Infectious pancreatic necrosis virus (IPNV) is the aetiological agent of infectious pancreatic necrosis (IPN), a disease associated with serious economic loss in Atlantic salmon (Salmo salar). The interaction between IPNV and the host is poorly characterised. IPNV has been detected within macrophages in natural and experimental infections. The macrophage is an important component of the host immune system, participating in innate and adaptive immune responses. The overarching objective of this project was to study aspects of the interaction between IPNV and innate immune responses in the Atlantic salmon macrophage. Methods were developed for the isolation and in vitro culture of Atlantic salmon macrophages. These cells were isolated from head kidney using percoll gradients and subsequently cultured in 24 well plates using Leibovitz L-15 medium containing penicillin, streptomycin and foetal calf serum. This procedure enabled the in vitro culture of macrophages for 9 days post isolation. Real time RT-PCR assays were developed to quantitate the expression of IPNV, Interferon (IFN), Mx, and Elongation factor 1 (ELF-1) in IPNV-infected macrophages and uninfected controls. ELF-1 is utilised as a control gene for relative quantitation in RT-PCR studies. The RT-PCR assays utilised targetspecific primers, and MGB probes. Assay efficiencies varied from 0.85 to 0.99, these were suitable for quantitative RT-PCR analyses. IPNV was demonstrated to replicate in macrophages cultured in vitro as assessed by quantitative RT-PCR. IPNV levels in macrophages were greatest at the early stages of infection. Virus was detected in infected macrophages throughout the nine day period of investigation. Quantitative RT-PCR analyses of the expression of the immune response genes IFN and Mx suggested that IPNV blocks IFN production, as opposed to blocking IFN signalling. The ability of three immunostimulants, Lipopolysaccharide (LPS), macrophage activating factor (MAF), and glucan to up regulate immune responses in IPNV-infected macrophages was also investigated. None of these immunostimulants were able to enhance expression of IFN and Mx, suggesting that these substances may not represent useful therapeutic means of mitigating IPN in Atlantic salmon.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:513598 |
| Date | January 2007 |
| Creators | McKinley, Gavin |
| Contributors | Starkey, William |
| Publisher | University of Stirling |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1893/243 |
Page generated in 0.002 seconds