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Chicken coccidiosis causes retarded growth and low production performance in poultry, resulting in huge economic losses to the poultry industry. In order to prevent and control chicken coccidiosis, great efforts have been made to develop new drugs and vaccines, which require pure isolates of Eimeria spp. In this study, we obtained the Eimeira tenella Xiantao isolate by single oocyst isolation technology and compared its genome with the reference genome GCF_000499545.2_ETH001 of the Houghton strain. The results of the comparative genomic analysis indicated that the genome of this isolate contained 46,888 single nucleotide polymorphisms (SNPs). There were 15,107 small insertion and deletion variations (indels), 1693 structural variations (SV), and 3578 copy number variations (CNV). In addition, 64 broilers were used to determine the resistance profile of Xiantao strain. Drug susceptibility testing revealed that this isolate was completely resistant to monensin, diclazuril, halofuginone, sulfachlorpyrazine sodium, and toltrazuril, but sensitive to decoquinate. These data improve our understanding of drug resistance in avian coccidia., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Chicken coccidiosis is a protozoan disease that leads to considerable economic losses in the poultry industry. Live oocyst vaccination is currently the most effective measure for the prevention of coccidiosis. However, it provides limited protection with several drawbacks, such as poor immunological protection and potential reversion to virulence. Therefore, the development of effective and safe vaccines against chicken coccidiosis is still urgently needed., Methods: In this study, a novel oral vaccine against Eimeria tenella was developed by constructing a recombinant Lactobacillus plantarum (NC8) strain expressing the E. tenella RON2 protein. We administered recombinant L. plantarum orally at 3, 4 and 5 days of age and again at 17, 18 and 19 days of age. Meanwhile, each chick in the commercial vaccine group was immunized with 3 × 10 2 live oocysts of coccidia. A total of 5 × 10 4 sporulated oocysts of E. tenella were inoculated in each chicken at 30 days. Then, the immunoprotection effect was evaluated after E. tenella infection., Results: The results showed that the proportion of CD4 + and CD8 + T cells, the proliferative ability of spleen lymphocytes, inflammatory cytokine levels and specific antibody titers of chicks immunized with recombinant L. plantarum were significantly increased (P < 0.05). The relative body weight gains were increased and the number of oocysts per gram (OPG) was decreased after E. tenella challenge. Moreover, the lesion scores and histopathological cecum sections showed that recombinant L. plantarum can significantly relieve pathological damage in the cecum. The ACI was 170.89 in the recombinant L. plantarum group, which was higher than the 150.14 in the commercial vaccine group., Conclusions: These above results indicate that L. plantarum expressing RON2 improved humoral and cellular immunity and enhanced immunoprotection against E. tenella. The protective efficacy was superior to that of vaccination with the commercial live oocyst vaccine. This study suggests that recombinant L. plantarum expressing the RON2 protein provides a promising strategy for vaccine development against coccidiosis., (© 2024. The Author(s).)

Avian coccidiosis, caused by Eimeria spp., is one of the major parasitic diseases in chicken. Aquaporins (AQP) are essential mediators of water regulation and nutritional intake in parasites, and it may be a suitable molecule for chemotherapeutic target and vaccine candidate. We identified two aquaporin genes in Eimeria tenella (EtAQP1 and EtAQP2) with their full sequence, and the expression profiles were analyzed across different stages of E. tenella life cycle. The expression of EtAQP1 and EtAQP2 in Xenopus oocytes renders them highly permeable for both water and glycerol. Sugar alcohols up to five carbons and urea pass the pore. The immunohistochemical analysis confirms the restriction of antiserum staining to the surface of transfected Xenopus oocytes. Like other AQP family, EtAQPs are transmembrane proteins that are likely important molecules that facilitate solute uptake for parasite intracellular growth and therapeutic targets., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In prokaryotes and lower eukaryotes, 2-methylcitrate cycle (2-MCC) is the main pathway for propionate decomposition and transformation, but little is known about the 2-MCC pathway of Eimeria tenella. The analysis of genomic data found that the coding gene of 2- methylcitrate synthase (EC 2.3.3.5, PrpC) exists in E. tenella, which is a key enzyme of 2-MCC pathway. Through the search analysis of the database (ToxoDB), it was found that ETH&#95; 00026655 contains the complete putative sequence of EtprpC. In this study, we amplified the ORF sequence of EtprpC based on putative sequence. Then, prokaryotic expression, enzyme activity and kinetic analysis was performed. The results showed that the EtprpC ORF sequence was 1272 bp, encoding a 46.3 kDa protein comprising 424 amino acids. Enzyme activity assays demonstrate linearity between the initial reaction rate (OD/min) and EtPrpC concentration (ranging from 1.5 to 9 µg/reaction), with optimal enzyme activity observed at 41°C and pH 8.0. The results of enzymatic kinetic analysis showed that the Km of EtPrpC for propionyl-CoA, oxaloacetic acid, and acetyl-CoA was 5.239 ± 0.17 mM, 1.102 ± 0.08 μM, and 5.999 ± 1.24 μM, respectively. The Vmax was 191.11 ± 19.1 nmol/min/mg, 225.48 ± 14.4 nmol/min/mg, and 370.02 ± 25.8 nmol/min/mg when EtPrpC concentration at 4, 6, and 8 μg, respectively. Although the ability of EtPrpC to catalyze acetyl-CoA is only 0.11% of its ability to catalyze propionyl-CoA, it indicates that the 2-MCC pathway in E. tenella is similar to that in bacteria and may have a bypass function in the TCA cycle. This study can provide the theoretical foundation for the new drug targets and the development of new anticoccidial drugs., Competing Interests: Declaration of Competing Interest Authors (Zhenxing Gong, Zigang Qu, and Jianping Cai) declare that there are no conflict of interests either financially or otherwise as relating the publication of the manuscript or whatever benefits that may result thereafter., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

