U-Taynapun Kittichon, Chirapongsatonkul Nion, Itami Toshiaki, Tantikitti Chutima
Aquatic Animal Health Management Research Unit, Department of Fisheries, Faculty of Agriculture, Rajamangala University of Technology Srivijaya, Nakhon Si Thammarat, 80110, Thailand; Aquatic Animal Health Research Center, Department of Aquatic Science, Faculty of Natural Resources, Prince of Songkla University, Songkhla, 90110, Thailand.
Aquatic Animal Health Management Research Unit, Department of Fisheries, Faculty of Agriculture, Rajamangala University of Technology Srivijaya, Nakhon Si Thammarat, 80110, Thailand; Department of Biochemistry, Faculty of Science, Prince of Songkla University, Songkhla, 90110, Thailand.
Fish Shellfish Immunol. 2016 Nov;58:116-124. doi: 10.1016/j.fsi.2016.09.005. Epub 2016 Sep 11.
Oligodeoxynucleotides (ODNs) containing unmethylated cytosine-phosphate-guanine CpG dinucleotides within specific sequence contexts (CpG motifs) have been reported as pathogen-associated molecular patterns (PAMPs). Its immunostimulatory effects have been demonstrated in diverse vertebrate models. CpG ODN is typically found in bacterial or viral genome and recognized by a non-self recognition receptor Toll-like receptor9 (TLR9). Here, a new CpG ODN 1013 which mimics sequence of SSU rDNA of early eukaryotic organism myxosporidia, Myxobolus supamattayai, was employed to stimulate the immune responses of Asian sea bass Lates calcarifer. Its immunostimulant potentiality was comparatively compared with that of CpG ODN 1668, a widely used as functional immunostimulant. Both unmethylated CpG ODNs with some modified phosphorothioated positions were intraperitoneally injection (5 μg/fish). Hematological examination, immunological assays and immune-related genes expression were evaluated 12 h, 1, 3 and 5 d after post CpG ODN challenge. The immunosimulatory effect of these CpG ODNs on fish immunity to protect the bacterial pathogen Streptococcus iniae was also determined. The results demonstrated that these two CpG ODNs could induce immune responses in Asian sea bass including the significant (P < 0.05) increase level of WBC, peroxidase activity and oxidative radicals in head kidney (HK) leukocyte, serum innate immune parameters and up-regulation of four immune responsive genes compared with the control group. Most of immune responses induced by ODN 1668 were strong within 1 d but lesser extended while ODN 1013 prolonged the stimulatory effects during the whole experimental period. After challenge with S. iniae, the survival proportion in ODN 1013-treated fish was apparently higher than that treated with ODN 1668 and PBS, respectively. The results together suggested that CpG ODN 1013 enhanced innate immune responses, including humoral and cellular responses, through TLR9 mediated signaling pathway which is mainly contribute to the protective immunity in Asian sea bass against S. iniae infection. These findings can lead to a new approach in immunostimulant development by using the novel CpG ODN originating from the parasite M. supamattayai, besides those from bacterial and viral genomes, for disease control in fish host.
在特定序列背景（CpG基序）中含有未甲基化胞嘧啶-磷酸-鸟嘌呤（CpG）二核苷酸的寡脱氧核苷酸（ODN）已被报道为病原体相关分子模式（PAMP）。其免疫刺激作用已在多种脊椎动物模型中得到证实。CpG ODN通常存在于细菌或病毒基因组中，并被非自我识别受体Toll样受体9（TLR9）识别。在此，一种新的CpG ODN 1013被用于刺激尖吻鲈（Lates calcarifer）的免疫反应，该ODN模拟了早期真核生物黏液孢子虫（Myxobolus supamattayai）的小亚基核糖体DNA（SSU rDNA）序列。将其免疫刺激潜力与广泛用作功能性免疫刺激剂的CpG ODN 1668进行了比较。两种在某些位置经过硫代磷酸化修饰的未甲基化CpG ODN均通过腹腔注射（5μg/鱼）。在CpG ODN刺激后的12小时、1天、3天和5天评估血液学检查、免疫学检测和免疫相关基因表达。还确定了这些CpG ODN对鱼类抵抗细菌病原体海豚链球菌免疫的免疫刺激作用。结果表明，与对照组相比，这两种CpG ODN均可诱导尖吻鲈的免疫反应，包括白细胞水平显著（P<0.05）升高、头肾（HK）白细胞中的过氧化物酶活性和氧化自由基增加、血清固有免疫参数以及四个免疫反应基因的上调。ODN 1668诱导的大多数免疫反应在1天内较强，但持续时间较短，而ODN 1013在整个实验期间延长了刺激作用。在用海豚链球菌攻击后，ODN 1013处理的鱼的存活比例明显分别高于用ODN 1668和PBS处理的鱼。结果共同表明，CpG ODN 1013通过TLR9介导的信号通路增强了固有免疫反应，包括体液和细胞反应，这主要有助于尖吻鲈抵抗海豚链球菌感染的保护性免疫。这些发现可能导致一种新的免疫刺激剂开发方法，即除了使用源自细菌和病毒基因组的新型CpG ODN外，还使用源自寄生虫Myxobolus supamattayai的新型CpG ODN来控制鱼类宿主中的疾病。
