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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.)

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.)

The widespread use of drugs has exacerbated the resistance of Eimeria tenalla to anti-coccidial drugs. Using RNA-seq, we previously found the ATPase ASNA1 homolog of E. tenella (EtASNA1) was differentially expressed in resistant strains and drug sensitive (DS) strain. In our study, we used western blotting and quantitative real-time PCR (qRT-PCR) to analyze the translational and transcriptional levels of EtASNA1 in a diclazuril-resistant (DZR) strain, maduramicin-resistant (MRR) strain, salinomycin-resistant (SMR) strain, and DS strain and found EtASNA1 was highly expressed in three drug-resistant strains. The qRT-PCR and western blotting results also showed that the expression levels of EtASNA1 increased with increasing drug concentration, and the transcription levels of the DZR strains isolated from the field were higher than those of the DS strain. In addition, we used in vivo and in vitro tests to analyze the changes of EtASNA1 expression after DZR, MRR, and DS strain infections in chickens, and in vitro inoculation of DF-1 cells in the presence of drugs. The addition of drugs caused expression to be upregulated. The results of qRT-PCR and western blotting also showed that the expression levels of EtASNA1 in second-generation merozoites (SM) and unsporulated oocysts (UO) were significantly higher than those in the other two developmental stages. The immunofluorescence localization of EtASNA1 indicated that the protein was distributed throughout the sporozoites (SZ) and SM, except for the refractile bodies of SZ. In vitro inhibition experiments showed that anti-EtASNA1 antibody incubation significantly inhibited SZ invasion of DF-1 cells. The above results showed that EtASNA1 may be related to host cell invasion of E. tenella and may be involved in the development of E. tenella resistance to some drugs., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Eimeria tenella is an obligate intracellular parasite in the phylum Apicomplexa. As described for other members of Apicomplexa, apical membrane antigen 1 (AMA1) has been shown to be critical for sporozoite invasion of host cells by E. tenella. Recently, an E. tenella paralogue of AMA1 (EtAMA1), dubbed sporoAMA1 (EtAMA3), was identified in proteomic and transcriptomic analyses of E. tenella, but not further characterized. Here, we show that EtAMA3 is a type I integral membrane protein that has 24% -38% identity with other EtAMAs. EtAMA3 has the same pattern of Cys residues in domains I and II of AMA1 orthologs from apicomplexan parasites, but high variance in domain III, with all six invariant Cys residues absent. EtAMA3 expression was developmentally regulated at the mRNA and protein levels. EtAMA3 protein was detected in sporulated oocysts and sporozoites, but not in the unsporulated oocysts or second-generation merozoites. EtAMA3 is secreted by micronemes and is primarily localized to the apical end of sporozoites during host-cell invasion. Additionally, pretreatment of sporozoites with rEtAMA3-specific antibodies substantially impeded their invasion into host cells. These results suggest EtAMA3 is a sporozoite-specific protein that is involved in host-cell sporozoite invasion., (© 2020 International Society of Protistologists.)

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

Eimeria tenella is an apicomplexan, parasitic protozoan known to infect poultry worldwide. An important calcium-dependent protein kinase (CDPK) has been identified in plants, green algae, ciliates and apicomplexan, such as E. tenella . CDPKs are effector molecules involved in calcium signaling pathways, which control important physiological processes such as gliding motility, reproduction, and host cell invasion. Given that CDPKs are not found in the host, studying the functions of CDPKs in E. tenella may serve as a basis for developing new therapeutic drugs and vaccines. To assess the function of CDPK4 in E. tenella ( Et CDPK4), a putative interactor, translation initiation factor eIF-5A ( Et eIF-5A), was screened by both co-immunoprecipitation (co-IP) and His pull-down assays followed by mass spectrometry. The interaction between Et eIF-5A and Et CDPK4 was determined by bimolecular fluorescence complementation (BiFC), GST pull-down, and co-IP. The molecular characteristics of Et eIF-5A were then analyzed. Quantitative real-time polymerase chain reaction and western blotting were used to determine the transcription and protein levels of Et eIF-5A in the different developmental stages of E. tenella . The results showed that the transcription level of Et eIF-5A mRNA was highest in second-generation merozoites, and the protein expression level was highest in unsporulated oocysts. Indirect immunofluorescence showed that the Et eIF-5A protein was found throughout the cytoplasm of sporozoites, but not in the refractile body. As the invasion of DF-1 cells progressed, Et eIF-5A fluorescence intensity increased in trophozoites, decreased in immature schizonts, and increased in mature schizonts. The secretion assay results, analyzed by western blotting, indicated that Et eIF-5A was a secreted protein but not from micronemes. The results of invasion inhibition assays showed that rabbit anti-r Et eIF-5A polyclonal antibodies effectively inhibited cell invasion by sporozoites, with an inhibition rate of 48%., 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 © 2021 Liang, Dong, Zhu, Zhao, Huang, Yu, Wang, Wang, Yu and Han.)

