a CAP domain from Eimeria necatrix and its role in invasion and immunoprotection

Eimeria necatrix is a high pathogenic pathogen, which seriously endangers the poultry industry. The surface antigens (SAGs) of Apicomplexa are a kind of membrane protein anchored on the surface of the parasites through its carboxyl terminal glycosylphosphatidylinositol (GPI) structure. However, little is known about GPI-linked surface proteins in E. necatrix. In the present work, the E. necatrix sag gene (Ensag-CAP) was amplified and cloned for expression of the recombinant protein (rEnSAG-CAP). The full length Ensag-CAP gene was 813 bp, coding 270 amino acids with a predicated molecular weight of 28.86 kDa and contained a CAP domain with four sequence motifs CAP1, CAP2, CAP3 and CAP4. The rEnSAG-CAP was about 32 kDa and mainly expressed in a soluble form. Western blot analysis indicated that the rEnSAG-CAP could be recognized by anti-rEnSAG-CAP monoclonal antibody (anti-rEnSAG-CAP McAb) and the convalescent serum of chicken infected with E. necatrix. Native protein of EnSAG-CAP was detected in second-generation merozoites (MZ-2) using anti-rEnSAG-CAP polyclonal antibody (anti-rEnSAG-CAP pAb). The findings from the indirect immunofluorescence assay and enzyme digestion utilizing Bacillus cereus phosphoinositide-specific phospholipase C (PI-PLC) revealed that EnSAG-CAP predominantly localized at the surfaces of SZ and MZ-2 via a GPI anchor. It was observed that EnSAG-CAP can be cleaved from MZ-2 by PI-PLC. Real-time quantitative PCR (qPCR) analysis showed that transcript levels of Ensag-CAP in MZ-2 was significantly higher than that in SZ (P < 0.05). The anti-rEnSAG-CAP McAb in vitro could significantly inhibit the sporozoite invasion into MDBK cells (P < 0.01), which suggests that the protein might participate in sporozoite invasion into MDBK cells. rEnSAG-CAP afforded an immune protection against E. necatrix. The ACI value was 164.99 in the chickens immunized with 200 µg rEnSAG-CAP. Chickens immunized with rEnSAG-CAP had a significantly higher antigen-specific serum IgY response (P < 0.0001). The data indicates that EnSAG-CAP could serve as a potential candidate antigen for the development of a recombinant coccidiosis vaccine.

Molecular characterization of methionine aminopeptidase1 from Eimeria tenella

Coccidiosis, a serious intestinal parasitic disease caused by Eimeria spp., can result in huge annual economic losses to the poultry industry worldwide. At present, coccidiosis is mainly controlled by anticoccidial drugs. However, drug resistance has developed in Eimeria because of the long-term and unreasonable use of the drugs currently available. In our previous study, RNA-seq showed that the expression of methionine aminopeptidase1 (EtMetAP1) was up-regulated in diclazuril-resistant (DZR) and maduramicin-resistant (MRR) strains compared to drug-sensitive (DS) strain of Eimeria tenella. In this study, EtMetAP1 was cloned and expressed, and the function and characteristics of the EtMetAP1 protein were analyzed. The transcription and translation levels of EtMetAP1 in DS strain of E. tenella at different developmental stages were analyzed by qPCR and western blotting. We found that the transcription and translation levels of EtMetAP1 in second-generation merozoites (SM) were higher than those of the other three stages (unsporulated oocyst, sporulated oocyst, and sporozoites). Simultaneously, qPCR was used to analyze the mRNA transcription levels of EtMetAP1 in DS, DZR, MRR, and salinomycin-resistant (SMR) strain. The results showed that compared to the sensitive strain, the transcription levels of EtMetAP1 in DZR and MRR were up-regulated. There was no significant difference in transcription level in SMR. Indirect immunofluorescence localization showed that the protein was mainly localised in the cell membrane and cytoplasm of sporozoites and SM. An invasion inhibition test showed that anti-rEtMetAP1 polyclonal antibody could effectively inhibit the sporozoite invasion of host cells. These results suggest that the protein may be involved in the growth and development of parasites in host cells, the generation of drug resistance, and host cell invasion.

