Introduction
The occurrence of fish bacterial diseases is one of the biggest challenges of world aquaculture intensification. Photobacteriosis is a septicemic disease caused by Photobacterium damselae subsp. piscicida (Phdp), affecting a wide range of marine species and leading to relevant economic losses. Although several studies have yet been performed regarding the pathogen features and virulence factors, few information about the host defence mechanisms activated after infection is available. The present study aimed to evaluate gilthead seabream (Sparus aurata) innate immune response to infection with Phdp.
Materials and methods
A time-course study was performed at CETEMARES (Politécnico de Leiria, Peniche, Portugal) facilities with 72 seabream juveniles (9.8 ± 2.2 g). After 2 weeks of acclimation, 12 fish were sampled before infection (time 0). The remaining animals were randomly selected and intraperitoneally (i.p.) injected with 100 µl PBS (placebo group) or 100 µl of exponentially growing Phdp (106 CFU mL-1; infected group) and distributed as a complete randomized design in 6 recirculating seawater systems (i.e. triplicates per experimental condition). Two animals per tank (n = 6 per treatment) were sampled at 3, 6, 9, 24 and 48 h after i.p. injection. Fish were euthanized with 2-phenoxyethanol (0.5 mL L-1) and blood samples were collected for haematological procedures. The remaining blood was centrifuged for plasma collection and innate humoral parameters (i.e. peroxidase, protease and antiprotease activities) were evaluated. Liver and head-kidney were also collected for oxidative stress (lipid peroxidation, total glutathione content, catalase, superoxide dismutase and glutathione-S-transferase activities) and gene expression analyses. Head-kidney total RNA isolation was performed with NZY Total RNA Isolation kit and first-strand cDNA synthesized with NZY First-Strand cDNA Synthesis Kit. Gene expression of innate immune selected genes was analysed by RT-qPCR and normalized by Pfaffl method using EF-1α as housekeeping gene. Although both placebo and infected groups were analysed, significant differences were mainly registered on infected groups against unstimulated animals and therefore are highlighted for a better understanding of host response to Phdp infection.
Results
Infection produced a host anaemic state since haemoglobin concentration and mean corpuscular haemoglobin concentration decreased in infected animals 48 h after bacterial challenge. Also total erythrocyte levels decreased 9 h after infection, while haematocrit presented lower values 48 h post-infection. In contrast, total peripheral leucocytes produced few differences throughout the time course. Circulating neutrophil and monocyte numbers augmented since the first hours of infection compared to non-infected animals. Regarding humoral responses, infected animals presented increased antiproteases activity 48 h post-infection. Hepatic oxidative stress biomarkers resulted on increased lipid peroxidase and glutathione-S-transferase 9 hours after pathogen inoculation, whereas catalase produced a later but similar variation. Moreover, hepatic total glutathione decreased 24 hours after bacterial challenge. Molecular findings go along with the cellular, humoral and oxidative stress results, with increased expression of interleukin1β, interleukin-34, interleukin-10 and major histocompatibility complex I, which are recognized genes related to inflammatory response and phagocytic processes. Also genes related to the iron metabolism such as haptoglobin and transferrin increased mRNA expression during infection, sugesting haemolysis and presenting a possible mechanism from host to avoid the bacterial uptake of iron.
Discussion and conclusion
The outputs of this study include several pathways that contribute for a steady immune response. Cellular mechanisms are driven by peripheral monocytes and neutrophils that migrate for the infection site and release antioxidant and antibacterial substances trying to cope with the infection. Inflammation develops through a balanced production of anti and pro-inflammatory cytokines that result on a sustained but directed response. It is also remarkable the selective differentiation of macrophages through the interleukin-34/colony stimulating factor 1 receptor route and the increase of transferrin production that might be considered preferred routes to avoid bacterial survival and proliferation.
A broad view of the host mechanisms involved on other bacterial infections are essential for the definition of health biomarkers that can be suitable for an early disease detection.
Keywords
Fish health, photobacteriosis, immune response, anaemia, macrophages, interleukin-34
Acknowledgements
This work was supported by project BE4AQUAHEALTH: RASTREIO NACIONAL DE PATOLOGIAS DE PEIXES DE AQUACULTURA: UMA APOSTA NA PREVENÇÃO (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. DP, IAF and BC were supported by FCT (UI/BD/150900/2021, SFRH/BD/147750/2019 and IF/00197/2015, respectively).