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Molecular characterization and functional analysis of Eimeria tenella malate dehydrogenase

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Abstract

Eimeria tenella is a serious intracellular parasite that actively invades cecal epithelial cells of chickens. The widespread use of drugs causes severe resistance to Eimeria tenella. We detected that malate dehydrogenase (MDH), one of the differentially expressed genes, was upregulated in diclazuril-resistant and maduramicin-resistant strains through transcriptome sequencing. In this study, we cloned and expressed MDH of E. tenella (EtMDH). Quantitative real-time polymerase chain reactions (qPCR) and Western blots were used to analyze the expression of EtMDH in resistant and sensitive strains, indicating EtMDH was upregulated in two resistant strains at the messenger RNA and protein levels. Enzyme activity was tested through absorbance measurement and the EtMDH activity increased in two resistant strains. Expression levels of EtMDH in four developmental stages of E. tenella were tested through qPCR and Western blot. Invasion inhibition assays explored if EtMDH was involved in invasion of DF-1 cells by E. tenella sporozoites. Indirect immunofluorescence assays investigated EtMDH distribution during parasite development in DF-1 cells invaded by E. tenella sporozoites. Experimental results showed that EtMDH may be related to drug resistance of E. tenella during its development and invasion. EtMDH may be an effective molecular marker for detection of E. tenella drug resistance.

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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 the National Natural Science Foundation of China (Grant No. 31572266, No. 31272557), Central Public Welfare Research Institutions and Basic Scientific Research Business Expenses (No. 2016JB10), National Sharing Service Platform for Parasite Resources (No. TDRC-22), and Shanghai Minhang District talent development special funds.

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  1. Ting Chen and Bing Huang contributed equally to this work.

Authors and Affiliations

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

    Ting Chen, Bing Huang, Qiping Zhao, Hui Dong, Shunhai Zhu, Zongping Zhao, Ling Lv, Ming Yan & Hongyu Han

  2. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, People’s Republic of China

    Bing Huang

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  1. Ting Chen
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Correspondence to Hongyu Han.

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This research with animals was approved by the Shanghai Administration Committee of Laboratory Animals (GB14925-2010) and was used according to the protocol approved by the Chinese Academy of Agricultural Sciences Institutional Animal Care and Use Committee guidelines.

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Chen, T., Huang, B., Zhao, Q. et al. Molecular characterization and functional analysis of Eimeria tenella malate dehydrogenase. Parasitol Res 117, 2053–2063 (2018). https://doi.org/10.1007/s00436-018-5875-x

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