Vibrio vaccination and protective effect of serum transfer in cod
1 Formål
Atlantic cod has a highly unusual immune system due to loss of Major Histocompatibility Complex (MHC) class II, normally used by antigen-presenting cells to activate CD4-positive T cells. CD4 is also lost in cod. Because of this defect, cod have poor and variable antibody responses. Vibrio anguillarum is the causative agent of a fatal haemorrhagic septicaemia and a commonly encountered disease in unvaccinated cold-water fish species such as Atlantic cod and salmon. Despite its lack of CD4 T cells, protective immunity against vibriosis can be achieved by immunisation in cod.
Published reports have suggested that specific antibodies are not an important factor in protection against vibrio infection after immunisation in cod. However, our serum transfer experiment in January 2021 showed a higher survival rate in fish that received serum from hyper-immune fish, compared to fish that received naïve serum. Encouraged by this promising result, which suggests that antibodies may have a role in vibrio protection after all, we would like to repeat the serum transfer experiment with larger group sizes. Furthermore, to determine whether it is the IgM antibody or other factors in immune sera that is protective we will purify IgM from immunised cod and use it in transfer experiments.
The protective effect of immunisation could be mediated by reduced internalisation of the vibrio bacteria due to neutralisation by specific IgM, or by specific immune response from lymphocytes that effectively fight off invasive vibrio bacteria, or a combination of these two effects. Infecting fish via i.p. injection will circumvent the mucosal protection offered by specific IgM. We wish therefore to compare the protective effect of vibrio vaccination on bath versus i.p. infection.
2 Skadevirkninger
Humane endpoints will be used for any fish that shows signs of disease or abnormal behaviour (lethargy, bloating, disoriented swimming).
3 Forventet nytteverdi
Little is known about how the cod immune system works. Vaccination with certain pathogens, such as Vibrio gives protective immunity, whereas the effect of vaccination is meagre for other pathogens such as Francisella noatunensis. Our pilot study indicates protective effect of immune serum transfer, which is in contrast to the leading literature on the field. Understanding the details of the protective response to Vibrio is important for the development of vaccines in this species.
4 Antall dyr og art
Atlantic cod (Gadus morhua), n=300
5 Hvordan etterleve 3R
Replacement is not possible due to the special immune system in cod. The number of fish used is optimised based on our prior experience with similar experiments. We got a p-value of 0.06 when we used 16 fish in each group, and now we are going to have 25 fish in each experimental group. The number of fish that need to be vaccinated in order to be serum and IgM donor, is also carefully calculated based on prior experience of how much serum and IgM we could harvest from a fish with the expected size in October 2021.
Atlantic cod has a highly unusual immune system due to loss of Major Histocompatibility Complex (MHC) class II, normally used by antigen-presenting cells to activate CD4-positive T cells. CD4 is also lost in cod. Because of this defect, cod have poor and variable antibody responses. Vibrio anguillarum is the causative agent of a fatal haemorrhagic septicaemia and a commonly encountered disease in unvaccinated cold-water fish species such as Atlantic cod and salmon. Despite its lack of CD4 T cells, protective immunity against vibriosis can be achieved by immunisation in cod.
Published reports have suggested that specific antibodies are not an important factor in protection against vibrio infection after immunisation in cod. However, our serum transfer experiment in January 2021 showed a higher survival rate in fish that received serum from hyper-immune fish, compared to fish that received naïve serum. Encouraged by this promising result, which suggests that antibodies may have a role in vibrio protection after all, we would like to repeat the serum transfer experiment with larger group sizes. Furthermore, to determine whether it is the IgM antibody or other factors in immune sera that is protective we will purify IgM from immunised cod and use it in transfer experiments.
The protective effect of immunisation could be mediated by reduced internalisation of the vibrio bacteria due to neutralisation by specific IgM, or by specific immune response from lymphocytes that effectively fight off invasive vibrio bacteria, or a combination of these two effects. Infecting fish via i.p. injection will circumvent the mucosal protection offered by specific IgM. We wish therefore to compare the protective effect of vibrio vaccination on bath versus i.p. infection.
2 Skadevirkninger
Humane endpoints will be used for any fish that shows signs of disease or abnormal behaviour (lethargy, bloating, disoriented swimming).
