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 tonnes 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 Due to this, a fully controlled farming environment has recently superseded fisheries and became the main source of seafood for human consumption1.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 a land space saving solution 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 cause 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 seen, it only takes 24-72 hours until mortality rates reach upwards of 70% of the infected fish population and cause grave 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 samples obtained from live 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 could also be used by farmers with no scientific knowledge. To reach this aim, in this test, we combined isothermal helicase-dependent amplification (HDA) and 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, whose consumption (by mass) in 2007 reached 821.997 tons. Other commonly used antibiotics in farms are oxolinic acid and florfenicol, whose 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 antibiotic consumption levels in the future. The main target of our treatment system became biofilms, which is being formed on fish fins or gills. These structures use autoinducer-2 (AI-2) 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 killing itself by using a kill-switch system.
However, even if rapid and accurate pathogen detection and fish treatment strategies are effective 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, in particular immunogenic proteins, which are immobilized in 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 were 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.
Despite the many goals of FlavoFlow, the whole year was shrouded in uncertainty due to the pandemic. At first, we had a lot of plans for the implementation of this project in the fish farm. Unfortunately, we were not able to fulfil our goals. Luckily, COVID-19 pandemic opened up new waters to modern and engaging platforms in the human practices area. Before the quarantine set, we had a lot of plans for activities that were traditional and required contact. However, new circumstances forced us to think outside the box. This is how the best of our human practices projects, that joined mutual learning spaces and interactivity, were born! The 6th SynBio Sense, educational colouring book, "BioBlox" and online lessons - all of these activities were designed to be accessible from anywhere and at any time. This way we were able to reach a lot of people and spread knowledge about science and synthetic biology without the need to leave the house, which keeps us and the others safe!
- Guillen, J. et al. Global seafood consumption footprint. Ambio 48, 111–122 (2019).
- The State of World Fisheries and Aquaculture 2020. The State of World Fisheries and Aquaculture 2020 (FAO, 2020). doi:10.4060/ca9229en.
- Badiola, M., Mendiola, D. & Bostock, J. Recirculating Aquaculture Systems (RAS) analysis: Main issues on management and future challenges. Aquacultural Engineering 51, 26–35 (2012).
- Yanong, R. P. E. Fish Health Management Considerations in Recirculating Aquaculture Systems-Part 2: Pathogens 1. http://edis.ifas.ufl.edu.
- Loch, T. P. & Faisal, M. Emerging flavobacterial infections in fish: A review. Journal of Advanced Research vol. 6 283–300 (2015).
- Starliper, C. E. Bacterial coldwater disease of fishes caused by Flavobacterium psychrophilum. Journal of Advanced Research vol. 2 97–108 (2011).
- Strepparava, N., Wahli, T., Segner, H. & Petrini, O. Detection and quantification of Flavobacterium psychrophilum in water and fish tissue samples by quantitative real time PCR. BMC Microbiology 14, (2014).
- Manage, P. M. Heavy use of antibiotics in aquaculture: Emerging human and animal health problems – A review. Sri Lanka J. Aquat. 23, 13 (2018).
- Burridge, L., Weis, J. S., Cabello, F., Pizarro, J. & Bostick, K. Chemical use in salmon aquaculture: A review of current practices and possible environmental effects. Aquaculture 306, 7–23 (2010).
- Stephens, K. & Bentley, W. E. Synthetic Biology for Manipulating Quorum Sensing in Microbial Consortia. Trends in Microbiology 28, 633–643 (2020).
- Waters, C. M. & Bassler, B. L. QUORUM SENSING: Cell-to-Cell Communication in Bacteria. 31 (2005).
- Ahmer, B. M. M. Cell-to-cell signaling in Escherichia coli and Salmonella enterica: Quorum sensing in E. coli and Salmonella. Molecular Microbiology 52, 933–945 (2004).
- Maurice, S., Nussinovitch, A., Jaffe, N., Shoseyov, O. & Gertler, A. Oral immunization of Carassius auratus with modified recombinant A-layer proteins entrapped in alginate beads. Vaccine 23, 450–459 (2004).
- Nelson, S. S., Bollampalli, S. & McBride, M. J. SprB Is a Cell Surface Component of the Flavobacterium johnsoniae Gliding Motility Machinery. Journal of Bacteriology 190, 2851–2857 (2008).
- Shin, C., Kang, Y., Kim, H.-S., Shin, Y. K. & Ko, K. Immune response of heterologous recombinant antigenic protein of viral hemorrhagic septicemia virus (VHSV) in mice. Anim Cells Syst (Seoul) 23, 97–105 (2019).
- Xu, F., Wang, P., Zhang, Y.-Z. & Chen, X.-L. Diversity of Three-Dimensional Structures and Catalytic Mechanisms of Alginate Lyases. Appl. Environ. Microbiol. 84 (2018).