On October 6^th, 2006 a group of experts on fish disease, diagnosis and treatment assembled in London to share recent advances in the field. Fish disease significantly impacts environmental balance and commercial productivity on a global basis. Losses have high economic consequences, especially in areas where fishing or aquatic culture are important community-supporting industries.

Dr. David Graham from the Fish Disease Unit of the Agri-food and Biosciences Institute in Belfast acted as chair of the proceedings and opened the day with a talk entitled: ‘Detection of virus-neutralizing antibodies and viremia: Complementary tools for diagnosis of salmonid alphavirus (SAV) infections. Across Europe, salmonid alphaviruses are a major problem where they infect farmed Atlantic salmon and rainbow trout. Otherwise, know as pancreas disease and sleeping disease, SAV exists in subtypes 1,2 and 3 and has been the most important pathogen affecting marine farming in Ireland, with recent outbreaks in Norway and Scotland. Current diagnostic methods rely on time-consuming pathology examination. Dr. Graham’s work focuses on the utilisation of serological methods to detect SAV. This assay capitalizes on the humoral immune response of the fish (IgM) via a virus neutralization test. The assay involves mixture of virus with a serum sample of the fish, which would contain antibodies if the fish was exposed to the pathogen. When the mixture is cultured with cells and incubated at room temperature, if antibodies are present in the serum, the virus would not grow and the cells will progress in a normal, healthy manner. If there are no antibodies present in the serum (no infection or exposure), the virus would then not be neutralized and would infect the cells killing them which is detected as a cytopathic effect (rounding of cells, cell death). This assay can then be followed up by immunostaining of the cultures. Utilization of this assay also identifies current infection. Approximately 3 weeks post infection, Abs can be detected and the virus is not detectable, thus seroconversion has occurred and the fish is no longer in the viremic stage. This assay has multiple advantages; it can be performed quickly and is very easy to read. In addition, it can be performed in a high throughput manner in microtiter plates thus requiring only small volumes.

The second talk of the day, entitled ‘Design of DNA vaccines for fish’ was presented by Dr Felicity D’Mello of the Royal Veterinary College in London. Because of the rapid growth currently experienced by the aquaculture industry, DNA vaccination is being evaluated as a means to protect fish. Currently there are several vaccines available for bacterial pathogens, a few for viral and none for parasitic pathogens. Viral vaccines can exist as inactivated or recombinant forms which must be injected in high doses or as live virus, which is an environmental concern. Dr D’Mello’s laboratory performed a study using DNA constructs containing the spring viremia of carp virus (SVCV) glycoprotein gene. These contracts were injected into myofibres of goldfish resulting in an antibody response in more than 50% of the fish. A large scale trial in carp was then initiated, involving a single immunization with a plasmid containing different glycoprotein gene constructs under control of a range of promoters, followed by SVCV challenge 6 weeks later. Protection was conferred via a cell mediated immune response and the strongest protection resulted from DNA vaccines containing the full length glycoprotein gene driven by the CMV-Intron A promoter.

Probiotics were the focus of the talk by Professor Brian Austin, Dean of the University and Professor of microbiology at Heriot-Watt University in Edinburgh. Probiotics, micro-organisms or their products which have health benefits to the host organism, have increased in use recently in aquaculture. These benefits are thought to result from improved nutrition, stimulation of immune response, alteration of microbial metabolism and competitive exclusion of detrimental pathogens. Use of probiotics in aquaculture (ideally 10⁶ – 10⁸ bacteria/gram food) can potentially increase the need for antimicrobial compounds and thus there is a significant market opportunity.

Dr David Hoole, of the University of Keele discussed the the utlisation of serum pentraxins as a diagnostic method to monitor the health status of fish. This evolutionarily conserved group of serum proteins includes the C-reactive protein (CRP) and serum amyloid protein (SAP), which have been demonstrated in various species to act in an acute phase immune response. Carp and other cyprinids constitute 40% of world aquaculture, thus understanding cyprinid disease and disease response is very important. Dr Hoole’s laboratory analysed CRP in carp to determine whether or not the protein is an acute phase reactant during disease by isolating the protein, raising polyclonal antibodies against CRP, and then developing an Elisa assay. With collaborators at the Institute of Ichthyobiology and aquaculture in Golysz, Poland, they exposed cultured carp to insults such as bacterial pathogens and then showed that CRP levels increased. Thus, the assay could be used as a very rapid diagnostic to screen and monitor the health of cultured fish populations.