Diclazuril-induced expression of CDK-related kinase 2 in the second-generation merozoites of Eimeria tenella

Diclazuril is a classic anticoccidial drug. The key molecules of diclazuril in anticoccidial action allows target screening for the development of anticoccidial drugs. Cyclin-dependent kinases (CDK) are prominent target proteins in apicomplexan parasites. In this study, a diclazuril anticoccidiosis animal model was established, and the transcription and translation levels of the CDK-related kinase 2 of Eimeria tenella (EtCRK2) were detected. mRNA and protein expression levels of EtCRK2 decreased in the infected/diclazuril group compared with those in the infected/control group. In addition, immunofluorescence analysis showed that EtCRK2 was localised in the cytoplasm of the merozoites. The fluorescence intensity of EtCRK2 in the infected/diclazuril group was significantly weaker than that in the infected/control group. The anticoccidial drug diclazuril against E.tenella affects the expression pattern of EtCRK2 molecule, and EtCRK2 is a potential target for new drug development.

ITRAQ-based proteomic analysis reveals invasion-related proteins among three developmental stages of Eimeria necatrix

Eimeria necatrix is an obligate intracellular parasite that has a complex life cycle and causes significant economic losses to the poultry industry. To better understand the cellular invasion mechanism of E. necatrix and develop new measures against its infection, we conducted isobaric tags for relative and absolute quantitation (iTRAQ) proteomic analysis to investigate protein abundance across different life cycle stages, including unsporulated oocysts (UO), sporozoites (SZ) and second-generation merozoites (MZ-2). Our analysis identified a total of 3606 proteins, among which 1725, 1724, 2143 and 2386 were annotated by the Gene Ontology (GO), EuKaryotic Orthologous Groups (KOG), Kyoto Encyclopedia of Genes and Genomes (KEGG) and InterPro (IPR) databases, respectively. We also identified 388, 300 and 592 differentially abundant proteins in SZ vs UO, SZ vs MZ-2 and MZ-2 vs UO, respectively. Further analysis revealed that 118 differentially abundant proteins were involved in cellular invasion and could be categorized into eight groups. These findings provide valuable insights into protein abundance across the different life cycle stages of E. necatrix and offer candidate proteins for future studies on cellular invasion and other biological processes. SignificanceEimeria necatrix is an obligate intracellular parasite results in huge economic losses to the poultry industry. Understanding proteomic variations across the life cycle stages of E. necatrix may offer proteins related to cellular invasion of E. necatrix, and provide resources for the development of new treatment and prevention interventions against E. necatrix infection. The current data provide an overall summary of the protein abundance across the three life cycle stages of E. necatrix. We identified differentially abundant proteins potential related to cellular invasion. The candidate proteins we identified will form the basis of future studies for cellular invasion. This work also will help in the development of novel strategies for coccidiosis control.

Molecular characterization and analysis of drug resistance-associated protein enolase 2 of Eimeria tenella

Eimeria tenella, an intestinal parasite, has brought huge economic losses to the poultry industry. The prevalence and severity of the development of drug resistance has increased the challenge of coccidiosis control. We previously identified the enolase 2 of E. tenella (EtENO2) was differentially expressed in drug-sensitive (DS) and drug-resistant strains using RNA-seq. In this study, the expression of EtENO2 in diclazuril-resistant (DZR), maduramicin-resistant (MRR), and salinomycin-resistant (SMR) strains was analyzed by quantitative real-time PCR (qRT-PCR) and western blots. EtENO2 was highly expressed in several drug-resistant strains compared with the DS strain. The qRT-PCR showed that the transcription level of EtENO2 in the field-isolated resistant strains was upregulated compared with the DS strain. The enzyme activity results indicated that the catalytic activity of EtENO2 in the drug-resistant strains was higher than in the DS strain. In addition, qRT-PCR and western blots showed that the expression level of EtENO2 was higher in second generation merozoites (SM) and unsporulated oocysts (UO) than that in sporozoites (SZ) and sporulated oocysts (SO). Immunofluorescence localization revealed that EtENO2 was distributed throughout SZ and SM and on the surface of the parasites. After the SZ invasion DF-1 cells, it was also observed on the parasitophorous vacuole membrane. Our secretion experiments found that EtENO2 could be secreted outside the SZ. This study indicated that EtENO2 might be related to the interaction between E. tenella and host cells and be involved in the development of E. tenella resistance to some anticoccidial drugs.

Nano-encapsulation of halofuginone hydrobromide enhances anticoccidial activity against Eimeria tenella in chickens.