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., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest Authors (Zigang Qu, Zhenxing Gong, Joshua Seun Olajide, Jing Wang, and Jianping Cai) declare that there are no conflict of interests either financially or otherwise as relating the publication of the manuscript or whatever benefits that may result thereafter., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Han, Li, Wu and Liao.)

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., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023. Published by Elsevier Inc.)

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., (© 2024 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd.)

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., (© 2024 John Wiley & Sons Ltd.)

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., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

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., (© 2023. The Author(s).)

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., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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 ( Et GAM56, Et SWP1) and two cysteine-rich proteins ( Et OWP2, Et OWP6). 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.

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&#95;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&#95;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&#95;MIT00100) as the sole mutated gene shared between the DecR&#95;H and XJ strains and also detected this gene in the DecR&#95;SC strain. Distinct non-synonymous mutations, namely Gln131Lys in DecR&#95;H, Phe263Leu in DecR&#95;XJ, and Phe283Leu in DecR&#95;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 Q o . 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., (© 2023. BioMed Central Ltd., part of Springer Nature.)

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., (© 2023. BioMed Central Ltd., part of Springer Nature.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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.

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, IL1β, IL6 , the receptor CCR2 , and the anti-inflammatory mediators TGFβ1 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Tomal, Sadrin, Gaboriaud, Guitton, Sedano, Lallier, Rossignol, Larcher, Rouille, Ledevin, Guabiraba, Silvestre, Lacroix-Lamandé, Schouler, Laurent and Bussière.)

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., Competing Interests: The authors declare no conflict of interest.

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., (Copyright © 2023 Elsevier GmbH. All rights reserved.)

Worldwide distributed coccidiosis is caused by infection of both Eimeria species and Cystoisospora in the host intestine and causes huge economic losses to the livestock industry, especially the poultry industry. The control of such diseases relies mainly on chemoprophylaxis with anticoccidials, which has led to a very common drug resistance in this field. However, the genetic mechanisms underlying resistance to many anticoccidial drugs remain unknown. In this study, strains of E. tenella resistant to 250 mg/kg monensin were generated and characterized. Forward genetic approaches based on pooled genome sequencing, including experimental evolution and linkage group selection, were used to locate candidate targets responsible for resistance to monensin and diclazuril in E. tenella. A total of 16 nonsynonymous mutants in protein-coding genes were identified in monensin-resistant strains, and two genomic regions with strong selection signals were also detected in diclazuril-resistant strains. Our study reveals the genetic characterization of the experimental evolution and linkage group selection in Eimeria species, and also provides important information that contributes to the understanding of the molecular mechanism of drug resistance in coccidia., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Shi, Li, Hao, Sun, Fu, Yan and Hao.)

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., Competing Interests: Hongsheng Wang is employed by Jiangsu Jinghai Poultry Group Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Yu, Wang, Tang, Dong, Dai, Zhang, Zhang, Xie, Wang and Zhao.)

