Immunological responses and disease resistance of rainbow trout (Oncorhynchus mykiss) juveniles following dietary administration of stinging nettle (Urtica dioica).

The present study investigated the effects of dietary supplementation of stinging nettle (Urtica dioica) on growth performance, skin mucus, immune response and disease resistance of rainbow trout (Oncorhynchus mykiss) fed with diets supplemented with U. dioica at 0, 1, 2 and 3%. After 8 weeks of feeding, the addition of U. dioica at 3% level resulted in improved weight gain, specific growth rate and feed conversion ratio significantly when compared to the other groups (P < 0.05). Hematological responses including: hematocrit (Htc), hemoglobin (Hb), lymphocyte and neutrophil populations enhanced significantly in fish fed 3% of stinging nettle when measured after 4 weeks; while, total red blood cells, white blood, Htc, Hb, lymphocyte and neutrophil populations significantly increased after 8 weeks in the same group (P < 0.05). Total serum protein and glucose contents increased significantly in fish fed stinging nettle at 3% when compared to the other groups after 8 weeks; however, triglycerides decreased significantly in the same group on the 4th and 8th week (P < 0.05). Additionally, several immune parameters, namely, IgM, lysozyme, complement components C3 and C4, and respiratory burst of blood leukocytes significantly increased in the 3% fed group on the 4th week; while, after 8 weeks the immune responses enhanced in fish fed 2 and 3% diets (P < 0.05). At the end of the feeding trial, mucus samples obtained from the fish fed stinging nettle supplementation exhibited improved antagonistic activities against several bacterial pathogens (Streptococcus iniae, Yersinia ruckeri, Vibrio anguillarum and Lactococcus garviae), skin mucus enzymes activities (alkaline phosphatase, lysozyme, protease and esterase) and protein levels in 2 and 3% groups with the highest being in case of 3% group when compared to the other groups (P < 0.05). The cumulative mortality of rainbow trout subjected to Y. ruckeri infectious exhibited relatively low mortality levels in all supplemented groups with the lowest being in fish fed 3% stinging nettle. The present findings demonstrated that dietary administration of U. dioica enhanced growth and stimulated fish immunity; thus, enabling the fish to be more resistant against bacterial infections.

Host-derived probiotics Enterococcus casseliflavus improves resistance against Streptococcus iniae infection in rainbow trout (Oncorhynchus mykiss) via immunomodulation.

The present study evaluated the benefits of dietary administration of host-derived candidate probiotics Enterococcus casseliflavus in juvenile rainbow trout Oncorhynchus mykiss. Experimental diets were prepared by incorporating the microorganisms in the basal feed at 3 inclusion levels (i.e. 10(7) CFU g(-1) of feed [T1], 10(8) CFU g(-1) of feed [T2], 10(9) CFU g(-1) of feed [T3]). The probiotic feeds were administered for 8 weeks, with a group fed with the basal diet serving as control. The effects on growth performance, gut health, innate immunity and disease resistance were evaluated. Results showed that growth performance parameters were significantly improved in T2 and T3 groups. Activities of digestive enzymes such as trypsin and lipase were significantly higher in these two groups as well. Gut micro-ecology was influenced by probiotic feeding as shown by the significant increase in intestinal lactic acid bacteria and total viable aerobic counts in T2 and T3. Humoral immunity was impacted by dietary probiotics as total serum protein and albumin were significantly elevated in T3. The levels of serum IgM significantly increased in all probiotic fed groups at week 8; with the T3 group registering the highest increment. Respiratory burst activity of blood leukocytes were significantly improved in T2 and T3. Hematological profiling further revealed that neutrophil counts significantly increased in all probiotic fed groups. Challenge test showed that probiotic feeding significantly improved host resistance to Streptococcus iniae infection, specifically in T2 and T3 where a considerable modulation of immune responses was observed. Taken together, this study demonstrated E. casseliflavus as a potential probiotics for rainbow trout with the capability of improving growth performance and enhancing disease resistance by immunomodulation.

