World fish consumption is expected to reach c. 180 million tons by 2015, most of which will have to come from farmed fish, as the majority of wild fisheries are either stagnant or grossly over-exploited. To meet future global food demands, aquaculture is expected to intensify production, delivering fish that will have to thrive at high densities on less water, less food and less space. However, stress during intensification can compromise the capacity of organisms to respond to pathogens, making them more susceptible to infectious diseases, though the underlying mechanisms are poorly understood. We will combine the expertise of salmon biologists, geneticists, bioinformaticians and parasitologists to determine how stress experienced during early life affects immune-competence and fitness of Atlantic salmon later in life, enabling them to cope with pathogens and respond to subsequent stressors. To achieve this we will manipulate stress during embryo development combining a brief cold shock and air exposure, and assess the relative roles of immune-related genes and epigenetic programming (DNA methylation) on subsequent resistance to Saprolegnia parasitica, a pervasive fish pathogen that costs the salmon farming industry tens of millions of pounds in losses every year and that can also cause considerable damage to wild salmon populations. Ultimately, the aim of our research is to investigate how knowledge of the effects of early life conditions on the epigenome could be incorporated into programmes for stress management and disease resistance in fish farming, thereby facilitating aquaculture intensification while minimising impacts on wild stocks.

Over the last 12 months anaemia has emerged as a significant threat to fish health in salmon aquaculture in Scotland. Anaemia occurs when there is a lack of red blood cells, reducing the oxygen carrying capacity of the blood and negatively impacting on fish health and production. Through recent SAIC project meetings with the aquaculture partners it was highlighted that the industry has limited practical methods to define the type of anaemia occurring, identify its source and subsequently develop a preventative strategy against it. The Scottish aquaculture industry is looking to establish a haematology monitoring programme, to characterise and quantify the impact of anaemia, but does not currently have access to automated technologies for fish haematology and is reliant on slow, labour intensive and subjective manual techniques. This lack of technological advancement has also resulted in a lack of reference data for the establishment of appropriate 'normal' background levels. The overall aim of this project is the development and validation of efficient methods to assess anaemia in Atlantic salmon in aquaculture that can be adopted by the industry and included into their regular fish health management programmes. This is being undertaken by the validation of existing veterinary and medical haematology devices for assessment of fish blood, which is considerably more difficult owing to fish blood cells being nucleated. However once the relevant techniques are established and validated they shall be used to investigate anaemia in fish and to put those results into the wider context of fish health measured using high throughput, automated medical technologies to assess clinical chemistry and immunology endpoints validated for use with fish in a separate (SAIC funded) project involving the same collaborative partners. The main benefit and impact of this research shall be improvements in fish health management, increasing fish welfare and growth, subsequently benefiting the industry by a reduction in costs, increased yield and improved product quality. The development of an appropriate diagnostic system to assess anaemia in salmon aquaculture requires; a) the development and validation of rapid, automated haematological methods for salmon blood, and b) the integration of this haematology data with relevant endpoints on fish health and disease. A holistic diagnostic approach is taken, placing the haematology findings in the context of fish health investigated by the high throughput assessment of biochemistry and immunology endpoints and histological analysis. This innovative approach provides information on the cause of anaemia, thereby allowing identification of solutions. Innovation is needed in the area of blood sample preparation and storage, data interpretation and particularly with the integration of physiological impacts associated with anaemia. There is a current lack of reference values in fish haematology and this work shall contribute towards the establishment of reference data of benefit to the salmon aquaculture industry in Scotland. In the final stage of the project anaemia in Atlantic salmon from various sites around Scotland shall be identified, defined, quantified and its impact measured using haematological, clinical chemistry and immunology high-throughput analysers integrated into a pro-active mechanised technologically advanced method to assess fish heath. These techniques are relevant to the salmon aquaculture industry in the UK and internationally and user friendly practical guides and protocols shall be developed along with an engagement workshop to offer hands on training and practice to encourage the inclusion of these techniques throughout the industry, greatly increasing the impact of this research.

