Correlation of caecal microbiome endotoxins genes and intestinal immune cells in Eimeria tenella infection based on bioinformatics.

Introduction: The infection with Eimeria tenella (ET) can elicit expression of various intestinal immune cells, incite inflammation, disrupt intestinal homeostasis, and facilitate co-infection with diverse bacteria. However, the reciprocal interaction between intestinal immune cells and intestinal flora in the progression of ET-infection remains unclear. Objective: The aim of this study was to investigate the correlation between cecal microbial endotoxin (CME)-related genes and intestinal immunity in ET-infection, with subsequent identification of hub potential biomarker and immunotherapy target. Methods: Differential expression genes (DEGs) within ET-infection and hub genes related to CME were identified through GSE39602 dataset based on bioinformatic methods and Protein-protein interaction (PPI) network analysis. Moreover, immune infiltration was analyzed by CIBERSORT method. Subsequently, comprehensive functional enrichment analyses employing Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis along with Gene Ontology (GO), gene set enrichment analysis (GSEA), and gene set variation analysis (GSVA) were performed. Results: A total of 1089 DEGs and 25 hub genes were identified and CXCR4 was ultimately identified as a essential CME related potential biomarker and immunotherapy target in the ET-infection. Furthermore, activated natural killer cells, M0 macrophages, M2 macrophages, and T regulatory cells were identified as expressed intestinal immune cells. The functional enrichment analysis revealed that both DEGs and hub genes were significantly enriched in immune-related signaling pathways. Conclusion: CXCR4 was identified as a pivotal CME-related potential biomarker and immunotherapy target for expression of intestinal immune cells during ET-infection. These findings have significant implications in elucidating the intricate interplay among ET-infection, CME, and intestinal immunity.

Recombinant production of SAG1 fused with xylanase in Pichia pastoris induced higher protective immunity against Eimeria tenella infection in chicken.

Chicken coccidiosis is an intestinal disease caused by the parasite Eimeria, which severely damages the growth of chickens and causes significant economic losses in the poultry industry. Improvement of the immune protective effect of antigens to develop high efficiency subunit vaccines is one of the hotspots in coccidiosis research. Sporozoite-specific surface antigen 1 (SAG1) of Eimeria tenella (E. tenella) is a well-known protective antigen and is one of the main target antigens for the development of subunit, DNA and vector vaccines. However, the production and immunoprotective effects of SAG1 need to be further improved. Here, we report that both SAG1 from E. tenella and its fusion protein with the xylanase XynCDBFV-SAG1 are recombinant expressed and produced in Pichia pastoris (P. pastoris). The substantial expression quantity of fusion protein XynCDBFV-SAG1 is achieved through fermentation in a 15-L bioreactor, reaching up to about 2 g/L. Moreover, chickens immunized with the fusion protein induced higher protective immunity as evidenced by a significant reduction in the shedding of oocysts after E. tenella challenge infection compared with immunized with recombinant SAG1. Our results indicate that the xylanase enhances the immunogenicity of subunit antigens and has the potential for developing novel molecular adjuvants. The high expression level of fusion protein XynCDBFV-SAG1 in P. pastoris holds promise for the development of effective recombinant anti-coccidial subunit vaccine.

CRISPR-Cas9-based method for isolating microgametes of Eimeria tenella.

Eimeria tenella infections are known to cause severe caecal damage and death of the infected chicken. Gamogony is an essential stage in E. tenella life cycle and in the establishment of coccidiosis. Prior research had extensively explored isolation and separation of the parasite gametes - microgamete (male) and macrogamete (female). However, there is little information on the efficient, highly purified and distinctly separated male and female gametes. In this study, we generated a genome editing line expressing mCherry fluorescent protein fused with GCS1 protein in E. tenella by using Toxoplasma gondii CRISPR-Cas9 system, flow cytometry and fluorescence microscopy. This allowed precise separation of E. tenella male and female gametes in the transgenic parasite population. The separation of male and female gametes would not only build on our understanding of E. tenella transmission, but it would also facilitate development of gametocidal compounds as drug targets for E. tenella infection.

Integrated application of transcriptomics and metabolomics provides insight into the mechanism of Eimeria tenella resistance to maduramycin.

