Understanding that disease is a limiting factor to the aquaculture industry together with the knowledge that drugs and chemotherapeutics can cause newly resistant bacterial strains, has driven attention to finding new prophylactic measures to control diseases that include vaccination and the use of “functional feeds” to modulate the fish immune system. The supplementation of immunostimulants into fish diets is already widely used in aquaculture. However, searching for new and effective substances is one of the targets of many fish feed suppliers, including EWOS Ltd. who have co-funded the work presented in this study. There are a number of immunostimulant molecules used at present. Some bacterial components such as LPS are used to enrich fish diets and have been described to improve the natural immune defences. In contrast, peptidoglycan (PG), another ubiquitous component of the bacterial cell wall, has so far received less attention and is therefore investigated in this present study. Its ability to stimulate innate immunity is assessed using antimicrobial peptides (AMPs) as molecular markers, which are known to be involved in the early response against a broad range of pathogens. To date, AMPs in fish are not well characterised and in most cases the mechanisms of pathogen killing as well as the pathways inducing their expression still remain to be elucidated. Initially the cloning and characterisation of three novel trout β-defensin genes (omDB-2, omDB-3, omBD-4) was performed, and the molecules compared to the previously reported omDB-1. Each β-defensin gene was fully cloned and preliminary expression work in vivo and in vitro revealed the ability of these genes to be induced by bacteria and viruses. Analysis of the gene organization found that all three new genes contained three exons divided by two introns. Constitutive expression of these genes was detected by real time PCR ofmucosal and systemic tissues from healthy fish, with omDB-3 and omDB-4 showing the highest expression levels. Following bacterial challenge in vivo, the defensin genes were induced at the three mucosal sites examined (skin, gill, gut), with levels of omDB-2 and omDB-3 increased some 16-fold in gut and gill respectively. Using polyinosinic polycytosinic RNA (polyI:C) as a viral mimic, all of the four trout -defensin genes were induced in head kidney primary leucocyte cultures at 4h post-stimulation, with omDB-1 and omDB-3 showing particularly high expression. To determine the -defensin spectrum of activity against 10 strains of Gram negative and Gram positive bacteria, transfected RTG-2 cell lines over expressing GFP and the target genes omDB-1, omDB-3 and omDB-4 -defensins were produced and their supernatants used. Results showed highest bioactivity against Gram negative bacteria, in particular the supernatant from omDB-1 transfected cells showed the widest range of activity towards the majority of selected bacteria. In addition immune relevant genes (Toll-like receptors, genes involved in the anti-inflammatory response and in the apoptosis process) were screened in normal cell lines stimulated with the supernatant of omDB-1, as well as in the RTG-2 cells transfected with the three different defensins. Results showed for all the cell lines, a clear link with the viral recognition receptors TLR 3 and TLR 9, which supported the poly I:C data reported in Chapter 2 and by the induction in omDB-1 and omDB-3 transfected cell lines of the IFN- gene known to be involved in the antiviral response. Trout β-defensins also up-regulated MHC II and the CCR6 receptor. To determine the effects of fish diets enriched with different concentrations of PG, three in vivo feeding trial experiments in rainbow trout were carried out. Effectiveness of the diets was assessed using gene expression of selected AMPs, including β-defensins, cathelicidins and liver expressed antimicrobial peptide molecules. Fish fed with diets containing either 10 mg/Kg or 50 mg/Kg of PG respectively, showed the highest up-regulation of AMPs at 14 days of feeding. Data showed omDB-2 in the gut as the most inducible gene in agreement with the results obtained in the first experiment and omDB-3 was the fastest to respond in skin and gill. In addition, after ceasation of feeding the enriched diet, modulation of AMP expression was still detectable 28 days later, although a lower degree of induction was found in such fish relative to those maintained on the enriched diet. A final PG feeding trial was combined with a Yersinia ruckeri bacterial challenge which used two PG supplemented diets containing 10 mg/Kg and 50 mg/Kg of immunostimulant, and a commercial β-glucan supplemented diet (as a positive control), and fed to trout for 7 and 14 days before intraperitoneal injection challenge of the fish. Only a delay in the mortality rate was found in fish fed for 14 days with the 10 mg/Kg diet, with no clear protection from any of the functional feeds assessed. Finally, at least 500 bp of the regulatory 5’ end flanking region of two defensin (omDB-1 and omDB-2) and two liver expressed (hepcidin and LEAP-2A) genes were cloned and sequenced. In addition, the promoter sequence already known for the cathelicidin-1 gene was used in this study. Bioinformatic tools were used to search for putative transcription factor binding sites, and revealed the presence in all promoters of regulatory elements which could enhance or inhibit the expression of these genes, in response to different stimuli.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:554280 |
| Date | January 2011 |
| Creators | Casadei, Elisa |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=179548 |
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