3 Forventet nytteverdi
Little is known about how the cod immune system works. Vaccination with certain pathogens, such as Vibrio gives protective immunity, whereas the effect of vaccination is meagre for other pathogens such as Francisella noatunensis. Our pilot study indicates protective effect of immune serum transfer, which is in contrast to the leading literature on the field. Understanding the details of the protective response to Vibrio is important for the development of vaccines in this species.
4 Antall dyr og art
Atlantic cod (Gadus morhua), n=300
5 Hvordan etterleve 3R
Replacement is not possible due to the special immune system in cod. The number of fish used is optimised based on our prior experience with similar experiments. We got a p-value of 0.06 when we used 16 fish in each group, and now we are going to have 25 fish in each experimental group. The number of fish that need to be vaccinated in order to be serum and IgM donor, is also carefully calculated based on prior experience of how much serum and IgM we could harvest from a fish with the expected size in October 2021.
Etterevaluering
The Norwegian Food Safety Authority must retrospectively assess all severe experiments.
Begrunnelse for etterevalueringen
This project is a follow-up and extension of previous experiments where the main aim is to investigate the immune response and effect of vibrio vaccination in cod. The results showed that protective immunity against Vibrio anguillarum after vaccination can be transferred in serum, more specifically by specific IgM antibodies in serum. In addition, the immune protection in cod against vibrio by vaccination consists of both an increased barrier function as well as a more vigorous and effective immune response against the bacterium. Overall, the aims of the experiments were achieved. The study showed that previous findings were reproduceable regarding the protective effective of serum transfer and the results showed that bath vaccination also offers some protection against i.p. infection.
In total 364 fish were used in the experiments. The severity was classified as mild for 52 cod, moderate for 143, and severe for 169 cod. However, the applicant used 64 more fish than the Norwegian Food Safety Authority had approved. We must remind both the applicant and the laboratory that before increasing the number of animals in an experiment, the applicant must send a change application (endringssøknad) to get this approved. This did not happen in this case.
There is virtually no cod cell line or other specific reagents available for the study of cod. Vaccination efficacy experiments are very difficult to simulate by in vitro models, especially given the unique immune system of cod.
Retrospectively, the applicant concluded that more fish should have been included for molecular analysis of the immune response. The number of animals used is still the most suitable for statistical analysis. The surveillance of the fish, and method for euthanasia worked well.
According to the applicant, it would have been possible to reduce the number of fish used in the i.p. dose titration experiment. However, this regime would have been prohibitively costly due to the time aspect and the high fee for access to infection unit. The extra monetary cost for infection unit would have been avoided if one did not need to disinfect spill water from the infection trial. The applicant states that V. anguillarum is universally present in salt water and the number of bacteria introduced by the experiment into the environment would have been insignificant. The NFSA (Mattilsynet) assess this argument as not relevant because all fish pathogenic organisms used in experimental infection trials exist in the marine environment. Proper disinfection of spill water is necessary and mandatory for all laboratories with approval for conduction of infection trials with fish pathogens.
In total 364 fish were used in the experiments. The severity was classified as mild for 52 cod, moderate for 143, and severe for 169 cod. However, the applicant used 64 more fish than the Norwegian Food Safety Authority had approved. We must remind both the applicant and the laboratory that before increasing the number of animals in an experiment, the applicant must send a change application (endringssøknad) to get this approved. This did not happen in this case.
There is virtually no cod cell line or other specific reagents available for the study of cod. Vaccination efficacy experiments are very difficult to simulate by in vitro models, especially given the unique immune system of cod.
Retrospectively, the applicant concluded that more fish should have been included for molecular analysis of the immune response. The number of animals used is still the most suitable for statistical analysis. The surveillance of the fish, and method for euthanasia worked well.
According to the applicant, it would have been possible to reduce the number of fish used in the i.p. dose titration experiment. However, this regime would have been prohibitively costly due to the time aspect and the high fee for access to infection unit. The extra monetary cost for infection unit would have been avoided if one did not need to disinfect spill water from the infection trial. The applicant states that V. anguillarum is universally present in salt water and the number of bacteria introduced by the experiment into the environment would have been insignificant. The NFSA (Mattilsynet) assess this argument as not relevant because all fish pathogenic organisms used in experimental infection trials exist in the marine environment. Proper disinfection of spill water is necessary and mandatory for all laboratories with approval for conduction of infection trials with fish pathogens.