Culture and Identification of Rickettsia-like Organisms in Maryland

描述(由申请人提供)：在过去的十年里，在美国，壁虱传播的感染有所增加。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落基山斑点热病(RMSF)的罪魁祸首，即使在现代医疗条件下，这种疾病仍然很严重，死亡率为3%-10%。RMSF的病原体里氏立克次体是立克次体斑点热群(SFG)的18个公认成员之一，是NIAID C类优先病原体。在RMSF流行的地区，立克次体的壁虱感染率据信约为1-3%。此外，许多其他潜在的致病细菌在这些壁虱种群中传播和维持，在一些研究中感染率高达36%。尽管使用分子和目视方法进行非特异性检测，但这些生物中的大多数都未能准确识别。具体感染率和感染这些硬蜱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)和类似立克次体的生物体(分类未知)。对其中许多病原体的准确鉴定仍然难以捉摸，可能是因为对已知分子靶标的保存不力，对通常从现场收集的扁虱中提取的数量非常有限的“未知”遗传物质的保护很差。该项目的重点将是优化从野外采集的美洲Amblyomma americanum ticks中培养立克次体和类似立克次体的方法，以便利用现有技术对它们进行表征和鉴定。这一目标物种被选为这项小型赠款提案的原则性证明，因为这些扁虱在马里兰州很常见，在马里兰州对已知的人类病原体的感染率较低，但对可识别的非致病性立克次体和类似立克次体的生物的感染率相对较高，但未被识别。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：贝壳瓶接种将导致从马里兰州的硬蜱中分离出已知的和新的立克次体感染，这些分离株的纯化和分子分析可以导致对这些微生物的阳性鉴定。这项提议的具体目标是：1.使用贝壳培养技术，从马里兰州咬人的扁虱中培养/分离已知和未知的类似立克次体的生物体。我们将从哺乳动物和节肢动物细胞系中分离和培养类似立克次体的生物，这些细胞系以前被证明允许感染不同的立克次体物种。2.鉴定和表征在目标1中发现的立克次体和类立克次体。对立克次体和类立克次体的初步特征将涉及区分斑点热组和非斑点热组立克次体和美洲立克次体。随后的鉴定将包括对柠檬酸合成酶和其他目标基因的测序，以进行分子鉴定和系统发育重建。与公共卫生相关：该项目的主要目标是使用贝壳培养技术从马里兰州咬人的扁虱中培养/分离已知和未知的立克次体和类似立克次体的生物体。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用现有工具无法识别的新生物。

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.