Centre for Environment, Fisheries and Aquaculture Science, Barrack Road, The Nothe, Weymouth, Dorset, DT4 8UB, United Kingdom.
Centre for Environment, Fisheries and Aquaculture Science, Barrack Road, The Nothe, Weymouth, Dorset, DT4 8UB, United Kingdom.
Fish Shellfish Immunol. 2019 Mar;86:287-300. doi: 10.1016/j.fsi.2018.11.029. Epub 2018 Nov 17.
An in vitro model to study the host response to Neoparamoeba perurans, the causative agent of amoebic gill disease (AGD), was evaluated. The rainbow trout gill derived cell line, RTgill-W1, was seeded onto permeable cell culture supports and maintained asymmetrically with apical seawater. Cells were inoculated with either a passage attenuated or a recent wild clone of N. perurans. Amoebae, loaded with phagocytosed fluorescent beads, were observed associated with host cells within 20 min post inoculation (pi). By 6 h small foci of cytopathic effect appeared and at 72 h cytolysis was observed, with total disruption of the cell monolayer at 96 h pi. Due to cell monolayer disruption, the platform could not support proliferation of amoebae, which showed a 3-log reduction in parasite 18S rRNA mRNA after 72 h (10 copies at 1 h to 10 at 72 h pi). SEM observations showed amoebae-like cells with either short pseudopodia and a malleiform shape, or, long pseudopodia embedded within the gill cells and erosion of the cell monolayer. To study the host immune response, inoculated gill cells were harvested from triplicate inserts at 0, 1, 3, 6, 24 and 48 h pi, and expression of 12 genes involved in the Atlantic salmon response to AGD was compared between infected and uninfected cells and between amoebic clones. Both clones induced similar host inmate immune responses, with the up-regulation of proinflammatory cytokine IL1β, complement C3 and cell receptor MHC-1. The Th2 pathway was up-regulated, with increased gene expression of the transcription factor GATA3, and Th2 cytokines IL10, IL6 and IL4/13A. PCNA and AG-2 were also up-regulated. The wild clone induced significantly higher up-regulation of IL1β, MHC-1, PCNA, lysozyme and IL10 than the attenuated clone for at least some exposure times, but AG-2 gene expression was higher in cells inoculated with the attenuated one. A principal component analysis showed that AG-2 and IL10 were key genes in the in vitro host response to N. perurans. This in vitro model has proved to be a promising tool to study host responses to amoebae and may therefore reduce the requirement for in vivo studies when evaluating alternative therapeutants to AGD control.
建立了一种体外模型来研究虹鳟鱼鳃源细胞系 RTgill-W1 对导致鳜鱼传染性造血器官坏死病（IGHN）的虹彩病毒的宿主反应，该细胞系被不对称地接种在可渗透的细胞培养支持物上，其顶部为海水。用传代减毒株或最近的野生克隆 Neoparamoeba perurans 感染细胞。感染后 20min 内，用荧光珠负载的变形虫即可观察到与宿主细胞相关的现象。感染后 6h 出现小的细胞病变效应焦点，72h 时观察到细胞溶解，96h 时细胞单层完全破坏。由于细胞单层的破坏，该平台不能支持变形虫的增殖，在 72h 时寄生虫 18S rRNA mRNA 减少了 3 个对数级（1h 时为 10 拷贝，72h 时为 10 拷贝）。扫描电镜观察显示，变形虫样细胞要么有短的伪足和变形的形状，要么有长的伪足嵌入鳃细胞中并侵蚀细胞单层。为了研究宿主的免疫反应，在感染后 0、1、3、6、24 和 48h 时从三个重复的插入物中收获感染的鳃细胞，并比较感染细胞和未感染细胞以及两种变形虫克隆之间 12 个与大西洋鲑对 IGHN 反应相关的基因的表达。两种克隆都诱导了相似的宿主先天免疫反应，促炎细胞因子 IL1β、补体 C3 和细胞受体 MHC-1 的表达上调。Th2 途径被上调，转录因子 GATA3 和 Th2 细胞因子 IL10、IL6 和 IL4/13A 的基因表达增加。PCNA 和 AG-2 也上调。野生克隆诱导的 IL1β、MHC-1、PCNA、溶菌酶和 IL10 的上调至少在某些暴露时间明显高于减毒株，而感染减毒株的细胞中 AG-2 基因的表达更高。主成分分析表明，AG-2 和 IL10 是宿主对 Neoparamoeba perurans 反应的关键基因。该体外模型已被证明是研究宿主对变形虫反应的一种很有前途的工具，因此在评估替代 IGHN 控制治疗方法时，可能减少对体内研究的需求。