Coccidiosis is a worldwide epidemic intestinal disease with high incidence, which causes huge economic losses. Halofuginone hydrobromide (HF) is widely applied as an effective anticoccidial drug in the poultry industry. However, its therapeutic efficacy is severely restrained due to toxic effects, poor aqueous solubility and low permeability. Nanotechnology can improve the biological effect of drugs, and thus, reduce administered doses and toxic effects. The objective of this study was to investigate the therapeutic and preventive potential of novel HF-loaded D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) polymer micelles (HTPM) for preventing coccidiosis in chickens. The HTPM were approximately spherical with a hydrodynamic diameter of 12.65 ± 0.089 nm, a zeta potential of 8.03 ± 0.242 mV, a drug loading of 14.04 ± 0.12%, and an encapsulation efficiency of 71.1 ± 4.15%. HF was encapsulated in the polymer micelles through interactions with TPGS, as characterized by X-ray diffraction (XRD) and Fourier transform infrared (FT-IR) spectroscopy. Cellular take up assays showed that TPGS polymer micelles could enhance drug internalization to alleviate intestinal apoptosis induced by coccidiosis and promote the necrosis of second-generation merozoites of E. tenella. Notably, clinical trials proved that 1.5 mg L-1 HTPM had a stronger anticoccidial effect on E. tenella than that of 3 mg kg-1 HF premix. Amplicon sequencing identified that HTPM could alleviate coccidiosis by restoring the structure of the gut microbiome. These findings indicated that the anticoccidial efficacy of HF was significantly enhanced after being encapsulated in polymer micelles, and further demonstrated the potential protective application of nano-encapsulating anticoccidial drugs as a promising approach to control coccidiosis in poultry. In summary, HTPM hold huge potential as an effective therapeutic agent for coccidiosis.

Inhibitory effect of morin on aldolase 2 from Eimeria tenella

Eimeria tenella (E. tenella) is a protozoal parasite that can cause severe cecal lesions and death in chickens, seriously harming the chicken industry. Conventional control strategies mainly rely on anticoccidial drugs. However, the emerging problems of anticoccidial resistance and drug residues necessitate exploring potential drug targets for developing new anticoccidial drugs. Fructose-1,6-bisphosphate aldolase (ALD) is an essential enzyme for parasite energy metabolism that has been considered a potential drug target. In this study, we analyzed the molecular and biochemical properties of E. tenella ALD2 (EtALD2). EtALD2 mRNA expression was highest in second-generation merozoites, whereas the protein level was highest in unsporulated oocysts. Indirect immunofluorescence showed that EtALD2 was mainly distributed in sporozoite’ cytoplasm. The natural product inhibitor (morin) was screened by computer-aided drug screening. Enzyme kinetic and inhibition kinetic assays showed that morin had a good inhibitory effect on EtALD2 activity (IC50 = 10.37 μM, Ki = 48.97 μM). In vitro inhibition assay demonstrated that morin had an inhibitory effect on E. tenella development, with an IC50 value of 3.98 μM and drug selection index of 177.49. In vivo, morin significantly improved cecal lesions (p < 0.05) and reduced oocyst excretion (p < 0.05) in E. tenella-infected chickens compared with the untreated group. The anticoccidial index of the group receiving 450 mg morin per kg feed was 162, showing a good anticoccidial effect. These findings suggest that EtALD2 could be a novel drug target for E. tenella treatment, and morin should be further evaluated as a therapeutic candidate for chicken coccidiosis.

Characteristics analyses of Eimeria tenella 14-3-3 protein and verification of its interaction with calcium-dependent protein kinase 4