Avian coccidiosis, caused by Eimeria parasites, continues to devastate the poultry industry and results in significant economic losses. Ionophore coccidiostats, such as maduramycin and monensin, are widely used for prophylaxis of coccidiosis in poultry. Nevertheless, their efficacy has been challenged by widespread drug resistance. However, the underlying mechanisms have not been revealed. Understanding the targets and resistance mechanisms to anticoccidials is critical to combat this major parasitic disease. In the present study, maduramycin-resistant (MRR) and drug-sensitive (DS) sporozoites of Eimeria tenella were purified for transcriptomic and metabolomic analysis. The transcriptome analysis revealed 5016 differentially expressed genes (DEGs) in MRR compared to DS, and KEGG pathway enrichment analysis indicated that DEGs were involved in spliceosome, carbon metabolism, glycolysis, and biosynthesis of amino acids. In the untargeted metabolomics assay, 297 differentially expressed metabolites (DEMs) were identified in MRR compared to DS, and KEGG pathway enrichment analysis indicated that these DEMs were involved in 10 pathways, including fructose and mannose metabolism, cysteine and methionine metabolism, arginine and proline metabolism, and glutathione metabolism. Targeted metabolomic analysis revealed 14 DEMs in MRR compared to DS, and KEGG pathway analysis indicated that these DEMs were involved in 20 pathways, including fructose and mannose metabolism, glycolysis/gluconeogenesis, and carbon metabolism. Compared to DS, energy homeostasis and amino acid metabolism were differentially regulated in MRR. Our results provide gene and metabolite expression landscapes of E. tenella following maduramycin induction. This study is the first work involving integrated transcriptomic and metabolomic analyses to identify the key pathways to understand the molecular and metabolic mechanisms underlying drug resistance to polyether ionophores in coccidia.

Expression of Toll-like receptors and host defence peptides in the cecum of chicken challenged with Eimeria tenella.

Chicken coccidiosis, caused by Eimeria protozoa, affects poultry farming. Toll-like receptors (TLRs) and host defence peptides (HDPs) help host innate immune responses to eliminate invading pathogens, but their roles in Eimeria tenella infection remain poorly understood. Herein, 14-day-old chickens were treated orally with 50,000 E. tenella oocysts and the cecum was dissected at different timepoints. mRNA expression of 10 chicken TLRs (chTLRs) and five HDPs was measured by quantitative real-time PCR. chTLR7 and chTLR15 were upregulated significantly at 3 h post-infection while other chTLRs were downregulated (p < .05). chTLR1a, chTLR1b, chTLR2b and chTLR4 peaked at 36 h post-infection, chTLR3, chTLR5 and chTLR15 peaked at 72 h post-infection and chTLR21 expression was highest among chTLRs, peaking at 48 h post-infection (p < 0.05). For HDPs, cathelicidin (CATH) 1 to 3 and B1 peaked at 48 h post-infection, liver-expressed antimicrobial peptide 2 peaked at 96 h post-infection, and CATH 2 expression was highest among HDPs. CATH2 and CATH3 were markedly upregulated at 3 h post-infection (p < .05). The results provide insight into innate immune molecules during E. tenella infection in chicken, and indicate that innate immune responses may mediate resistance to chicken coccidiosis.

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.

Phylogenetic analysis of Eimeria tenella isolates from chicken of sub-tropical mountains of Meghalaya, India.

