Introduction
One of the most devastating bacterial diseases, associated with high mortality and economic losses, of wild and farmed marine fish is tenacibaculosis, caused by Gram-negative bacterium Tenacibaculum maritimum (Avendaño-Herrera et al., 2006). Knowledge regarding the pathogenesis of T. maritimum is scarce and can only be improved through the identification of the key virulence factors required for host colonization and disease progression and the host-pathogen interactions occurring during infection. The present study was conceived to evaluate European seabass (Dicentrarchus labrax) systemic and mucosal immune responses against T. maritimum infection. For that, seabass juveniles were bath-infected and changes in blood and plasma parameters as well as in the expression of immune-related genes in skin, distal gut and head-kidney were evaluated.
Materials and methods
A time-course trial was performed, in which groups of seabass (31.9 ± 6.9 g) were bath-challenged for 2 h in aerated seawater with 5 x 105 CFU mL-1 T. maritimum (challenged fish) or marine broth instead of bacteria (mock-challenged fish). Undisturbed fish randomly selected from the groups just before infection were used as controls (time 0). Following 4, 8, 24 and 48 h post-challenge, 8 fish from each treatment were randomly selected, euthanized and head-kidney (HK), distal gut and skin collected for total RNA extraction. RNA was retro-transcribed to cDNA and expression of immune-related genes was analysed by RT-qPCR and normalized with 40s and ef1b. Blood samples were also collected for assessing haematological parameters (i.e., total white and red blood cells counts, haematocrit, haemoglobin, mean corpuscular volume, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration) and for collecting plasma to evaluate innate humoral parameters (i.e., peroxidase, protease, antiprotease, bactericidal and nitric oxide activities). To determine the severity of the challenge, a lethality trial was performed in parallel, using the same bacterial inoculum/challenge protocol used for the time-course trial.
Results
Challenge with T. maritimum induced 40% mortality, whereas no mortality occurred in mock-challenged fish. The number of erythrocytes decreased in the blood of challenged fish at 8 h post-infection, when compared to control (time 0). Regarding leucocyte numbers, the levels of circulating neutrophils in challenged fish increased at 8 h post-challenge compared to control fish and mock-challenged individuals. Lymphopenia was observed at 8 h and 24 h in challenged fish compared to the controls and mock-challenged. However, no significant differences were observed for the remaining evaluated haematological parameters. Similarly, no significant changes were observed among the plasma humoral parameters evaluated. At the transcriptional level, an up-regulation of il-1β gene expression was detected in the distal gut and HK tissues at 8 h post-challenge, whereas il-10 transcripts, were up-regulated at 8 h in the HK and at 24 h in skin and distal gut for the challenged fish. The expression of mmp9 gene also increased at 8 and 24 h post-challenge in distal gut and skin, respectively. Moreover, cxcr4 and il-8 transcripts increased at 8 and 24 h in skin whilst in the HK the response was delayed for il-8, with an up-regulation at 48 h for the challenged fish.
Discussion and conclusion
The up-regulation of some innate immune-related genes such as il-1β, il-8, mmp9 and cxcr4 in mucosal tissues (i.e. skin and gut) following T. maritimum infection by bath, suggest that a local response was triggered. This immune response can be explained by T. maritimum capacity to adhere, colonize and degrade mucosal tissues (Avendaño-Herrera et al., 2006). The abrasion and damage in the tissues can subsequently lead to a systemic infection. However, the absence of changes in the plasma innate humoral parameters supports the occurrence of a poor immune response at the systemic level. Nevertheless, it cannot be disregarded that cytokines il-1β and il-10 were up-regulated in the HK as early as 8 h post-challenge, suggesting signs of a response at a systemic level. The neutrophilia in challenged fish is likely due to the migration of these cells to peripheral infected tissues. This interpretation is further supported by the enhancement of mmp9 and cxcr4 transcripts in distal gut and skin. The observed lymphopenia may be related with the immune response to T. maritimum. An acute challenge, such as an infection, can lead to the redistribution of lymphocytes, from the blood to other body compartments where they are needed (Davis et al., 2008), resulting in a decrease in circulating lymphocytes. Further work is required to disclosure the dynamics stablished between the local and systemic immune responses triggered by T. maritimum infection, as well as to better understand T. maritimum - host interactions.
Keywords
Tenacibaculosis, Gene expression, Immune response, Mucosal Immunity, Aquaculture
References
Avendaño-Herrera, R., Toranzo, A. E., Magariños, B. (2006). A challenge model for Tenacibaculum maritimum infection in turbot (Scophthalmus maximus). Journal of Fish Diseases, 29, 1-4. https://doi.org/10.1111/j.1365-2761.2006.00712.x
Davis, A. K., Maney, D. L., Maerz, J.C. (2008). The use of leukocyte profiles to measure stress in vertebrates: a review for ecologists. Functional Ecology, 22, 760-772. https://doi.org/10.1111/j.1365-2435.2008.01467.x
Acknowledgements
This work is partially supported by project BE4AQUAHEALTH (16-02-05-FMP-0013), funded by MAR2020 Operational Programme and the European Union through FEDER, and by national funds through FCT - Foundation for Science and Technology within the scope of UIDB/04423/2020 and UIDP/04423/2020. I. Ferreira, A. do Vale and B. Costas benefited from grants by FCT (SFRH/BD/147750/2019, L57/2016/CP1355/CT0010 and IF/00197/2015, respectively).