Isolation of novel rodent chlamydiae

新型啮齿动物衣原体的分离

• 批准号: 9035821
• 负责人: Kyle H. Ramsey
• 金额: $ 23.62万
• 依托单位: MIDWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9035821
• 关键词: Animal Model; Antigens; Biological; Biological Response Modifiers; Breeding; Cell Culture Techniques; Chlamydia; Chlamydia Infections; Chlamydia muridarum; Chlamydiaceae; Chronic; Data; Environment; Evolution; Foxes; Future; Genitourinary system; Genome; Genomics; Genotype; Growth; Hamsters; Health Sciences; Human; Immunity; In Vitro; Inbred DBA Mice; Incubators; Infection; Infertility; Interferon Type II; Investigation; Laboratories; Laboratory mice; Lead; Life; Livestock; Maintenance; Mammalian Cell; Mediating; Methods; Modeling; Molecular; Mus; Nature; New Agents; Nucleic Acids; Outcome; Pathologic; Pathology; Peromyscus; Personal Communication; Phenotype; Plaque Assay; Polymerase Chain Reaction; Population; Population Dynamics; Population Heterogeneity; Psittacosis; Pulmonary Inflammation; Reporting; Research Personnel; Rodent; Scientist; Serial Passage; Serological; Shipping; Ships; Source; Specificity; Specimen; Staging; Surveys; T-Lymphocyte; Testing; Time; Tissue Sample; Tissues; Tubal Occlusion; Universities; Variant; Virulence; Work; attenuation; base; companion animal; comparative genomics; egg; functional genomics; genetic variant; genome sequencing; genomic biomarker; high reward; high risk; improved; in vivo; insight; major outer membrane protein; mouse model; novel; nucleic acid detection; pathogen; public health relevance; reproductive tract

 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.

 描述（由申请人提供）：衣原体科是人类、牲畜、伴侣动物和许多野生动物物种的病原体。衣原体疾病的全球影响是巨大的。小鼠通常用于人类感染模型，并且小鼠模型有几种优势。有新的信息表明，C有多种变体。在我们目前可用的菌株的群体中的muridarum。然而，我们对C的范围知之甚少。在过去的60年里，muridarum适应了无数的世代，自从它最初被分离出来以来，它在鸡蛋和细胞培养中的传代次数不计其数。“新鲜的”和更多样化的分离物的定义和更广泛的剧目啮齿动物适应衣原体研究人员，更好的建模人类衣原体感染和免疫力可以实现。新的啮齿动物衣原体的分离也可以提高我们的衣原体变异体的稳定性或变化，在体外和体内，以及衣原体在各种主机的人口动态的理解。我们目前的血清学和分子证据表明，啮齿动物衣原体循环的鹿鼠属。在自然界中的小鼠和一些驯化的Peromyscus spp实验室小鼠中。在这个提议中，我们假设可以成功地从这些来源分离出新型啮齿动物衣原体。我们将在两个目标中检验这个假设。在Aim 1（Ramsey，Midwestern University）中，我们将诱捕Peromyscus spp.小鼠，并通过聚合酶链反应（PCR）筛选衣原体核酸。将对小鼠实施安乐死，并选择PCR阳性的小鼠进行进一步研究。在目标2中，目标1中选择的阳性组织样本将被运送到田纳西大学的区域生物防护实验室。健康科学中心（Peters，UTHSC），在ABSL 3条件下将其接种到高度衣原体易感的DBA小鼠中。来自PCR阳性驯化的Peromyscus小鼠的组织也将用于对DBA小鼠进行PCR。重复用于分离C的原始方法。我们将在小鼠中进行连续传代以扩增病原体。衣原体分离株将在培养物中扩增，并以低体外传代次数维持。分离株最初将通过培养物中的表型生长来表征（例如，生长速率、空斑测定/效率和内含物的显微形态计量分析）。将对克隆的候选分离株（最多10株）的基因组进行测序。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 小鼠进一步的工作可以表征体内和体外的变异群体动态。我们认为，该提案的目的和范围构成了一项具有高回报潜力的探索性研究，因为它们将为通过确定新试剂在新方向上扩展研究奠定基础，这些新试剂将改善人类衣原体感染的建模，提供对衣原体基因组学、种群动力学、进化和适应以及宿主-衣原体相互作用的深入了解。