Whole transcriptome analysis of the Atlantic cod vaccine response reveals subtle changes in adaptive immunity.

Atlantic cod has lost the Major Histocompatibility complex class II pathway - central to pathogen presentation, humoral response and immunity. Here, we investigate the immunological response of Atlantic cod subsequent to dip vaccination with Vibrioanguillarum bacterin using transcriptome sequencing. The experiment was conducted on siblings from an Atlantic cod family found to be highly susceptible towards vibriosis where vaccination has demonstrated improved pathogen resistance. Gene expression analyses at 2, 4, 21 and 42 days post vaccination revealed GO-term enrichment for muscle, neuron and metabolism-related pathways. In-depth characterization of immune-related GO terms demonstrated down-regulation of MHCI antigen presentation, C-type lectin receptor signaling and granulocyte activation over time. Phagocytosis, interferon-gamma signaling and negative regulation of innate immunity were increasingly up-regulated over time. Individual differentially expressed immune genes implies weak initiation of acute phase proteins with little or no inflammation. Furthermore, gene expression indicates presence of T-cells, NK-like cells, B-cells and monocytes/macrophages. Three MHCI transcripts were up-regulated with B2M and SEC61. Overall, we find no clear immune-related transcriptomic response which could be attributed to Atlantic cod's alternative immune system. However, we cannot rule out that this response is related to vaccination protocol/sampling strategy. Earlier functional studies demonstrate significant memory in Atlantic cod post dip vaccination and combined with our results indicate the presence of other adaptive immunity mechanisms. In particular, we suggest that further investigations should look into CD8+ memory T-cells, γδ T-cells, T-cell independent memory or memory induced through NK-like/other lymphoid cells locally in the mucosal lining for this particular vaccination strategy.

Disentangling the immune response and host-pathogen interactions in Francisella noatunensis infected Atlantic cod.

The genetic repertoire underlying teleost immunity has been shown to be highly variable. A rare example is Atlantic cod and its relatives Gadiformes that lacks a hallmark of vertebrate immunity: Major Histocompatibility Complex class II. No immunological studies so far have fully unraveled the functionality of this particular immune system. Through global transcriptomic profiling, we investigate the immune response and host-pathogen interaction of Atlantic cod infected with the facultative intracellular bacterium Francisella noatunensis. We find that Atlantic cod displays an overall classic innate immune response with inflammation, acute-phase proteins and cell recruitment through up-regulation of e.g. IL1B, fibrinogen, cathelicidin, hepcidin and several chemotactic cytokines such as the neutrophil attractants CXCL1 and CXCL8. In terms of adaptive immunity, we observe up-regulation of interferon gamma followed by up-regulation of several MHCI transcripts and genes related to antigen transport and loading. Finally, we find up-regulation of immunoglobulins and down-regulation of T-cell and NK-like cell markers. Our analyses also uncover some contradictory transcriptional findings such as up-regulation of anti-inflammatory IL10 as well as down-regulation of the NADPH oxidase complex and myeloperoxidase. This we interpret as the result of host-pathogen interactions where F. noatunensis modulates the immune response. In summary, our results suggest that Atlantic cod mounts a classic innate immune response as well as a neutrophil-driven response. In terms of adaptive immunity, both endogenous and exogenous antigens are being presented on MHCI and antibody production is likely enabled through direct B-cell stimulation with possible neutrophil help. Collectively, we have obtained novel insight in the orchestration of the Atlantic cod immune system and determined likely targets of F. noatunensis host-pathogen interactions.

Francisella noatunensis subsp. noatunensis invades, survives and replicates in Atlantic cod cells.

Systemic infection caused by the facultative intracellular bacterium Francisella noatunensis subsp. noatunensis remains a disease threat to Atlantic cod Gadus morhua L. Future prophylactics could benefit from better knowledge on how the bacterium invades, survives and establishes infection in its host cells. Here, facilitated by the use of a gentamicin protection assay, this was studied in primary monocyte/macrophage cultures and an epithelial-like cell line derived from Atlantic cod larvae (ACL cells). The results showed that F. noatunensis subsp. noatunensis is able to invade primary monocyte/macrophages, and that the actin-polymerisation inhibitor cytochalasin D blocked internalisation, demonstrating that the invasion is mediated through phagocytosis. Interferon gamma (IFNγ) treatment of cod macrophages prior to infection enhanced bacterial invasion, potentially by stimulating macrophage activation in an early step in host defence against F. noatunensis subsp. noatunensis infections. We measured a rapid drop of the initial high levels of internalised bacteria in macrophages, indicating the presence and action of a cellular immune defence mechanism before intracellular bacterial replication took place. Low levels of bacterial internalisation and replication were detected in the epithelial-like ACL cells. The capacity of F. noatunensis subsp. noatunensis to enter, survive and even replicate within an epithelial cell line may play an important role in its ability to infect live fish and transverse epithelial barriers to reach the bacterium's main target cells-the macrophage.

