Introduction
Vaccination is a common and necessary practice in aquaculture, since it is one of the most effective tools to prevent infectious disease outbreaks. However, vaccination practices can be fastidious and time consuming, where fish have to be netted and handled by different operators, causing stress to the animals and disrupting normal daily operations. Amongst different vaccination approaches, dip vaccination is minimally invasive, avoids the use of anaesthetics and requires a lesser amount of human intervention and handling than intraperitoneal vaccines. Nonetheless, both the vaccine itself, normally consisting in highly concentrated viscous substances immiscible in water which reduce the efficacy of oxygen capture and exchange, the netting and crowding required for fish immunization, create stressful conditions that can impact fish health. The present work summarizes the main results from three studies aimed to evaluate the physiologic and metabolic effects of acute stress induced by dip vaccination in three juvenile fish species: European seabass (Dicentrarchus labrax), rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar) pre-smolts.
Material and Methods
Briefly, European seabass (32 g IBW), Atlantic salmon (6-7 g IBW) and rainbow trout (4-5 g IBW) were randomly distributed between tanks and acclimated for one week, all fish were fed commercial diets. Environmental conditions were kept constant, with a temperature of 20°C and salinity of 35 ppt for seabass, whereas salmonid species were maintained at 16°C (rainbow trout) and 14°C (Atlantic salmon) in freshwater. Sampling procedures were transversal to all trials. Before vaccination, samples were collected constituting the time 0 (pre-challenge). Afterwards, fish were netted and transferred into a smaller tank containing the vaccine solution in the case of the vaccine group (VAC) or only water in the case of sham group (SHAM). European seabass were vaccinated with an experimental vaccine containing Tenacibaculum maritimum antigens, whereas trout and salmon fingerlings were vaccinated with a commercial one (Aquavac ERM) against Yersinia ruckeri. Both groups remained in the tank with aeration for 30 seconds (European seabass) or 1 min (salmonid species). Fish were returned to their initial tanks and samples were collected at 1-, 6-, and 24-hours post-vaccination. At every sampling point, blood, plasma and liver were sampled for the analysis of humoral parameters, metabolites and hepatic oxidative stress response. Brain, hypophysis and head-kidney were collected to assess neuroendocrine and immune responses according to previously defined molecular markers. Skin mucus and water were sampled both for proteomic and transcriptomic (miRNA) analysis, while skin tissue was collected solely for transcriptomics (RNAseq).
Results and discussion
European seabass plasma cortisol levels increased in both vaccinated and sham groups 1-hour after stress and gradually decreased reaching basal levels after 24h. Plasma glucose showed a similar pattern to cortisol, however, glucose levels remained higher in the VAC group compared to SHAM, returning to baseline after 24 hours. Finally, after 1 hour of vaccination plasma lactate levels increased in both groups, but the increase was significantly higher in vaccinated fish. However, this effect disappeared after 6 hours, with lactate levels returning to their baseline. In trout juveniles, following the vaccination procedure, cortisol levels increased in VAC group at 6 hours post-vaccination returning to basal levels after 24h, SHAM group levels remained unchanged. In addition to that, lactate and glucose followed a similar pattern, showing an increase at 6 hours post-vaccination in VAC group. Regarding, those same parameters in SHAM group, glucose increased after 1 hour and 24 hours post-vaccination, while lactate remained unaffected when compared to CTRL group.
Monocyte counts and plasma nitric oxide (NO) levels in European seabass progressively decreased over time in stressed groups (VAC and SHAM). High glucocorticoid levels can trigger monocyte migration from blood to peripheral tissues. Contrarily, in rainbow trout there is an increase in monocytes 24h post-vaccination in SHAM group, which coincided with a concomitant increase in NO production. Monocytes are NO producing cells and higher or lower NO levels are directly related to cell activation and migration, possibly mediated by cortisol.
Seabass hepatic GSH/GSSG ratio showed a significant drop at 24h in both SHAM and VAC groups, with significantly lower values when compared to earlier time points and to CTRL. The decrease of hepatic GSH/GSSG ratio at 24 h, indicates oxidative stress in the liver. Lower GSH/GSSG ratio values could point to excess ROS production. The decline was higher in vaccinated fish than in SHAM group, suggesting that the immune activation triggered by the antigen increased oxidative demands beyond handling stress alone. On the other hand, trout hepatic GSH/GSSG ratio remained unaffected throughout the experiment for both groups (SHAM and VAC).
These findings indicate an acute stress response immediately after the vaccination procedure in European seabass with a subsequent recovery overtime and a delayed and milder stress response in rainbow trout. The integration of all generated data, is expected to provide better understanding of stress and health related mechanisms involved in vaccination induced stress, namely the relation between the HPI axis and the immune mechanisms. Atlantic salmon samples are currently under evaluation.
Acknowledgments
The present work is supported by IGNITION (co-funded by the European Union, with the GA 101084651, and the UKRI https://ignition-project.eu/). Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union or REA. Neither the European Union nor the granting authority can be held responsible for them