Growth Dynamics and Tissue Tropism of Rickettsia buchneri, The Endosymbiont of The Blacklegged Tick, Ixodes scapularis

Abstract

Abstract Ch1: I analyzed the growth dynamics of R. buchneri in vivo and in vitro. The prevalence and quantification of R. buchneri in laboratory colonized and field-collected I. scapularis ticks was examined by qPCR. Rickettsia buchneri has been shown to be mainly restricted to the ovaries of females, however, PCR has demonstrated that larvae, nymphs, and males also harbor R. buchneri. For in vitro experiments, the growth of wildtype and transformant R. buchneri (GFPuv) grown in Ixodes ricinus cells (line IRE11) was measured via qPCR to determine the copy number of the rickettsial citrate synthase (gltA) gene to estimate numbers of R. buchneri. Additionally, a novel fluorescent plate reader method was utilized to measure the growth of transformant R. buchneri. In vitro qPCR results indicated the doubling time of wildtype and transformant R. buchneri in the IRE11 cell line was approximately 8 and 7 days, respectively. In vitro experiments utilizing a novel fluorescent plate reader methodology confirmed the slow growth rate of the transformant. The in vivo doubling time of R. buchneri in field-collected ticks measured approximately 6 and 11 days for larvae and nymphs, respectively. Nymphs and males had the highest instances of negative detections with larvae and females having 100% prevalence. Additionally, there was limited growth of R. buchneri in males after molting from engorged nymphs. Females contained the highest and least variable quantities of R. buchneri and experienced just under an additional 4 doublings after the molt from engorged nymphs. The findings from this study help strengthen the contemporary understanding of R. buchneri as a transovarially transmitted rickettsial endosymbiont. Abstract Ch 2: The blacklegged tick, Ixodes scapularis, is a prominent pathogen vector found throughout much of the Eastern and Midwestern states, including Minnesota, which carries significant medical importance to human health. Ixodes scapularis harbors an endosymbiotic bacterium known as Rickettsia buchneri with non-pathogenic and transovarial transmission characteristics. To this day, the biological role of R. buchneri in I. scapularis remains undefined. Historically, R. buchneri has been shown to be restricted to the ovaries of females, however, further quantitative exploration via quantitative PCR (qPCR) has determined all life stages can harbor various quantities or lack R. buchneri entirely indicating that R. buchneri is not restricted to ovarian tissues. Our primary objective was to evaluate the tissue tropism of R. buchneri in flat and partially engorged female I. scapularis ticks collected at Camp Ripley, Minnesota via RNA fluorescent in situ hybridization (FISH). A fluorescent DNA probe, which targets Rickettsia 23S ribosomal RNA, was applied to dissected whole organs of flat females while the addition of a dual-plex assay utilizing the 23S Rickettsia probe coupled with a 16S eubacterial probe was performed on dissected whole ovaries of partially engorged females. We visualized strong positive signals of R. buchneri in the developing oocytes of the ovary and of flat females. Reduced detection of R. buchneri was seen in the ovarian lumen of partially engorged females due to oviposition to facilitatate transovarial transmission. The tissue localization of R. buchneri supports its physiological specialization in a medically important tick vector.