Intestinal coccidiosis is a common parasitic disease in livestock, caused by the infection of Eimeria and Cystoisospora parasites, which results in great economic losses to animal husbandry. Triazine compounds, such as toltrazuril and diclazuril, are widely used in the treatment and chemoprophylaxis of coccidiosis. Unfortunately, widespread drug resistance has compromised their effectiveness. Most studies have focused on prophylaxis and therapeutics with toltrazuril in flocks, while a comprehensive understanding of how toltrazuril treatment alters the transcriptome of E. tenella remains unknown. In this study, merozoites of E. tenella were treated in vitro with 0.5 μg/mL toltrazuril for 0, 1, 2 and 4 h, respectively. The gene transcription profiles were then compared by high-throughput sequencing. Our results showed that protein hydrolysis genes were significantly upregulated after drug treatment, while cell cycle-related genes were significantly downregulated, suggesting that toltrazuril may affect parasite division. The expression of redox-related genes was upregulated and elevated levels of ROS and autophagosomes were detected in the parasite after toltrazuril treatment, suggesting that toltrazuril may cause oxidative stress to parasite cells and lead to its autophagy. Our results provide basic knowledge of the response of Eimeria genes to toltrazuril and further analysis of the identified transcriptional changes can provide useful information for a better understanding of the mechanism of action of toltrazuril against Eimeria .

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., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests. The funding agencies played no role in the design or implementation of the study, analysis or interpretation of the data, or the preparation and submission of the manuscript., (Copyright © 2023. Published by Elsevier Ltd.)

Eimeria tenella ( E. tenella ), an important intestinal parasite of chicken caeca, causes coccidiosis and brings large economic losses to the poultry industry annually. Gut microorganismal alterations directly affect the health of the body. To understand how E. tenella affects its host, we analysed the changes in caecal microbial diversity and the physiological and morphological changes during the peak of oocyst shedding. Infected and healthy chickens differed significantly in caecal pathology and blood indicators. At the genus level, the abundances of Faecalibacterium , Clostridium , Lachnoclostridium , Gemmiger , Flavonifractor , Pseudoflavonifractor and Oscillibacter were significantly decreased in the infected samples, whereas Escherichia , Nocardia and Chlamydia were significantly increased. Functional gene pathways related to replication, recombination and repair, and transcription were significantly decreased, and functional genes related to metabolism were highly significantly reduced in the infected samples. Furthermore, in the infected samples, E. tenella reduced the haemoglobin levels and red blood cell counts, greatly reduced the beneficial bacteria and increased the potentially pathogenic bacteria. This study provides a research basis for further understanding the pathogenic mechanisms of E. tenella and provides insight for potential new drug development. RESEARCH HIGHLIGHTS First simultaneous description of caecal microbiota and physiological indicators during E. tenella infection.Metagenomics used to explore functional properties of chicken caecal microbiota during E. tenella infection.Caecal microbial compositions and functional genes altered significantly after infection.Blood indicators and caecal morphology were significantly altered in the infected group.

