Title page for ETD etd-03242009-155921


Type of Document Master's Thesis
Author Zanetti, Andrea S.
Author's Email Address azanetti@vetmed.lsu.edu
URN etd-03242009-155921
Title Quantitative Real-Time Polymerase Chain Reaction(QPCR) Assay as a Molecular Tool to Assess Rickettsial Replications in Tick Hosts
Degree Master of Science (M.S.)
Department Pathobiological Sciences (Veterinary Medical Sciences)
Advisory Committee
Advisor Name Title
Kevin R. Macaluso Committee Chair
James E. Miller Committee Member
John B. Malone Committee Member
Keywords
  • Rickettsia spp
  • Amblyomma americanum
  • quantitative real time polymerase chain reaction
  • Spotted Fever Group Rickettsia
Date of Defense 2009-02-17
Availability unrestricted
Abstract
During the past century, many species of the Spotted Fever Group Rickettsia (SFGR)have been described, especially, through the introduction of a variety of molecular techniques applied to detect rickettsiae inside of their host. In this study we developed a quantitative realtime polymerase chain reaction (qPCR) assay (1) to characterize the growth and the distribution of a SFGR of unrecognized pathogenicity in naturally infected Amblyomma americanum ticks during physiological events; and (2) to terminate the influence of the host cell specificity in the replication patterns of recognized and unrecognized SFGR during a reciprocal rickettsiae challenge in both mammalian and tick cell lines. Rickettsia amblyommii was identified in the tissue samples of naturally infected A. americanum ticks at ratios of 1 rickettsiae per tick cell. Significant variability in the ratio of ickettsial to tick gene copy numbers between the tissues was identified; however, no single tissue was consistently observed to have the greatest rickettsial burden throughout the feeding event. Furthermore, the ratio of rickettsial to tick gene copy numbers did not significantly differ between eggs, immature ticks, and feeding events. In the in vitro study, differences in the ratio of rickettsiae per cell were observed within each cell line. The ratio of rickettsiae per host cell was greatest in Rickettsia-infected ISE6 cells, compared to Vero cells. Rickettsia parkeri infection load was consistently greater in both cell lines compared to R. amblyommii and Rickettsia montanensis; and considerable variability between these last two Rickettsia species was observed when the ratio of ickettisae per host cell was calculated for each individual cell line. The implications of the use of this technique to understand the pathogenic nature of some SFGR and to investigate the host specificity in the tick-SFGR interactions is further presented and discussed.
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