Avian coccidiosis is a common disease caused by Eimeria spp. In the genus Eimeria, the species Eimeria tenella is an obligate intracellular parasite that invades mostly chicken cecal epithelial cells. The 14-3-3 protein is one of the most common adaptor proteins. It is involved in regulating protein phosphorylation and is associated with phosphorylated proteins to regulate signal transduction. Previous reports have shown that 14-3-3 protein has a direct regulatory effect on calcium-dependent protein kinases (CDPKs) activity by interacting with CDPKs. In this study, the characteristics of the E. tenella 14-3-3 protein including transcription and translation analyses, localization in different developmental stages etc were analyzed. The interaction between E. tenella 14-3-3 (Et14-3-3) and E. tenella calcium-dependent protein kinase 4 (EtCDPK4) which is a critical molecule in E. tenella invasion of host cells was verified by Bimolecular Fluorescent Complimentary (BiFC), Co-Immunoprecipitation (co-IP), and Glutathione S-transferase (GST) pull-down. The transcription and translation levels were analyzed using real-time quantitative PCR and western blot. The results showed that the mRNA transcription level of Et14-3-3 was highest in the sporozoite, and the translation level was higher in the unsporulated oocyst than in the other stages. Indirect immunolocalization found that Et14-3-3 was located mainly at the anterior of sporozoites and on the surface of second-generation merozoites. As the sporozoites developed in cells, the fluorescence intensity of Et14-3-3 gradually darkened. BiFC results showed green fluorescence under microscopy in 293T cells co-transfected with pBiFC-VN155-Et14-3-3 and pBiFC-VC155-EtCDPK4. Co-IP and GST pull-down showed that Et14-3-3 interacted with EtCDPK4, which is consistent with the BiFC results. These results indicated that Et14-3-3 had significant interactions with EtCDPK4. Co-localization of Et14-3-3 with EtCDPK4 in sporozoites revealed that they were located in the same position. The secretion assay results indicated that Et14-3-3 was a secreted protein but was not secreted from micronemes. These results lay the foundation for further research on the mechanism of action of EtCDPK4 with Et14-3-3 and the functions of Et14-3-3 in the lifecycle of E. tenella.

Comparison of endogenous development, invasion ability and apoptotic features between diclazuril resistant and sensitive strains of Eimeria tenella

Diclazuril (DIC) is widely used in the poultry industry to control coccidiosis. However, drug resistance makes it less effective, and the underlying mechanism remains unclear. One DIC-resistant E. tenella (RE) isolate and one sensitive E. tenella (SE) isolate were used to compare the differences in their endogenous development, pathogenicity, invasion-related gene expression and apoptotic characteristics. Chickens were allocated into four groups to receive RE or SE strain and their corresponding DIC treatment or not. Caeca tissues were sampled at 96 h, 120 h and 144 h post-infection (PI) for pathological analysis. Meanwhile, second-generation merozoites (Mz2) were separated at 120 h PI to detect alterations in mitochondrial membrane potential (MMP), apoptotic rate and caspase-3 activity and mRNA expression of protein phosphatase 5 (PP5), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), actin depolymerizing factor (ADF) and microneme proteins (MICs). Haematoxylin and eosin staining revealed that DIC treatment strictly blocked the development of the SE strain but slightly affected the RE strain. Meanwhile, the number of SE Mz2 and their MMP decreased at the same time the apoptotic rate increased after DIC treatment. Real-time quantitative PCR and caspase-3 activity studies demonstrated that Mz2 from the RE strain had higher mRNA expression of ADF and MICs along with no significant changes in GAPDH and caspase-3 activity under DIC pressure compared to its control; in contrast, the mRNA expression of ADF, MICs and PP5 was markedly suppressed in Mz2 from SE with upregulated caspase-3 activity and GAPDH transcription. In addition, the mRNA expression of GAPDH and PP5 in Mz2 from RE was remarkably higher than that of SE. Taken together, the higher mRNA expression of invasion-related genes and almost unaffected endogenous development provide a better understanding of coccidian resistance to DIC.

Molecular characterization of glyceraldehyde-3-phosphate dehydrogenase from Eimeria tenella.

Chicken coccidiosis is an extremely common and lethally epidemic disease caused by Eimeria spp. The control measures of coccidiosis depend mainly on drugs. However, the ensuing drug resistance problem has brought considerable economic loss to the poultry industry. In our previous study, comparative transcriptome analyses of a drug-sensitive (DS) strain and two drug-resistant strains (diclazuril-resistant (DZR) and maduramicin-resistant (MRR) strains) of Eimeria tenella were carried out by transcriptome sequencing. The expression of glyceraldehyde-3-phosphate dehydrogenase of E. tenella (EtGAPDH) was upregulated in the two resistant strains. In this study, we cloned and characterized EtGAPDH. Indirect immunofluorescence localization was used to observe the distribution of EtGAPDH in E. tenella. The results showed that the protein was distributed mainly on the surface of sporozoites and merozoites, and in the cytoplasm of merozoites. qPCR was performed to detect the transcription level of EtGAPDH in the different developmental stages of the E. tenella DS strain. The transcription level of EtGAPDH was significantly higher in second-generation merozoites than in the other three stages. The transcription level of EtGAPDH in the different drug-resistant strains and DS strain of E. tenella was also analyzed by qPCR. The results showed that the transcription level was significantly higher in the two drug-resistant strains (MRR and DZR) than in the DS strain. As the concentration of diclazuril and maduramicin increased, the transcription levels also increased. Western blot results showed that EtGAPDH protein was upregulated in the DZR and MRR strains. Enzyme activity showed that the enzyme activity of EtGAPDH was higher in the two resistant strains than in the DS strain. These results showed that EtGAPDH possess several roles that separate and distinct from its glycolytic function and maybe involved in the development of E. tenella resistance to anticoccidial drugs.