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 (IC 50  = 10.37 μM, Ki = 48.97 μM). In vitro inhibition assay demonstrated that morin had an inhibitory effect on E. tenella development, with an IC 50 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., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interests regarding the publication of this paper., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Coccidiostat and antibiotics are widely used in poultry industry, but their effects on cecum microbial community and metabolomics in chickens infected with coccidia have been rarely studied. In this study, we analyzed the changes of microbiota and metabolomic which associated with Eimeria tenella infection in 8 days of age chickens in the presence or absence of ethanamizuril, sulfachlorpyridazine or their combinations treatment for 3 consecutive days. 16S rRNA gene sequencing and LC-MS/MS analyses were used to profile the cecal microbiome and metabolome in each group of chickens on 7 days post-infection. The results showed that coccidial infection induced significant perturbations in the distribution of microbial taxonomy and the metabolism of physiological functional molecules in cecal contents. Ethanamizuril treatment seemed to transform microbiota into a steady state conducive to animal health, and sulfachlorpyridazine treatment alleviated the growth of potentially harmful bacteria such as Escherichia-Shigella. The change trends of metabolites such as n-carbamoylglutamic acid were consistent with the anticoccidial effect of ethanamizuril. The combinations of ethanamizuril and sulfachlorpyridazine at low-dose had little effect on gut microbiota, metabolism and anticoccidial effect. These data indicate that the cecal microbiota and metabolic status of chickens infected with E. tenella following ethanamizuril treatment could be used to monitor the response to drug efficacy. This study provides a new system approach to elucidate the microbiota, metabolic and therapeutic effects of the combination of coccidiostat and antibiotics in the context of avian coccidiosis., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Though genome sequencing of Eimeria tenella predicts more than 8,000 genes, the molecular functions of many proteins remain unknown. In this study, the coding region corresponding to the mature peptide of a hypothetical protein of E. tenella (ETH&#95;00023950) was amplified and expressed in a bacterial system. Following preparation of polyclonal antibody that recognizes ETH&#95;00023950, the expression of ETH&#95;00023950 in merozoites was examined. Meanwhile, we determined the transcriptomic responses of the leghorn male hepatoma (LMH) cells to its expression. Sequencing analysis showed that one single nucleotide polymorphism and one indel of ETH&#95;00023950 of E. tenella SD-01 strain were found compared with that of the UK reference Houghton strain, leading to a frame shift and a premature stop codon. The expression of ETH&#95;00023950 in E. tenella merozoites was confirmed by indirect immunofluorescence and Western blot analysis. Transcriptomic analysis showed that ETH&#95;00023950 altered the expression of 2,680 genes (321 downregulated genes and 2,359 upregulated genes) in LMH cells. The RNA-sequencing data were consistent with the results of the quantitative real-time polymerase chain reaction (qRT-PCR). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that differentially expressed transcripts were significantly related to 8 pathways, including oxidative phosphorylation and TGF-beta signaling pathway. These findings contribute to understanding host-pathogen interaction and secondary bacterial infections related to E. tenella., (Copyright © 2022. Published by Elsevier Inc.)

Lactobacillus strains exhibit preferable properties that make them attractive candidates for vaccine delivery systems because of their ability to regulate intestinal mucosal immunity in the body. To date, live Lactobacillus delivery vaccines reported for the defense against Eimeria tenella have been inducer-dependent systems whose applications are significantly limited due to their unattainable induction conditions in vivo. Here, a constitutive expression of Lactobacillus plantarum NC8 surface display system was constructed. Then, this system was used to prepare a live oral vaccine to constitutively express the E. tenella U6L5H2 (EtU6) protein on the NC8 surface and to evaluate its protective efficacy against E. tenella challenge in chickens. The results showed that the heterologous protein (EGFP or EtU6) was successfully expressed on the surface of L. plantarum NC8 without any inducer. The immunoprotection of EtU6 with constitutive expression in L. plantarum NC8 system (NC8/Pc-EtU6) was significantly stronger than that of EtU6 with induced expression of L. plantarum NC8 system (NC8/Pi-EtU6) (ACI: 168.28 vs. 152.74) as evidenced by increased body weight, decreased oocyst output and lesion scores. Furthermore, the constitutive system NC8/Pc-EtU6 produced higher levels of specific cecal SIgA, serum IgG, transcription of cytokines IFN-γ and IL-2, and lymphocyte proliferation than the induced system NC8/Pi-EtU6. These results indicate that, compared to the inducible system, the constitutive surface display system of L. plantarum has the advantages of continuously expressing antigens in vivo and stimulating the host immune system. It could be an ideal platform for vaccine expression. The live vector vaccine for coccidiosis constructed by this constitutive system greatly improves the application potential in chicken production and provides a novel platform for the prevention of coccidiosis in chickens., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Thioredoxin (Trx) is a widespread protein regulator of redox reactions in all organisms. It operates together with NADPH and thioredoxin reductase as a general protein disulfide catalytic system. Recently, Trx has been found to be related to the process by which apicomplexan protozoa invade host cells. In this study, Eimeria tenella thioredoxin (EtTrx1) was identified and its gene structural features, expression levels at different developmental stages, localization in sporozoites, roles in adhesion and invasion, and immunogenicity were investigated. Sequence analysis indicated that EtTrx1 contains a Trx domain with a WCGPC motif in 29-33 aa and a typical Trx fold, and belongs to thioredoxin family. EtTrx1 was detected on the surface of sporozoites using anti-EtTrx1 polyclonal antibodies under non-permeabilized conditions by indirect immunofluorescence assay (IFA) and also in a secretion form. EtTrx1 protein was highly transcribed and expressed in merozoites and sporozoites by quantitative PCR and western blot. The attachment assay showed that the adherence rates of yeast cells expressing EtTrx1 on the surface to host cells were 3.1-fold higher than those of the blank control. Specific anti-EtTrx1 antibodies inhibited the invasion of sporozoites into DF-1 cells. The highest inhibition rate was up to 36.75% compared to the control group. Immunization with recombinant EtTrx1 peptides also showed significant protection against lethal infections in chickens. It could offer moderate protective efficacy (Anticoccidial Index [ACI]: 163.70), induce humoral responses, and be an effective candidate for the development of new vaccines., Competing Interests: Declaration of Competing Interest The authors declare that they have no known personal relationships or competing financial interests that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Coccidiosis is a major recurring problem in the poultry industry and is caused by infection of one or more of the seven Eimeria species known to infect chickens, with Eimeria tenella among the best studied and economically important. Studies on the genetic diversity of E. tenella strains is essential for the development of universally acceptable diagnostic markers and vaccines against the disease. Eimeria tenella internal transcribed spacer-1 (ITS-1) and apical membrane antigen-1 (AMA-1) sequences from different parts of the world are available in the public domain and therefore provide suitable comparative markers for genetic diversity study. In this study, the ITS-1 and AMA-1 sequence diversity of two local E. tenella strains, namely EtNSN6 and EtSGR6 were characterized. Both ITS-1 and AMA-1 sequences for EtNSN6 and EtSGR6 were retrieved by mapping to their respective genome sequences generated using next generation sequencing. Multiple sequence alignment of the ITS-1 and AMA-1 sequences with selected homologous sequences revealed the presence of a total of five and 13 single nucleotide polymorphisms (SNPs) respectively. All SNPs appeared to occur at random and did not show any unique pattern based on geographical regions while no insertions and deletions (indels) was found to occur in the aligned sequences. However, unique bases that defined certain strains were detected. Phylogenetics analyses performed with Maximum Parsimony (MP) and Maximum Likelihood (ML) methods revealed similar topology for the internal groups with all the E. tenella ITS-1 and AMA-1 sequences grouped in the same clade supported by high bootstrap confidence. This confirmed that both EtNSN6 and EtSGR6 samples are E. tenella strains. Sequence comparison and phylogenetics analyses further suggest the possibility of low genetic diversity among E. tenella strains.