Multiple specialised goose-type lysozymes potentially compensate for an exceptional lack of chicken-type lysozymes in Atlantic cod.

Previous analyses of the Atlantic cod genome showed unique combinations of lacking and expanded number of genes for the immune system. The present study examined lysozyme activity, lysozyme gene distribution and expression in cod. Enzymatic assays employing specific bacterial lysozyme inhibitors provided evidence for presence of g-type, but unexpectedly not for c-type lysozyme activity. Database homology searches failed to identify any c-type lysozyme gene in the cod genome or in expressed sequence tags from cod. In contrast, we identified four g-type lysozyme genes (LygF1a-d) constitutively expressed, although differentially, in all cod organs examined. The active site glutamate residue is replaced by alanine in LygF1a, thus making it enzymatic inactive, while LygF1d was found in two active site variants carrying alanine or glutamate, respectively. In vitro and in vivo infection by the intracellular bacterium Francisella noatunensis gave a significantly reduced LygF1a and b expression but increased expression of the LygF1c and d genes as did also the interferon gamma (IFNγ) cytokine. These results demonstrate a lack of c-type lysozyme that is unprecedented among vertebrates. Our results further indicate that serial gene duplications have produced multiple differentially regulated cod g-type lysozymes with specialised functions potentially compensating for the lack of c-type lysozymes.

Ultrapure LPS induces inflammatory and antibacterial responses attenuated in vitro by exogenous sera in Atlantic cod and Atlantic salmon.

Phagocyte recognition of lipopolysaccharide (LPS) is an early key event for triggering the host innate immune response necessary for clearance of invading bacteria. The ability of fishes to recognise LPS has been questioned as contradictory results have been presented. We show here that monocyte/macrophage cultures from Atlantic cod (Gadus morhua) and Atlantic salmon (Salmo salar) respond with an increased expression of inflammatory and antibacterial genes to both crude and ultrapure Escherichia coli LPS. Crude LPS produces higher induction than the ultrapure LPS type in both species in vitro as well as in vivo in cod injected with LPS. Crude LPS gave, in contrast to ultrapure LPS, an additional weak up-regulation of antiviral genes in salmon macrophages, most likely because of contaminants in the LPS preparation. Increased levels of chicken (c)-type lysozyme transcripts and enzyme activity were measured in salmon macrophages following ultrapure LPS stimulation demonstrating not only increased transcription but also translation. Simultaneous use and even pre-treatment with bovine sera suppressed the LPS-induced expression thereby reflecting the presence of transcription inhibitory components in sera. Together, these findings show that both cod and salmon recognise LPS per se and that the observed induction is highly dependent on the absence of sera.

Co-infection of Atlantic salmon (Salmo salar), by Moritella viscosa and Aliivibrio wodanis, development of disease and host colonization.

Two species of bacteria are repeatedly isolated from farmed fish with winter-ulcer disease. Moritella viscosa is the aetiological agent of the disease; the significance of Aliivibrio wodanis is uncertain but has not been related to the primary pathogenesis. A cell culture infection model showed that A. wodanis adhered to, but did not invade the fish cells. Exposure to culture supernatant of A. wodanis caused the fish cells to vacoulate, retract, round up and detach from the surface, and rearrange the actin filaments of the cytoskeleton. These observations suggest that the bacterium secretes toxins into the extracellular environment. Any pathologic effect of A. wodanis and the effect of co-culturing with M. viscosa was studied in Atlantic salmon (Salmo salar) bath challenged with; only M. viscosa or only A. wodanis or both bacteria together. Both M. viscosa and A. wodanis were re-isolated from external surfaces and internal organs from live and deceased co-infected fish. It is further hypothesized that A. wodanis colonization might influence the progression of a M. viscosa infection. This is to our knowledge the first study that reproduces field observations where both bacteria infect Atlantic salmon.

Response to vaccination of Atlantic cod (Gadus morhua L.) progenies from families with different estimated family breeding values for vibriosis resistance.