BACKGROUND: Coccidiosis is the most common and pathogenic intestinal disease caused by different species of Eimeria is chicken. In this study, we describe the prevalence, molecular diagnosis and evolutionary insight of Eimeria tenella in chicken of Meghalaya's sub-tropical mountainous area. METHODS AND RESULTS: Faecal samples (337 no.) and dead chicks (298 no.) were collected every month from January to July' 2023 from poultry farms (4nos.) in and around Umiam, Ri-Bhoi, Meghalaya. The chicks were categorized into different age groups viz. < 3, 3-6 and > 6 weeks. Samples were examined by flotation techniques and post-mortem. The oocysts were sporulated in 2.5% potassium dichromate solution. Eimeria tenella's 18 S rRNA gene genomic DNA was extracted, amplified, and sequenced. Fecal sample and postmortem examinations revealed 24.04% and 33.22% infections of Eimeria sp., respectively. Oocyst per gram (OPG) was recorded highest and lowest in July (26,500) and February (9800), respectively. Amplification of the 18 S rRNA small subunit gene (SSU) by Polymerase Chain Reaction (PCR) revealed a 1790 bp band size. The amplicon was sequenced and deposited in the NCBI database. BLAST analyses of the SSU rRNA gene of E. tenella, Umiam, Meghalaya isolate (OR458392.1) revealed sequence similarities of more than 99% with SSU rRNA gene sequences available in the NCBI database. Pair wise alignment exhibited nucleotide homology ranging from 71.59 to 100.0% with the maximum sequence homology (100.0%) shared with the E. tenella isolate from Turkey (HQ680474.1) and the lowest homology of 95.6% with UK (HG994972.1). Umiam isolate were found to have 97.08% and 100.0% nucleotide similarities with E. tenella from both the UK (AF026388.1) and the USA (U40264.1), respectively. However, nucleotide similarities of 98.24%, 85.33%, 84.75% and 81.35% were observed with E. tenella strain Bangalore (JX312808.1), E. tenella isolate Kerala-1 (JX093898.1), E. tenella isolate Kerala-3 (JX093900.1) and E. tenella isolate Kerala-2 (JX093899.1), respectively. Phylogenetic analysis of SSU rRNA sequences of E. tenella Umiam, Meghalaya isolate with cognate sequences throughout the world revealed these sequences are distinct but at the same time share a close phylogenetic relationship with Indian isolates from Bangalore and Andhra Pradesh. In addition, the distant phylogenetic relationship was observed with cognate gene sequences of United States of America, Canada, China. CONCLUSION: Phylogenetic analysis of SSU rRNA sequences of E. tenella Umiam, Meghalaya isolate with cognate sequences throughout the world revealed these sequences are distinct but at the same time share a close phylogenetic relationship with Indian isolates from Bangalore and Andhra Pradesh. Distant phylogenetic relationship was observed with cognate gene sequences of United States of America, Canada, China.

Protective efficacy of multiepitope vaccines constructed from common antigens of Eimeria species in chickens.

Clinical avian coccidiosis is typically caused by coinfection with several Eimeria species. Recombinant protein and DNA vaccines have shown promise in controlling coccidiosis. On this basis, DNA vaccines that encode multiple epitopes from different Eimeria species may provide broad protection against coinfections. In this study, we designed a fusion gene fragment, 14EGT, that contained concentrated T-cell epitopes from four common antigens of Eimeria species (14-3-3, elongation factor 2, glyceraldehyde-3-phosphate dehydrogenase, and transhydrogenase). The multiepitope DNA vaccine pVAX1-14EGT and recombinant protein vaccine pET-32a-14EGT (r14EGT) were then created based on the 14EGT fragment. Subsequently, cellular and humoral immune responses were measured in vaccinated chickens. Vaccination-challenge trials were also conducted, where the birds were vaccinated with the 14EGT preparations and later exposed to single or multiple Eimeria species to evaluate the protective efficacy of the vaccines. According to the results, vaccination with 14EGT preparations effectively increased the proportions of CD4+ and CD8+ T cells and the levels of Th1 and Th2 hallmark cytokines. The levels of serum IgG antibodies were also significantly increased. Animal vaccination trials revealed alleviated enteric lesions, weight loss, and oocyst output compared to those of the control groups. The preparations were found to be moderately effective against single Eimeria species, with the anticoccidial index (ACI) ranging from 160 to 180. However, after challenge with multiple Eimeria species, the protection provided by the 14EGT preparations was not satisfactory, with ACI values of 142.18 and 146.41. Collectively, the results suggest that a multiepitope vaccine that encodes the T-cell epitopes of common antigens derived from Eimeria parasites could be a potential and effective strategy to control avian coccidiosis.

A candidate virulence factor of Eimeria tenella (EtROP30) predicted by virulence enhancement of transgenic Toxoplasma gondii.