This study analyzed the large-subunit (60S) ribosomal protein L12 of Eimeria tenella (Et60s-RPL12). A full-length cDNA was cloned, and the recombinant protein was expressed in E. coli BL21 and inoculated in rabbits to produce the polyclonal antibody. Quantitative real-time polymerase chain reaction and western blotting were used to analyze the transcription levels of Et60s-RPL12 and translation levels in different developmental stages of E. tenella. The results showed that the mRNA transcription level of Et60s-RPL12 was highest in second-generation merozoites, whereas the translation level was highest in unsporulated oocysts. Indirect immunofluorescence showed that Et60s-RPL12 was localized to the anterior region and surface of sporozoites, except for the two refractile bodies. As the invasion of DF-1 cells progressed, fluorescence intensity was increased, and Et60s-RPL12 was localized to the parasitophorous vacuole membrane (PVM). The secretion assay results using staurosporine indicated that this protein was secreted, but not from micronemes. The role of Et60s-RPL12 in invasion was evaluated in vitro. The results of the invasion assay showed that polyclonal antibody inhibited host cell invasion by the parasite, which reached about 12%. However, the rate of invasion was not correlated with the concentration of IgG., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Avian coccidiosis is a widespread and economically significant poultry disease caused by several Eimeria species, including Eimeria tenella. Previously, E. tenella serine/threonine protein phosphatase (EtSTP) was found to be differentially expressed in drug-sensitive (DS) and drug-resistant strains using RNA-seq. In the present study, we found that transcription and translation levels of EtSTP were higher in diclazuril-resistant (DZR) strains and maduramicin-resistant (MRR) strains than in DS strains using quantitative real-time PCR (qPCR) and Western blotting. Enzyme activity results indicated that the catalytic activity of EtSTP was higher in the two drug-resistant strains than in DS strains. Western blot and qPCR analysis also showed that expression levels of EtSTP were higher in unsporulated oocysts (UO) and second-generation merozoites (SM). Indirect immunofluorescence localization showed that EtSTP was located in most areas of the parasite with the exception of refractile bodies, and fluorescence intensity was enhanced during development. In vitro inhibition experiments showed that the ability of sporozoites (SZ) to invade cells was significantly decreased after treatment with anti-rEtSTP antibody. These results indicated that EtSTP acted mainly during the developmental and reproductive stages of the parasite and may be related to the resistance of coccidia to external drug pressure., (© 2020 International Society of Protistologists.)

Apical membrane antigen 1 (AMA1) is a type I integral membrane protein that is highly conserved in apicomplexan parasites. Previous studies have shown that Eimeria tenella AMA1 (EtAMA1) is critical for sporozoite invasion of host cells. Here, we show that EtAMA1 is a microneme protein secreted by sporozoites, confirming previous results. Individual and combined treatment with antibodies of EtAMA1 and its interacting proteins, E. tenella rhoptry neck protein 2 (EtRON2) and Eimeria-specific protein (EtESP), elicited significant anti-invasion effects on the parasite in a concentration-dependent manner. The overexpression of EtAMA1 in DF-1 cells showed a significant increase of sporozoite invasion. Isobaric tags for relative and absolute quantitation (iTRAQ) coupled with LC-MS/MS were used to screen differentially expressed proteins (DEPs) in DF-1 cells transiently transfected with EtAMA1. In total, 3953 distinct nonredundant proteins were identified and 163 of these were found to be differentially expressed, including 91 upregulated proteins and 72 downregulated proteins. The DEPs were mainly localized within the cytoplasm and were involved in protein binding and poly(A)-RNA binding. KEEG analyses suggested that the key pathways that the DEPs belonged to included melanogenesis, spliceosomes, tight junctions, and the FoxO and MAPK signaling pathways. The data in this study not only provide a comprehensive dataset for the overall protein changes caused by EtAMA1 expression, but also shed light on EtAMA1's potential molecular mechanisms during Eimeria infections., (© Q. Wang et al., published by EDP Sciences, 2020.)