Bacterial Fish Diseases Research Articles

Temperature-dependent expression of virulence genes in fish-pathogenic bacteria.

Virulence gene expression in pathogenic bacteria is modulated by environmental parameters. A key factor in this expression is temperature. Its effect on virulence gene expression in bacteria infecting warm-blooded hosts is well documented. Transcription of virulence genes in these bacteria is induced upon a shift from low environmental to a higher host temperature (37°C). Interestingly, host temperatures usually correspond to the optimum for growth of these pathogenic bacteria. On the contrary, in ectothermic hosts such as fish, molluscs, and amphibians, infection processes generally occur at a temperature lower than that for the optimal growth of the bacteria. Therefore, regulation of virulence gene expression in response to temperature shift has to be modulated in a different way to that which is found in bacteria infecting warm-blooded hosts. The current understanding of virulence gene expression and its regulation in response to temperature in fish-pathogenic bacteria is limited, but constant extension of our knowledge base is essential to enable a rational approach to the problem of the bacterial fish diseases affecting the aquaculture industry. This is an interesting issue and progress needs to be made in order to diminish the economic losses caused by these diseases. The intention of this review is, for the first time, to compile the scattered results existing in the field in order to lay the groundwork for future research. This article is an overview of those relevant virulence genes that are expressed at temperatures lower than that for optimal bacterial growth in different fish-pathogenic bacteria as well as the principal mechanisms that could be involved in their regulation.

Differential solvent extraction of two seaweeds and their efficacy in controlling Aeromonas salmonicida infection in Oreochromis mossambicus: A novel therapeutic approach

Abstract The present study uses two differential extraction methods (sequential, maceration) to identify bioactive compounds present in two seaweeds Gracilaria folifera and Sargassum longifolium . Five different solvents namely Petroleum ether, Dichloromethane, Chloroform, ethanol and water were used. The bioactive compounds present in the extracts showed antioxidant and antibacterial activities against Aeromonas salmonicida . Ethanol and aqueous extracts of these seaweeds were found to have better antibacterial activity against bacterial diseases in fish when compared to the other extracts. Toxicological and in vivo pathogenicity studies were performed with the highest concentration of 250 mg/l of extracts. The compounds identified by GC–MS which were found to have antibacterial activity were prepared in the form of a colloidal suspension for oral treatment. The use of algal extracts in aquaculture disease management is a cost effective and eco-friendly approach. These extracts could be used as prophylactics.

Identification of Bacterial Fish Pathogens in Brazil by Direct Colony PCR and 16S rRNA Gene Sequencing

Intensive fish farming systems in Brazil have increased the disease incidence, mainly of bacterial origin, due to higher stocking density, high organic matter levels and poor quality of the aquatic environment that causes high mortality rates during outbreaks. The identification of pathogenic species using a fast and reliable method of diagnosis is essential for successful epidemiological studies and disease control. The present study evaluated the use of direct colony PCR in combination with 16S rRNA gene sequencing to diagnose fish bacterial diseases, with the goal of reducing the costs and time necessary for bacterial identification. The method was successful for all 178 isolates tested and produced bands with the same intensity as the standard PCR performed using pure DNA. In conclusion, the genetics methods allowed detecting the most common and important pathogens in Aquaculture, including 12 species of occurrence in Brazilian fish farms. The results of the present study constitute an advance in the available diagnostic methods for bacterial pathogens in fish farms.

Invasion ability and disease dynamics of environmentally growing opportunistic pathogens under outside-host competition.

Most theories of the evolution of virulence concentrate on obligatory host-pathogen relationship. Yet, many pathogens replicate in the environment outside-host where they compete with non-pathogenic forms. Thus, replication and competition in the outside-host environment may have profound influence on the evolution of virulence and disease dynamics. These environmentally growing opportunistic pathogens are also a logical step towards obligatory pathogenicity. Efficient treatment methods against these diseases, such as columnaris disease in fishes, are lacking because of their opportunist nature. We present a novel epidemiological model in which replication and competition in the outside-host environment influences the invasion ability of a novel pathogen. We also analyze the long-term host-pathogen dynamics. Model parameterization is based on the columnaris disease, a bacterial fresh water fish disease that causes major losses in fish farms worldwide. Our model demonstrates that strong competition in the outside-host environment can prevent the invasion of a new environmentally growing opportunist pathogen and long-term disease outbreaks.

