Withering syndrome (WS) is a chronic bacterial disease that affects numerous northeastern Pacific abalone Haliotis spp. The causative agent of WS is an obligate intracellular Rickettsiales-like bacterium (WS-RLO) that remains unculturable, thereby limiting our understanding of WS disease dynamics. The objectives of our study were to (1) determine the temporal stability of WS-RLO DNA outside of its abalone host in 14°C and 18°C seawater, (2) develop a standardized protocol for exposing abalones to known concentrations of WS-RLO DNA, and (3) calculate the dose of WS-RLO DNA required to generate 50% infection prevalence (ID50) in the highly cultured red abalone Haliotis rufescens. The WS-RLO stability trials were conducted in October 2016, February 2017, and June 2017. A quantitative PCR (qPCR) analysis was used to quantify bacterial DNA for 7 d in seawater collected at an abalone farm in southern California, where the pathogen is now endemic. For all trials and temperature treatments, WS-RLO DNA was unstable in seawater for longer than 2 d. To determine an ID50, groups of uninfected juvenile red abalone were subjected to 3-h bath exposures with four concentrations of WS-RLO at 0, 103 , 104 , and 105 DNA copies/mL. Abalone feces were tested biweekly for the presence of WS-RLO DNA, and abalone tissues were sampled 9 weeks postinfection for histological and qPCR analyses. The ID50 results indicated that our protocol was successful in generating WS-RLO infections; a pathogen dose of 2.3 × 103 DNA copies/mL was required to generate a 50% infection prevalence in red abalone tissue. These findings are critical components of disease dynamics that will help assess WS transmission risk within and among abalone populations and facilitate appropriate management and restoration strategies for both wild and cultured abalone species in WS-endemic areas.

中文翻译：

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鲍鱼萎蔫综合征疾病动态：海水中的感染剂量和时间稳定性

枯萎综合征 (WS) 是一种慢性细菌性疾病，会影响许多东北太平洋鲍鱼 Haliotis spp。WS 的病原体是一种专性细胞内立克次体样细菌 (WS-RLO)，它仍然无法培养，从而限制了我们对 WS 疾病动态的理解。我们研究的目的是 (1) 确定 WS-RLO DNA 在鲍鱼寄主外在 14°C 和 18°C 海水中的时间稳定性，(2) 制定标准化方案，将鲍鱼暴露于已知浓度的 WS- RLO DNA，和 (3) 计算在高度培养的红鲍鱼 Haliotis rufescens 中产生 50% 感染率 (ID50) 所需的 WS-RLO DNA 剂量。WS-RLO 稳定性试验分别于 2016 年 10 月、2017 年 2 月和 2017 年 6 月进行。使用定量 PCR (qPCR) 分析对在加利福尼亚南部一个鲍鱼养殖场收集的海水中的细菌 DNA 进行 7 天的定量分析，该养殖场现在病原体已成为地方性流行病。对于所有试验和温度处理，WS-RLO DNA 在海水中的不稳定时间超过 2 天。为了确定 ID50，将未感染的幼年红鲍鱼在 0、103、104 和 105 DNA 拷贝/mL 的四种浓度的 WS-RLO 中浸泡 3 小时。每两周检测一次鲍鱼粪便中 WS-RLO DNA 的存在，并在感染后 9 周对鲍鱼组织进行取样进行组织学和 qPCR 分析。ID50 结果表明我们的协议成功地产生了 WS-RLO 感染；需要 2.3 × 103 DNA 拷贝/mL 的病原体剂量才能在红鲍鱼组织中产生 50% 的感染率。