Vaccines development is targeted at the complete neutralization of pathogens via binding to antibodies or TCR. However to date this aim has not been achieved for many diseases of Atlantic salmon including furunculosis. Vaccination reduces mortality but fails to provide a complete protection as confirmed in this study. Vaccinated HR fish that survived to the end of challenge test still showed relatively high levels of infection in the liver and spleen and induction of genes known for strong responses to bacterial pathogens. For example NFkB was induced 1000-fold with respect to the uninfected control. Markedly lower pathogen loads in HR suggested that survival of the vaccinated salmon was most likely determined with the ability to suppress and clear bacteria. Comparisons of gene expression within the groups of vaccinated and naïve fish found large scale differences between HR and LR. It is noteworthy to mention that analyses with vaccinated fish did not find significant role of genes involved in adaptive immune responses. This was in contrast with our studies of salmon challenges with virus (ISAV), which showed clear dependence between resistance evaluated by time of survival and activation of adaptive immunity [59
]. Rapid stimulation of Igs was also seen in salmon challenged with the parasite, salmon louse [60
]. Apparently, pathogen did not stimulate further activation of adaptive immunity in the vaccinated fish. Therefore one may assume that vaccine-dependent protection of salmon infected with A. salmonicida
was determined mainly with the events that take place after recognition of the pathogen. Given high complexity and diversity of this network and limited level of knowledge, multiple gene expression profiling provides an efficient approach to search for the protective mechanisms.
The gene expression analyses were designed with focus on the mechanisms of vaccine-dependent protection against furunculosis. Genes with greater expression changes in HR can be regarded as candidate markers of protection while opposite regulation may indicate either pathology or unsuccessful defense. Overall, expression changes that can be interpreted as active anti-bacterial responses tended to be greater in LR. We produced an indirect evidence for larger regulation of immune cells recruitment in LR. More detailed study of the immune cells populations is complicated with shortage of cell-specific markers for salmon. Interaction of recruited and resident cells with pathogen components and cytokines activates signal transducers and other regulators of gene expression. We found increase of several genes known for their key roles in responses to pathogens and the magnitude was either greater in LR or equal in LR and HR (NFkB).
A panel of genes for proteins involved in metabolism of iron had higher expression in LR. These regulations indicate the need to reduce the levels of free inorganic iron and heme. HO was activated in all infected fish but the magnitude was greater in LR. HO is the rate-controlling enzyme of the degradation of heme into iron, carbon monoxide, and biliverdin, which is subsequently converted to antioxidant bilirubin [61
]. Extracellular iron ions bind to transferrin, which delivers it into cells. Within cells iron is stored as complex with ferritin. Damage of erythrocytes is one of the symptoms of furunculosis. Heme and iron catalyze production of free radicals through Fenton's reaction thus increasing risk of oxidative stress. Shortage of iron may suppress proliferation of pathogenic bacteria and therefore sequestration of bioavailable iron and heme is regarded as anti-bacterial defense. In case of furunculosis this strategy is obviously unsuccessful.
The complement system was the only group of immune genes that showed strong association with survival and several genes presented in Figure can be considered as candidate markers of vaccine protection against furunculosis. The complement system is the major link between the effector anti-bacterial mechanisms of adaptive and innate arms of immunity. The role of complement in antibody mediated defense against A. salmonicida
was demonstrated in rainbow trout. Combination of specific IgM and complement accelerated ingestion of bacteria and ingestion-activated respiratory burst in phagocytes [62
]. The complement pathways converge at the level of C3 convertase, which initiates the anti-bacterial effector mechanisms. C3 and the downstream genes, the parts of membrane attack complex had higher expression levels in HR, especially C5. Mobilization of the effector complement mechanisms correlated with survival, however it remained unknown, which of the complement pathways was responsible for these changes. Increase of C1QBP, the negative regulator of CP was greater in LR. This could be regarded as indirect evidence for higher activity of CP in HR, however, the expression difference between the study groups was relatively small. Furthermore, it remains unknown if CP provides the only or the major connection between the complement system and acquired immunity in salmonid fish. Homolog to mammalian C1Q was found in lampreys, primitive vertebrates that lack adaptive immunity [63
]. The link between the classical pathway and antibodies appeared in the course of vertebrate evolution but the timing of this event remains undefined. C1Q is the member of a large multi-gene family with diverse functions [64
]. Analysis of non-redundant Atlantic salmon mRNA sequences with blastx revealed 17 distinct C1Q related transcripts. To our knowledge, the antigen-antibody complex binding protein of salmonid CP has not been identified so far. In theory, interaction between the fish complement and acquired immunity molecules can be mediated by MP. MBL interacts with immunoglobulins in mammals [65
] despite the principal role of CP in recognition of antibody antigen complexes. The qPCR analyses showed dramatic up-regulation of MBL in HR. Given the lack of changes in LR, this gene is one of the most promising markers of the vaccine-dependent protection. However taking into account the presence of multiple lectins with unknown roles in Atlantic salmon, induction of MBL does not necessarily mean activation of MP. Furthermore, MASP showed no increase in HR in comparison with uninfected control. Expression profiles of the complement factors Bf and B/C2-B suggested higher activity of AP in HR, however, differences between HR and LR were minor. Diversity of putative sensor proteins in salmonid fish and limited knowledge on their functions impede interpretation of the gene expression data.
Importantly, HR showed greater expression of factor H, the negative regulator of AP, and vitronectin (VTN), a protein that protects host tissues from the complement damages. Similarly, the transcript encoding alpha antiproteinase-like protein was up-regulated in HR relative to LR. Vaccine-dependent resistance to furunculosis was clearly associated with the abilities to prevent and repair damages from pathogen and immune responses and to neutralize and clear toxic compounds of endogenous and exogenous origin. The genes shown in Figure can be divided in three functional groups - tissue reparation, clearance and xenobiotic metabolism - each represented with a gene whose markedly greater up-regulation in HR was confirmed with two methods. Elevated levels of fibronectin can be beneficial since FN1 is involved in reparation of tissues at the wound contraction stage of wound healing [66
]. Due to its opsonic properties, fibronectin takes part in the removal of unwanted substances in the liver by phagocytic Kupffer cells [67
]. CYP3A4 metabolizes a strikingly large number of xenobiotics including bacterial toxins and more than half of modern prescription drugs. This gene is down-regulated in inflammatory conditions by a range of cytokines and NFkB [68
]. Hence individuals with strong innate immune responses may have a higher risk of bacterial intoxication. The role of ABCB4 in bacterial diseases has not been reported so far. One may speculate that the transporter protein that directs phospholipids from liver to bile can be important for clearance of remains of killed cells and bacteria. Transcriptomic comparison of salmon with high and low resistance to pathogen indicated pivotal importance of processes that deserve more attention in studies of fish diseases.