Increased stocking density causes changes in expression of selected stress- and immune-related genes, humoral innate immune parameters and stress responses of rainbow trout (Oncorhynchus mykiss).

The present study investigated the effects of various stocking densities on the health status (stress and immune responses) of rainbow trout (Onchorhynchus mykiss). Juvenile rainbow trout were acclimated, placed in circular tanks under stocking densities of 10, 40 and 80 kg m(-3) and reared for 30 days. The relative expression of genes involved in stress and immunity such as HSP70, LyzII, TNF-1α, IL-1ß, IL-8 and IFN-γ1 in the head kidney was determined. Serum cortisol, ACTH, total antioxidant capacity, osmolality and lactate were measured after 30 days of culture at different stocking densities (D1:10 kg m(-3), D2: 40 kg m(-3) and D3: 80 kg m(-3)) as indices of stress responses. In addition, the effects of stocking densities on serum complement, bactericidal activity, agglutinating antibody titers, serum IgM, anti-protease activity, serum total protein and alkaline phosphatase of the fish were measured. HSP70 gene expression was significantly density-dependent upregulated in D2 and D3 densities compared to D1 (P < 0.05). Also, there was significant downregulation in expression of LyzII, TNF-1α, IL-1ß, IL-8 and IFN-γ1 in fish reared at density of either D2 or D3 (P < 0.05). In terms of stress responses, serum ACTH, cortisol and lactate level showed significant density-dependent increase (P < 0.05) while serum osmolality and total antioxidant capacity showed significant decline (P < 0.05) in fish reared at higher densities (D2 and D3) compared to fish reared at lower density (D1) (P < 0.05). Concordant with the expression of the immune-related genes, the serum complement and bactericidal activity as well as specific antibody titer against Aeromonas hydrophila, IgM and anti-protease activity decreased along with elevation of stocking density from D1 to D3 (P < 0.05). However, different stocking densities had no significant effect on serum total protein level and alkaline phosphatase activity. These results suggested that elevation of stocking densities and crowding resulted in the increase in HSP70 gene expression and the levels of selected stress responses in the serum. However, there was down-regulation of immune genes expression and decreased innate immune responses in the fish. The mRNA expression of the genes and immune parameters that were measured in this study could be helpful in monitoring the health status and welfare of the fish in aquaculture systems particularly in relation to increased stocking densities.

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 and ex vivo models indicate that the molecular clock in fast skeletal muscle of Atlantic cod is not autonomous.

The notion that the circadian rhythm is exclusively regulated by a central clock has been challenged by the discovery of peripheral oscillators. These peripheral clocks are known to have a direct influence on the biological processes in a tissue or cell. In fish, several peripheral clocks respond directly to light, thus raising the hypothesis of autonomous regulation. Several clock genes are expressed with daily rhythmicity in Atlantic cod (Gadus morhua) fast skeletal muscle. In the present study, myosatellite cell culture and short-term cultured fast skeletal muscle explant models were developed and characterized, in order to investigate the autonomy of the clock system in skeletal muscle of Atlantic cod. Myosatellite cells proliferated and differentiated in vitro, as shown by the changes in cellular and myogenic gene markers. The high expression of myogenic differentiation 1 during the early days post-isolation implied the commitment to myogenic lineage and the increasing mRNA levels of proliferating cell nuclear antigen (pcna) indicated the proliferation of the cells in vitro. Transcript levels of myogenic marker genes such as pcna and myogenin increased during 5 days in culture of skeletal muscle explants, indicating that the muscle cells were proliferating and differentiating under ex vivo conditions. Transcript levels of the clock gene aryl hydrocarbon receptor nuclear translocator-like 2 (arntl2) in myosatellite cells showed no daily oscillation regardless of photoperiod manipulation. On the other hand, mRNA levels of the clock gene circadian locomotor output cycles kaput (clock) showed circadian rhythmicity in 5-day-old skeletal muscle explant under different photoperiod regimes. The expression of arntl2, cryptochrome2 (cry2), period 2a (per2a) and nuclear receptor subfamily 1, group D, member 1 was not rhythmic in muscle explants but photoperiod manipulation had a significant effect on mRNA levels of cry2 and per2a. Taken together, the lack of rhythmicity of molecular clocks in vitro and ex vivo indicate that the putative peripheral clock in Atlantic cod fast skeletal muscle is not likely to be autonomous.

