Introduction
Evidence from fish studies suggests that dietary methionine supplementation can support both innate (Machado et al., 2021, Machado et al., 2018) and adaptive (Martin et al., 2023) immune mechanisms. It is therefore hypothesized that dietary methionine supplementation could be a promising strategy to enhance vaccine efficacy when administered prior vaccination procedures. The present study aimed to evaluate the combined effects of dietary methionine supplementation and vaccination on immune status, antioxidant capacity and response to Yersinia ruckeri in rainbow trout (Oncorhynchus mykiss).
Material and Methods
Juvenile trout (5.9 ± 0.9 g) were randomly distributed across 32 tanks (n=35) in a semi-open recirculating freshwater system (14 ± 0.5°C) and fed either a control diet (CTRL, 8.0 mg/g DW) or a CTRL-based diet supplemented with methionine (MET, 17.8 mg/g DW). After 4 weeks, half of the fish within each dietary group were either dip-vaccinated (AquaVac® ERM Concentrate, MSD Animal Health) against Y. ruckeri or sham-vaccinated to serve as control. At 21 days post-vaccination, samples were collected from two fish per tank (n=16) to assess bacterial load, hematological parameters, differential cell counts, hepatic metabolites, antioxidant defenses and gene expression, head-kidney gene expression and plasma proteomic profiles. The remaining fish were intraperitoneally challenged with Y. ruckeri (5 x 105 cfu/ml) or injected with HBSS as a control. The same parameters were evaluated in 9 fish per group at 4, 24 and 48 hours post-infection. Mortality was monitored daily for 13 days. Fish remained on their respective diets throughout the entire trial.
Results and Discussion
Prior to infection, MET-fed fish showed decreased hepatic amd1 expression compared to CTRL-fed fish, a difference that persisted following infection. In unvaccinated fish, post-infection analyses revealed that methionine supplementation appeared to compromise antioxidant defenses, evidenced by decreased hepatic SOD activity, GSH levels, and reduced plasma GPx abundance, and to prolong immune activation, indicated by increased expression of saa and cd8b in the head-kidney and elevated plasma levels of C1q domain-containing protein and chemotaxin. These alterations coincided with the exclusive detection of Y. ruckeri in MET-fed fish at 48 hours post-infection and a lower survival rate (75%) compared to CTRL-fed fish (92.5%). In vaccinated fish, no mortality was observed in either dietary group. However, proteomic analyses at 24 hours post-infection revealed distinct responses: MET-vaccinated fish showed increased abundance of hemostasis-related proteins, whereas CTRL-vaccinated fish maintained a proteomic profile more similar to that of pre-infection fish. This difference may have contributed to the observed trend of slightly faster pathogen clearance in the MET vaccinated fish.
Acknowledgements
This work was supported by the European Union’s Horizon Europe research and innovation programme (GA No.101079467 project GRINNAQUA) and national funds by Fundação para a Ciência e a Tecnologia (FCT, Portugal) through project IMMUNAA (reference PTDC/CVT-CVT/7741/2020). IC, DP, MM and BC were supported by FCT, Portugal (2021.04867.BD, UI/BD/150900/2021, 2022.03304.CEECIND and 2020.00290.CEECIND, respectively).
References
Machado, M., Azeredo, R., Fontinha, F., Fernandez-Boo, S., Conceição, L. E. C., Dias, J. & Costas, B. 2018. Dietary methionine improves the European seabass (Dicentrarchus labrax) immune status, inflammatory response, and disease resistance. Frontiers in Immunology, 9, 2672.
Machado, M., Moura, J., Peixoto, D., Castro-Cunha, M., Conceição, L. E. C., Dias, J. & Costas, B. 2021. Dietary methionine as a strategy to improve innate immunity in rainbow trout (Oncorhynchus mykiss) juveniles. General and Comparative Endocrinology, 302, 113690.
Martin, D., Ordas, M. C., Carvalho, I., Diaz-Rosales, P., Nunez-Ortiz, N., Vicente-Gil, S., Arrogante, A., Zarza, C., Machado, M., Costas, B. & Tafalla, C. 2023. L-methionine supplementation modulates IgM(+) B cell responses in rainbow trout. Frontiers in Immunology, 14, 1264228.