Development of immunological tools for monitoring the immune response of Nile tilapia

Project summary

Aquaculture is the fastest growing animal food production sector globally. Fish are an import source of protein and as such aquaculture has great potential to play a key role in future food security programmes. Because of their rapid growth and high protein content, tilapia is an attractive species for aquaculture, reaching harvest size after only 6-7 months; tilapia are now in fact the second most farmed species after carp. They are farmed in many low and middle-income countries (LMIC) and provide an important source of revenue for many low income families. Disease in tilapia culture is associated with intensification of the farming system, and both bacterial and viral diseases are severely impacting on the expansion of tilapia farming; in particular Streptococcus spp. There is increasing concern about the use of antibiotics to control disease outbreaks and attention is focusing on the use of vaccination for disease control. Vaccination exposes fish to a non-infectious dose of the pathogen, so when they come into contact with the pathogen at a later date, memory cells of their immune system stimulate a response to combat the disease. We need a better understanding of how tilapia respond to infection and vaccination to be able to develop and formulate effective vaccine products for tilapia. We currently have few reagents available for investigating the immune response of tilapia. Through a collaboration of scientists in Vietnam, Canada and the UK, we plan to develop and apply novel tools (synthetic antibodies) for studying the immune response of this important aquaculture species, using Streptococcus agalactiae as our infection model. Synthetic antibodies are made in the laboratory, unlike conventional antibodies which are produce in animals, thus eliminating the need to use animals to make these reagents. This work will ultimately lead to the development of more effective strategies for managing disease in tilapia aquaculture systems.

Workshop

In October 2018, Dr Nguyen Ngoc Phuoc and Dr Kim Thompson hosted a workshop on the use of vaccines in tilapia aquaculture at Hue University of Agriculture and Forestry in Vietnam. You can read about their workshop in this blog post.

Project outcomes

With the world population projected to increase to over 8.5 billion by 2030, aquaculture has great potential to meet the world’s increasing demands for protein. It is the fastest growing animal food production sector globally and now accounts for over 50 percent of all fish consumed. Tilapia is a very attractive species for aquaculture because it can reach harvest size in just 6 months. It is the second most predominant fish species farmed globally after carp. They are farmed in many low and middle-income countries (LMIC) and provide an important source of revenue for low income families. Disease is associated with intensification of aquaculture systems, and both bacterial and viral diseases are severely impacting on the expansion of tilapia farming in LMICs. There is increasing concern about the use of antibiotics to control disease outbreaks and attention is focusing on the use of vaccination for disease control. We need a better understanding of how the cells of the immune response of tilapia respond to infection and vaccination to be able to develop and formulate effective vaccine products for this species. Few reagents are available to allow us do this, however. This project was a collaboration between scientists from Vietnam, Canada and the UK with the aim of developing a suite of synthetic antibodies (sAbs) for key immunological markers to study the response of different immune cell populations in tilapia. Synthetic antibodies are made in the laboratory unlike conventional antibodies, which are produced in animals, thus eliminating the need to use animals to make these reagents. The cell marker targets used for antibody production in this project included CD3Ɛ, CD4, CD8, CD172 (also known as SIRPα), CD45 and CD163. The DNA sequence of each target was successfully identified in the tilapia genome and Fc-fusion proteins produced for each target in HEK293 mammalian cells. As larger quantities of SIRPα, CD45 and CD3ε Fc-fusion proteins were produced compared to the other three targets, Fab-phage binding selection was initially performed for these markers, screening with an antibody phage-display library. Two unique binding phages were identified for tSIRPα, two for CD3 and 19 for CD45. So far five of the 19 phages for CD45 have been cloned into a human IgG vector and transfected into HEK293 mammalian cells to produce anti-CD45 sAbs. The other unique binding phages for SIRPα, CD3ε and CD45 remain to be cloned, and unique binding phages identified for CD4, CD8 and CD163. Three of the five anti-CD45 IgGs work in flow cytometry and indirect immunofluorescence (IIF), but not immunohistochemistry (IHC). These were subsequently used to analyse tilapia leukocyte populations sampled from a vaccination and challenge experiment performed by Vietnamese partners against Streptococcus agalactiae serotype III, sequence type 283, which gave a relative percent survival value of 62.5% in vaccinated fish. Interestingly, greater amounts of leukocytes were detected in the spleen of non-vaccinated fish compared to vaccinated fish with the CD45 sAbs by flow cytometry. Although further development and optimisation of the sAbs is continuing, it is clear that such tools are important for monitoring the development of adaptive immunity and establishing correlates of protection for the vaccine.