Culture and Identification of Rickettsia-like Organisms in Maryland

马里兰州类立克次体生物的培养和鉴定

• 批准号: 7849908
• 负责人: DOUGLAS E NORRIS
• 金额: $ 8.2万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7849908
• 关键词: Amblyomma; Applications Grants; Arthropods; Bacteria; Biological; Bite; Caring; Categories; Cell Line; Citrate (si)-Synthase; Clinical; Complex Mixtures; Culture Techniques; Detection; Diagnostic tests; Disease; Fever; Gene Targeting; Genetic Materials; Goals; Hemolymph; Human; Infection; Knowledge; Lead; Maryland; Medical; Methods; Molecular; Molecular Analysis; Molecular Target; National Institute of Allergy and Infectious Disease; Organism; Phylogenetic Analysis; Population; Prevalence; Relative (related person); Rickettsia; Rickettsia rickettsii; Risk; Rocky Mountain Spotted Fever; Sampling; Serologic tests; Spottings; Techniques; Tick-Borne Infections; Ticks; United States; Vial device; Visual; interest; meetings; member; novel; pathogen; pathogenic bacteria; public health relevance; reconstruction; success; tool

DESCRIPTION (provided by applicant): In the United States there has been an increase in tick-borne infections over the last decade. Among the most threatening of these are the rickettsial infections. The best known of the human rickettsial infections is responsible for Rocky Mountain Spotted Fever (RMSF), a disease that has remained severe with a fatality rate of 3-10%, even with modern medical care. The etiologic agent of RMSF, Rickettsia rickettsii, is one of 18 recognized members of the Rickettsia Spotted Fever Group (SFG) and a NIAID Category C Priority Pathogen. Tick infection rates for R. rickettsii are believed to be approximately 1-3% where RMSF is endemic. In addition, many other potentially pathogenic bacteria circulate and are maintained in these tick populations with infection rates in some studies as high as 36%. Despite non-specific detection using molecular and visual methods, the majority of these organisms have eluded accurate identification. Specific infection rates and the potential human pathogens with which these ticks are infected, are poorly documented, but could include several Rickettsia (NIAID Category C Priority Pathogens) and Rickettsia-like organisms (categorization unknown). Accurate identification of many of these agents has remained elusive, likely due to poor conservation of known molecular targets to a very limited amount of "unknown" genetic material that is routinely recovered from field-collected ticks. This project will focus on optimization of methods for culturing of Rickettsia and Rickettsia-like organisms from field-collected Amblyomma americanum ticks, such that they can be characterized and identified using existing techniques. This target species was selected for this Small Grant proposal for proof of principle because these ticks are common in Maryland, have low infection rates with known human pathogens in Maryland, but have relatively high infection rates with identifiable non-pathogenic Rickettsia and Rickettsia-like organisms that have evaded identification. Knowledge of what agents are components of the natural ecological background is integral to the accurate assessment of the introduction and spread of native and non-native pathogens, either through incidental or more malicious means. [PARAGRAPH] Hypotheses: Shell-vial inoculation will result in isolation of known and novel Rickettsial infections from Maryland ticks and purification and molecular analysis of these isolates can lead to positive identification of these organisms. [PARAGRAPH] Specific aims of this proposal are to: [PARAGRAPH] 1. Use shell-vial culture techniques to culture/isolate known and unknown Rickettsia-like organisms from man-biting ticks of Maryland. We will isolate and culture Rickettsia-like organisms from tick hemolymph in mammalian and arthropod cell lines that have been previously shown to be permissive to infection with diverse Rickettsial species. [PARAGRAPH] 2. Identify and characterize Rickettsia and Rickettsia-like organisms recovered in Aim #1. Preliminary characterization of the Rickettsia and Rickettsia-like organisms will involve differentiation between Spotted Fever Group and non-Spotted Fever Group Rickettsia from A. americanum. Subsequent identification will involve sequencing of the citrate synthase and other target genes for molecular identifications and phylogenetic reconstructions. PUBLIC HEALTH RELEVANCE: The primary goal of this project is to use shell-vial culture techniques to culture/isolate known and unknown Rickettsia and Rickettsia-like organisms from man-biting ticks of Maryland. Additionally, we will utilize the genetic material recovered via culture, to develop, optimize and utilize molecular methods to characterize these novel organisms that have eluded identification with existing tools.

描述（由申请人提供）：在美国，过去十年蜱媒感染有所增加。其中最具威胁性的是立克次体感染。最著名的人类立克次体感染是导致落基山斑点热（RMSF）的原因，这种疾病仍然很严重，即使有现代医疗护理，死亡率也只有3- 10%。RMSF的病原体，立克次体，是立克次体斑点热组（SFG）的18个公认成员之一，也是NIAID C类优先病原体。蜱感染率为R。在RMSF流行的地方，立克次氏体被认为约为1-3%。此外，许多其他潜在的致病细菌在这些蜱虫种群中传播和维持，在一些研究中感染率高达36%。尽管使用分子和视觉方法进行非特异性检测，但这些微生物中的大多数都无法准确识别。具体的感染率和这些蜱虫感染的潜在人类病原体的记录很少，但可能包括几种立克次体（NIAID C类优先病原体）和立克次体样生物（分类未知）。许多这些代理人的准确鉴定仍然难以捉摸，可能是由于已知的分子靶向非常有限的数量的“未知”的遗传物质，这是常规回收从现场收集的蜱保存不良。该项目将重点关注从野外收集的美洲钝眼蜱中培养立克次体和立克次体样生物的方法的优化，以便使用现有技术对其进行表征和鉴定。选择该目标种属用于本小额赠款提案的原则证明，因为这些蜱在马里兰州很常见，在马里兰州对已知人类病原体的感染率较低，但对可识别的非致病性立克次体和立克次体样微生物的感染率相对较高，这些微生物已逃避识别。了解哪些病原体是自然生态背景的组成部分，对于准确评估本地和非本地病原体的引入和传播（无论是通过偶然的还是更恶意的手段）是不可或缺的。假设：壳瓶接种将导致从马里兰州蜱中分离出已知和新型立克次体感染，这些分离株的纯化和分子分析可导致这些微生物的阳性鉴定。本建议的具体目标是：使用壳瓶培养技术从马里兰州的咬人蜱中培养/分离已知和未知的立克次体样微生物。我们将从哺乳动物和节肢动物细胞系中的蜱血淋巴中分离和培养立克次体样生物，这些细胞系先前已被证明允许感染不同的立克次体物种。[段落] 2.鉴别和表征目标1中回收的立克次体和立克次体样微生物。立克次体和立克次体样微生物的初步鉴定将涉及斑点热群和非斑点热群立克次体与A。美国。随后的鉴定将涉及柠檬酸合酶和其他靶基因的测序，用于分子鉴定和系统发育重建。公共卫生关系：本项目的主要目标是使用壳瓶培养技术从马里兰州的咬人蜱中培养/分离已知和未知的立克次体和立克次体样生物。此外，我们将利用通过培养回收的遗传物质，开发，优化和利用分子方法来表征这些无法用现有工具鉴定的新生物。