Probiotics, immunostimulants, plant products and oral vaccines, and their role as feed supplements in the control of bacterial fish diseases.

There is a rapidly increasing literature pointing to the success of probiotics, immunostimulants, plant products and oral vaccines in immunomodulation, namely stimulation of the innate, cellular and/or humoral immune response, and the control of bacterial fish diseases. Probiotics are regarded as live micro-organisms administered orally and leading to health benefits. However, in contrast with the use in terrestrial animals, a diverse range of micro-organisms have been evaluated in aquaculture with the mode of action often reflecting immunomodulation. Moreover, the need for living cells has been questioned. Also, key subcellular components, including lipopolysaccharides, have been attributed to the beneficial effect in fish. Here, there is a link with immunostimulants, which may also be administered orally. Furthermore, numerous plant products have been reported to have health benefits, namely protection against disease for which stimulation of some immune parameters has been reported. Oral vaccines confer protection against some diseases, although the mode of action is usually linked to humoral rather than the innate and cellular immune responses. This review explores the relationship between probiotics, immunostimulants, plant products and oral vaccines.

Candidate Probiotic Bacteria의 어류병원성 Streptococcus sp. 성장에 대한 억제 효과

International Commerce and Trade Division, Ministry of Oceans and Fisheries, Sejong si 339-012, Korea Department of Aqualife Medicine, Chonnam National University, Yeosu 550-749, KoreaFor the treatments and protection of bacterial fish disease, many requirements are needed for aquatic probiotics so that they are effective in aquaculture animals but are also harmless to humans. In the present study, among 17 candidate probiotic bacteria (CPB) obtained from the edible part of the shellfish, Bacillus sp. CPB-St (CPB-St) were selected and in vitro evaluated for the possibility as a probiotic strain for the control of fish streptococcosis which frequently occurs in the olive flounder farms. CPB-St showed inhibitory effects on the growth of various fish pathogenic bacteria, Streptococcus sp., S. parauberis, S. iniae, Lactococcus garvieae and L. piscium by the double layer test ranging about 18～26 mm of clear zone. Inhibitory activity of CPB-St to Streptococcus sp. was observed 6 hours after and the growth of Streptococcus sp. was decreased to 8～55 folds in the co-culture of CPB-St with Streptococcus sp.. The safety of CPB-St to fish and survival of CPB-St in the intestine were assessed in the olive flounder, Paralichthys olivaceus. Fish mortality was not observed in artificial infection with CPB-St for 2 weeks. CPB-St was entirely excreted from the stomach and intestine 24 hours after oral injection. This results indicate that CPB-St has potential applications as a probiotic for the control of fish streptococcosis in aquaculture. Key words: Candidate probiotic bacteria (CPB), Bacillus sp., Oyster, Streptococcus spp., Antibacterial effect

Molecular cloning, expression analysis and functional characterization of interleukin-22 in So-iny mullet, Liza haematocheila

In the present study, interleukin-22 (IL-22) from So-iny mullet (Liza haematocheila) was identified, and its tissue expression in both healthy and Streptococcus dysgalactiae-infected fish was examined. The full length cDNA sequence of mullet IL-22 was 1070bp, containing an open reading frame of 555bp. The deduced amino acid sequence shared high similarity (45.1–67.9%) with IL-22 from other fish species. Mullet IL-22 also contained an IL-10 family signature and four cysteine residues that were well conserved in other vertebrate IL-22 molecules. Mullet IL-22 mRNA was highly expressed in kidney, moderately expressed in liver and gut, and relatively weakly expressed in spleen, and its expression was significantly up-regulated in all the examined tissues following S. dysgalactiae infection. Furthermore, recombinant mullet IL-22 protein was shown to promote the expression of β-defensin in the four tissues and to increase the survival rate of the fish infected with S. dysgalactiae. Our results suggest mullet IL-22 plays an important role in the immune defense against bacterial infection and has the potential to be used to treat bacterial diseases in fish.

Quorum quenching bacteria Bacillus sp. QSI-1 protect zebrafish (Danio rerio) from Aeromonas hydrophila infection.

