Christopher Marlowe A. Caipang

Intraperitoneal vaccination of Atlantic cod, Gadus morhua with heat-killed Listonella anguillarum enhances serum antibacterial activity and expression of immune response genes.

Serum-mediated reduction in bacterial count and expression of a number of immune response genes in the blood of Atlantic cod, Gadus morhua were investigated following intraperitoneal vaccination with heat-killed Listonella (Vibrio) anguillarum. Blood was collected from the caudal vein of both vaccinated and non-vaccinated (PBS-injected) fish at 0, 1, 3, 7 and 10 days post-vaccination (dpv). Serum protein concentration and antibacterial activity of the serum samples were determined. Whole blood was used for semi-quantitative RT-PCR of immune-related genes. Total serum protein was not significantly different between the vaccinated and non-vaccinated groups. Sera from the vaccinated fish significantly reduced L. anguillarum count on 3 dpv, with reductions of at least 2 log colony forming units per ml (CFU/ml) relative to the non-vaccinated fish. Expression of antibacterial genes, bactericidal/permeability-increasing protein/lipopolysaccharide-binding protein (BPI/LBP), g-type lysozyme and transferrin was significantly upregulated in the vaccinated fish, with maximum expression within 7 dpv. Cytotoxic-related and cell-mediated immunity genes such as, apolipoprotein A-I and the non-specific cytotoxic cell receptor protein (NCCRP-1) had maximum expression at 3 and 7 dpv, respectively. Significant upregulation in expression of pro-inflammatory cytokines, IL-1 beta and IL-8 was also observed in the vaccinated fish at 1 dpv. The upregulation of immune response genes following vaccination provides valuable information in the understanding of immune mechanisms against vibriosis in Atlantic cod particularly on the acute phase response during bacterial infection.

Proteome reference map of the skin mucus of Atlantic cod (Gadus morhua) revealing immune competent molecules

The skin mucosal proteome of Atlantic cod (Gadus morhua) was mapped using a 2D PAGE, LC-MS/MS coupled approach. Mucosal proteins from naive fish were identified primarily by similarity searches across various cod EST databases. The identified proteins were clustered into 8 groups based on gene ontology classification for biological process. Most of the proteins identified from the gel are hitherto unreported for cod. Galectin-1, mannan binding lectin (MBL), serpins, cystatin B, cyclophilin A, FK-506 binding protein, proteasome subunits (alpha-3 and -7), ubiquitin, and g-type lysozyme are considered immune competent molecules. Five of the aforementioned proteins were cloned and their tissue distribution was analysed by RT-PCR.

Infection-induced changes in expression of antibacterial and cytokine genes in the gill epithelial cells of Atlantic cod, Gadus morhua during incubation with bacterial pathogens.

The molecular processes of immune responses in mucosal tissues, such as the gills, during infection with bacterial pathogens are poorly understood. In the present study, we analyzed the transcriptional profiles of selected antibacterial genes and cytokines in the gills of a cold-water fish, Atlantic cod, Gadus morhua following in vitro infection with bacterial pathogens, Vibrio anguillarum and atypical Aeromonas salmonicida using semi-quantitative RT-PCR. There was significant upregulation in the transcripts of the antibacterial genes: bactericidal permeability-increasing protein/lipopolysaccharide-binding protein (BPI/LBP), g-type lysozyme, transferrin, metallothionein, galectin and hepcidin at 3h post-incubation with the two pathogens. The expression of cathelicidin in the gills was significantly enhanced by A. salmonicida, but not by V. anguillarum. At 24h post-incubation, most of these genes were still significantly upregulated, although some genes returned to their basal expression levels. The transcription levels of cytokines such as interleukin (IL)-1beta, IL-8 and interferon (IFN)-gamma significantly increased at 3h post-incubation with the pathogens. IL-22 and CC-chemokine type 1 transcripts were enhanced by A. salmonicida, but not by V. anguillarum. There was down-regulation of expression in CC-chemokine type-2 and -3 by V. anguillarum, while the expression levels of IL-10 remained unchanged upon infection with either of the two bacterial pathogens. The early upregulation of antibacterial genes in the gills could signal the onset of the acute phase response following bacterial infection and the differential modulation of some cytokine genes could be related to host-pathogen interactions that trigger immune response cascades in mucosal tissues of the host.

