Open Access

Bacterial kidney disease (BKD) is a chronic bacterial disease affecting both wild and farmed salmonids. The causative agent for BKD is the Gram-positive fish pathogen Renibacterium salmoninarum. As treatment and prevention of BKD have proven to be difficult, it is important to know and identify the key bacterial proteins that interact with the host. We used subcellular fractionation to report semi-quantitative data for the cytosolic, membrane, extracellular, and membrane vesicle (MV) proteome of R. salmoninarum. These data can aid as a backbone for more targeted experiments regarding the development of new drugs for the treatment of BKD. Further analysis was focused on the MV proteome, where both major immunosuppressive proteins P57/Msa and P22 and proteins involved in bacterial adhesion were found in high abundance. Interestingly, the P22 protein was relatively enriched only in the extracellular and MV fraction, implicating that MVs may play a role in host–pathogen interaction. Compared to the other subcellular fractions, the MVs were also relatively enriched in lipoproteins and all four cell wall hydrolases belonging to the New Lipoprotein C/Protein of 60 kDa (NlpC/P60) family were detected, suggesting an involvement in the formation of the MVs.

The aquaculture industry has been consistently and successfully growing over the years, supplying over 50% of the fish humans consume. A large part of this success is due to the implementation of vaccination, which is by far the most reliable prophylactic method in large-scale fish farming. Nonetheless, although recent fish vaccines have greatly contributed to the development and sustainability of the aquaculture industry, they not always offer sufficient protection to provide acceptable survival rates when infectious diseases outbreaks occur. Therefore, infectious diseases and effective vaccines still constitute major problems for aquaculture. Different practical aspects and biological factors of fish have also contributed to the unsuccessful outcome of fish vaccines. To date, many of the most effective vaccines for fish are injectable, and their formulation includes aluminum or oil emulsion adjuvants. Both facts constitute a major issue for animal welfare due to the stress and side effects they trigger. Great strides have been made in innovative technologies for fish vaccines. However, as of today, they are not available on the market. Thus, improvements in vaccine formulations and delivery routes remain an open topic and leads the to-do list of science with the aquaculture of the future. Vaccination provides immunity against a determined pathogen, and this is inherent to the immune system. Therefore, thorough knowledge about the fish immune system and how it is influenced by internal and external factors will certainly support rational vaccine design. Thereby, the immune responses triggered by a vaccine can be exhaustively characterized, and the formulations improved in case it is needed. Hence, the goal of this PhD thesis, is to provide knowledge to improve fish vaccination, both in its formulation and in its efficacy, aiming to promote the rational design of fish vaccines. Additionally, this work proposes a holistic view of fish, where the physiology and culture conditions of the fish are the starting points for the development and application of vaccines. Thus, concepts and considerations for rational vaccine design specific for fish are presented here. Article I of this thesis offers a comprehensive review on the current situation in Chile, but also worldwide aquaculture and the challenges it must face in the future. Namely, recurrent pathogenic outbreaks and sub-optimal levels of protection due to inefficient vaccination. This article established an open and flexible ground upon which to reflect on how and what to improve in fish vaccines, leading the efforts towards rational vaccine design. In Article II, we investigated whether the current most used vaccination route, intraperitoneal, can be improved by reducing the side effects of adjuvants, replacing them with in the vaccine formulations with Poly-(D,L-lactic-co-glycolic) acid (PLGA) microparticles, that serve simultaneously as vaccine vehicle and adjuvants. Article III summarizes the scientific literature about what is known about the teleost thymus. From this, it became clear how external factors such as photoperiod and seasonality can modulate this primary lymphatic organ, and probably, immune responses. These are essential factors to consider if effective and protective vaccines are needed in species highly influenced by the environment such as fish. As discussed in Article III, fish are poikilotherm animals, highly sensitive to environmental factors like light. In Article IV, we reported for the first time, light generates daily rhythms in cells’ circulation and gene expression, entraining the trout immune response. Therefore, “when” (time of the day) we stimulate fish matters in order to get optimal immune responses. Article V provides valuable knowledge about what happens with fish immune responses, against a bacterial agent, under constant cues like light/dark cycles and temperature. Once again, “when” we stimulate fish (season), influences the fish immune status and therefore, their immune responses. Finally, Article VI reports, for the first time, leukocytes extracted from fins of trout directly respond to a parasitic infection. This article supports the idea that further research must be done on fish mucosal surfaces, since they are key to stimulating/vaccinating fish, as they are a natural entry route for pathogens and modulate the immune responses mounted. Overall, the information provided by these articles is highly relevant for the aquaculture industry. Firstly, because the vaccine platform based on PLGA microparticles is promising for the future of fish vaccination, harmful adjuvants can be avoided, while still providing enhanced stimulation thanks to the timed-released capacity of the particles. Additionally, they offer the possibility to adapt them to in-feed vaccine pellets, which is the ideal delivery route for fish. Secondly, accurate vaccination protocols can be established; vaccination should be done during daytime, and preferably during the morning, where the physiological status of fish provide optimal conditions for induction of an ultimately protective immune response after vaccination. Furthermore, vaccination should be done during warm months, spring, or summertime, as apparently fish have free-run internal clocks that negatively modulate adaptive immune responses during wintertime. In summary, the present thesis provides a novel concept for vaccination of aquacultured species based on new data for rational vaccine design, with optimal application procedures based on the optimal timing (season and daytime), reduced stress by oral application and considerations about improving “first-line defenses” by vaccination via mucosal surfaces of gut or skin.