Coccidiosis is a widespread parasitic disease that causes serious economic losses to the poultry industry every year. Long noncoding RNAs (lncRNAs) play important roles in transcriptional regulation and are involved in a variety of diseases and immune responses. However, the lncRNAs associated with Eimeria tenella ( E. tenella ) resistance have not been identified in chickens. In addition, the expression profiles and functions of lncRNAs during E. tenella infection remain unclear. In the present study, high-throughput sequencing was applied to identify lncRNAs in chicken cecal tissues from control (JC), resistant (JR), and susceptible (JS) groups on day 4.5 post-infection (pi), and functional tests were performed. A total of 564 lncRNAs were differentially expressed, including 263 lncRNAs between the JS and JC groups, 192 between the JR and JS groups, and 109 between the JR and JC groups. Functional analyses indicated that these differentially expressed lncRNAs were involved in pathways related to E. tenella infection, including the NF-kappa B signaling, B cell receptor signaling and natural killer cell-mediated cytotoxicity pathways. Moreover, through cis regulation network analysis of the differentially expressed lncRNAs, we found that a novel lncRNA termed lncRNA BTN3A2 was significantly increased in both cecum tissue and DF-1 cells after coccidia infection or sporozoite stimulation. Functional test data showed that the overexpression of lncRNA BTN3A2 reduced the production of inflammatory cytokines, including IL-6, IL-1β, TNF-α and IL-8, while lncRNA BTN3A2 knockdown promoted the production of these inflammatory cytokines. Taken together, this study identify the differentially expressed lncRNAs during E. tenella infection in chickens for the first time and provide the direct evidence that lncRNA BTN3A2 regulates the host immune response to coccidia infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Yu, Mi, Wang, Dai, Zhang, Zhang, Xie and Zhao.)

