Title page for ETD etd-0414102-234031


Type of Document Dissertation
Author Elkamel, Ahmad A.
Author's Email Address aelkamel@hotmail.com
URN etd-0414102-234031
Title Pathogenic Mechanisms of Photobacterium Damselae Subspecies Piscicida in Hybrid Striped Bass
Degree Doctor of Philosophy (Ph.D.)
Department Veterinary Microbiology and Parasitology (Veterinary Medical Sciences)
Advisory Committee
Advisor Name Title
Ronald L. Thune Committee Chair
David W. Horohov Committee Member
John Hawke Committee Member
Philip H. Elzer Committee Member
Richard Cooper Committee Member
Douglas Allen Rutherford Dean's Representative
Keywords
  • pathogenic
  • fish
  • invasion
  • virulence
  • photobacterium damselae subspecies piscicida
  • macrophages
  • hybrid striped bass
  • apoptosis
  • intracellular
Date of Defense 2002-03-28
Availability unrestricted
Abstract
Photobacterium damselae subspecies piscicida, previously known as Pasteurella piscicida, is an important pathogen of hybrid striped bass and many fish species cultured in brackish water in the United States, Japan, Europe, and the Mediterranean. The purpose of this study is to investigate virulence mechanisms that contribute to the pathogenesis of this organism.

The ability of P. damselae to survive/replicate within hybrid striped bass macrophages was evaluated with an in vitro killing assay. Results indicated that the numbers of bacteria recovered from macrophages at 3, 6, 12, and 18 hours of incubation increased significantly over time. In contrast, the numbers of Escherichia coli control strain recovered from macrophages declined at the same designated incubation times. Light and electron microscopy confirmed internalization, uptake, and multiplication of bacteria within spacious, clear vacuoles in the macrophages. Using acid phosphatase as a lysosomal marker, it was shown that P. damselae inhibits phagolysosomal fusion.

Invasion and replication of P. damselae within epithelioma papillosum carpio (EPC), channel catfish ovary (CCO), and fathead minnow (FHM) cells lines was also evaluated using an in vitro invasion assay. All three cell lines were susceptible to invasion and supported replication of P. damselae. Fathead minnow cells were more susceptible to invasion than the other two cell lines as indicated by greater numbers of infected cells and recovered bacteria at time 0. Using light and electron microscopy, invasion of cells by bacteria was observed as early as 30 minutes after infection, and intracellular bacteria were observed in large, clear, membrane-bound vacuoles. The intracellular location of P. damselae was confirmed using ruthenium red staining to discriminate between the extra- and intra-cellular spaces.

Using flow cytometry, results indicated that P. damselae induces apoptosis in phagocytes obtained from hybrid striped bass head kidney and after 12, 18, and 24 hours of incubation, the relative numbers of cells infected with P. damselae showing signs of apoptosis increased over time and were significantly greater than the controls. The relative numbers of apoptotic cells that were infected with the formalin-killed strain increased, but not significantly, above the control after the same designated times of incubation.

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