Quorum Sensing (QS) is a bacterial regulatory mechanism, which is responsible for controlling the expression of various biological macromolecules such as the virulence factors in a cell density-dependent manner. Disruption of the QS system of pathogens has been proposed as a new anti-infective strategy. Biodegradation of AHLs proves to be an efficient way to interrupt QS, since AHLs are the main family of QS autoinducers used in Gram negative bacteria. In this study, the effect of Bacillus sp. QSI-1 as an efficient quorum quencher on virulence factors production and biofilm formation of fish pathogen Aeromonas hydrophila was investigated. QSI-1 reduced the accumulation of AHLs but did not affect the growth of A. hydrophila YJ-1 when cocultured. In the result, the supernatant of QSI-1 showed significant inhibition of protease production (83.9%), hemolytic activity (77.6%) and biofilm formation (77.3%) in YJ-1. In biocontrol experiment, QSI-1 significantly reduced the pathogenicity of A. hydrophila strain YJ-1 in zebrafish (Danio rerio). The fish fed with QSI-1 was observed to have a relative percentage survival of 80.8%. Our results indicate that AHLs degrading bacteria should be considered as an alternative for antibiotics in aquaculture for the biocontrol of bacterial fish diseases.

Combined effects of Iranian heavy crude oil and bacterial challenge (Streptococcus iniae) on biotransformation and innate immune responses in rockfish (Sebastes schlegeli).

To clarify the effects of spilled crude oil on fish bacterial disease resistance, rockfish (Sebastes schlegeli) were exposed to Iranian Heavy crude oil (IHCO) and Streptomyces iniae in combination. Hepatic biotransformation enzymes (ethoxyresorufin O-de-ethylase, glutathione-S-transferase) and plasma biochemical parameters (glutamic oxaloacetic transaminase, glutamic pyruvic transaminase and glucose) in fish exposed to IHCO were not significantly different from those in unexposed fish. The level of biliary 1-OH-pyrene and cytochrome P4501A mRNA expression increased in a dose-dependent manner with IHCO exposure. The interferon stimulated gene 15, interleukin-1beta and cathepsin L were increased significantly in the liver in IHCO-exposed fish, but not dose-dependently, but the granulocyte colony stimulating factor was not related to IHCO exposure. The percentage mortality in fish following a single exposure to S. iniae was positively correlated with IHCO exposure concentration. We concluded that IHCO exposure exacerbates fish mortality following environmental bacterial infection.

Oral and Anal Vaccination Confers Full Protection against Enteric Redmouth Disease (ERM) in Rainbow Trout

The effect of oral vaccines against bacterial fish diseases has been a topic for debate for decades. Recently both M-like cells and dendritic cells have been discovered in the intestine of rainbow trout. It is therefore likely that antigens reaching the intestine can be taken up and thereby induce immunity in orally vaccinated fish. The objective of this project was to investigate whether oral and anal vaccination of rainbow trout induces protection against an experimental waterborne infection with the pathogenic enterobacteria Yersinia ruckeri O1 biotype 1 the causative agent of enteric redmouth disease (ERM). Rainbow trout were orally vaccinated with AquaVac ERM Oral (MERCK Animal Health) or an experimental vaccine bacterin of Y. ruckeri O1. Both vaccines were tested with and without a booster vaccination four months post the primary vaccination. Furthermore, two groups of positive controls were included, one group receiving the experimental oral vaccine in a 50 times higher dose, and the other group receiving a single dose administered anally in order to bypass the stomach. Each group was bath challenged with 6.3×108 CFU/ml Y. ruckeri, six months post the primary vaccination. The challenge induced significant mortality in all the infected groups except for the groups vaccinated anally with a single dose or orally with the high dose of bacterin. Both of these groups had 100% survival. These results show that a low dose of Y. ruckeri bacterin induces full protection when the bacterin is administered anally. Oral vaccination also induces full protection, however, at a dose 50 times higher than if the fish were to be vaccinated anally. This indicates that much of the orally fed antigen is digested in the stomach before it reaches the second segment of the intestine where it can be taken up as immunogenic antigens and presented to lymphocytes.

