Unit of Molecular Zoology, Department of Zoology, Technical University of Munich, Freising, Germany.
Department of Fish, Wildlife and Conservation Ecology, New Mexico State University, Las Cruces, NM, United States of America.
PLoS One. 2018 Oct 22;13(10):e0206164. doi: 10.1371/journal.pone.0206164. eCollection 2018.
The proliferative darkening syndrome (PDS) is an annually recurring disease that causes species-specific die-off of brown trout (Salmo trutta fario) with a mortality rate of near 100% in pre-alpine rivers of central Europe. So far the etiology and causation of this disease is still unclear. The objective of this study was to identify the cause of PDS using a next-generation technology detection pipeline. Following the hypothesis that PDS is caused by an infectious agent, brown trout specimens were exposed to water from a heavily affected pre-alpine river with annual occurrence of the disease. Specimens were sampled over the entire time period from potential infection through death. Transcriptomic analysis (microarray) and RT-qPCR of brown trout liver tissue evidenced strong gene expression response of immune-associated genes. Messenger RNA of specimens with synchronous immune expression profiles were ultra-deep sequenced using next-generation sequencing technology (NGS). Bioinformatic processing of generated reads and gap-filling Sanger re-sequencing of the identified pathogen genome revealed strong evidence that a piscine-related reovirus is the causative organism of PDS. The identified pathogen is phylogenetically closely related to the family of piscine reoviruses (PRV) which are considered as the causation of different fish diseases in Atlantic and Pacific salmonid species such as Salmo salar and Onchorhynchus kisutch. This study also highlights that the approach of first screening immune responses along a timeline in order to identify synchronously affected stages in different specimens which subsequently were ultra-deep sequenced is an effective approach in pathogen detection. In particular, the identification of specimens with synchronous molecular immune response patterns combined with NGS sequencing and gap-filling re-sequencing resulted in the successful pathogen detection of PDS.
增生性黑化综合征（PDS）是一种每年都会发生的疾病，它会导致中欧高山前河流中特定物种的褐鳟（Salmo trutta fario）死亡，死亡率接近 100%。到目前为止，这种疾病的病因和发病机制尚不清楚。本研究的目的是使用下一代技术检测管道来确定 PDS 的原因。根据 PDS 是由传染性病原体引起的假设，将褐鳟标本暴露于受疾病影响严重的高山前河流的水中。标本在从潜在感染到死亡的整个时间段内进行采样。对褐鳟肝脏组织进行转录组分析（微阵列）和 RT-qPCR 表明，免疫相关基因的表达水平有强烈的反应。使用下一代测序技术（NGS）对具有同步免疫表达谱的标本的信使 RNA 进行超深度测序。对生成的读数进行生物信息处理，并对鉴定病原体基因组的缺口进行 Sanger 重测序，强烈表明一种鱼类相关呼肠孤病毒是 PDS 的致病病原体。鉴定出的病原体与鱼类呼肠孤病毒（PRV）家族密切相关，PRV 被认为是大西洋和太平洋鲑鱼物种（如 Salmo salar 和 Onchorhynchus kisutch）中不同鱼类疾病的病因。本研究还强调，首先沿着时间线筛选免疫反应，以识别不同标本中同时受到影响的阶段，然后对其进行超深度测序，是一种有效的病原体检测方法。特别是，结合 NGS 测序和缺口填补重测序，鉴定出具有同步分子免疫反应模式的标本，成功地检测到 PDS 的病原体。
