Antibody ELISAs - an alternative to challenge trials for batch potency testing of fish vaccines

The main objective of the project was to provide the scientific basis for using antibody response after vaccination for quality control of combination vaccines for Atlantic salmon, so that today's most widely used method (challenge testing) can be phased out. The results show that the antibody method was able to reveal experimental vaccine formulations with reduced levels of M. viscosa after 6 weeks, and experimental vaccines with reduced V. salmonicida levels after 9 weeks of immunization at 15oC. Current challenge methods for batch potency testing take 3 months. There is reason to believe that the time needed for quality control using antibody based tests can be further reduced by new and improved laboratory methods, improved reagents, and increased group size. A new, automated immunological technology (Luminex xMAP) was shown to be suitable for simultaneous measurement of antigen-specific antibodies in salmon blood, as is needed in routine quality control of fish vaccines. Following immunization with vaccine formulations that had reduced antigen content, parallel infection experiments tests were conducted. Neither intraperitoneal nor intramuscular infection could uncover experimental vaccines with reduced amounts of V. salmonicida. However, a clear dose response was observed when fish that had received test vaccines with reduced M. viscosa antigen were exposed by bath challenge. This indicates that antibody-based tests can also provide a more reliable quality control than current infection challenge tests. Fish that fell ill after experimental infection had the same level of serontonin (associated with prolonged stress or fear) in the brain as fishes that were only vaccinated and blood sampled. In contrast, fishes showing disease symptoms had lower levels of stress hormone cortisol in the blood, and lower levels of dopamine (a "reward" substance) in the brain than clinically healthy individuals. Reduced consciousness as a result of generalized infection may explain this observation. The project has further developed a new model for documenting welfare-relevant behavioural and disease signs in fish that have been used in vaccination or infection trials (so-called retrospective assessment of procedure severity). The project's results will stimulate efforts to replace challenge tests in routine quality control of fish vaccines both nationally and internationally, and to increase the use of physiological indicators when assessing the actual welfare status of experimental fish used for vaccine and infection studies.

This project aims to create a firm scientific basis for the discontinuation of infection experiments as the method for routine quality testing of fish vaccines. In a series of trials it will demonstrate the main feasibility of an alternative method (antibody tests), that only involves vaccination and blood sampling under anesthesia, without additional procedures for the test fish. The project will also utilize the samples from the experiments to determine whether one can measure the degree of welfare impact on salmon using a neurotransmitter substance in the fish brain (serotonin). Last but not least, the project will develop a clinical welfare assessment scheme for recording how much welfare strain fish included in batch potency tests have experienced, and to demonstrate how large welfare gains the new test method represents. The project results will be used by both business and government, and representatives of fish vaccine industry and regulatory authorities will therefore be invited to follow the project closely through a reference group.