Transcriptome response following administration of a live bacterial vaccine in Atlantic salmon (Salmo salar)
Introduction
Genomic approaches are enabling the analysis of complex genomic responses that may lead to both novel gene discovery and coordinated transcriptional responses. Nowhere is this information more relevant than in the inflammatory immune response. During the early stage of an infection the host needs to determine if the invading pathogen is bacterial, viral or parasitic and activate the relevant immune response accordingly (Janeway and Medzhitov, 2002). The immune response to pathogens by teleost fish have many common features with other higher vertebrates, but the ability of the fish to respond depends on the environmental conditions (Bols et al., 2001, Nikoskelainen et al., 2004) giving unique features to the lower vertebrate immune system. A particular difference is the dependence on the innate immune system at lower environmental temperatures experienced by fish (Le Morvan et al., 1998, Pylkko et al., 2002). Following infection a number of different defence mechanisms come into action, which include production of antibacterial peptides, the acute phase response and finally targeted killing of the pathogen which includes elements of the adaptive immune response. The acute phase response is characterised by the production of major plasma proteins to defend against the pathogen (Bayne et al., 2001). Such proteins have been studied in detail in salmonids and are known to include complement factors, serum amyloid A, transferrin, lectins, α microglobulin, haptaglobin and anti proteases (reviewed by Bayne and Gerwick, 2001). Lectins and their receptors (Soanes et al., 2004) are also produced in large quantities during the early infection to identify bacterial surfaces and aid neutralization of pathogens and foreign bodies in the host (Holmskov et al., 2003). In mammals, this response is stimulated by factors such as LPS, which triggers the production of proinflammatory cytokines including IL-1, IL-6 and TNFα that in turn are responsible for production of acute phase proteins (Mizel, 1989), similar mechanisms are thought to occur in fish. In parallel to the increase in acute phase proteins, there is a decrease in production of other “normal” plasma proteins such as apolipoproteins (Hoffman and Benditt, 1982).
Specific to the bacterial responses is the production of antibacterial peptides. These proteins are synthesised as preproproteins that are cleaved to produce a mature peptide that can bind to and kill invading bacterial pathogens (Boman, 2003). These proteins have been found in all animal and plant phyla to date and about 800 have so far been discovered (Bulet et al., 2004). There is a growing number of antibacterial peptides being characterised in fish, that include hepcidin (Douglas et al., 2003), cathelicidin (Chang et al., 2005) oncorhyncin (Fernandes et al., 2004), and liver expressed antimicrobial peptide 2 (Zhang et al., 2004) amongst others. Further antimicrobial peptides are reviewed by Patrzykat and Douglas (2003). Other enzymes that destroy invading bacteria include lysozymes (Mitra et al., 2003) and enzymes with lytic properties such as cathepsins (Tahtinen et al., 2002).
In recent years, the genomic information for Atlantic salmon has increased dramatically, with >110,000 EST sequences now publicly available (NCBI, 2005). However, there are still many differentially expressed genes not within the data bank. In this paper, we report on the construction and bioinformatics analysis of enriched cDNA libraries from liver, head kidney and gill tissues following exposure to a genetically attenuated strain of Aeromonas salmonicida (Marsden et al., 1996). In addition, the same RNA has been used to probe an Atlantic salmon cDNA micro array (Rise et al., 2004a, Rise et al., 2004b) to allow the expression in parallel of large numbers of mRNAs to be monitored. The array analysis served two purposes, firstly to confirm, using a limited number of genes cloned from the enriched libraries, that they are indeed up-regulated in response to bacterial exposure and secondly to observe expression of other genes not identified in our suppressive subtraction hybridization (SSH) study, as only a fraction of the libraries were sequenced. Lastly, a selected number of genes were chosen to perform temporal mRNA expression analysis following bacterial exposure, by reverse transcription-PCR. The strain of A. salmonicida used in this study is rapidly cleared from the host between 5 and 9 days following injection (Marsden et al., 1996) and elicits a protective immune response. Therefore, this study aims to identify molecules induced during the early stages of a bacterial infection of potential relevance to protective responses.
Section snippets
Bacterial exposure
Atlantic salmon (mean weight 147.4 g ± 5.0 S.E.M.) were maintained in freshwater 250 l tanks at the University of Aberdeen, UK, fish facilities. The water temperature was maintained at 12 °C and the fish were fed 1% body weight per day of a commercial pelleted diet (Nutreco). On the day of exposure fish were anesthetised with benzocaine (Sigma 20 mg l−1) and injected intraperitoneally with 100 μl of a genetically attenuated strain (aroA−) of A. salmonicida (Brivax II, Marsden et al., 1996) (109 CFU ml−1)
Library construction
During all challenges no fish died or showed gross pathology. Swabs from the peritoneum showed that the bacteria were present at high numbers in the fish injected with the bacteria. Three subtracted cDNA libraries were constructed from head kidney, gill and liver RNA from fish that had been exposed to the aroA− A. salmonicida bacteria. The libraries were constructed from pooled RNA from 20 bacterially exposed fish; 10 from 24 h and 10 from 48 h after injection. Following sequence analysis only
Discussion
Functional genomic analysis of the host response to fish pathogens is advancing at an increasing rate since large collections of salmon ESTs (Rise et al., 2004a) are now available in public access databases. However, the majority of the sequences are unannotated and these database sequences are often generated from fish that have not been subjected to infections. As a consequence, relatively few genes that encode immune responsive proteins and defence proteins are present in these gene
Acknowledgements
This work was supported by BBSRC Grant EGA17675 (Salmon TRAITS). We thank Ben Koop and Willie Davidson, GRASP (Genomics Research in Atlantic Salmon Project), Canada, for providing the microarays. We thank Gordon Richie (Nutreco Aquaculture Research Centre) for providing the salmon.
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