Enhanced detection of Rickettsia species in Ixodes pacificus using highly sensitive fluorescence in situ hybridization coupled with Tyramide Signal Amplification

Abstract

Ixodes pacificus is a host of many bacteria including Rickettsia species phylotypes G021 and G022. As part of the overall goal of understanding interactions of phylotypes with their tick host, this study focused on molecular detection of rickettsiae in ovarian and midgut tissue of I. pacificus by fluorescent in situ hybridization (FISH), PCR, and ultrastructural analysis. Of three embedding media (Technovit 8100, Unicryl, and paraffin) tested for generating thin sections, tissues embedded in paraffin resulted in the visualization of bacteria with low autofluorescence in FISH. Digoxigenin-labeled probes were used in FISH to intensify bacterial hybridization signals using Tyramide Signal Amplification reaction. Using this technique, rickettsiae were detected in the cytoplasm of oocytes of I. pacificus. The presence of rickettsiae in the ovary and midgut was further confirmed by PCR and transmission electron microscopic analysis. Overall, the methods in this study can be used to identify locations of tick-borne bacteria in tick tissues and understand transmission routes of bacterial species in ticks.

The folA gene from the Rickettsia endosymbiont of Ixodes pacificus encodes a functional dihydrofolate reductase enzyme

“…The described work uses molecular biology and biochemistry to narrow the gap of knowledge in interactions involving bacteria-tick relationships, particularly folate production by phylotype G021. With the genetic capacity of de novo biosynthesis of folate (Hunter et al, 2015), the production of a functional dihydrofolate reductase enzyme, the predominant infection in the midgut and ovary tissues of I. pacificus (Phan et al, 2011; Bagheri et al, 2017), and the increased bacterial burden in I. pacificus following a blood meal (Cheng, et al, 2013a), Rickettsia species phylotype G021 may significantly contribute to its host’s physiological homeostasis.…”

Isolation and characterization of a Rickettsia from the ovary of a Western black-legged tick, Ixodes pacificus

“…We have previously identified Rickettsia species phylotype G021 and Rickettsia species phylotype G022 via PCR amplification using genomic DNA extracted from I. pacificus ticks; of which, the bacterial prevalence was found to be 100% and 2%, respectively (Phan et al, 2011;Cheng et al, 2013b). Transmission electron microscope and Fluorescence in situ hybridization assays detected the presence of Rickettsia species in midgut and ovaries of I. pacificus (Phan et al, 2011;Bagheri et al, 2017). It was later noted that Rickettsia species phylotype G021 was found to possess 100% transovarial and transstadial efficiency as well as the genetic capacity to synthesize tetrahydrofolate, indicating a possible nutritional relationship between the host and endosymbiont (Bodnar et al, 2018;Cheng et al, 2013a;Kurlovs et al, 2014;Hunter et al, 2015).…”