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Molecular Interaction Between Fish Pathogens and Host Aquatic Animals

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Molecular Interaction Between Fish Pathogens and Host Aquatic Animals K. Tsukamoto, T. Kawamura, T. Takeuchi, T. D. Beard, Jr. and M. J. Kaiser, eds. Fisheries for Global Welfare and Environment, 5th World Fisheries Congress 2008, pp. 277–288. © by TERRAPUB 2008. Molecular Interaction between Fish Pathogens and Host Aquatic Animals Laura L. Brown* and Stewart C. Johnson National Research Council of Canada Institute for Marine Biosciences 1411 Oxford Street Halifax, NS, B3H 3Z1, Canada Present address: Fisheries and Oceans Canada Pacific Biological Station 3190 Hammond Bay Road Nanaimo, NS, V9T 6N7, Canada *E-mail: [email protected] We have studied the host-pathogen interactions between Atlantic salmon (Salmo salar L.) and Aeromonas salmonicida. Sequencing the genome of the bacterium allowed us to investigate virulence factors and other gene products with potential as vaccines. Using knock-out mutants of A. salmonicida, we identified key virulence factors. Proteomics studies of bacterial cells grown in a variety of media as well as in an in vivo implant system revealed differential protein production and have shed new light on bacterial proteins such as superoxide dismutase, pili and flagellar proteins, type three secretion systems, and their roles in A. salmonicida pathogenicity. We constructed a whole ge- nome DNA microarray to use in comparative genomic hybridizations (M-CGH) and bacterial gene expression studies. Carbohydrate analysis has shown the variation in LPS between strains and reveals the importance of LPS in viru- lence. Salmon were challenged with A. salmonicida and tissues were taken to construct suppressive subtractive hybridization libraries to investigate differ- ential host gene expression. We constructed an Atlantic salmon cDNA microarray to investigate the host response to A. salmonicida. Real-Time qPCR and NMR-based metabolomics have revealed important information about host responses to infection and to chronic stress. By linking genome sequencing, functional genomics, proteomics, carbohydrate analysis, metabolomics, and whole animal assays, we took integrated and innovative approach to pathogenesis research. KEYWORDS host-pathogen interactions; genomics; proteomics; furunculo- sis; DNA microarrays; knock-out mutants 278 L. L. BROWN AND S. C. JOHNSON it is notable that several problems are asso- 1. Introduction ciated with commercially available vaccines for fish. Oil-based and formalin-based The development of genomics, proteomics vaccines have been associated with adhe- and metabolomics, have enabled the transi- sions and other pathologies including weight tion from sequential studies of single genes, loss (Midtlyng and Lillehaug 1998), second- proteins or metabolites to the simultaneous ary infections (Bruno and Brown 1999), in- study of many components and their inter- jury and inflammation (Mutoloki et al. actions at a variety of biological levels (from 2004). Therefore, research into vaccines and pathways to cells, tissues and whole organ- vaccine delivery systems is ongoing. isms). The development of these fields has Similarly, effective husbandry tools and been supported by the concurrent develop- practices have been used since the inception ment of new technologies and methods such of aquaculture. Fallowing farm sites, sepa- as quantitative PCR or Real Time PCR, RNA ration of year classes, culling of infected interference (RNAi) assays, and in situ hy- broodstock, and reduction of anthropogenic bridization. These fields and their support- stressors are all examples of successful hus- ing technologies and methods are now bandry techniques. However, these are widely used in the study of disease of higher largely based on trial and error and on em- vertebrates as well as in the development of pirical observations. new therapeutants and vaccines. The field In order to devise effective aquatic ani- of aquatic animal health has only recently mal health management tools in a timely and begun to benefit from their application. cost-effective fashion, it is essential that we It is well known that farmed aquatic ani- identify meaningful biological targets, by mals are susceptible to a wide variety of in- knowing which antigens, host immune fac- fectious diseases. Direct and indirect costs tors, and stress response genes to target. In of preventing and controlling infectious dis- order to do that, it is necessary to increase eases in aquaculture are estimated to exceed our understanding of the interactions be- 10% of total production costs. This repre- tween host aquatic animals and pathogens. sents an appreciable loss of profits to the Genomics, proteomics, and other biotechno- aquaculture industry. Coupled with this is an logical approaches help us to do