The purpose of the study was to elucidate whether responses to vibriosis vaccination and gene expressions in parts of the innate immune system were different in families of Atlantic cod (Gadus morhua). The fish were progenies of families with differences in estimated breeding values (EBV) for vibriosis resistance. Families of coastal cod (CC) and northeast Arctic cod (AC) responded well to vaccination with a relative percent survival of 72-95. No correlation between response to vaccination and vibriosis resistance were found (p = 0.146). The AC family with medium low (M) resistance had significant (p ≤ 0.019) lowest mortality among all the unvaccinated fish but the CC-M family. Further, when comparing the vaccinated fish the AC family with very high (VH) resistance had significant (p ≤ 0.004) higher mortality than all except the CC-VL and CC-H families. Parts of the innate immune response were studied by measuring the gene expression of innate immune genes 2 and 4 days post dip vaccination. Vaccinated fish from two families had a weak but significant higher innate immune response compared to control fish of the same family. In vaccinated fish, the gene expression of interleukin (IL) 1b, IL-10, IL-12p40 and hepcidin were significant up-regulated. While, no measureable activations of interferon gamma (IFNγ), IL-8, cathelicidin, LBP/BPI and G-type lysozyme were found.

Intracellular localisation and innate immune responses following Francisella noatunensis infection of Atlantic cod (Gadus morhua) macrophages.

The facultative intracellular bacterium Francisella noatunensis causes francisellosis in Atlantic cod (Gadus morhua), but little is known about its survival strategies or how these bacteria evade the host immune response. In this study we show intracellular localisation of F. noatunensis in cod macrophages using indirect immunofluorescence techniques and green fluorescent labelled bacteria. Transmission electron microscopy revealed that F. noatunensis was enclosed by a phagosomal membrane during the initial phase of infection. Bacteria were at a later stage of the infection found in large electron-lucent zones, apparently surrounded by a partially intact or disintegrated membrane. Immune electron microscopy demonstrated the release of bacterial derived vesicles from intracellular F. noatunensis, an event suspected of promoting phagosomal membrane degradation and allowing escape of the bacteria to cytoplasm. Studies of macrophages infected with F. noatunensis demonstrated a weak activation of the inflammatory response genes as measured by increased expression of the Interleukin (IL)-1ß and IL-8. In comparison, a stronger induction of gene expression was found for the anti-inflammatory IL-10 indicating that the bacterium exhibits a role in down-regulating the inflammatory response. Expression of the p40 subunit of IL-12/IL-17 genes was highly induced during infection suggesting that F. noatunensis promotes T cell polarisation. The host macrophage responses studied here showed low ability to distinguish between live and inactivated bacteria, although other types of responses could be of importance for such discriminations. The immunoreactivity of F. noatunensis lipopolysaccharide (LPS) was very modest, in contrast to the strong capacity of Escherichia coli LPS to induce inflammatory responsive genes. These results suggest that F. noatunensis virulence mechanisms cover many strategies for intracellular survival in cod macrophages.

Vibriosis vaccines based on various sero-subgroups of Vibrio anguillarum O2 induce specific protection in Atlantic cod (Gadus morhua L.) juveniles.

The purpose of this study was to investigate the efficacy of three monovalent and a trivalent vibriosis dip vaccines in juvenile Atlantic cod (Gadus morhua L.), examine whether the responses were specific and study the expression of selected immune genes after dip vaccination. In addition, the study addressed whether the deviating isolates of Vibrio anguillarum serotype O2 belongs to another sero-subgroup than the previously established sero-subgroups O2a, O2b and O2c. Rabbit V. anguillarum serotype O2 antiserum adsorbed with V. anguillarum O2a O-antigen was shown, by both ELISA and immunoblotting, to still contain serotype O2 specific antibodies. Cod V. anguillarum serotype O2 antiserum reacted only with isolate of homologous serotype and not with heterologous sero-subgroups. This indicates that the deviating V. anguillarum O2 isolates represent a new sero-subgroup differing from sero-subgroup O2a. The monovalent vaccines included formalin inactivated cultures of V. anguillarum sero-subgroup O2a, O2b or serotype O2, while the trivalent vaccine contained all three sero-subgroups. Cod mounted high protection 7 weeks post dip vaccination with monovalent vaccines when challenged with homologous isolates and significantly lower when challenged with heterologous isolates, regardless of sero-subgroups. The trivalent vaccine resulted in efficient protection against all sero-subgroups tested. Dip vaccination of cod juveniles did not result in detectable antibody production or alteration in gene expression of the heavy chain of IgM and IgD. In the trivalent vaccine group expression of IFNγ and IL-12p40 were significantly up-regulated 3 days post vaccination. However, in groups vaccinated against V. anguillarum sero-subgroups O2b or O2, IL-12p40 and IFNγ gene expression were slightly increased 3 and 55 days post vaccination, respectively.