Difficulties of in vitro culture and genetic manipulation of Eimeria tenella have hindered the screening of virulence factors in this parasite. In this study, the E. tenella rhoptry protein 30 (EtROP30) was expressed in Toxoplasma gondii (RH∆Ku80-EtROP30), and its effect on the proliferation and virulence of parasites was investigated. The results revealed that the expression of EtROP30 had no impact on the invasion and egress processes. However, the RH∆Ku80-EtROP30 strain formed larger plaques compared to the RH∆Ku80, indicating that the EtROP30 expression promotes T. gondii proliferation. Furthermore, the RH∆Ku80-EtROP30 strain exhibited greater pathogenicity, resulting in earlier mortality and shorter overall survival time compared to RH∆Ku80. These results imply that EtROP30 expression facilitates parasite intracellular proliferation and virulence in mice, suggesting that EtROP30 might be a candidate virulence factor of E. tenella.

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.

Chromosomal scale assembly reveals localized structural variants in avian caecal coccidian parasite Eimeria tenella.

Eimeria tenella is a major cause of caecal coccidiosis in commercial poultry chickens worldwide. Here, we report chromosomal scale assembly of Eimeria tenella strain APU2, a strain isolated from commercial broiler chickens in the U.S. We obtained 100× sequencing Oxford Nanopore Technology (ONT) and more than 800× Coverage of Illumina Next-Seq. We created the assembly using the hybrid approach implemented in MaSuRCA, achieving a contiguous 51.34 Mb chromosomal-scale scaffolding enabling identification of structural variations. The AUGUSTUS pipeline predicted 8060 genes, and BUSCO deemed the genomes 99% complete; 6278 (78%) genes were annotated with Pfam domains, and 1395 genes were assigned GO-terms. Comparing E. tenella strains (APU2, US isolate and Houghton, UK isolate) derived Houghton strain of E. tenella revealed 62,905 high stringency differences, of which 45,322 are single nucleotide polymorphisms (SNPs) (0.088%). The rate of transitions/transversions among the SNPs are 1.63 ts/tv. The strains possess conserved gene order but have profound sequence heterogeneity in a several chromosomal segments (chr 2, 11 and 15). Genic and intergenic variation in defined gene families was evaluated between the two strains to possibly identify sequences under selection. The average genic nucleotide diversity of 2.8 with average 2 kb gene length (0.145%) at genic level. We examined population structure using available E. tenella sequences in NCBI, revealing that the two E. tenella isolates from the U.S. (E. tenella APU2 and Wisconsin, "ERR296879") share a common maternal inheritance with the E. tenella Houghton. Our chromosomal level assembly promotes insight into Eimeria biology and evolution, hastening drug discovery and vaccine development.

Distinct non-synonymous mutations in cytochrome b highly correlate with decoquinate resistance in apicomplexan parasite Eimeria tenella.

BACKGROUND: Protozoan parasites of the genus Eimeria are the causative agents of chicken coccidiosis. Parasite resistance to most anticoccidial drugs is one of the major challenges to controlling this disease. There is an urgent need for a molecular marker to monitor the emergence of resistance against anticoccidial drugs, such as decoquinate. METHODS: We developed decoquinate-resistant strains by successively exposing the Houghton (H) and Xinjiang (XJ) strains of E. tenella to incremental concentrations of this drug in chickens. Additionally, we isolated a decoquinate-resistant strain from the field. The resistance of these three strains was tested using the criteria of weight gain, relative oocyst production and reduction of lesion scores. Whole-genome sequencing was used to identify the non-synonymous mutations in coding genes that were highly associated with the decoquinate-resistant phenotype in the two laboratory-induced strains. Subsequently, we scrutinized the missense mutation in a field-resistant strain for verification. We also employed the AlphaFold and PyMOL systems to model the alterations in the binding affinity of the mutants toward the drug molecule. RESULTS: We obtained two decoquinate-resistant (DecR) strains, DecR_H and XJ, originating from the original H and XJ strains, respectively, as well as a decoquinate-resistant E. tenella strain from the field (DecR_SC). These three strains displayed resistance to 120 mg/kg decoquinate administered through feed. Through whole-genome sequencing analysis, we identified the cytochrome b gene (cyt b; ETH2_MIT00100) as the sole mutated gene shared between the DecR_H and XJ strains and also detected this gene in the DecR_SC strain. Distinct non-synonymous mutations, namely Gln131Lys in DecR_H, Phe263Leu in DecR_XJ, and Phe283Leu in DecR_SC were observed in the three resistant strains. Notably, these mutations were located in the extracellular segments of cyt b, in close proximity to the ubiquinol oxidation site Qo. Drug molecular docking studies revealed that cyt b harboring these mutants exhibited varying degrees of reduced binding ability to decoquinate. CONCLUSIONS: Our findings emphasize the critical role of cyt b mutations in the development of decoquinate resistance in E. tenella. The strong correlation observed between cyt b mutant alleles and resistance indicates their potential as valuable molecular markers for the rapid detection of decoquinate resistance.