Molecular characterization and immune protection by cystathionine β-synthase from Eimeria tenella.

Eimeria tenella is an obligate intracellular apicomplexan parasite that causes avian coccidiosis and leads to severe economic losses in the global poultry industry. Cystathionine β-synthase (CBS) and cystathionine γ-lyase (CGL) act together to generate H2S in the reverse transsulfuration pathway. In this study, E.tenella Cystathionine β-synthase (EtCBS) was cloned using rapid amplification of cDNA 5'-ends (5'RACE) and characterized, and its immunoprotective effects were evaluated. The recombinant EtCBS protein (rEtCBS) was expressed and successfully recognized by anti-sporozoites (Spz) protein rabbit serum. EtCBS mRNA levels were highest in Spz by qPCR, and the protein expression levels were higher in unsporulated oocysts (UO) than in other stages by Western blot. Indirect immunofluorescence showed that EtCBS protein was found on the surface of Spz and second-generation merozoites (Mrz). The invasion inhibition assays showed that rabbit anti-rEtCBS polyclonal antibodies effectively inhibited parasite invasion host cells. Chickens immunized with rEtCBS protein showed prominently increased weight gains and decreased oocyst output compared to nonimmunized and infected control group. The results suggest that EtCBS could be a potential vaccine candidate against E.tenella.

Genome-Wide Expression Patterns of Rhoptry Kinases during the Eimeria tenella Life-Cycle.

Kinome from apicomplexan parasites is composed of eukaryotic protein kinases and Apicomplexa specific kinases, such as rhoptry kinases (ROPK). Ropk is a gene family that is known to play important roles in host–pathogen interaction in Toxoplasma gondii but is still poorly described in Eimeria tenella, the parasite responsible for avian coccidiosis worldwide. In the E. tenella genome, 28 ropk genes are predicted and could be classified as active (n = 7), inactive (incomplete catalytic triad, n = 12), and non-canonical kinases (active kinase with a modified catalytic triad, n = 9). We characterized the ropk gene expression patterns by real-time quantitative RT-PCR, normalized by parasite housekeeping genes, during the E. tenella life-cycle. Analyzed stages were: non-sporulated oocysts, sporulated oocysts, extracellular and intracellular sporozoites, immature and mature schizonts I, first- and second-generation merozoites, and gametes. Transcription of all those predicted ropk was confirmed. The mean intensity of transcription was higher in extracellular stages and 7–9 ropk were specifically transcribed in merozoites in comparison with sporozoites. Transcriptional profiles of intracellular stages were closely related to each other, suggesting a probable common role of ROPKs in hijacking signaling pathways and immune responses in infected cells. These results provide a solid basis for future functional analysis of ROPK from E. tenella.

Molecular characterization and analysis of the ATPase ASNA1 homolog gene of Eimeria tenella in a drug sensitive strain and drug resistant strains

