With a rapidly growing human population, it became crucial to produce as much additional food as possible. Keeping in mind that in many countries, seafood protein represents an essential nutritional component, massive fish consumption has led to an increased aquaculture production1. Scientific data shows that in 2018 almost 156 million of global fish production ended up in our plates2.

As the scale of the aquacultural output rises, it becomes impossible to provide enough fish only from capture fisheries. Due to this, a fully controlled farming environment has recently superseded fisheries and became the main source of seafood for human consumption1. One such system is the Recirculating Aquaculture System (RAS), also known as a water reuse system. RAS is often being described as the solution for land space-saving and it could be used where freshwater sources are limited3. However, due to continuous reuse of water, bacterial, viral and fungal infections in RAS can all become concentrated 4.

One of the broadest host and geographic ranges of any fish bacterial pathogens constitutes Flavobacterium columnare, Flavobacterium psychrophilum and Flavobacterium branchiophilum bacteria, which accordingly causes columnaris, bacterial cold water disease or rainbow trout fry syndrome and bacterial gill disease5.

Typically, these bacteria can be induced and recognised in healthy fish of various ages. In this way, fish themselves are the most important reservoirs and can act as asymptomatic carriers of the pathogen until the predisposing factors such as overcrowding, reduced dissolved oxygen, temperature changes or increased ammonia amount in water enhances the stress level of fish6. After physical changes in fish appearance and behaviour are being seen, it only takes 24-72 hours until mortality rates reach upwards of 70% of the infected fish population and cause hundreds of thousands of devastating financial losses for aquaculture farms 5.

After research on flavobacterial diseases in a global perspective, it became interesting to see the situation of these infections in Lithuania. During phone calls with aquaculture farmers, we found a rainbow trout aquaculture farm FishNet which had encountered Flavobacterium spp. directly and lost more than 50 tonnes of fish only in two weeks.

However, these high mortality rates were only the tip of the iceberg. During the conversation with farms, as well as with the National Food and Veterinary Risk Assessment Institute of Lithuania, we were informed that there are no available detection tools for Flavobacterium species identification in our country. So it became clear that there is a huge demand in Lithuania, as well as all over the world, to have point-of-care detection tools that could help aquaculture farms to reduce financial losses as much as possible.

Despite the economic impact, nowadays, an exact flavobacterium species identification is mainly based on a qPCR technique. However, even though this method is sensitive and quite rapid, it requires special laboratory equipment and protocols, as well as there is a need to obtain samples from alive fish7.

Based on this knowledge, our project’s first goal was to develop a flavobacterium species detection kit, which would be not only cost-effective, robust and fully portable, but also it can be used by farmers with no scientific knowledge. To reach this aim, in this test, we combined isothermal helicase-dependent amplification (HDA) with a lateral flow assay (LFA) methods.

But what happens when we detect an exact pathogenic bacteria? In order to start an effective treatment process as soon as possible, gallons of different types of antibiotics are being used8. Scientific data 9 shows that the most abundant antibiotic used for salmon cultivation is quinolone, which consumption (by mass) in 2007 reached 821.997 tons. Other commonly used antibiotics in farms are oxolinic acid and florfenicol, which consumption reached 681 kg in 2008 and 166 kg in 2010 respectively. This enormous usage of a wide variety of antibiotics forces the evolution of antibiotic-resistant bacterial fish pathogens.

Keeping in mind that some F. psychrophilum isolates already have a susceptibility to quinolones, oxolinic acid and enrofloxacin10, our project’s second goal became the development of a new exogenous fish infection treatment strategy, which will help to reduce antibiotics consumption levels in the future. The main target in our treatment system became biofilms, which is being formed on fish fins or gills. These structures use autoinducer-2 (AI2) signalling molecules for cell-cell communication10-12.

Based on this action, known as quorum sensing, we decided to build two genetic circuits under AI-2 inducible promoters. Both of these synthetic biological systems are based on exolysin synthesis. The main differences of these systems are its lysis mechanisms that result in the genetically modified bacteria to kill itself by using a kill-switch system.

However, even if rapid and accurate pathogen detection and fish treatment strategies are crucial in disease control, they do not guarantee that the infection will not reoccur. The most effective and promising solution which could help to prevent these diseases is vaccination. On the other hand, methods of immunisation used nowadays require physical intervention which causes even more stress to the animals and weakens their immune system13.

Due to this reason, our third goal of the project became the development of a prevention system based on orally administered subunit vaccines based on immunogenic proteins which are immobilized into calcium alginate beads. In the beginning, the main aim was to create a subunit vaccine against columnariosis disease by using immunogenic bacterial outer membrane protein GldJ14.

Nonetheless, after integrated meetings with specialists from governmental institutions and companies, we got informed that viral infections in fish farms are sometimes even more dangerous. Because of that, we added VHSV Glycoprotein G which induces an immune response against the VHSV virus in fish15. These immunogenic proteins have to be enveloped into calcium alginate because we chose a non-invasive oral delivery route, where proteins travel through the digestive tract until they get absorbed into the bloodstream. The envelope protects proteins from degradation in the stomach and allows the protein to be released only in the midgut where bacteria lyse the alginate16.