Impact of reattaching various Aeromonas salmonicida A-layer proteins on vaccine efficacy in Atlantic cod (Gadus morhua).

Atypical Aeromonas salmonicida causes atypical furunculosis in a whole range of farmed fish species. The bacterium comprises a heterogeneous group differing in surface components such as the A-layer protein and O-chain polysaccharide structures. Previously, the A-layer protein was shown to contribute to protective immunity as a vaccine with A. salmonicida cells with reattached A-layer protein protecting significantly better than the corresponding A-layer deficient bacteria used in the Atlantic cod (Gadus morhua) vaccine. In the present study, genetically different A. salmonicida A-proteins, either as preformed A-layer sheets from culture supernatants or as purified preparations, were shown to attach to A-layer deficient isolates with a different O-chain structure. Only vaccines containing A. salmonicida cells with reattached A-protein genetically homologous to the challenge isolate, elicited protection comparable to that of the homologous vaccine.

Atypical furunculosis vaccines for Atlantic cod Gadhus morhua: impact of reattached Aeromonas salmonicida A-layer protein on vaccine efficacy.

Atypical furunculosis caused by atypical Aeromonas salmonicida bacteria is reported as an increasing problem in farmed Atlantic cod Gadus morhua in Norway. At present, furunculosis vaccines adapted for cod or other marine fish species are not available. To identify bacterial components important for inducing protection in cod, we compared oil-adjuvanted vaccines based on A. salmonicida isolates phenotypically differing in their major cell surface constituents, such as the A-layer protein and lipopolysaccharide O-chains. Also included was an A-layer-deficient isolate with physically reattached A-layer protein. Vaccines containing A. salmonicida A-layer-producing cells elicited significantly better protection than vaccines with A-layer-deficient cells or with a supernatant with secreted A-layer protein. The A. salmonicida cells with reattached A-layer-protein resulted in significant and equal protection to the A-layer-producing cells and protected significantly better than the A-layer-deficient isolate. These results indicate that the A-layer protein when attached to the cell surface plays a role in inducing protective immunity in cod.

Comparison of antibody responses in Atlantic cod (Gadus morhua L.) to Vibrio anguillarum, Aeromonas salmonicida and Francisella sp.

Bacterial diseases such as vibriosis, atypical furunculosis and francisellosis, are registered as an increasing problem in cod farming in Norway. In order to develop efficient vaccines against diseases it is of interest to investigate if the cod immune system differentiates between various serotypes of Vibrio anguillarum and variants of Aeromonas salmonicida associated with the diseases by raising specific antibody responses. Cod of the same origin were shown to raise significant responses to V. anguillarum, A. salmonicida and the intracellular bacteria Francisella sp. Individual responses to V. anguillarum or A. salmonicida varied from none to high responses, while all individuals immunised with Francisella revealed a significant response. The cod immune system appeared in some degree to distinguish between V. anguillarum serotypes and A. salmonicida variants. Although all bacteria had induced significant antibody responses detectable in whole cell ELISA, only some had induced antibodies with specificity to linear O-polysaccharide epitopes on blot.

Atypical furunculosis vaccines for Atlantic cod (Gadus morhua); vaccine efficacy and antibody responses.

Atypical furunculosis caused by atypical Aeromonas salmonicida, is an emerging problem in farming of Atlantic cod (Gadus morhua) in Norway, and vaccines are needed. Atypical A. salmonicida comprises a heterogeneous group of bacteria differing in surface antigens such as the A-layer protein and lipopolysaccharides (LPS). Except for one of the experimental oil-adjuvanted whole cell vaccines based on various isolates they all resulted in moderate protection. No clear correlation between vaccine efficacies and the A-protein group or LPS type of the vaccine isolates was revealed, while a correlation between efficacy and the presence of cross-reacting LPS-specific antibodies is indicated.

Comparison of atypical furunculosis vaccines in spotted wolffish (Anarhicas minor O.) and Atlantic halibut (Hippoglossus hippoglossus L.).

Atypical furunculosis caused by atypical Aeromonas salmonicida is a problem in farming of Atlantic halibut (Hippoglossus hippoglossus) and spotted wolffish (Anarhicas minor) in Norway, and vaccines for marine fish species are not available. Susceptibility to atypical A. salmonicida infection and efficacy of furunculosis vaccines in various fish species indicate that the host responses differ. Here, spotted wolffish was shown to be highly susceptible to intraperitoneal challenge with atypical A. salmonicida compared to halibut, and the two species appeared to respond differently to identical vaccines. Grouping of the atypical A. salmonicida isolates used as vaccines was based on a variable region in the surface A-layer protein, but no clear correlation between vaccine efficacy and A-protein group was found.

