Date of Award

8-2010

Level of Access Assigned by Author

Campus-Only Thesis

Degree Name

Master of Science (MS)

Department

Marine Biology

Advisor

Ian Bricknell

Second Committee Member

Sharon Ashworth

Third Committee Member

Carol H. Kim

Abstract

Bacterial kidney disease (BKD), caused by the bacterium Renibacterium salmoninarum, is a global disease problem in both wild and cultured salmonids. The disease is extremely difficult to control, information regarding the pathogenesis of R. salmoninarum is incomplete, and there is no effective treatment. Salmonids are the natural host of R. salmoninarum, but it is challenging and costly to work with these animals in the laboratory. Zebrafish have become an important tool in which to study the genetics underlying development, normal body function, and disease. This is the first study that investigates the potential use of the zebrafish as a model for BKD. A zebrafish model for BKD would provide a tool to study the pathogenesis of R. salmoninarum. A better understanding of the mechanisms involved in the virulence and intracellular survival of R. salmoninarum could lead to better preventative treatments and vaccines for BKD. There are two major hurdles with using the zebrafish as a model for BKD: there is a temperature difference between the pathogen and zebrafish of more than 10 °C and R. salmoninarum is believed to be specific to salmonids. A zebrafish cell culture line was infected with R. salmoninarum to determine whether or not the bacterium is capable of infecting zebrafish cells. Zebrafish liver cells were acclimated to 15-, 22-, or 26 °C and inoculated with bacteria every hour, for 8 hours. R. salmoninarum was able to successfully infect zebrafish cells in vitro. There were a significant proportion of infected cells at 22 °C, 8 hours post infection. Once it was determined that R. salmoninarum could infect zebrafish cells, 2 sets of adult zebrafish were challenged with the pathogen. Both groups of fish were acclimated down to 18 °C because outbreaks of BKD are typically observed between 15–18 °C. Fish were challenged via intraperitoneal injection and left for 12 weeks. Fish were sampled every 2 weeks for confirmation by culture, immunofluorescence (DFAT), and histology. In addition, fish were sampled for PCR in the second trial. The first group of fish was challenged with doses of R. salmoninarum ranging from 1–105 cells/mL to determine the median lethal dose (LD50). No fish died of R. salmoninarum and a disease response curve could not be determined. Some liver, spleen, and kidney tissues came back positive for R. salmoninarum via DFAT. Based on these results, another group of fish was inoculated with a low (102 cells/mL) and high (105 cells/mL) dose of R. salmoninarum to evaluate progression of the disease in vivo. One fish was suspicious for R. salmoninarum by DFAT, but all samples were negative via PCR and culture. Histological examination showed Gram-positive bacteria in normally sterile organs and revealed a typical disease-related trend in tissue degradation. This is the first time the zebrafish has been evaluated as a model for BKD and modifications to methods used in this study could lead to an effective model for BKD.

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