FoN Zf Infection 2
The SFI "Foods of Norway" is developing experimental diets for farm animals and fish using sustainable local feed ingredients to reduce the reliance on imported feed ingredients. These experimental diets contain varying levels of protein derived from yeast that are produced from sugars and other nutrients derived from natural resources such as wood, grass and seaweed. The goal of Foods of Norway is to evaluate the potential of new, yeast-based diets to replace at least partially currently used soybean-based feeds, and to evaluate the potential of certain yeast strains to promote resistance to bacterial infections.
1 Purpose
The purpose of this project will be to evaluate the potential of zebrafish and medaka as infection models for the salmon pathogen Aeromonas salmonicida and to assess the performance of yeast-containing diets in the zebrafish infection model.
2 Distress
A low mortality infection model will be used. Fish will be exposed to level of bacteria that results in less than 20% mortality. Surviving fish would experience moderate distress. Moribund or fish experiencing severe distress are to be euthanazed in accordance with defined humane endpoints.
3 Expected benefit
It is expected that the effective screening in zebrafish and medaka will greatly enhance the identification of the health-related bioactivity of diets. The benefit will be that fewer diets (but with a known bioactive profile) are tested in production animals and fish. This will reduce the number of large animal experiments that need to be performed
4 Number of animals, and what kind
The project will use 1350 zebrafish and 360 medaka. The initial infection experiments will be performed to compare directly the virulence of Aeromonas salmonicida in zebrafish and in medaka, in the latter at 2 different temperatures (20°C and 12°C). In each experiment, surviving fish will be collected at 1, 2 and 4 weeks after infection for (a) group weighing, (b) histopathology analysis, (c) immunohistochemistry analysis using an antibody specific to Aeromonas salmonicida, (d) qPCR analysis using primers specific to inflammation and infection response markers as well as to Aeromonas salmonicida.
The diets to be tested in the zebrafish infection model are: 6 different diets with different yeast-based ingredients, in comparison with 1 standard zebrafish diet. Adult zebrafish will be fed for 4 weeks, and will be challenged with bacterial infections (Aeromonas salmonicida) after 4 weeks, after which survival rates will be determined. Surviving fish will be sacrificed at 1, 2, and 4 weeks after infection, after which they will analyzed as described above.
5 How to adhere to 3R
Based on the results of these experiments, future studies of yeast-based diets in zebrafish and/or medaka will be designed accordingly to minimize the number of animals used in each study.
1 Purpose
The purpose of this project will be to evaluate the potential of zebrafish and medaka as infection models for the salmon pathogen Aeromonas salmonicida and to assess the performance of yeast-containing diets in the zebrafish infection model.
2 Distress
A low mortality infection model will be used. Fish will be exposed to level of bacteria that results in less than 20% mortality. Surviving fish would experience moderate distress. Moribund or fish experiencing severe distress are to be euthanazed in accordance with defined humane endpoints.
3 Expected benefit
It is expected that the effective screening in zebrafish and medaka will greatly enhance the identification of the health-related bioactivity of diets. The benefit will be that fewer diets (but with a known bioactive profile) are tested in production animals and fish. This will reduce the number of large animal experiments that need to be performed
4 Number of animals, and what kind
The project will use 1350 zebrafish and 360 medaka. The initial infection experiments will be performed to compare directly the virulence of Aeromonas salmonicida in zebrafish and in medaka, in the latter at 2 different temperatures (20°C and 12°C). In each experiment, surviving fish will be collected at 1, 2 and 4 weeks after infection for (a) group weighing, (b) histopathology analysis, (c) immunohistochemistry analysis using an antibody specific to Aeromonas salmonicida, (d) qPCR analysis using primers specific to inflammation and infection response markers as well as to Aeromonas salmonicida.
The diets to be tested in the zebrafish infection model are: 6 different diets with different yeast-based ingredients, in comparison with 1 standard zebrafish diet. Adult zebrafish will be fed for 4 weeks, and will be challenged with bacterial infections (Aeromonas salmonicida) after 4 weeks, after which survival rates will be determined. Surviving fish will be sacrificed at 1, 2, and 4 weeks after infection, after which they will analyzed as described above.