Adaptive response to environmental changes in the fish pathogen Moritella viscosa.

The marine psychrophilic bacterium Moritella viscosa is the causative agent of winter ulcer in farmed Atlantic salmon and cod. In this study, the growth requirements of the pathogen were established. The effects of changes in salinity and temperature on growth, surface features and proteomic regulation were also investigated. The genome of this bacterium has not yet been sequenced; therefore, comparative two-dimensional gel electrophoresis (2-DE) was used, coupled with high performance tandem mass spectrometry (MS/MS), to perform cross-species protein identification. Results from this study establish that M. viscosa is a true marine psychrophilic bacterium capable of surviving and proliferating in an oligotrophic and cold environment. Low temperature combined with 3-4% NaCl resulted in significantly higher cell yields and stability compared to high temperature and 1% NaCl. Nine cytoplasmic proteins were shown to be regulated by temperature and 12 by salinity. Several of the regulated proteins indicated a stressful situation at 15 degrees C compared to 4 degrees C, consistent with the growth characteristics observed. Furthermore, temperature and salinity were demonstrated to be important determinants of motility and viscosity of M. viscosa.

Comparison of antibody responses in Atlantic cod (Gadus morhua L.) to Aeromonas salmonicida and Vibrio anguillarum.

The immune system of Atlantic cod (Gadus morhua L.) differ from other bony fish species in that no or only very low increases in antibody levels are detected post-immunization with Vibrio salmonicida or V. anguillarum. Here, we report the results from the first study on comparison of antibody responses in cod to Aeromonas salmonicida and V. anguillarum. A. salmonicida appear to induce a stronger antibody response in cod compared to V. anguillarum, and more individuals immunized with A. salmonicida revealed a response compared to those immunized with V. anguillarum. The antibody responses to both bacterial species were mainly toward LPS, and the results indicate that cod antibodies are able to differentiate between LPS antigens from typical and atypical A. salmonicida strains.

Genetic diversity among A-proteins of atypical strains of Aeromonas salmonicida.

The virulence array protein gene A (vapA) encoding the A-protein subunit of the surface layer of 23 typical and atypical strains of Aeromonas salmonicida from salmonids and marine fish species were sequenced, and the deduced A-protein sequences compared. The A-proteins of the typical A. salmonicida ssp. salmonicida strains were shown to be identical, while amino acid variability was revealed among A-proteins of atypical strains. The highest amino acid variability appears to be in a predicted surface exposed region and is believed to result in antigenic differences among the atypical strains of A. salmonicida.

Vaccine efficacy in spotted wolffish Anarhichas minor: relationship to molecular variation in A-layer protein of atypical Aeromonas salmonicida.

Atypical Aeromonas salmonicida strains comprise a heterogeneous group in terms of molecular and phenotypic characteristics. They cause various conditions of ulcer diseases or atypical furunculosis and are being isolated in increasing number from various fish species and geographical areas. Several marine fish species susceptible to atypical A. salmonicida, including spotted wolffish Anarhichas minor O., are now being farmed and new vaccines may be needed. A commercial furunculosis vaccine for salmon is reported to protect wolffish poorly against experimental challenge with atypical A. salmonicida. The protective antigen(s) in furunculosis vaccines is still unclear, but in oil-adjuvanted vaccine for Atlantic salmon Salmo salar L., the surface A-layer was shown to be important for protection. In spotted wolffish, the efficacy of atypical furunculosis vaccines seems to vary with the atypical A. salmonicida strains used as bacterin in the vaccine. In the present study we investigated whether differences in the A-layer protein among atypical strains might be responsible for the observed variation in vaccine efficacy. Atypical A. salmonicida strains from 16 fish species in 11 countries were compared by genome polymorphism analysis using amplified fragment length polymorphism (AFLP) fingerprinting and by comparative sequencing of the vapA genes encoding the A-protein. The A-protein sequences appeared to be highly conserved except for a variable region between Residues 90 to 170. Surprisingly, the grouping of strains based on AFLP- or A-protein sequence similarities was consistent. In addition, serological differences in the A-protein among the strains were demonstrated by an A-protein-specific monoclonal antibody. Vaccines based on atypical A. salmonicida strains possessing genetically and serologically different A-layer proteins were shown to result in significantly different protection in spotted wolffish.