Title page for ETD etd-06252010-160336

Type of Document Dissertation
Author Soto, Esteban
Author's Email Address esoto1@lsu.edu, balasotom@gmail.com
URN etd-06252010-160336
Title In vivo and In vitro Pathogenesis of Francisella asiatica in Tilapia Nilotica(Oreochromis niloticus)
Degree Doctor of Philosophy (Ph.D.)
Department Pathobiological Sciences (Veterinary Medical Sciences)
Advisory Committee
Advisor Name Title
Hawke, John P. Committee Chair
Elzer, Philip H. Committee Member
Macaluso, Kevin R. Committee Member
Thune, Ronald L. Committee Member
Baltz, Donald M. Dean's Representative
  • Macrophages
  • Vaccine
  • Tilapia
  • Francisella
Date of Defense 2010-05-06
Availability unrestricted
Francisella asiatica is a Gram negative, facultative intracellular pathogen that causes fish francisellosis. In this project, homologues to the F. tularensis iglABCD genes were found present in F. asiatica. As few as 23 F. asiatica bacteria injected in the peritoneum were found capable of causing mortalities in tilapia nilotica (Oreochromis niloticus), and even fewer were enough to cause pathological changes. We examined the in vivo and in vitro interaction of F. asiatica wild type (WT) and a ÄiglC strain with tilapia and tilapia head kidney derived macrophages (HKDM). The ÄiglC was found to be attenuated following intraperitoneal and immersion challenges in tilapia. The WT was found to be able to invade HKDM and replicate vigorously within them, causing apoptosis and cytotoxicity in the macrophages. The ÄiglC, however is defective for survival, replication and the ability to cause cytotoxicity in HKDM. We further characterize the efficacy of the ÄiglC as a live attenuated vaccine against subsequent immersion challenge with the WT. Tilapia vaccinated by immersion with a suspension of the ÄiglC and subsequently challenged with WT were protected (90% survival) from the lethal challenges. F. asiatica-specific antibodies produced in response to immunization with the ÄiglC were subsequently found to protect tilapia against WT challenge in passive immunization experiments. The lack of effective treatments led us to investigate the efficacy of florfenicol for treatment of F. asiatica in vitro and in vivo. Addition of florfenicol to the medium at 10 µg/ml resulted in uptake of the drug by HKDM and significantly reduced bacterial loads in vitro. Fish fed medicated feed 1 and 3 days post infection showed significantly higher survival rates, and significantly lower numbers of bacteria than controls. Finally, a real time polymerase chain reaction assay was developed to rapidly and accurately detect and quantify F. asiatica from fish tissue. Probe specificity was confirmed by the lack of signal and cross-reactivity with twelve common fish pathogens, two subspecies of F. tularensis, F. noatunensis, and tilapia tissue. The limit of detection was 50 fg of DNA (~25 genome equivalents).

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