Related Experiment Video
Updated: Oct 7, 2025
Isolation, Fixation and Characterization of Juvenile Gilthead Seabream Head Kidney Leukocytes by Flow Cytometry
Published on: May 9, 2025
In vitro modulation of gilthead seabream (Sparus aurata L.) leukocytes by Bacillus spp. extracellular molecules upon
Rafaela A Santos1, Nuno Mariz-Ponte2, Nicole Martins3
1Departamento de Biologia, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre s/n, Ed. FC4, 4169-007, Porto, Portugal; CIIMAR - Centro Interdisciplinar de Investigação Marinha e Ambiental, Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos s/n, 4450-208, Matosinhos, Portugal; CITAB - Centro de Investigação e Tecnologias Agroambientais e Biológicas, Universidade de Trás-os-Montes e Alto Douro, Quinta de Prados, 5000-801, Vila Real, Portugal; CECAV - Centro de Ciência Animal e Veterinária, Universidade de Trás-os-Montes e Alto Douro, P.O. Box 1013, 5001-801, Vila Real, Portugal.
Abstract:
Stimulation of the fish immune system using immunostimulants is an environmentally friendly strategy to minimize bacterial outbreaks in aquaculture. Different biological and synthetic immunostimulants can enhance non-specific innate immune responses by directly activating immune cells. An example are Bacillus spp., known for their immunostimulatory effects, although the exact mechanisms by which Bacillus spp. offer protection against diseases remains to be elucidated. Furthermore, most studies have focused on Bacillus spp. cells, while the immunostimulant effect of their extracellular metabolome, known to harbour biologically important metabolites, including antimicrobial molecules, has been scarcely evaluated. Here, we evaluated the in vitro immune-modulatory properties of extracellular extracts of three Bacillus spp. strains (B. subtilis FI314, B. vezelensis FI436 and B. pumilus FI464), previously isolated from fish-guts and characterized for their in vitro and in vivo antimicrobial activity against a wide range of fish pathogens. Bacillus spp. extracellular extracts did not affect immune cells viability, but remarkably increased pathogens' phagocytosis when seabream head-kidney leukocytes were challenged with Vibrio anguillarum and Edwardsiella tarda. All extracts significantly increased the engulfment of bacterial pathogens 1 h post-infection. Cells stimulated with the extracellular extracts showed an up-regulation of the expression of immune-relevant genes associated with inflammation, including IL-1β, IL-6, and COX-2. In cells challenged with E. tarda, FI314 extracellular extract significantly increased the expression of IL-1β, IL-6, and COX-2, while FI436 and FI464 significantly increased IL-6 expression. The results of this study revealed that the extracellular molecules from Bacillus spp. fish isolates improved the in vitro response of gilthead seabream immune cells and are thus promising candidates to act as immunostimulants, helping fish fight diseases.
