Section of Animal and Veterinary Sciences, Department of Agricultural, Food, Environmental and Animal Sciences (DI4A), University of Udine, 33100, Udine, Italy.
Section of Animal and Veterinary Sciences, Department of Agricultural, Food, Environmental and Animal Sciences (DI4A), University of Udine, 33100, Udine, Italy.
Vet Immunol Immunopathol. 2021 Apr;234:110217. doi: 10.1016/j.vetimm.2021.110217. Epub 2021 Feb 21.
The ectoparasite protozoan Amyloodinium ocellatum (AO) is the causative agent of amyloodiniosis in European seabass (ESB, Dicentrarchus labrax). There is a lack of information about basic molecular immune response mechanisms of ESB during AO infestation. Therefore, to compare gene expression between experimental AO-infested ESB tissues and uninfested ESB tissues (gills and head kidney) RNA-seq was adopted. The RNA-seq revealed multiple differentially expressed genes (DEG), namely 679 upregulated genes and 360 downregulated genes in the gills, and 206 upregulated genes and 170 downregulated genes in head kidney. In gills, genes related to the immune system (perforin, CC1) and protein binding were upregulated. Several genes involved in IFN related pathways were upregulated in the head kidney. Subsequently, to validate the DEG from amyloodiniosis, 26 ESB (mean weight 14 g) per tank in triplicate were bath challenged for 2 h with AO (3.5 × 10/tank; 70 dinospores/mL) under controlled conditions (26-28 °C and 34‰ salinity). As a control group (non-infested), 26 ESB per tank in triplicate were also used. Changes in the expression of innate immune genes in gills and head kidney at 2, 3, 5, 7 and 23 dpi were analysed using real-time PCR. The results indicated that the expression of cytokines (CC1, IL-8) and antimicrobial peptide (Hep) were strongly stimulated and reached a peak at 5 dpi in the early infestation stage, followed by a gradual reduction in the recovery stage (23 dpi). Noticeably, the immunoglobulin (IgM) expression was higher at 23 dpi compared to 7 dpi. Furthermore, in-situ hybridization showed positive signals of CC1 mRNA in AO infested gills compared to the control group. Altogether, chemokines were involved in the immune process under AO infestation and this evidence allows a better understanding of the immune response in European seabass during amyloodiniosis.
体外寄生虫原生动物奥氏拟阿脑虫（AO）是欧洲鲈鱼（ESB，Dicentrarchus labrax）阿脑虫病的病原体。关于 ESB 在 AO 感染期间基本分子免疫反应机制的信息还很缺乏。因此，为了比较 AO 感染 ESB 组织和未感染 ESB 组织（鳃和头肾）之间的基因表达情况，采用了 RNA-seq 技术。RNA-seq 揭示了多个差异表达基因（DEG），即在鳃中 679 个上调基因和 360 个下调基因，在头肾中 206 个上调基因和 170 个下调基因。在鳃中，与免疫系统（穿孔素、CC1）和蛋白质结合相关的基因上调。头肾中几个与 IFN 相关途径相关的基因上调。随后，为了验证阿脑虫病中的 DEG，在受控条件（26-28°C 和 34‰盐度）下，用 AO（3.5×10/池；70 个孢子/mL）对 26 条 ESB（每条重 14 克）进行 3 小时的浴式攻毒，每条 ESB 放在三个重复的池中。作为对照组（未感染），也使用三个重复的池中 26 条 ESB。用实时 PCR 分析感染后 2、3、5、7 和 23 天鳃和头肾中固有免疫基因的表达变化。结果表明，细胞因子（CC1、IL-8）和抗菌肽（Hep）的表达在早期感染阶段被强烈刺激，并在 5 天达到高峰，随后在恢复阶段（23 天）逐渐降低。值得注意的是，与 7 天相比，23 天时免疫球蛋白（IgM）的表达更高。此外，原位杂交显示 AO 感染的鳃中 CC1 mRNA 的阳性信号高于对照组。总之，趋化因子参与了 AO 感染下的免疫过程，这一证据使我们更好地理解了欧洲鲈鱼在阿脑虫病期间的免疫反应。