Identification of Vibrio harveyi, Vibrio ichthyoenteri, and Photobacterium damselae isolated from olive flounder Paralichthys olivaceus in Korea by multiplex PCR developed using the rpoB gene

Major fish bacterial diseases in Korea are edwardsiellosis, streptococcosis, and vibriosis. Among vibrionaceae, Listonella anguillarum, Vibrio harveyi, V. ichthyoenteri, and Photobacterium damselae were identified as causative organisms of vibriosis in flounder. In this study, we developed a multiplex PCR method using the RNA polymerase β subunit (rpoB) gene, known as a housekeeping gene for identification of Vibrio spp. causing vibriosis in flounder. Three pairs of PCR primers were designed based on the rpoB sequence of three species, V. harveyi, V. ichthyoenteri, and P. damselae. The PCR assay, using a mixture of six primers, yielded amplicons of 601, 434, and 533 bp in V. harveyi, V. ichthyoenteri, and P. damselae. None of the untargeted species yielded an amplicon. The detection limits for pure culture in kidney were 2.5 × 104 cfu/g kidney for V. harveyi, 2.5 × 105 cfu/g kidney for V. ichthyoenteri, and 2.5 × 106 cfu/g kidney for P. damselae. From the colonies on TCBS agar plates of different samples, 632 Vibrio spp. isolated from aquacultured flounder between 2004 and 2010 were identified by the multiplex PCR method. As a result, 265 strains (41.9 %) were V. ichthyoenteri; 115 strains (18.2 %) were V. harveyi and 72 strains (11.4 %) were P. damselae.

The use of eugenol against Aeromonas hydrophila and its effect on hematological and immunological parameters in silver catfish (Rhamdia quelen)

The aim of this study was to evaluate the activity of eugenol against the fish pathogen Aeromonas hydrophila and eugenol's effect on hematological and natural immune parameters in silver catfish (Rhamdia quelen). In vitro, eugenol showed weak activity against A. hydrophila, but in vivo, at a subinhibitory concentration (10mgL−1), it promoted survival in infected silver catfish. Eugenol (50μgmL−1) reduced the hemolytic activity of A. hydrophila supernatant in vitro in fish erythrocytes. Subjecting catfish to eugenol baths (5 and 10mgL−1) for five days did not alter the hematological and immunological parameters studied in this work. Based on these results, eugenol can be used to treat or prevent bacterial diseases in fish.

Immune responses evoked by infection with Vibrio anguillarum in zebrafish bath-vaccinated with a live attenuated strain

Live attenuated vaccines are a promising application to control bacterial fish diseases. A live attenuated Vibrio anguillarum vaccine candidate was established in our laboratory to protect fish against vibriosis. To elucidate the mechanism of immunoprotection, it is necessary to compare the different immune responses to infection between vaccinated and non-vaccinated fish. In this study, the expression levels of pathogen-specific antibodies and immune-related genes upon challenge at 28 days post-vaccination were compared between vaccinated and non-vaccinated zebrafish. In the results, the specific antibody levels against virulent V. anguillarum in the vaccinated group did not rise significantly following infection, which suggested that high-affinity antibodies were induced by the vaccine. In the non-vaccinated group, the specific IgM response was triggered at 3 days post-infection and showed a delayed antibody response. Meanwhile, the transcription levels of the genes encoding the pro-inflammatory cytokine IL-1β and the chemokine IL-8 were more highly up-regulated in non-vaccinated fish than in vaccinated fish. This suggests that the overwhelming inflammatory response trigged by infection in non-vaccinated zebrafish was controlled in vaccinated zebrafish. Interestingly, the expression levels of adaptive immune-related genes were increased in vaccinated fish after challenge, compared to the non-vaccinated fish. These results suggest that inoculation with the live attenuated vaccine triggered protection by curbing inflammation and strengthening the adaptive immune response.

Immune modulatory effects of Aloe arborescens extract on the piscine SAF-1 cell line

The pharmacological potential of Aloe arborescens Miller leaf components was investigated, with special attention deserved to immune modulatory effects on the Sparus aurata fibroblast cell line SAF-1. The cells were treated with Aloe extract at different concentrations (1.2–4.8 mg ml−1) for various times (24–72 h). The lowest concentration did not provoke any cellular damage observable by SEM and did not affect ATP amounts after 24 and 48 h, while even induced a significant increase over controls after 72 h. We next examined the transcription kinetics of different immune-related genes (IL-1β, TGF-β, TNF-α, COX-2, IFN-I, Mx and MHCI-α) in SAF-1 cells stimulated with LPS or poly I:C. The Aloe extract (1.2 mg ml−1) acted as a powerful immune stimulant in LPS- or poly I:C-activated SAF-1 cells, inducing a synergic effect on interconnected genes that are expected to be involved in different aspects of the immune responses.These reports provide a new perspective for the use of A. arborescens to prevent or oppose bacterial and viral fish diseases and to face, as an alternative strategy based on natural plant extracts, the growing unwillingness to rely upon standard solutions involving antibiotics or antimicrobial chemicals.