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., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

Coccidiosis caused by Eimeria spp. incurs significant morbidity and mortality in chickens, and is thus of great economic importance. Post-mortem intestinal lesion scoring remains one of the most common means of diagnosis; therefore alternative, non-invasive methods of diagnosis and monitoring would be highly desirable. Micro-RNAs (miRNAs) have been shown to be stable in faeces of human and animal species with expression altered in gastrointestinal disease. We hypothesized that miRNA is stable in caecal content of chickens, and that differential miRNA expression patterns would be seen in Eimeria -infected versus uninfected individuals. Initially, RNA was extracted from Eimeria tenella -infected ( n  = 3; 7 days post infection) and uninfected ( n  = 3) chicken caecal content to demonstrate miRNA stability. Subsequently, next-generation miRNA sequencing was performed on caecal content from E. tenella -infected chickens with high (lesion score (LS) 3-4; n  = 3) or low (LS1; n  = 3) levels of pathology, and uninfected controls ( n  = 3). Comparative analysis identified 19 miRNAs that exhibited significantly altered expression in the caecal content of E. tenella , infected chickens versus uninfected chickens ( t -test, False Discovery Rate (FDR) < 0.05). Eight of these miRNAs showed significant up-regulation in infection (fold change of 9.8-105, FDR <0.05). Quantitative PCR was performed using separate biological replicates to confirm differential regulation in eight of these miRNA candidates in caecal and faecal content. This work has identified a panel of miRNA candidates which may be appropriate for use as non-invasive faecal markers of active caecal coccidiosis without the need for culling. RESEARCH HIGHLIGHTS E. tenella induced differential miRNA expression in caecal content and faeces.

Background: Eimeria tenella is the most pathogenic intracellular protozoan parasite of seven Eimeria species causing chicken coccidiosis around the world. This species is particularly responsible for caecal coccidiosis leading to serious morbidity-mortality and financial loss in poultry production., Methods: The present study explored the genetic diversity of E. tenella. Litter slurry was collected from 18 broiler farms located in Mymensingh district, Bangladesh. Litter samples were processed for oocyst isolation-identification using parasitological techniques followed by genomic DNA extraction from sporulated oocysts. For molecular analysis, the internal transcribed spacer 1 gene of E. tenella was amplified using species-specific primers and sequenced. After editing and alignment, 263 bp sequences were used for analysis., Results: Genetic analysis showed seven distinct genotypes and detected six single nucleotide polymorphisms among the 18 E. tenella isolates. The nucleotide and genotype diversity were 0.00507 and 0.8235, respectively. A phylogenetic tree was constructed with 66 sequences (seven studied genotypes and 59 reference sequences from GenBank database). The neighbour-joining tree represented that the studied E. tenella isolates were grouped with reference E. tenella isolates with strong nodal support (100%) and the nucleotide sequences of E. tenella, E. necatrix, E. acervulina, E. brunetti, E. maxima, E. mitis and E. praecox formed separate clusters without any geographical boundaries., Conclusions: This is the first study on the genetic analysis of E. tenella from Mymensingh district, Bangladesh. These findings will provide baseline data on the species conformation and genetic variations of E. tenella. Further extensive investigation will be needed to reveal the population genetic structure of this parasite and thus will facilitate the planning of effective control strategies., (© 2022 The Authors. Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

This study investigated the role of calpain in Eimeria tenella-induced host cell apoptosis. Chick embryo cecal epithelial cell culture technology, flow cytometry, enzyme-linked immunosorbent assays, and fluorescence quantitative PCR were used to detect the E. tenella host cell apoptotic rate, Bax and Bid expression levels, and calpain activity. The results demonstrated that Bax, Bid, and calpain levels were upregulated and apoptosis was increased following E. tenella infection at 24-120 h. Calpain levels were reduced by pharmacological inhibition of calpain using SJA6017 or by blocking Ca 2+ entry into the cell using BAPTA/AM at 24-120 h. The mRNA and protein levels of Bax and Bid, the E. tenella infection rate, and the early apoptotic and late apoptotic (necrosis) rates were decreased by using SJA6017 at 24-120 h. These results indicated that E. tenella-promoted host cell apoptosis is regulated by calpain via Bid and Bax at 24-120 h. Thus, manipulation of calpain levels could be used to manage E. tenella infection in chickens in the middle and late developmental stages., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The purpose of this study was to compare micro-oocyst counts of Eimeria to PCR analysis of intestinal DNA from smears of duodenum, jejunum/ileum, and cecum of chickens infected with Eimeria acervulina , Eimeria maxima , or Eimeria tenella oocysts. Broiler chicks were infected in triplicate with various doses of E. acervulina , E. maxima , or E. tenella oocysts and were necropsied 5-6 days later to recover duodenal, jejunal, or cecal tissue for micro-oocyst count and for DNA recovery. Micro-oocyst counts were done independently by three individuals. Micro-oocyst counts and PCR directed to ITS1 rDNA or to a single-copy orthologue (SCO 5995) displayed a linear relationship with oocyst dose for each Eimeria species. A strong correlation was found between mean micro-oocyst counts and both PCR assays for E. acervulina ( r = 0.78-0.94), E. maxima ( r = 0.79-0.91), and E. tenella ( r = 0.85-0.96). There was good agreement between ITS1 and SCO 5995 PCR assays: E. acervulina ( r = 0.92), E. maxima ( r = 0.79), and E. tenella ( r = 0.93). However, only ITS1 PCR analysis corroborated micro-oocyst counts of Eimeria oocyst DNA recovered from Eimeria -infected broiler chickens submitted to a poultry diagnostic laboratory. These findings suggest that ITS1 PCR or SCO PCR can validate traditional micro-oocyst counts used in quantifying Eimeria infection in chickens. Additional studies may provide a method for estimating the relative abundance of each Eimeria species in a natural infection.

