Amblyomma americanum, the lone star tick, is an indigenous tick species in southern Indiana that harbors a diverse group of pathogenic and nonpathogenic microorganisms, including Borrelia lonestari, the putative agent for the southern tick associated rash illness (START) and a spotted fever group rickettsial endosymbiont. The purpose of this study was to implement the real-time polymerase chain reaction (real-time PCR) as a molecular technique to examine the microbial diversity in A. americanum ticks by estimating abundances of different microorgansisms. A SYBR Green real-time PCR assay was designed to detect and quantify B. lonestari in A. americanum ticks, and a previously published TaqMan real-time PCR assay, designed to detect (not quantify) Rickettsia species in ticks, was validated for the detection and quantification of the spotted fever group rickettsial endosymbiont in A. americanum ticks. Many pitfalls associated with real-time PCR were experienced in this study, such as difficulties in assay design and problems with contamination, and appropriate modifications are recommended to laboratories routinely performing real-time PCR. / Department of Biology

Amblyomma americanum is a hard tick species found in southern Indiana. Once a notorious pest to humans and livestock, A. americanum has now taken on a role as vector to pathogenic organisms. This study aimed to detect and quantify three microbes in A. americanum: Rickettsia amblyommii, Ehrlichia chaffeensis, and Borrelia lonestari. A primary objective of this study was to determine microbial interaction within a single A. americanum tick through quantification of each microbe within a co-infected tick. A second objective was to determine the density of R. amblyommii within the salivary glands of A. americanum ticks. Infection rates were 44%, 1%, and 0% for R. amblyommii, E. chaffeensis, and B. lonestari respectively. This study found no co-infected ticks, therefore no microbial interaction was determined. This study also found multiple drawbacks with utilizing quantitative real-time PCR to determine the density of R. amblyommii within the salivary glands of A. americanum ticks. / Department of Biology