Gene expression profiling in live attenuated Edwardsiella tarda vaccine immunized and challenged zebrafish: Insights into the basic mechanisms of protection seen in immunized fish

Despite the importance and success of vaccine immunization against bacterial diseases in fish, little is known about the molecular mechanisms of vaccine-induced immune protection in teleost fish. In this study, the live attenuated Edwardsiella tarda vaccine strain WED, which has been shown to evoke efficacious protection against edwardsiellosis and ascites diseases in fish, was extensively evaluated for multiple parameters in a 5-week immunization and challenge experiment in zebrafish. The parameters evaluated included the immunologic potency (relative percent survival, RPS), the specific IgM antibody titers and the expression profiles of multiple immune-related gene markers at multiple time points following immunization and challenge. During the 4-week immunization phase, the toll-like receptor (TLR) 5 signaling pathway, the MHC-I antigen processing pathway and cytotoxic T lymphocyte (CTL) responses were activated in succession. In contrast, the MHC-II antigen processing pathway and the markers of CD4+ T lymphocyte activation were down-regulated, and IgM transcription and specific IgM antibody titers were not significantly induced following immunization. During the 1-week challenge phase, the induction of MHC-I and CTL responses and the inhibition of MHC-II and CD4+ T cell responses were similarly observed in immunized zebrafish following challenge with wild E. tarda. With the 5-week immunization and challenge model, our data suggest the basic mechanism that underlying the long-lasting protective immunity elicited by WED in zebrafish. This mechanism involved the induction of the TLR-5 signaling pathway, the MHC-I antigen processing pathway and CTL effector function, and CTL function seems play a major role in the protection against E. tarda infection in zebrafish.

Isolation and Identification of Flavobacterium columnare and Streptococcus iniae Antibacterial Compounds from the Terrestrial Plant Atraphaxis laetevirens

Columnaris disease, enteric septicemia of catfish, and streptococcosis are common bacterial diseases of certain freshwater fish and are caused by Flavobacterium columnare , Edwardsiella ictaluri , and Streptococcus iniae , respectively. During the process of evaluating several species of plants to isolate and identify compounds with toxicity against these bacteria, a promising extract from the aerial parts of the terrestrial plant Atraphaxis laetevirens (Ledeb.) Jaub. et Spach (Polygonaceae Juss.) was selected for bioassay-guided fractionation using a rapid microplate bioassay. The active dichloromethane extract was subjected to liquid-liquid partitioning, and active fractions were further separated by normal-phase column chromatography and normal-phase high-performance liquid chromatography (HPLC). Nepodin (3) and emodin (4) were isolated from two fractions with strong toxicities against S. iniae . A chloroform fraction was further separated by normal-phase column chromatography to yield two active fractions against F. columnare , and these fractions contained chrysophanol (1), physcion (2), and nepodin (3). Compound 1 had strong activity, and compound 3 had moderate activity against F. columnare , while compounds 2 and 4 were not toxic at the concentrations tested.

수입산 담수관상어 및 양식 해산어의 장내세균에서 나타나는 내성균 위험성 비교

Various antibiotics, that could induce the appearance of resistant microorganisms, have been used for treatment or prevention of bacterial diseases in marine and ornamental fish. We determined and characterized the level of antibiotic-resistant bacteria and proportion of multi-drug resistant bacteria in intestinal microflora of both marine fish cultured in Korea and imported ornamental freshwater fish. For this the bacterial species and resistance to antibiotics were investigated in intestine of rock bream Oplegnathus fasciatus cultured in Korea and pearl gourami Trichogaster leeri imported from Singapore to characterise. Although the bacterial species were different, proportions of resistant bacteria to single antibiotics or multi-drug were higher in intestinal microflora of pearl gourami Trichogaster leeri imported from Singapore than in rock bream Oplegnathus fasciatus cultured in Korea. These results indicate that various antibiotics have been being used before trading without measures in the market of asian ornamental fishes, providing high risks for the emergence of multi-drug resistant bacteria.

Identification of surrogates of protection against yersiniosis in immersion vaccinated Atlantic salmon.

