Detection and quantification of Borrelia lonestari and a rickettsial endosymbiont in Amblyomma americanum ticks from southern Indiana using real-time PCR

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 BiologyThesis (M.S.