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., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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., (© 2021 International Society of Protistologists.)

Apicomplexans are important pathogens that cause severe infections in humans and animals. The biology and pathogeneses of these parasites have shown that proteins are intrinsically modulated during developmental transitions, physiological processes and disease progression. Also, proteins are integral components of parasite structural elements and organelles. Among apicomplexan parasites, Eimeria species are an important disease aetiology for economically important animals wherein identification and characterisation of proteins have been long-winded. Nonetheless, this review seeks to give a comprehensive overview of constitutively expressed Eimeria proteins. These molecules are discussed across developmental stages, organelles and sub-cellular components vis-à-vis their biological functions. In addition, hindsight and suggestions are offered with intention to summarise the existing trend of eimerian protein characterisation and to provide a baseline for future studies., (© 2022. The Author(s).)

Protein phosphorylation is an important post-translational modification (PTM) involved in diverse cellular functions. It is the most prevalent PTM in both Toxoplasma gondii and Plasmodium falciparum , but its status in Eimeria tenella has not been reported. Herein, we performed a comprehensive, quantitative phosphoproteomic profile analysis of four stages of the E. tenella life cycle: unsporulated oocysts (USO), partially sporulated (7 h) oocysts (SO7h), sporulated oocysts (SO), and sporozoites (S). A total of 15,247 phosphorylation sites on 9514 phosphopeptides corresponding to 2897 phosphoproteins were identified across the four stages. In addition, 456, 479, and 198 differentially expressed phosphoproteins (DEPPs) were identified in the comparisons SO7h vs. USO, SO vs. SO7h, and S vs. SO, respectively. Gene Ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of DEPPs suggested that they were involved in diverse functions. For SO7h vs. USO, DEPPs were mainly involved in cell division, actin cytoskeleton organization, positive regulation of transport, and pyruvate metabolism. For SO vs. SO7h, they were related to the peptide metabolic process, translation, and RNA transport. DEPPs in the S vs. SO comparison were associated with the tricarboxylic acid metabolic process, positive regulation of ATPase activity, and calcium ion binding. Time course sequencing data analysis (TCseq) identified six clusters with similar expression change characteristics related to carbohydrate metabolism, cytoskeleton organization, and calcium ion transport, demonstrating different regulatory profiles across the life cycle of E. tenella . The results revealed significant changes in the abundance of phosphoproteins during E. tenella development. The findings shed light on the key roles of protein phosphorylation and dephosphorylation in the E. tenella life cycle.

Parasitic diseases are major trouble in many parts of the world. We consider that if a chemical can break a DNA barcode sequence, it might be used to develop a species-specific anti-parasitic agent. To examine this hypothesis, we constructed sgRNAs that target both the control (5.8S rDNA) and a DNA barcode (ITS) sequence in Eimeria tenella. In vitro experiment showed that Cas9 mRNA combined with sgRNAs could reduce the sporulation percentage of oocysts and the survival rate of sporulated oocysts and sporozoites. Quantitative real-time PCR showed that the DNAs of parasites exposed to Cas9 mRNA and sgRNAs were significantly affected, regardless of whether they were exposed to a combination of two sgRNAs or just a single sgRNA. The DNA sequencing also indicated that the experimental group exposed to two sgRNAs mixed with Cas9-induced deletion of large parts and a single sgRNA mixed with Cas9-induced mutation at sgRNA targeted fragments. In vivo trial, the effect of sgRNA and Cas9 RNA on the pathogenicity of E. tenella in chicken showed less lesion score and oocysts score (P < 0.05) in experimental groups than control groups. The results and concepts presented in this research can lead to discovering novel nucleic acid therapeutic drugs for Eimeriasis and other parasitic infections, which provide insights into the development of species-specific anti-parasitic agents., (Copyright © 2021. Published by Elsevier Inc.)