that. increasing global concern about the use of Aeromonas salmonicida is a non-motile, therapeutants (antibiotics and chemical treat- Gram-negative bacterium that is the ments) in the production of food animals and etiological agent of furunculosis. Furuncu- issues related to animal welfare. Develop- losis has been recognized as a widespread ment of more effective vaccines for aqua- important disease in wild and farmed fish culture is necessary to reduce losses due to since the 1890s (Emmerich and Weibel 1894). disease, to enable further reductions in the Due to its early recognition as an impor- use of therapeutants, and to improve animal tant pathogen it is arguably the best studied welfare. bacterial pathogen of fish. Several excellent Vaccines against infectious diseases in reviews of earlier research on A. salmonicida fish have been effective and available for and furunculosis are available (Bernoth years, For example, multi-valent vaccines 1997; Wiklund and Dalsgaard 1998; Hiney based on whole bacterins in formalin- or oil- and Olivier 1999). based preparations have provided protection In this chapter we will demonstrate how to salmon against Aeromonas salmonicida, a multi-disciplinary approach, combining Listonella anguillarum. Vaccines are also genomics, proteomics and metabolomics available against viral pathogens. However, with more traditional methods was used Molecular interaction between fish pathogens and host aquatic animals 279 within the National Research Council in infectious disease research. Genomic Genomics and Health Initiative Program sequencing has facilitated the development “Genomics Approaches to Aquatic Animal of new methods of pathogen detection, our Health Management,” to understand the bi- understanding of genetic relationships be- ology of the bacterial pathogen Aeromonas tween species and investigations into the salmonicida and its interactions with one of nature of host-pathogen interactions. its hosts, the Atlantic salmon (Salmo salar Through the use of bioinformatics methods, Linnaeus). We will also show how this ap- which allow for genomic comparisons be- proach was used to identify candidate pro- tween species, putative virulence factors, as tein and carbohydrate antigens for use in well as genes that encode proteins that may vaccines, in conjunction with whole animal be suitable for use in vaccines can be identi- assays. fied. Genomic sequencing also facilitates the use of other technologies such as the devel- 2. Development of an in-vivo Growth opment of knock-out mutants, and other System for Aeromonas salmonicida genomics (e.g., microarray), and proteomic studies, all of which serve to increase our It is well known that the conditions under understanding of host-pathogen interactions which the bacterium are grown influence and aid in the rational design of effective their gene expression and ultimately their vaccines phenotype (Dalsgaard et al. 1998). This was At the initiation of our research program originally demonstrated for A. salmonicida there was little genomic information avail- grown within chambers in the host by Garduño able for A. salmonicida or closely related et al. (1993). These authors demonstrated species. In order to understand the infection that cells grown under in vivo growth condi- process and possible interactions between tions had increased resistance to bacterioly- A. salmonicida and its hosts, as well as to sis, phagocytosis and oxidative killing. We aid in the rational design of new vaccines developed a method for the in vivo growth the whole genome of wild-type strain A449 of large volumes of A. salmonicida (Dacanay was sequenced and assembled (GenBank Ac- et al. 2003). Briefly this method involves the cession #s NC_009348-009350). The A449 use of growth chambers made of autoclaved genome consists of a circular chromosome 12 to 14-kDa molecular-mass-cutoff dialy- and five plasmids that encode more than sis tubes that are filled with A. salmonicida 4700 genes (Boyd et al. 2003; Reith et al. suspensions and surgically implanted into the unpublished). During this program a full abdominal cavity of Atlantic salmon for a genomic sequence became available for period of 24 hours, then harvested. Bacteria Aeromonas hydrophila (Seshadri et al. grown within these implants have been used 2006). Comparison of the Aeromonas in studies of gene expression, for proteomics, salmonicida genome with genomes of related carbohydrate analysis and for studying the organisms such as A. hydrophila enabled the host response to infection as described be- identification of large numbers of genes en- low. coding potential virulence factors (Boyd et al. 2003; Nash et al. 2006; Reith et al. un- 3. Development of Genomics Resources published). Virulence genes were targeted for and Tools for Aeromonas salmonicida future study and for use as vaccine candi- subsp. salmonicida
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