Expression profiles of genes associated with immune response and oxidative stress in Atlantic cod, Gadus morhua head kidney leukocytes modulated by live and heat-inactivated intestinal bacteria

The molecular mechanisms of immune response and antioxidant defense in Atlantic cod Gadus morhua head kidney (HK) leukocytes to live and heat-inactivated intestinal bacteria were investigated by transcriptome analyses. The HK leukocytes were incubated with Pseudomonas sp. (GP21) and Psychrobacter sp. (GP12), which are intestinal bacteria of Atlantic cod. The responses of the defense-associated genes at 3 and 24h post-incubation (hpi) were assayed by semi-quantitative RT-PCR. Live and heat-inactivated GP21 caused a significant increase in the transcript levels of bacterial defense genes in the HK leukocytes: BPI/LBP and g-type lysozyme were highest at 24hpi. The levels of BPI/LBP were significantly upregulated at 24hpi by live GP12 but not by the heat-inactivated type. The expression of g-type lysozyme was significantly elevated regardless of the type of GP12. IL-1beta was significantly upregulated by live GP21 and GP12, with maximum expression observed at 3hpi. In contrast, the expression levels of IL-8 in the HK leukocytes were not augmented by both types of GP21 and GP12. A significant upregulation of the non-specific cytotoxic cell receptor protein-1 (NCCRP-1) was observed with live GP12 at 3hpi, whereas in the case of GP21 such a change was noted only with the heat-inactivated type at 24hpi. A definite pattern of granzyme expression was not observed with both the live and heat-inactivated GP21 and GP12. The levels of antioxidant genes (catalase and GSH-Px) remained unchanged except in cells incubated with heat-inactivated GP21, where a significant elevation of GSH-Px was seen at 24hpi. Thus, this in vitro study has revealed that the defense mechanisms in the HK leukocytes can be modulated by the commensal intestinal bacteria of Atlantic cod. The extent of this activation is dependent on the bacterial species and its viability.

Profiling gene expression in the spleen of Atlantic cod, Gadus morhua upon vaccination with Vibrio anguillarum antigen.

The mechanisms of physiological responses in Atlantic cod, Gadus morhua following vaccination with a heat-killed Vibrio anguillarum were investigated by transcriptome analysis of spleen tissues. Semi-quantitative RT-PCR of several genes involved in immune defense, inflammation, antioxidant defense and glucose transport were determined in vaccinated fish at 1, 3, 7 and 10 days after vaccination (dpv)and compared with sham-injected fish. Transcript levels of the selected genes involved in bacterial defense such as the bactericidal permeability-increasing protein/lipopolysaccharide-binding protein (BPI/LBP), g-type lysozyme and transferrin, were significantly upregulated (P < 0.05) throughout the duration of sampling (1-10 dpv). There was differential expression of the genes involved in antiviral activity, cellular immunity, antioxidant defense and glucose transport, while the pro-inflammatory cytokines remained relatively unchanged in both the vaccinated and sham-vaccinated fish. The expressions of interferon stimulated gene-15 (ISG-15) and interferon regulatory factor-1 (IRF-1), which are involved in viral defense, were significantly enhanced (P < 0.05) after vaccination. Likewise, the transcript levels of the non-specific cytotoxic cell receptor protein-1 (NCCRP-1) and granzyme A/K, which are components of the cell-mediated immunity were upregulated. Among the antioxidants, the transcript levels of catalase and phospholipid hydroperoxide glutathione peroxidase (GSH-Px) significantly increased (P < 0.05) following vaccination, while glucose transporter-4 (GLUT-4) was enhanced among the genes involved in glucose transport. Our results indicate that the spleen of Atlantic cod is able to mount a potent physiological response through enhanced transcription of at least the mentioned genes, upon exposure to a bacterial antigen. These genes work synergistically to protect the fish during subsequent infection.

Short-term overcrowding of Atlantic cod, Gadus morhua: Effects on serum-mediated antibacterial activity and transcription of glucose transport and antioxidant defense related genes

Serum-mediated control of Listonella anguillarum and transcriptional profiles of selected glucose transport and antioxidant defense genes, following short-term overcrowding in Atlantic cod, Gadus morhua were determined. Fish were subjected to overcrowding by reducing the water level in the tank for 1 h and this was repeated thrice over a 12 h period. Blood samples were collected before overcrowding (initial group) and at 2, 24 and 72 h post-crowding. The sera from fish obtained at 2 h post-crowding caused a significant reduction in L. anguillarum counts compared to the initial samples. There was a transcriptional upregulation of the glucose transport-4 and glyceraldehyde-3-phosphate dehydrogenase genes at 2 h after crowding. Gene transcripts of the antioxidant enzymes, Cu/Zn superoxide dismutase (Cu/Zn SOD), catalase and phospholipid hydroperoxide glutathione peroxidase also significantly increased at 2 h post-crowding, but thereafter they returned to their pre-crowding levels with the exception of Cu/Zn SOD that remained significantly higher than the initial group until 72 h. Thus, short-term overcrowding of Atlantic cod leads to a transient enhancement of in vitro serum antibacterial activity and enhanced transcriptional activity of glucose transport and antioxidant defense genes.

Differential expression of immune and stress genes in the skin of Atlantic cod (Gadus morhua).

