Isolation of novel rodent chlamydiae

 描述(申请人提供)：衣原体科是人类、牲畜、伴生动物和许多野生动物的病原体。衣原体疾病对全球的影响是巨大的。小鼠通常被用来模拟人类感染，关于小鼠模型有几个优点。有新的信息出现，在我们目前可用的菌株的种群中存在多种不同的鼠疫杆菌变种。然而，我们对过去60年来小球藻的适应程度知之甚少，而且自从最初分离以来，在母鸡的鸡蛋中和细胞培养中传代的次数不计其数。如果定义了“新鲜的”和更多样的分离株，并为研究人员提供了更广泛的啮齿动物适应衣原体菌谱，就可以实现对人类衣原体感染和免疫的更好建模。新型啮齿动物衣原体的分离也有助于我们更好地了解衣原体在体内外的稳定性或变异，以及衣原体在不同宿主中的种群动态。我们提出了啮齿动物衣原体在Permyscus spp.中循环的血清学和分子证据。自然界中的小鼠和一些驯化的Permyscus spp实验室小鼠。在这项提议中，我们假设可以从这些来源中成功地分离出新的啮齿动物衣原体。我们将在两个目标中检验这一假设。在目标1(拉姆齐，中西部大学)，我们将诱捕Permyscus spp.并通过聚合酶链式反应(PCR)对这些样本进行衣原体核酸筛查。小鼠将被安乐死，那些聚合酶链式反应阳性的小鼠将被选作进一步研究。在目标2中，在目标1中选择的阳性组织样本将被运往位于大学的区域生物遏制实验室。田纳西州。它们将在ABSL3条件下接种到高度易感衣原体的DBA小鼠体内。来自PCR阳性的驯化Permyscus小鼠的组织也将被用于接种DBA小鼠。我们将复制最初用于分离小鼠隐孢子虫的方法，在小鼠身上进行连续传代，以扩增病原体。衣原体分离株将在培养中扩增，并保持较低的体外传代次数。分离物最初将以培养中的表型生长为特征(例如，生长速度、菌斑分析/效率和包裹体的微形态分析)。克隆的候选菌株(最多10个)的基因组将被测序。这些分离株的体外研究将为将来在实验室建立体内表型奠定基础。 老鼠。进一步的工作可以表征体内和体外的变异群体动力学。我们相信，这项建议的目标和范围构成了一项探索性研究，具有很高的回报潜力，因为它们将为在新方向上扩展研究奠定基础，确定将改善人类衣原体感染模型的新制剂，为深入了解衣原体基因组学、种群动力学、进化和适应以及宿主-衣原体相互作用提供依据。

 DESCRIPTION (provided by applicant): The Chlamydiaceae are pathogens of humans, livestock, companion animals and many species of wildlife. The global impact of chlamydial diseases is significant. Mice are quite commonly used to model human infection and there are several strengths in regard to the mouse model. There is new information emerging that there are multiple variants of C. muridarum within the populations of our present available strains. Yet we know little of the extent of C. muridarum adaptation over the past 60 years and untold numbers of passages in hen's eggs and then in cell culture since it was originally isolated. Were "fresh" and more varied isolates defined and a broader repertoire of rodent-adapted chlamydiae available to investigators, better modeling of human chlamydial infections and immunity could be realized. Isolation of novel rodent chlamydiae could also improve our understanding of chlamydial variant stability or variation in vitro and in vivo as well as chlamydial population dynamics in various hosts. We present serological and molecular evidence that rodent chlamydiae circulates in Peromyscus spp. mice in nature and in some domesticated Peromyscus spp laboratory mice. In this proposal, we hypothesize that one can successfully isolate novel rodent chlamydiae from these sources. We will test this hypothesis in 2 Aims. In Aim 1 (Ramsey, Midwestern University), we will trap Peromyscus spp. mice and screen these for chlamydial nucleic acids by polymerase chain reaction (PCR). Mice will be euthanized and those mice that are PCR-positive will be selected for further investigation. In Aim 2, positive tissue samples selected in Aim 1 will be shipped to the Regional Biocontainment Laboratory at the Univ. Tenn. Health Sciences Center (Peters, UTHSC) where they will be inoculated into highly chlamydia-susceptible DBA mice under ABSL3 conditions. Tissues from PCR-positive domesticated Peromyscus mice will also be used to inoculate DBA mice. Replicating the original methods used to isolate C. muridarum, we will conduct serial passage in mice to amplify the pathogen. Chlamydial isolates will be expanded in culture with maintenance at low in vitro passage number. Isolates will be initially characterized by phenotypic growth in culture (e.g., growth rate, plaque assay/efficiency, and micromorphometirc analysis of inclusions). The genome of cloned candidate isolates (up to 10) will be sequenced. These in vitro studies of isolates will set the stage for future ones involving establishing in vivo phenotypes in laboratory mice. Further work could characterize variant population dynamics in vivo and in vitro. We believe the aims and scope of this proposal constitute an exploratory study that is potential of high reward in that they will lay the ground work for extending studies in new directions by identifying new agents that will improve modeling of human chlamydial infections, provide insight into chlamydial genomics, population dynamics, evolution and adaptation, and host- chlamydia interactions.