Huang Xinmei, Liu Jianhua, Tian Di, Li Wenyu, Zhou Zhouyang, Huang Jianmei, Song Xiaokai, Xu Lixin, Yan Ruofeng, Li Xiangrui
MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, PR China; Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Science, Nanjing 210014, PR China.
MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, PR China.
Vet Parasitol. 2018 Jul 15;258:114-123. doi: 10.1016/j.vetpar.2018.06.020. Epub 2018 Jun 25.
E. mitis is ubiquitous in clinical coccidiosis caused by mixed infection of Eimeria species and the infection by E. mitis usually significantly impairs productivity of the infected chickens. To date, however, few protective antigens from E. mitis have been reported. In this study, the molecular characterization and protective efficacy of microneme 3 of Eimeria mitis (EmiMIC3) were analyzed. EmiMIC3 gene was cloned from sporozoites of E. mitis and its MARs (microneme adhesive repeats domain) were predicted. Recombinant EmiMIC3 (rEmiMIC3) was expressed in E. coli and purified and then was analyzed by western blot with anti-E. mitis chicken serum. Meanwhile, native EmiMIC3 from sporozoites was analyzed by anti-rEmiMIC3 rat serum. The expressions of EmiMIC3 in E. mitis sporozoites and merozoites were analyzed by immunofluorescence assay. The rEmiMIC3-induced changes of T lymphocytes subpopulation, serum cytokines and IgY levels and the protective efficacy of rEmiMIC3 were determined in animal experiments. The results showed that the deduced open reading frame (ORF) of EmiMIC3 was composed of 1145 amino acids, possessing 9 MARs. EmiMIC3 gene was submitted to GenBank (accession number: MG888670). EmiMIC3 could express in sporozoites and merozoites respectively and located at the apex of E. mitis sporozoite. Western blot assay revealed that the rEmiMIC3 could be recognized by serum of chicken infected by E. mitis and the native EmiMIC3 from sporozoites could also be recognized by rat serum against rEmiMIC3. Following vaccination with rEmiMIC3, higher levels of IL-10, IFN-γ, TGF-βand IL-17, higher proportions of CD4+/CD3+ and CD8+/CD3 + T lymphocytes and higher level of IgY antibody were induced compared to the controls. Vaccination with rEmiMIC3 prominently increased the weight gains and decreased oocyst output of the vaccinated chickens after challenge infection. Our result not only enriches protective candidate antigen of E. mitis, but also provides available protective antigen of E. mitis for the development of multivalent vaccines against infection caused by mixture of Eimeria species in clinical coccidiosis.
温和艾美耳球虫在由多种艾美耳球虫混合感染引起的临床球虫病中普遍存在，且温和艾美耳球虫感染通常会显著损害受感染鸡的生产性能。然而，迄今为止，关于温和艾美耳球虫的保护性抗原报道较少。在本研究中，对温和艾美耳球虫微线体蛋白3（EmiMIC3）进行了分子特征分析和保护性效力研究。从温和艾美耳球虫子孢子中克隆了EmiMIC3基因，并预测了其微线体黏附重复结构域（MARs）。重组EmiMIC3（rEmiMIC3）在大肠杆菌中表达并纯化，然后用抗温和艾美耳球虫鸡血清进行western blot分析。同时，用抗rEmiMIC3大鼠血清分析子孢子中的天然EmiMIC3。通过免疫荧光试验分析EmiMIC3在温和艾美耳球虫子孢子和裂殖子中的表达情况。在动物实验中测定了rEmiMIC3诱导的T淋巴细胞亚群、血清细胞因子和IgY水平的变化以及rEmiMIC3的保护效力。结果显示，EmiMIC3推导的开放阅读框（ORF）由1145个氨基酸组成，具有9个MARs。EmiMIC3基因已提交至GenBank（登录号：MG888670）。EmiMIC3可分别在子孢子和裂殖子中表达，且位于温和艾美耳球虫子孢子的顶端。western blot分析表明，rEmiMIC3可被温和艾美耳球虫感染鸡的血清识别，子孢子中的天然EmiMIC3也可被抗rEmiMIC3大鼠血清识别。与对照组相比，用rEmiMIC3免疫后，诱导产生了更高水平的IL-10、IFN-γ、TGF-β和IL-17，更高比例的CD4+/CD3+和CD8+/CD3 + T淋巴细胞以及更高水平的IgY抗体。用rEmiMIC3免疫显著增加了攻毒感染后免疫鸡的体重增加并减少了卵囊产量。我们的结果不仅丰富了温和艾美耳球虫的保护性候选抗原，也为开发针对临床球虫病中多种艾美耳球虫混合感染的多价疫苗提供了可用的温和艾美耳球虫保护性抗原。