The present study describes the transcriptional profiles of selected immune and stress genes with putative important roles in the cutaneous immune defense of Atlantic cod (Gadus morhua). In addition it shows differential expression of many genes at the dorsal and ventral sides of fish, in general having the highest expression at the latter side. Genes related to antibacterial activity, antiviral response, cytokine production, glucose transport, stress response and anti-apoptotic activity were monitored and bactericidal/permeability-increasing protein/lipopolysaccharide-binding protein (BPI-LBP), g-type lysozyme, transferrin, metallothionein, fortilin, interferon regulatory factor-1 (IRF-1), a CC chemokine isoform, interleukin-8 (IL-8), glucose transport (GLUT)-1, -3 and -4, Cu,Zn-superoxide dismutase (Cu,Zn-SOD), catalase and hsp 70 showed significantly higher expression at the ventral side. Further g-type lysozyme, metallothionein, fortilin, IRF-1, interferon γ, interleukin-1β (IL-1β), GLUT-3 and -4, catalase and anti apoptotic gene Bcl-X1 were highly expressed in adult cod skin. Therefore fish skin can be considered an immunological active site, especially at the ventral side of Atlantic cod.

Prospects of host-associated microorganisms in fish and penaeids as probiotics with immunomodulatory functions

Aquatic animals harbor a great number of microorganisms with interesting biological and biochemical diversity. Besides serving as the natural defense system of the host, the utilization potential of this microbial association has been identified particularly as reservoirs of candidate probiotics. Host-derived probiotics have gained popularity in recent years as they offer an alternative source of beneficial microbes to the industry that is customarily dependent on the use of terrestrial microorganisms. At present, there is an overwhelming number of candidate probiotics in aquaculture but their large-scale application is restricted by bio-technological concerns and fragmentary documented probiotic actions. This paper presents the current understanding on the use of probiotics as a sustainable alternative that promotes health and welfare in fish and penaeids. In particular, this paper discusses the relevance of host microbiota and its potential as a source of candidate probiotics. It also revisits the interaction between probiotics and host immunity to provide the foundation of the immunomodulatory functions of host-derived probiotics. Several studies demonstrating the immunomodulatory capabilities of host-derived candidate probiotics are given to establish the current knowledge and provide avenues for future research and development in this thematic area of probiotics research in aquaculture.

In vitro adherence of two candidate probiotics from Atlantic cod and their interference with the adhesion of two pathogenic bacteria

The potential of two candidate probiotic bacteria (GP21 and GP12), isolated from the gut of Atlantic cod, to adhere to primary cultures of the epithelial cells from the different regions of the intestine and to interfere with the adhesion of two pathogens, Vibrio anguillarum and Aeromonas salmonicida subsp. salmonicida were investigated. The intestinal isolates showed clear preference in adhering to the cells from the different intestine segments. GP12 adhered strongly to the fore- and mid intestine cells. The adherence of GP21 was most to the cells from the hind intestine followed by those from the mid-segment. The adhesion of V. anguillarum was affected by both GP21 and GP12; GP12 interfered through competition, but a specific mode of action was not observed for GP21. In the case of A. salmonicida, competition was the principal mechanism by which GP21 interfered with their adhesion, while exclusion mechanism was favoured by GP12. In addition, GP21 was more auto-aggregative than GP12, but the latter was more co-aggregative with both the pathogens. The isolates were also capable of lowering lactate dehydrogenase activity compared to that by the pathogen and they reduced the caspase-3 activity in the epithelial cells from the hind intestine, to which the pathogens adhered the most. Thus it could be concluded that the adhesion of the candidate probiotics is segment-specific and their interference with the adhesion of pathogens is dependent on both source of the epithelial cells and the mechanism adopted by the isolates. This information is novel in the case of fish and the manner in which potential probiotic organisms interfere with the pathogen adhesion provides supportive information for disease control.

Nutritional impacts on fish mucosa: immunostimulants, pre- and probiotics

Preventive health care by dietary manipulation has been regarded as a sustainable approach in modern aquaculture. This strategy is substantiated by remarkable evidence that nutrition is an important modulator of the fish immune system. From a plethora of feed supplements that are being utilized as health promoters in aquaculture, there are three major groups that have generated considerable attention: immunostimulants, prebiotics, and probiotics. These feed supplements vary in their modes of action in fish but their ability to boost not only the innate immunity but also the mucosal immunity positioned them as significant promoters of fish health. This chapter provides the basic concepts of immunostimulants, prebiotics, and probiotics and their contemporary importance in the health and welfare of aquacultured fish. Specifically, this chapter highlights the current understanding of the roles of each substance through dietary administration on the mucosal immunity of fish and provides a platform for the advancement and multi-contextual understanding of these feed supplements in future studies.