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Molecular characterization and functional analysis of Eimeria tenella citrate synthase

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Abstract

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.

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Acknowledgments

We would like to thank all organizations which funded this work and all the teachers who cooperated in technical assistance.

Funding

This work was supported by grants from the National Key R&D program of China (No. 2018YFD0500302) and the National Science Foundation of China (No. 31970420, 31572266) and the National Parasitic Resources Center (No. NPRC-2019-194-30).

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Author notes

  1. Haixia Wang and Qiping Zhao contributed equally to this paper and should be considered as co-first authors

Authors and Affiliations

  1. Key Laboratory of Animal Parasitology of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Minhang, Shanghai, 200241, People’s Republic of China

    Haixia Wang, Qiping Zhao, Shunhai Zhu, Hui Dong, Shuilan Yu, Qingjie Wang, Yu Yu, Shanshan Liang, Huanzhi Zhao, Bing Huang & Hongyu Han

  2. College of Life Sciences, Shanghai Normal University, Shanghai, 200234, People’s Republic of China

    Yu Yu & Shanshan Liang

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  1. Haixia Wang
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Correspondence to Hongyu Han.

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All animal procedures were approved by the Animal Ethics Committee of Shanghai Veterinary Institute, Chinese Academy of Agricultural Science. Experiments were conducted in accordance with animal ethics guidelines and approved protocols.

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Wang, H., Zhao, Q., Zhu, S. et al. Molecular characterization and functional analysis of Eimeria tenella citrate synthase. Parasitol Res 120, 1025–1035 (2021). https://doi.org/10.1007/s00436-020-07014-6

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