Transcriptomic, 16S ribosomal ribonucleic acid and network pharmacology analyses shed light on the anticoccidial mechanism of green tea polyphenols against Eimeria tenella infection in Wuliangshan black-boned chickens.

BACKGROUND: Eimeria tenella is an obligate intracellular parasitic protozoan that invades the chicken cecum and causes coccidiosis, which induces acute lesions and weight loss. Elucidating the anticoccidial mechanism of action of green tea polyphenols could aid the development of anticoccidial drugs and resolve the problem of drug resistance in E. tenella. METHODS: We constructed a model of E. tenella infection in Wuliangshan black-boned chickens, an indigenous breed of Yunnan Province, China, to study the efficacy of green tea polyphenols against the infection. Alterations in gene expression and in the microbial flora in the cecum were analyzed by ribonucleic acid (RNA) sequencing and 16S ribosomal RNA (rRNA) sequencing. Quantitative real-time polymerase chain reaction was used to verify the host gene expression data obtained by RNA sequencing. Network pharmacology and molecular docking were used to clarify the interactions between the component green tea polyphenols and the targeted proteins; potential anticoccidial herbs were also analyzed. RESULTS: Treatment with the green tea polyphenols led to a reduction in the lesion score and weight loss of the chickens induced by E. tenella infection. The expression of matrix metalloproteinase 7 (MMP7), MMP1, nitric oxide synthase 2 and ephrin type-A receptor 2 was significantly altered in the E. tenella infection plus green tea polyphenol-treated group and in the E. tenella infection group compared with the control group; these genes were also predicted targets of tea polyphenols. Furthermore, the tea polyphenol (-)-epigallocatechin gallate acted on most of the targets, and the molecular docking analysis showed that it has good affinity with interferon induced with helicase C domain 1 protein. 16S ribosomal RNA sequencing showed that the green tea polyphenols had a regulatory effect on changes in the fecal microbiota induced by E. tenella infection. In total, 171 herbs were predicted to act on two or three targets in MMP7, MMP1, nitric oxide synthase 2 and ephrin type-A receptor 2. CONCLUSIONS: Green tea polyphenols can directly or indirectly regulate host gene expression and alter the growth of microbiota. The results presented here shed light on the mechanism of action of green tea polyphenols against E. tenella infection in chickens, and have implications for the development of novel anticoccidial products.

The caecal microbiota promotes the acute inflammatory response and the loss of the intestinal barrier integrity during severe Eimeria tenella infection.