Simple cost-effective bacterins are the earliest and most successfully used commercial vaccines in fish. In particular, those prepared from Yersinia ruckeri have proven effective at controlling Enteric Red Mouth Disease (ERM) and yersiniosis in rainbow trout and Atlantic salmon, respectively. However, the emergence of outbreaks of ERM caused by atypical biotypes of Y. ruckeri and reports of vaccine failure resulting in mass mortality of hatchery Atlantic salmon has reinvigorated interest in vaccines against fish bacterial diseases. Therefore the objective of this study was to identify surrogates of protection against yersiniosis using cDNA microarray to characterise the response of host genes in the gills of unvaccinated and vaccinated Atlantic salmon challenged with Y. ruckeri. Differentially expressed genes were identified using two-way ANOVA and restricted to those with >2.5-fold change at P<0.05. Using cDNA microarray we identified the expression of 6 genes in response to infection and 4 genes associated with the protective host response to yersiniosis. Analysis by real-time PCR confirmed that three immunologically relevant genes, namely a cathelicidin (47-fold) and a C-type lectin (19-fold) increased in response to yersiniosis. Including collagenase (17-fold increase), an important tissue remodelling and repair enzyme, these genes represent 3 of 6 non-protective and/or pathological responses to yersiniosis. Genes associated with the protective host response included an immunoglobulin gene and a selenoprotein that showed significant fold changes (15-fold increases each), highlighting the importance of antibody-mediated protection against yersiniosis. These findings provide much needed knowledge of the host-pathogen interaction in response to bacterial infection and immunisation in fish. Significantly, we identified a transcriptional biosignature consisting of predominantly immune-relevant genes (14 up and 3 down-regulated) in the gills of Atlantic salmon after immersion vaccination and before bacterial challenge. This biosignature may be used as a surrogate of protection and therefore as a predictor of vaccine success against yersiniosis.

Characteristics of Disease Spectrum in relation to Species, Serogroups, and Adhesion Ability of Motile Aeromonads in Fish

An attempt was made to delineate the relationship between of Aeromonas species and/or serogroups and specific disease symptoms in common carp Cyprinus carpio L. and rainbow trout Oncorhynchus mykiss Walbaum. The adhesion of Aeromonas strains to various tissues in relation to disease spectrum was also tested. All strains of A. hydrophila caused skin ulcers as well as septicaemia in both carp and trout while the other strains were able to cause only skin ulcers or some specific internal lesions with or without septicaemia depending on which species and/or serogroup they represented. Disease symptoms depended also on fish species. It was found that adhesion intensity of Aeromonas strains tested was significantly higher to tissues, which were susceptible to infection with these strains. The results indicate that adhesion to various cells of the fish organism is principal marker to detect virulent Aeromonas strains. The findings presented in this study may be helpful in the appraisal of aeromonads disease risk and kind of the infection in particular fish farms by epizootiological studies or/and during routine fish examinations. They will also be useful to improve and facilitate diagnosis of bacterial fish disease.

Gene expression profiling in naïve and vaccinated rainbow trout after Yersinia ruckeri infection: Insights into the mechanisms of protection seen in vaccinated fish

Despite the importance and success of vaccination against bacterial diseases in fish, little is known about the mechanisms of vaccine-induced disease resistance. In this study a known efficacious bacterial vaccine, to Enteric Redmouth Disease (ERM), was used to vaccinate rainbow trout, and sixty days later the fish were challenged with the causative agent of the disease, Yersinia ruckeri. The bacterial burden in the spleen, the spleen index, and the expression profiles of pro- and anti-inflammatory cytokines and marker genes for T helper (Th) cells in the spleen and gills were analyzed, in comparison to the profiles in naïve/challenged fish. As expected, the bacterial burden in the spleen of naïve fish increased over time and was correlated with the spleen index after Y. ruckeri challenge. The gene expression data showed that pro-inflammatory cytokines were upregulated post-infection in the spleen of both naïve and vaccinated fish after Y. ruckeri challenge although the pro-inflammatory cytokine expression was much lower in vaccinated fish compared to the naïve fish. A correlated expression between pro-inflammatory cytokines and anti-inflammatory cytokines was only seen in spleen of ERM vaccinated fish, where a Th1-like response was indicated by the correlated gene expression of IFN-γ, T-bet and IL-2. In contrast, in the gills, the inflammatory gene response was enhanced in vaccinated fish compared to naïve fish, but perhaps more importantly there was a strong upregulation of IL-22 which was negatively correlated with IFN-γ gene expression at this site. Thus, it is possible that different types of adaptive responses are on-going within the vaccinated fish during infection with Y. ruckeri, potentially affected by the site and stage of infection.