Abstract
Abstract
The disease rainbow trout fry syndrome (RTFS) is caused by the bacterial fish pathogen Flavobacterium psychrophilum, which for decades has been the cause of great losses of rainbow trout in aquacultures both in Denmark and around the world. Disease outbreaks are typically seen at water temperatures below 15°C and typically with fry mortality rates of 50-60%. Several attempts of vaccine development against RTFS have been made, but according to my knowledge no commercial vaccine is yet available. Bacterial chemotherapy is still the most effective and used treatment of RTFS today. However, the increasing problem with antibiotic resistance has led to increased attention to the use of phages for controlling F. psychrophilum infections in aquaculture. In a synopsis and four scientific papers, this PhD project studies the potential and optimizes the use of phage therapy for treatment and prevention of F. psychrophilum infections in rainbow trout fry.
In the first paper, studies of the controlling effect of different phages infecting F. psychrophilum in liquid cultures showed that a high initial phage concentration was crucial for fast and effective bacterial lysis in the cultures and sensitive cells could be maintained at a low level throughout the rest of the experiment. Surprisingly, no difference was observed between infection with single phages or phage cocktails. At the end of incubation phage-sensitive strains dominated in the cultures with low initial phage concentrations and phage-resistant strains dominated at high initial phage concentrations. Isolated strains showed great phenotypic diversity and differences in resistance patterns against a range of phages. Furthermore, some of the strains showed reduced growth rates and reduced ability to utilize different substrates.
In the second paper, studies of the genetic diversity and susceptibility patterns of F. psychrophilum strains and phages isolated in three geographically distinct areas (Chile, Denmark, and USA) showed that the strains and phages clustered into geographically distinct groups. However, cross-infectivity between Chilean phage isolates and Danish host isolates and vice versa were found. Furthermore, the development of phage resistance against certain phages led to increased susceptibility to other phages in various F. psychrophilum groups, and in some cases this shift in sensitivity was associated with the loss of a specific pro-phage.
In the third paper, a detailed analysis of the resistance mechanisms in F. psychrophilum and six phage resistant mutants was done. The results revealed unique changes in the genomes in all the phage resistant strains and that some of these changes were related to cell surface properties which were suggested to be phage receptors. The observed links between phage resistance and the genetic modifications were supported by direct measurements of bacteriophage adsorption rates, biofilm formation and secretion of extracellular enzymes, which were all impaired in the resistant strains.
In the fourth paper, the dispersal and survival of a F. psychrophilum phage in vivo in juvenile rainbow trout after administration by three different methods was examined. Following phage administration by bath and oral intubation into the stomach, phages could be detected in the intestine, spleen, brain and kidney for 24 h before the phage concentrations started to decline. Continuous administration of phages via phage coated feed resulted in a constant abundance of phages in the intestine and the spleen throughout the experimental period. Furthermore, we documented that phages can tolerate long periods of desiccation on feed pellets. We suggest that continuous delivery of phages via coated feed pellets constitutes a promising method of treatment and especially prevention of rainbow trout fry syndrome.
The disease rainbow trout fry syndrome (RTFS) is caused by the bacterial fish pathogen Flavobacterium psychrophilum, which for decades has been the cause of great losses of rainbow trout in aquacultures both in Denmark and around the world. Disease outbreaks are typically seen at water temperatures below 15°C and typically with fry mortality rates of 50-60%. Several attempts of vaccine development against RTFS have been made, but according to my knowledge no commercial vaccine is yet available. Bacterial chemotherapy is still the most effective and used treatment of RTFS today. However, the increasing problem with antibiotic resistance has led to increased attention to the use of phages for controlling F. psychrophilum infections in aquaculture. In a synopsis and four scientific papers, this PhD project studies the potential and optimizes the use of phage therapy for treatment and prevention of F. psychrophilum infections in rainbow trout fry.
In the first paper, studies of the controlling effect of different phages infecting F. psychrophilum in liquid cultures showed that a high initial phage concentration was crucial for fast and effective bacterial lysis in the cultures and sensitive cells could be maintained at a low level throughout the rest of the experiment. Surprisingly, no difference was observed between infection with single phages or phage cocktails. At the end of incubation phage-sensitive strains dominated in the cultures with low initial phage concentrations and phage-resistant strains dominated at high initial phage concentrations. Isolated strains showed great phenotypic diversity and differences in resistance patterns against a range of phages. Furthermore, some of the strains showed reduced growth rates and reduced ability to utilize different substrates.
In the second paper, studies of the genetic diversity and susceptibility patterns of F. psychrophilum strains and phages isolated in three geographically distinct areas (Chile, Denmark, and USA) showed that the strains and phages clustered into geographically distinct groups. However, cross-infectivity between Chilean phage isolates and Danish host isolates and vice versa were found. Furthermore, the development of phage resistance against certain phages led to increased susceptibility to other phages in various F. psychrophilum groups, and in some cases this shift in sensitivity was associated with the loss of a specific pro-phage.
In the third paper, a detailed analysis of the resistance mechanisms in F. psychrophilum and six phage resistant mutants was done. The results revealed unique changes in the genomes in all the phage resistant strains and that some of these changes were related to cell surface properties which were suggested to be phage receptors. The observed links between phage resistance and the genetic modifications were supported by direct measurements of bacteriophage adsorption rates, biofilm formation and secretion of extracellular enzymes, which were all impaired in the resistant strains.
In the fourth paper, the dispersal and survival of a F. psychrophilum phage in vivo in juvenile rainbow trout after administration by three different methods was examined. Following phage administration by bath and oral intubation into the stomach, phages could be detected in the intestine, spleen, brain and kidney for 24 h before the phage concentrations started to decline. Continuous administration of phages via phage coated feed resulted in a constant abundance of phages in the intestine and the spleen throughout the experimental period. Furthermore, we documented that phages can tolerate long periods of desiccation on feed pellets. We suggest that continuous delivery of phages via coated feed pellets constitutes a promising method of treatment and especially prevention of rainbow trout fry syndrome.
Originalsprog | Engelsk |
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Forlag | Department of Biology, Faculty of Science, University of Copenhagen |
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Status | Udgivet - 2014 |