Culture and Identification of Rickettsia-like Organisms in Maryland

描述（由申请人提供）：在过去的十年里，美国的蜱传感染有所增加。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引起落基山斑疹热（RMSF）的疾病，即使采用现代医疗保健，这种疾病仍然很严重，死亡率为3-10%。RMSF的病原体立克次体立克次体是立克次体斑疹热组（SFG）的18个公认成员之一，也是NIAID C类优先病原体。在RMSF流行的地方，立克次体蜱虫感染率据信约为1-3%。此外，许多其他潜在致病性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）。许多这些药剂的准确鉴定仍然是难以捉摸的，可能是由于已知分子靶点对非常有限的“未知”遗传物质的保护不力，这些遗传物质通常是从野外收集的蜱虫中恢复的。本项目将重点优化从野外采集的美洲钝体蜱中培养立克次体和立克次体样生物的方法，以便利用现有技术对其进行表征和鉴定。这个目标物种被选为这个小型拨款提案的原理证明，因为这些蜱在马里兰州很常见，在马里兰州对已知的人类病原体的感染率很低，但对可识别的非致病性立克次体和立克次体样生物的感染率相对较高。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是不可或缺的，无论是通过偶然的还是更恶意的手段。[段落]假设：壳瓶接种将导致从马里兰蜱中分离出已知和新型立克次体感染，对这些分离物的纯化和分子分析可导致这些生物体的阳性鉴定。【第1段】本提案的具体目的是：【第2段】使用壳瓶培养技术从马里兰州叮人蜱中培养/分离已知和未知的立克次体样生物。我们将从哺乳动物和节肢动物细胞系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多种立克次体物种感染。(段)2。鉴定和表征在Aim #1中恢复的立克次体和立克次体样生物。立克次体和立克次体样生物的初步鉴定将包括区分斑点热组和非斑点热组的立克次体。随后的鉴定将包括对柠檬酸合成酶和其他靶基因进行测序，以进行分子鉴定和系统发育重建。公共卫生相关性：本项目的主要目标是使用壳瓶培养技术从马里兰州叮人蜱虫中培养/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识别的新生物。

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.