Despite advances in vaccine development and chemotherapy, bacterial pathogens remain a significant constraint to aquaculture production, as well as a potential threat to wild stocks. In addition to so-called 'new' bacterial pathogens, the threat often comes from established pathogens evolving to circumvent developed control strategies. An example of such an evolving pathogen is the Gram-negative bacterium Yersinia ruckeri which causes economically-important infections of Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss). Y. ruckeri has been identified as the aetiological agent of enteric redmouth disease (ERM) of farmed rainbow trout in England and Wales for more than thirty years. The organism also causes significant outbreaks of disease in farmed Atlantic salmon but very little is known about the epidemiology of Y. ruckeri in this species. Until recently, Y. ruckeri posed a reduced risk to farmed trout and salmon due largely to the extensive use of vaccination. However, increasing worldwide reports of vaccine breakdown in the field are believed to be associated with the emergence of new pathogenic strains of Y. ruckeri. Traditionally, the majority of ERM infections in rainbow trout in the USA and Europe have been caused by a subgroup (biotype) of closely-related serotype O1 Y. ruckeri strains represented by the so-called Hagerman strain. However, a new and distinct serotype O1 biotype has emerged in recent years that was first identified in the UK but which is now increasingly affecting rainbow trout production across Europe and the USA. Isolates recovered from infected Atlantic salmon appear to be more diverse and represent a wider range of O-serotypes (O1, O2 and O5). Preliminary evidence suggests that salmon-pathogenic serotypes are significantly less pathogenic to rainbow trout than they are to Atlantic salmon. The outer membrane (OM) of Gram-negative bacteria is at the interface between pathogen and host and outer membrane proteins (OMPs) play key roles in host-pathogen interactions such as adherence and colonization, nutrient uptake (specifically iron acquisition), evasion of the host immune response and secretion of virulence factors. Because many of these OMPs are under strong selection pressures (e.g. from the host immune system) they can exhibit varying degrees of diversity within a single bacterial species. This diversity is likely to confer upon strains different virulence attributes and host tropisms. In addition, many OMPs are surface-exposed and immunogenic and are therefore likely to serve as protective antigens. Despite the importance of Y. ruckeri as a pathogen of farmed salmonids, very little is known about the diversity of its OMPs in different strains or their roles in the disease process. Furthermore, very little is known about the host immune response to Y. ruckeri in salmon and trout or about the bacterial factors responsible for providing protection against infection. The aims of this project are as follows: 1. To carry out an epidemiological survey of the prevalence of Y. ruckeri in Scottish salmon and assess the effectiveness of transmission of the organism in seawater. 2. To characterize the OM proteomes of key selected isolates of Y. ruckeri recovered from salmon and trout under different growth conditions and identify cross-protective antigens (and potential vaccine candidates) by immunoproteomics. 3. To compare the virulence of selected isolates for Atlantic salmon and trout. 4. To compare the kinetics of the immune response to selected isolates of Y. ruckeri in Atlantic salmon and trout. 5. To study serotype specificity of immunity and the protection afforded by vaccines prepared from different isolates of Y. ruckeri in Atlantic salmon and trout.

Fish diseases are a huge threat for the aquaculture industry and for global food security. Some of the most important disease-causing organisms in aquaculture are part of the oomycetes or watermoulds, in particular Saprolegnia parasitica, Saprolegnia diclina and Saprolegnia australis are causing serious fish losses. Collectively, these fungal-like organisms are responsible for at least 10% annual mortalities in most salmon hatcheries and freshwater sites. Consequently, Saprolegnia ranks among the most important pathogens of Atlantic salmon. Unfortunately, over the last few years the incidences of saprolegniosis outbreaks in Scottish farms have significantly increased. Indeed, some sites have had very high losses due to saprolegniosis. Whereas other farms have remained largely disease free. The reasons as to why some farms are badly affected and others seem to avoid disease outbreaks, with apparent identical welfare standards and husbandry management practises, are at present completely unclear and form the main rational for the current application. Our hypothesis is that several risk factors (pertaining to fish, pathogens and the environment) are playing a synergistic role in suppressing immunity in fish towards Saprolegnia, which lead to outbreaks of saprolegniosis. Therefore, we propose a concerted industry-wide, industry-led and industry-supported research programme to discover, map, model and understand the main drivers, risk factors, that allow saprolegniosis outbreaks. A "big data" resource will be created that will be scrutinised with statistical methods to identify the main risk factors and conditions for outbreaks of saprolegniosis. Undoubtedly, identifying the main, or a combination of, risk factors will greatly aid the salmon aquaculture industry to pre-empt any future outbreaks and would lead to an integrated approach to saprolegniosis management, which would result in increased welfare standards, improved fish health, fewer losses and a reduction in production and treatment costs.