Minimising fish use in immune studies

Dr Bertrand Collet, from Marine Scotland Science in Aberdeen, received an NC3Rs project grant in 2011 to develop a new refined sampling method in fish used for vaccine and immunology studies.  

Research details

Principal Investigator: Dr Bertrand Collet, Senior Research Scientist
Organisation: Marine Scotland Science
Award: £435,700, in 2011, over 24 months
Title: Development of a non-lethal sampling method to monitor immune response and disease progression in salmonid fish

Read more about Dr Collet's research

Case study

Growth of the salmon farming industry is driving an increase in fish disease research

In recent years the use of fish in scientific procedures has increased substantially. This is partly due to the increased investment in research to understand the fish immune system and to develop vaccines to support the fish farming industry. Each year in the UK, approximately 300,000 fish are used for fundamental or applied research, 10% of which are used in vaccine and immunology studies.

Sampling methods currently use large groups of fish and are limited scientifically

Evaluating pathogen virulence and testing the efficacy of vaccines is traditionally carried out in infection challenge experiments. These involve infection of groups of fish with the pathogen of interest with cohorts of fish killed at regular time-points for tissue collection in order to measure pathogen load and/or immune parameters as the infection progresses.

There is significant inter-fish variability as the animals are sourced from farms and are genetically diverse. Variability is also inherent in immune studies as the animals never get infected at the same time and this means that the expression of cytokines, key regulators of the immune response, is never synchronised. In order to minimise statistical ‘noise’ arising from the inter-fish variability large numbers of animals are used. The studies provide basic information on gene expression but cannot be used to address specific questions about whether vaccines are able to increase or decrease cytokine levels for example, which are essential for designing control measures against fish diseases.

A new sampling protocol to reduce fish use by 86%

In 2011 Dr Bertrand Collet at Marine Scotland Science was awarded NC3Rs funding to develop a method which would enable the immune response to various pathogens to be studied in individual fish longitudinally.

Studies were carried out using Atlantic salmon (Salmo salar) or Rainbow trout (Oncorhynchus mykiss) and a variety of pathogens causing or threatening significant economic loses to the UK fish farming industry, including salmon anaemia virus, salmon alphavirus and the bacterium Yersinia ruckeri. The fish were electronically tagged for identification purposes and pathogen load and immune markers were measured using small volumes of blood (around 100 microlitres) taken under general anaesthesia from the same individuals at various time-points throughout the infection cycle.

Using the traditional approach of pooling tissue from a group of fish requires 84 animals for a study looking at seven time-points. The equivalent study using the new sampling method uses 12 fish, representing an 86% reduction.

Repeat blood sampling does not affect fish welfare

To minimise the potential stress of repeated sampling, Dr Collet has developed an ‘in-tank’ general anaesthesia procedure which avoids having to ‘net’ the fish. Observation of fish behaviour after anaesthesia and sampling has shown no adverse effects. In a typical infection study, blood samples are taken every four days for up to 28 days. Analysis of the haematocrit, the volume percentage of erythrocytes in the blood and a physiological marker of stress, also shows no effect from repeated sampling, with animals being able to fully cover between time-points.

Innovation in bioanalytical tools for studying the fish immune response

Unlike in mammals, there is a dearth of reagents against fish immune molecules. A key challenge of using the blood sampling approach was the requirement for new analytical techniques which enable the use of small volumes. To address this, Dr Collet has developed for the first time a cell-based functional assay for cytokine analysis in fish. The assay uses a trout cell line expressing the luciferase gene under the control of the interferon-induced MX gene. Other assays are under development, which are likely to have even greater sensitivity. These include transgenic fish cell lines expressing fluorescent molecules, under the control of various interferon regulator factor genes, which translocate into nucleus upon activation.

Adoption of the new protocol by other research groups

The new method using significantly fewer fish has been adopted by Dr Collet’s collaborators at the Universities of Aberdeen and Stirling and is also being modified for the study of fish farming. The work has also been disseminated at the international workshops of the European Veterinary Immunology Group and the Danish Fish Immunology Research Network. They will also be used in the European Commission funded project TARGETFISH, which aims to develop targeted disease prophylactics for use in European fish farming.

Further NC3Rs funding to develop fish cell lines

Infection and immunology experiments are currently dependent on the use of live fish. There are few well-defined fish cell lines available and most of these are not susceptible to the virus strains responsible for fish diseases. In 2013, Dr Collet was awarded a pilot study grant to develop cell lines from the Atlantic salmon gill, heart, kidney and spleen.

This case study was published in a review of our research portfolio in November 2013.