The widespread use of drugs has exacerbated the resistance of Eimeria tenalla to anti-coccidial drugs. Using RNA-seq, we previously found the ATPase ASNA1 homolog of E. tenella (EtASNA1) was differentially expressed in resistant strains and drug sensitive (DS) strain. In our study, we used western blotting and quantitative real-time PCR (qRT-PCR) to analyze the translational and transcriptional levels of EtASNA1 in a diclazuril-resistant (DZR) strain, maduramicin-resistant (MRR) strain, salinomycin-resistant (SMR) strain, and DS strain and found EtASNA1 was highly expressed in three drug-resistant strains. The qRT-PCR and western blotting results also showed that the expression levels of EtASNA1 increased with increasing drug concentration, and the transcription levels of the DZR strains isolated from the field were higher than those of the DS strain. In addition, we used in vivo and in vitro tests to analyze the changes of EtASNA1 expression after DZR, MRR, and DS strain infections in chickens, and in vitro inoculation of DF-1 cells in the presence of drugs. The addition of drugs caused expression to be upregulated. The results of qRT-PCR and western blotting also showed that the expression levels of EtASNA1 in second-generation merozoites (SM) and unsporulated oocysts (UO) were significantly higher than those in the other two developmental stages. The immunofluorescence localization of EtASNA1 indicated that the protein was distributed throughout the sporozoites (SZ) and SM, except for the refractile bodies of SZ. In vitro inhibition experiments showed that anti-EtASNA1 antibody incubation significantly inhibited SZ invasion of DF-1 cells. The above results showed that EtASNA1 may be related to host cell invasion of E. tenella and may be involved in the development of E. tenella resistance to some drugs.

Eimeria tenella Translation Initiation Factor eIF-5A That Interacts With Calcium-Dependent Protein Kinase 4 Is Involved in Host Cell Invasion.

Eimeria tenella is an apicomplexan, parasitic protozoan known to infect poultry worldwide. An important calcium-dependent protein kinase (CDPK) has been identified in plants, green algae, ciliates and apicomplexan, such as E. tenella. CDPKs are effector molecules involved in calcium signaling pathways, which control important physiological processes such as gliding motility, reproduction, and host cell invasion. Given that CDPKs are not found in the host, studying the functions of CDPKs in E. tenella may serve as a basis for developing new therapeutic drugs and vaccines. To assess the function of CDPK4 in E. tenella (EtCDPK4), a putative interactor, translation initiation factor eIF-5A (EteIF-5A), was screened by both co-immunoprecipitation (co-IP) and His pull-down assays followed by mass spectrometry. The interaction between EteIF-5A and EtCDPK4 was determined by bimolecular fluorescence complementation (BiFC), GST pull-down, and co-IP. The molecular characteristics of EteIF-5A were then analyzed. Quantitative real-time polymerase chain reaction and western blotting were used to determine the transcription and protein levels of EteIF-5A in the different developmental stages of E. tenella. The results showed that the transcription level of EteIF-5A mRNA was highest in second-generation merozoites, and the protein expression level was highest in unsporulated oocysts. Indirect immunofluorescence showed that the EteIF-5A protein was found throughout the cytoplasm of sporozoites, but not in the refractile body. As the invasion of DF-1 cells progressed, EteIF-5A fluorescence intensity increased in trophozoites, decreased in immature schizonts, and increased in mature schizonts. The secretion assay results, analyzed by western blotting, indicated that EteIF-5A was a secreted protein but not from micronemes. The results of invasion inhibition assays showed that rabbit anti-rEteIF-5A polyclonal antibodies effectively inhibited cell invasion by sporozoites, with an inhibition rate of 48%.

Identification and Characterization of a Novel Apical Membrane Antigen 3 in Eimeria tenella.

Eimeria tenella is an obligate intracellular parasite in the phylum Apicomplexa. As described for other members of Apicomplexa, apical membrane antigen 1 (AMA1) has been shown to be critical for sporozoite invasion of host cells by E.tenella. Recently, an E.tenella paralogue of AMA1 (EtAMA1), dubbed sporoAMA1 (EtAMA3), was identified in proteomic and transcriptomic analyses of E.tenella, but not further characterized. Here, we show that EtAMA3 is a type I integral membrane protein that has 24% -38% identity with other EtAMAs. EtAMA3 has the same pattern of Cys residues in domains I and II of AMA1 orthologs from apicomplexan parasites, but high variance in domain III, with all six invariant Cys residues absent. EtAMA3 expression was developmentally regulated at the mRNA and protein levels. EtAMA3 protein was detected in sporulated oocysts and sporozoites, but not in the unsporulated oocysts or second-generation merozoites. EtAMA3 is secreted by micronemes and is primarily localized to the apical end of sporozoites during host-cell invasion. Additionally, pretreatment of sporozoites with rEtAMA3-specific antibodies substantially impeded their invasion into host cells. These results suggest EtAMA3 is a sporozoite-specific protein that is involved in host-cell sporozoite invasion.