Avian coccidiosis is a disease caused by members of the genus Eimeria . Huge economic losses incurred by the global poultry industry due to coccidiosis have increased the need for cost-effective and easily available recombinant vaccines. Microneme protein 2 (MIC2) and surface antigen 1 (SAG1) of E. tenella have been recognised as potential vaccine candidates. However, the genetic diversity of the antigens in field isolates, which affects vaccine efficacy, has yet to be largely investigated. Here, we analysed genetic diversity and natural selection of etmic2 and etsag1 in Korean E. tenella isolates. Both genes exhibited low levels of genetic diversity in Korean isolates. However, the two genes showed different patterns of nucleotide diversity and amino acid polymorphism involving the E. tenella isolates obtained from different countries including China and India. These results underscore the need to investigate the genetic diversity of the vaccine candidate antigens and warrant monitoring of genetic heterogeneity and evolutionary aspects of the genes in larger numbers of E. tenella field isolates from different geographical areas to design effective coccidial vaccines.

Background: Coccidiosis is an infectious disease with large negative impact on the poultry industry worldwide. It is an enteric infection caused by unicellular Apicomplexan parasites of the genus Eimeria. The present study aimed to gain more knowledge about interactions between parasites and the host immune system during the early asexual replication phase of E. tenella in chicken caeca. For this purpose, chickens were experimentally infected with E. tenella oocysts, sacrificed on days 1-4 and 10 after infection and mRNA from caecal tissues was extracted and sequenced., Results: Dual RNA-seq analysis revealed time-dependent changes in both host and parasite gene expression during the course of the infection. Chicken immune activation was detected from day 3 and onwards with the highest number of differentially expressed immune genes recorded on day 10. Among early (days 3-4) responses up-regulation of genes for matrix metalloproteinases, several chemokines, interferon (IFN)-γ along with IFN-stimulated genes GBP, IRF1 and RSAD2 were noted. Increased expression of genes with immune suppressive/regulatory effects, e.g. IL10, SOCS1, SOCS3, was also observed among early responses. For E. tenella a general up-regulation of genes involved in protein expression and energy metabolism as well as a general down-regulation genes for DNA and RNA processing were observed during the infection. Specific E. tenella genes with altered expression during the experiment include those for proteins in rhoptry and microneme organelles., Conclusions: The present study provides novel information on both the transcriptional activity of E. tenella during schizogony in ceacal tissue and of the local host responses to parasite invasion during this phase of infection. Results indicate a role for IFN-γ and IFN-stimulated genes in the innate defence against Eimeria replication., (© 2021. The Author(s).)

Microneme proteins (MICs) of Eimeria tenella play key roles in motility, migration, attachment, and invasion processes. More than 20 apicomplexan parasite's MICs have been identified, with nine Eimeria MICs being reported. In this study, a novel E. tenella MIC was identified, and its gene structural features, developmental expression levels, localization, role in adhesion and invasion, and immunogenicity were studied. The results showed that the open reading frame was 1,650 bp, encoding 550 amino acids. It contains a signal sequence, a transmembrane region, four low-complexity boxes, and five epidermal growth factor-like domains (EGF). Subcellular localization revealed its distribution on the membrane surface of the parasite. These characteristics are consistent with the common features of MICs and are named EtMIC8. Anti-EtMIC8 antibodies recognized a specific binding of about 100 kDa in E. tenella, which was twice as large as the prokaryotic expression (about 50 kDa), suggesting that MIC8 may exist naturally as a dimer. EtMIC8 was expressed at higher levels in sporozoites (3.08-fold) and merozoites (2.1-fold) than in sporulated oocysts. The attachment assays using a yeast surface display of MIC8 and its different domains showed that the adherence rates of EtMIC8 to host cells were significantly higher than those of the control (3.17-fold), which was the full contribution of EGF, but neither was alone. Anti-EtMIC8 antibodies significantly reduced the invasion rate of sporozoites into host cells compared to those of the control ( P  < 0.01). Recombinant EtMIC8-EGF peptides could provide moderate protective efficacy (anticoccidial index [ACI]: 169.7), induce humoral responses, and upregulate CD3 + CD8 + lymphocyte cells.

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 × 10 4 and 2 × 10 5 , 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.