Laboratory of Molecular Fish Immunology and Genetics, Department of Tropical Agriculture and International Cooperation, National Pingtung University of Science and Technology, Pingtung, Taiwan.
Research Center for Animal Biologics, National Pingtung University of Science and Technology, Pingtung, Taiwan.
Vet Res Commun. 2024 Feb;48(1):85-101. doi: 10.1007/s11259-023-10182-2. Epub 2023 Aug 2.
The cost of the purification process hinders the extensive use of cytosine phosphate guanosine-oligodeoxynucleotides (CpG-ODNs) for shrimp culture. Therefore, this study used a shuttle vector plasmid to carry 60 copies of CpG-ODN 1668 (pAD43-25_60CpG), which can replicate in Escherichia coli and Bacillus subtilis strain RIK1285. The first experiment used a reverse gavage procedure to deliver a substance (PBS [CK], pAD43-25 [P0], and pAD43-25_60CpG [P60], respectively) directly into the anterior midgut of Penaeus vannamei and transcriptome sequence analysis with a reference genome was performed to examine the expression of well-known immune-related genes. The results showed that the expression levels of immune-related genes in P60 group were significantly increased, particularly those associated with AMPs. In addition, using RT‒qPCR, the expression levels of AMP genes (LvALF, LvPEN-2, and LvPEN-3) in the P60 group may vary depending on the tissue and time point. The second experiment used dietary supplementation with three kinds of heat-killed B. subtilis (HKBS, HKBS-P0, and HKBS-P60) in 28 days of feeding experiments. The results showed that dietary supplementation with HKBS-P60 did not significantly improve shrimp growth performance and survival. However, on days 14 and 28 of the feeding regimens, alkaline phosphatase (AKP) and acid phosphatase (ACP) activity were considerably higher than in other treatments. In addition, following infection with Vibrio harveyi, AKP and ACP activity in the HKBS-P60 group was significantly higher than in other treatments, particularly at the early stage of bacterial infection. Moreover, HKBS-P60 was found to be better protected against V. harveyi infection with lower cumulative mortality (60%) compared to HKBS (90%) and HKBS-P0 (100%) at 7 days after infection. Overall, these findings confirmed that P60 could increase immunological responses in the shrimp midgut, and HKBS-P60 could be used as an effective tool to enhance the immune response and disease resistance in shrimp.
纯化过程的成本阻碍了胞嘧啶磷酸鸟苷寡脱氧核苷酸（CpG-ODN）在虾类养殖中的广泛应用。因此，本研究使用穿梭载体质粒携带 60 个 CpG-ODN 1668（pAD43-25_60CpG），其可以在大肠杆菌和枯草芽孢杆菌 RIK1285 中复制。第一项实验使用反转灌胃程序将物质（PBS[CK]、pAD43-25[P0]和 pAD43-25_60CpG[P60]）直接递送到凡纳滨对虾的前中肠，并进行参考基因组的转录组序列分析，以检查已知免疫相关基因的表达。结果表明，P60 组中免疫相关基因的表达水平显著增加，特别是与 AMPs 相关的基因。此外，使用 RT‒qPCR，P60 组中 AMP 基因（LvALF、LvPEN-2 和 LvPEN-3）的表达水平可能因组织和时间点而异。第二项实验使用三种热灭活枯草芽孢杆菌（HKBS、HKBS-P0 和 HKBS-P60）在 28 天的饲养实验中进行膳食补充。结果表明，膳食补充 HKBS-P60 并未显著提高虾的生长性能和存活率。然而，在饲养方案的第 14 天和第 28 天，碱性磷酸酶（AKP）和酸性磷酸酶（ACP）的活性明显高于其他处理。此外，在感染哈维弧菌后，HKBS-P60 组的 AKP 和 ACP 活性显著高于其他处理，特别是在细菌感染的早期阶段。此外，与 HKBS（90%）和 HKBS-P0（100%）相比，HKBS-P60 组在感染后 7 天的累积死亡率（60%）较低，对哈维弧菌感染的保护效果更好。总体而言，这些发现证实 P60 可以增加虾中肠的免疫反应，而 HKBS-P60 可以用作增强虾免疫反应和抗病能力的有效工具。
