Studies of the transmission routes of the Rickettsia symbiont in Ixodes pacificus ticks by fluorescent in situ hybridization and transmission electron microscopy

Graduation Date

2012

Document Type

Thesis

Program

Other

Program

Thesis (M.S.)--Humboldt State University, Biology, 2012

Committee Chair Name

Jianmin Zhong

Committee Chair Affiliation

HSU Faculty or Staff

Keywords

Ticks, Humboldt State University -- Theses -- Biology, Symbiont, Ixodes pacificus

Abstract

Ixodes pacificus is a primary vector of Lyme borreliosis and human anaplasmosis to people and animals in the Pacific states of the United States. This tick is also a host for a number of other bacterial species including members of the genus Rickettsia. Recently, our laboratory reported two new phylotypes (G021 and G022) of Rickettsia in Ixodes pacificus ticks based on PCR and sequence analysis. The prevalence, host specificity and biology of interaction of these new phylotypes with its tick host are a focus of analysis in the lab. As part of this overall goal, this study focused on the molecular detection of these new phylotypes of Rickettsia in the ovary and midgut of Ixodes pacificus ticks by in situ hybridization using Rickettsia-specific labeled probes and ultrastructural analysis. To identify the ideal medium for generating thin sections of detecting the intracellular bacteria without high fluorescence background, Technovit 8100, Unicryl and paraffin were used in in situ hybridization studies. The high background fluorescence of Technovit 8100 and Unicryl made it difficult to detect Rickettsia in these sections. Tissue embedded in paraffin gave lower autofluorescence. However, the signal generated by fluorescently labeled probes was not high enough to generate convincing images. Therefore, Digoxigenin-tagged probes were used for the hybridization and the signal was amplified using Tyramide Signal Amplification reaction. Using this technique, Rickettsia bacteria were detected in the ovary of Ixodes pacificus ticks. The presence of the Rickettsia species in ovary and midgut was further confirmed by transmission electron microscopic analysis and PCR. Overall, the methods in this study can be used to understand transmission routes of this bacterial species in ticks.

https://scholarworks.calstate.edu/concern/theses/v405sc67f

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