5 How to adhere to 3R
Based on the results of these experiments, future studies of yeast-based diets in zebrafish and/or medaka will be designed accordingly to minimize the number of animals used in each study.
Etterevaluering
Forøket er betydelig belastende.
Begrunnelse for etterevalueringen
As a result of delays in having the necessary laboratory staff available, the part of this experiment assessing the potential of yeast-based diet to promote resistance to bacterial infections was performed only with zebrafish. The initial part of the experiment was carried out both with zebrafish and medaka. Medaka were assessed for their tolerance of low water temperatures, but as mentioned before, an infection experiment was not performed with medaka. The results show that a low mortality Aeromonas salmonicida infection protocol in zebrafish revealed the ability of a yeast-based diet to increase resistance to bacterial disease. Thus, only some of the objectives of this experiment were achieved.
A low mortality Aeromonas salmonicida infection protocol was performed on zebrafish fed a control and a yeast-based diet. This experiment showed that the mortality rate in fish fed the yeast-based diet was <1% compared to a mortality rate of 7% in the control diet group.
The experiment used 691 zebrafish and 280 medaka in total, a lot fewer than applied for. The severity of the infection for the animals was as anticipated in the application. The severity was severe for 48 zebrafish, moderate for 603 and mild for 40. For the medaka, severity was categorized as severe for 6 of them, while moderate for 204 and mild for 70.
This experiment demonstrated that a yeast-based diet enhanced resistance to bacterial infection in a low-mortality infection protocol for Aeromonas salmonicida in zebrafish. The number of animals was appropriate and the continued use of a pilot infection prior to an infection study to confirm the virulence of the bacteria is appropriate. This model appears useful for screening diets in future experiments, to help reduce the number of larger fish (e.g. salmon) that would otherwise be needed for such experiments.
Work is in progress in the field to develop in vitro tests to assess the bioreactivity of feed ingredients. These tests currently do not replace animal experiments.
The design of the experiment performed was appropriate. The group sizes used approximate those that should be used for subsequent diet screening experiments using this infection model. Further experiments should continue to use a pilot infection before the main infection experiment to check the virulence of the strain of A. salmonicida to be used. Further experiments will be needed to determine how well this model approximates the pathogenicity of Aeromonas salmonicida infections of e.g. salmon.
The current score forms and welfare protocols functioned well. Both the humane endpoints and euthanasia method is appropriate and working well.
A low mortality Aeromonas salmonicida infection protocol was performed on zebrafish fed a control and a yeast-based diet. This experiment showed that the mortality rate in fish fed the yeast-based diet was <1% compared to a mortality rate of 7% in the control diet group.
The experiment used 691 zebrafish and 280 medaka in total, a lot fewer than applied for. The severity of the infection for the animals was as anticipated in the application. The severity was severe for 48 zebrafish, moderate for 603 and mild for 40. For the medaka, severity was categorized as severe for 6 of them, while moderate for 204 and mild for 70.
This experiment demonstrated that a yeast-based diet enhanced resistance to bacterial infection in a low-mortality infection protocol for Aeromonas salmonicida in zebrafish. The number of animals was appropriate and the continued use of a pilot infection prior to an infection study to confirm the virulence of the bacteria is appropriate. This model appears useful for screening diets in future experiments, to help reduce the number of larger fish (e.g. salmon) that would otherwise be needed for such experiments.
Work is in progress in the field to develop in vitro tests to assess the bioreactivity of feed ingredients. These tests currently do not replace animal experiments.
The design of the experiment performed was appropriate. The group sizes used approximate those that should be used for subsequent diet screening experiments using this infection model. Further experiments should continue to use a pilot infection before the main infection experiment to check the virulence of the strain of A. salmonicida to be used. Further experiments will be needed to determine how well this model approximates the pathogenicity of Aeromonas salmonicida infections of e.g. salmon.
The current score forms and welfare protocols functioned well. Both the humane endpoints and euthanasia method is appropriate and working well.