Laccases are regarded as versatile green biocatalysts, and recent scientific research has focused on improving their redox potential for broader industrial and environmental applications. The density functional theory (DFT) quantum mechanics approach, sufficiently rigorous and efficient for the calculation of electronic structures, is conducted to better comprehend the connection between the redox potential and the atomic structural feature of laccases. According to the crystal structure of wild type laccase CueO and its variant, a truncated miniature cluster model method was established in this research. On the basic of thermodynamic cycle, the overall Gibbs free energy variations before and after the one-electron reduction were calculated. It turned out that the trends of redox potentials to increase after variant predicted by the theoretical calculations correlated well with those obtained by experiments, thereby validating the feasibility of this cluster model method for simulating the redox potentials of laccases.

Bloodstream infections caused by Streptococcus pneumoniae induce strong inflammatory and procoagulant cellular responses and affect the endothelial barrier of the vascular system. Bacterial virulence determinants, such as the cytotoxic pore-forming pneumolysin, increase the endothelial barrier permeability by inducing cell apoptosis and cell damage. As life-threatening consequences, disseminated intravascular coagulation followed by consumption coagulopathy and low blood pressure is described. With the aim to decipher the role of pneumolysin in endothelial damage and leakage of the vascular barrier in more detail, we established a chamber-separation cell migration assay (CSMA) used to illustrate endothelial wound healing upon bacterial infections. We used chambered inlets for cell cultivation, which, after removal, provide a cell-free area of 500 μm in diameter as a defined gap in primary endothelial cell layers. During the process of wound healing, the size of the cell-free area is decreasing due to cell migration and proliferation, which we quantitatively determined by microscopic live cell monitoring. In addition, differential immunofluorescence staining combined with confocal microscopy was used to morphologically characterize the effect of bacterial attachment on cell migration and the velocity of gap closure. In all assays, the presence of wild-type pneumococci significantly inhibited endothelial gap closure. Remarkably, even in the presence of pneumolysin-deficient pneumococci, cell migration was significantly retarded. Moreover, the inhibitory effect of pneumococci on the proportion of cell proliferation versus cell migration within the process of endothelial gap closure was assessed by implementation of a fluorescence-conjugated nucleoside analogon. We further combined the endothelial CSMA with a microfluidic pump system, which for the first time enabled the microscopic visualization and monitoring of endothelial gap closure in the presence of circulating bacteria at defined vascular shear stress values for up to 48 h. In accordance with our CSMA results under static conditions, the gap remained cell free in the presence of circulating pneumococci in flow. Hence, our combined endothelial cultivation technique represents a complex in vitro system, which mimics the vascular physiology as close as possible by providing essential parameters of the blood flow to gain new insights into the effect of pneumococcal infection on endothelial barrier integrity in flow.