Introduction: Coccidiosis, a disease caused by intestinal apicomplexan parasites Eimeria, is a threat to poultry production. Eimeria tenella is one of the most pathogenic species, frequently causing a high prevalence of opportunistic infections. Objective: The objective of this study is to investigate the role of the microbiota in the pathogenesis of severe Eimeria tenella infection. Methods: We have previously shown that microbiota can promote parasite development. To study the effect of the microbiota on the pathogenesis of this infection, we used an experimental condition (inoculum of 10 000 oocysts E. tenella INRAE) in which the parasite load is similar between germ-free and conventional broilers at 7 days post-infection (pi). Thirteen conventional and 24 germ-free chickens were infected. Among this latter group, 12 remained germ-free and 12 received a microbiota from conventional healthy chickens at 4 days pi. Caeca and spleens were collected at 7 days pi. Results: Our results demonstrated caecal lesions and epithelium damage in conventional chickens at 7 days pi but not in germ-free infected chickens. Administration of conventional microbiota to germ-free chickens partially restored these deleterious effects. At day 7 pi, both infected conventional and germ-free chickens exhibited increased gene expression of inflammatory mediators, including IL15, IFNγ, TNFα and the anti-inflammatory mediator SOCS1, whereas the inflammatory mediators CXCLi2, CCL20, IL18, CSF1, NOS2, PTGS2, IL1ß, IL6, the receptor CCR2, and the anti-inflammatory mediators TGFß1 and IL10 were upregulated only in infected conventional chickens. Notably, the IL18, PTGS2 gene expression was significantly higher in the infected conventional group. Overall, the inflammatory response enhanced by the microbiota might be in part responsible for higher lesion scores. Epithelial tight junction protein gene expression analysis revealed a significant upregulation of CLDN1 with the infection and microbiota, indicating a potential loss of the intestinal barrier integrity. Conclusion: These observations imply that, during E. tenella infection, the caecal microbiota could trigger an acute inflammatory response, resulting in a loss of intestinal integrity. Increase in bacterial translocation can then lead to the likelihood of opportunistic infections. Hence, modulating the microbiota may offer a promising strategy for improving poultry gut health and limiting caecal coccidiosis.

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.

Label-free quantitative detection and comparative analysis of lysine acetylation during the different life stages of Eimeria tenella.

Proteome-wide lysine acetylation has been documented in apicomplexan parasite Toxoplasma gondii and Plasmodium falciparum. Here, we conducted the first lysine acetylome in unsporulated oocysts (USO), sporulated 7 h oocysts (SO 7h), sporulated oocysts (SO), sporozoites (S), and the second generation merozoites (SMG) of Eimeria tenella through a 4D label-free quantitative technique. Altogether, 8532 lysine acetylation sites on 2325 proteins were identified in E. tenella, among which 5445 sites on 1493 proteins were quantified. In addition, 557, 339, 478, 248, 241, and 424 differentially expressed proteins were identified in the comparisons SO7h vs USO, SO vs SO7h, SO vs USO, S vs SO, SMG vs S, and USO vs SMG, respectively. The bioinformatics analysis of the acetylome showed that the lysine acetylation is widespread on proteins of diverse functions. Moreover, the dynamic changes of lysine acetylome among E. tenella different life stages revealed significant regulation during the whole process of E. tenella growth and stage conversion. This study provides a beginning for the investigation of the regulate role of lysine acetylation in E. tenella and may provide new strategies for anticoccidiosis drug and vaccine development. Raw data are publicly available at iProX with the data set identifier PXD040368.

Molecular characterization and protective efficacy of vacuolar protein sorting 29 from Eimeria tenella.

Introduction: Vacuolar protein sorting 29 (VPS29) is a core component of the retromer-retriever complex and is essential for recycling numerous cell-surface cargoes from endosomes. However, there are no reports yet on VPS29 of Eimeria spp. Methods: Here, we cloned and prokaryotically expressed a partial sequence of Eimeria tenella VPS29 (EtVPS29) with RT-PCR and engineered strain of Escherichia coli respectively. The localization of the VPS29 protein in E. tenella sporozoites was investigated with immunofluorescence (IFA) and overexpression assays. And its protective efficacy against E. tenella infection was investigated in chickens with the animal protection test. Results: An EtVPS29 gene fragment with an ORF reading frame of 549 bp was cloned. The band size of the expressed recombinant protein, rEtVPS29, was approximately 39 kDa and was recognized by the chicken anti-E. tenella positive serum. EtVPS29 protein was observed widely distributing in the cytoplasm of E. tenella sporozoites in the IFA and overexpression assays. rEtVPS29 significantly increased average body weight gain and decreased mean lesion score and oocyst output in chickens. The relative weight gain rate in the rEtVPS29-immunized group was 62.9%, which was significantly higher than that in the unimmunized and challenged group (P < 0.05). The percentage of reduced oocyst output in the rEtVPS29 immunized group was 32.2%. The anticoccidial index of the rEtVPS29-immunized group was 144.2. Serum ELISA also showed that rEtVPS29 immunization induced high levels of specific antibodies in chickens. Discussion: These results suggest that rEtVPS29 can induce a specific immune response and is a potential candidate for the development of novel vaccines against E. tenella infections in chickens.