Environmental and climate changes potentially have drastic effects on the health status of many species. Professor Beth Okamura of the University of Reading, looked at proliferative kidney disease (PKD) in salmonid fish, which potentially can result in severe economic loss to the cultured trout and salmon industry. PKD is increasing in prevalence which could be a result of climate change; the number of fish have decreased as water temperature increases. The laboratory of Dr Okamura investigated the life cycle of the myxozoan endoparasite, Tetracapsuloides bryosalmonae that lives part of its lifecycle in the freshwater bryozoans, which are benthic invertebrates. The endoparasite exploits the clonal bryozoan lifecycle by persisting through cycles of degeneration and regeneration. Furthermore, the parasite develops spores within the bryozoan which are released and can infect fish in large numbers. Their studies demonstrated a complex host-parasite interaction: The bryozoans increased in abundance with temperature increase in addition to their level of covert infections by the parasite. Residual covert infections were maintained in the bryozoan host population as spores. Thus, as global climate increases or thermal inputs to freshwater continue via power plants, the aquaculture industry is at increased risk to PKD infection. A greater awareness of PKD infection and strategies to combat the disease are desperately needed.

Dr Ian Bricknell from the Marine Laboratory in Aberdeen addressed the use of immunostimulants, for health management and disease control in aquaculture. Immunostimulants may be synthetic or biological and include compounds such as levamisol, poly I:C, LPS, and glucan. Benefits of immunostimulant use in vivo include an increased survival rate following bacterial challenge, antiparasitic effects, viral resistance, growth enhancement, increased antibody production, a rise in serum lysozyme levels and degranulation of granulocytes. Immunostimulants activate the monocyte and macrophage system via the highly-conserved Toll-like receptors (TLR) inducing an immune response and eventually generating immunotolerance. Therefore immunostimulants are often administered in a pulse-dose manner during farmed fish development. Dr Bricknell’s laboratory treated halibut larvae with glucan and then challenged the immune system and demonstrated a greater survival rate in fish that were administered glucan before the challenge versus those that did not receive glucan. Clear benefits of immunostimulants have been demonstrated in adult fish, however, more studies are required using larval fish. Additionally, the optimum treatment differs among species and tolerance remains an issue.

Fish disease can result from the introduction of new fish species into an existing fish population. Dr Rodolphe Gozlan, of the CEH Dorset, Natural Environmental Research Council and Winfrith Technology Centre studied the invasion of the Topmouth gudgeon (Pseudorasbora parva) a cyprinid, into Europe from its native habitat of Asia. This introduction has impacted the sunbleak (Leucaspius delineatus), a cyprinid species endigenous to Europe which is now endangered. In a very demonstrative experiment, Dr Gozlans lab emptied a pond which contained 1200 L. delineatus, and introduced 600 individuals of each species resulting in a very significant decrease in the population of the native L. delineatus species. To determine the interaction, the next experiment was conducted in the laboratory where 3 tanks were set up with a common water recirculating system. The top tank contained the Asian species (Pseudorasbora parva) and the bottom two, the endigenous L. delineatus. A simultaneous control set-up contained only L. delineatus. The result was 16% mortality of L. delineatus and 1,596 eggs in the control tank versus 69% mortality and 0 eggs in the experimental tank. To determine the agent, PCR was employed resulting in the detection of a new species of rosette agent, Sphaerothecum destruens, an intracellular parasite. Many species of salmonid are suspetible to the rosette agent, which have been detected in Europe, the UK and the Western United States. Control measures are required to limit invasion of non-native species, especially those carrying the rosette agent, in order to protect biodiversity and aquaculture.

The final presentation of the day, by Professor Sandra Adams of the University of Stirling, addressed new technologies in fish immunodiagnostics. Rapid detection and characterization of pathogens can be accomplished morphologically, molecularly or immunologically, the latter being the focus of this session. Antibody based methods include agglutination, fluorescent AB tests, immunohistochemistry, ELISA, blots and lateral flow technology. Regardless of the method employed, standardisation is key: It must be from a reliable source, specific and performed via an SOP. They have developed a new immunochromatographic method utilising lateral flow technology called the RAPID test. This user-friendly, sensitive and specific assay is fairly cheap and amenable to long-term storage. The RAPID test was found to demonstrate equivalent results to PCR, a molecular method generally considered the most sensitive. Other commercial products such as the Luminex system for multiplexing – detection of multiple pathogens simultaneously or microarray technology are becoming more popular for aquaculture diagnostics. With all the new technologies available, the field of fish diagnostics will progress rapidly in the coming years.