Comprehensive analysis of gut microbiome and host transcriptome in chickens after Eimeria tenella infection.

Background: Coccidiosis is an intestinal parasitic disease caused by Eimeria protozoa, which endangers the health and growth of animals, and causes huge economic losses to the poultry industry worldwide every year. Studies have shown that poultry gut microbiota plays an important role in preventing the colonization of pathogens and maintaining the health of the host. Coccidia infection also affects host gene expression. However, the underlying potential relationship between gut microbiome and host transcriptome during E. tenella infection in chickens remain unclear. Methods: In this study, metagenomic and transcriptome sequencing were applied to identify microbiota and genes in cecal contents and cecal tissues of infected (JS) and control (JC) chickens on day 4.5 postinfection (pi), respectively. Results: First, microbial sequencing results of cecal contents showed that the abundance of Lactobacillus, Roseburia sp. and Faecalibacterium sp decreased significantly after E. tenella infection (P < 0.05), while the abundance of Alistipes and Prevotella pectinovora increased significantly (P < 0.05). Second, transcriptome sequencing results showed that a total of 434 differentially expressed mRNAs were identified, including 196 up-regulated and 238 down-regulated genes. These differentially expressed genes related to inflammation and immunity, such as GAMA, FABP1, F2RL1 and RSAD2, may play an important role in the process of host resistance to coccidia infection. Functional studies showed that the enriched pathways of differentially expressed genes included the TGF-beta signaling pathway and the ErbB signaling pathways. Finally, the integrated analysis of gut microbiome and host transcriptome suggested that Prevotella pectinovora associated with FABP1, Butyricicoccus porcorum and Colidextribacter sp. associated with RSAD2 were involved in the immune response upon E. tenella infection. Conclusion: In conclusion, this study provides valuable information on the microbiota and key immune genes after chicken E. tenella infection, with the aim of providing reference for the impact of coccidia infection on cecal microbiome and host.

Overexpression of Eimeria tenella Rhoptry Kinase 2 Induces Early Production of Schizonts.

Eimeria tenella is an obligate intracellular parasite responsible for avian coccidiosis. Like other apicomplexan parasites, such as Toxoplasma gondii, cell invasion and intracellular development rely on apical organelle content discharge, named micronemes and rhoptries. Some rhoptry (ROP) kinases (ROPK) are key virulence factors in T. gondii. To date, among the 28 ropk genes carried by E. tenella, only two to four were confirmed by proteomic analysis or immunostaining to be expressed at the sporozoite stage. We have previously shown that EtROP1 is implicated in the inhibition of host cell apoptosis by interacting with the cellular p53. This work functionally described the second ROP kinase expressed at the sporozoite stage in E. tenella. EtROP2 is an active kinase that phosphorylates cell substrates of approximately 50 kDa. Its overexpression leads to the shortening of the prepatent period and to the early development of first-generation schizonts. Conduction of RNA sequencing analysis and reverse transcriptase quantitative PCR (RT-qPCR) on the host cell allowed us to identify the mitogen-activated protein kinase (MAPK) pathway and the transcription factor cFos to be upregulated by EtROP2. We also showed by immunofluorescence assay that the active kinase EtROP2 is implicated in the p38 MAPK pathway activation. We established here that EtROP2 activates the p38 MAPK pathway through a direct or indirect phosphorylation, leading to the overexpression of the master transcription factor cFos known to be implicated in E. tenella development. IMPORTANCE Rhoptries are specialized secretory organelles found in zoite stages of apicomplexan parasites. In addition to well-conserved rhoptry neck proteins, their protein consists mostly of kinase proteins, highly divergent from eukaryotic kinases. Some of those kinases are described as major virulence factors in Toxoplasma gondii, secreted into the host cell to hijack signaling pathways. Most of those kinases remain to be characterized in Eimeria tenella. Deciphering their cellular function is a prerequisite to supporting their relevance as a druggable target in development of new means of Eimeria tenella control. Secreted divergent kinases that interact with host cell partners to modulate pathways are good candidates, as they coevolve with their host targets to ensure their function within the host and are less prone to mutations that would lead to drug resistance. The absence of any orthologous kinase in host cells makes these parasite kinases a promising drug target candidate.

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.