Mucosal immunity and probiotics in fish.

Teleost mucosal immunity has become the subject of unprecedented research studies in recent years because of its diversity and defining characteristics. Its immune repertoire is governed by the mucosa-associated lymphoid tissues (MALT) which are divided into gut-associated lymphoid tissues (GALT), skin-associated lymphoid tissues (SALT), and gill-associated lymphoid tissues (GIALT). The direct contact with its immediate environment makes the mucosal surfaces of fish susceptible to a wide variety of pathogens. The inherent immunocompetent cells and factors in the mucosal surfaces together with the commensal microbiota have pivotal role against pathogens. Immunomodulation is a popular prophylactic strategy in teleost and probiotics possess this beneficial feature. Most of the studies on the immunomodulatory properties of probiotics in fish mainly discussed their impacts on systemic immunity. In contrast, few of these studies discussed the immunomodulatory features of probiotics in mucosal surfaces and are concentrated on the influences in the gut. Significant attention should be devoted in understanding the relationship of mucosal immunity and probiotics as the present knowledge is limited and are mostly based on extrapolations of studies in humans and terrestrial vertebrates. In the course of the advancement of mucosal immunity and probiotics, new perspectives in probiotics research, e.g., probiogenomics have emerged. This review affirms the relevance of probiotics in the mucosal immunity of fish by revisiting and bridging the current knowledge on teleost mucosal immunity, mucosal microbiota and immunomodulation of mucosal surfaces by probiotics. Expanding the knowledge of immunomodulatory properties of probiotics especially on mucosal immunity is essential in advancing the use of probiotics as a sustainable and viable strategy for successful fish husbandry.

Probiotics-pathogen interactions elicit differential regulation of cutaneous immune responses in epidermal cells of Atlantic cod Gadus morhua.

Little is known on the cutaneous immune responses during probiotics-pathogen interactions in fish. Thus, this study employed Atlantic cod primary epidermal (EP) cell cultures as a model to understand this interaction. The probiotics-pathogen interactions in the EP cell cultures were elucidated using Vibrio anguillarum 2133 (VA) as the pathogen and two host-derived bacteria (GP21 and GP12) as the probiotics. There was a regional size difference on the EP cells; i.e., EP cells from the dorsal region were significantly larger than the EP cells at the ventral side. VA significantly decreased viability of EP cells. In the presence of probiotics, this inhibition was mitigated. The probiotics reduced VA-induced cellular apoptosis and the probiotics-pathogen interactions influenced cellular myeloperoxidase activity during the latter stage of co-incubation. The probiotics-pathogen interactions triggered differential regulation of immune-related genes and the effects of the interaction were dependent on the region where the cells were isolated and the length of the co-incubation period. In most cases, the presence of probiotics alone showed no significant change on the mRNA level of immune genes in the EP cells but triggered immunostimulatory activity when incubated with VA. This study showed that the virulence of VA in EP cells could be modulated by host-derived probiotics and the immunomodulatory characteristics of the two candidate probionts advanced their immune-related probiotic potential.

The Salmon Louse Lepeophtheirus salmonis (Copepoda: Caligidae) life cycle has only two Chalimus stages.

Each year the salmon louse (Lepeophtheirussalmonis Krøyer, 1838) causes multi-million dollar commercial losses to the salmon farming industry world-wide, and strict lice control regimes have been put in place to reduce the release of salmon louse larvae from aquaculture facilities into the environment. For half a century, the Lepeophtheirus life cycle has been regarded as the only copepod life cycle including 8 post-nauplius instars as confirmed in four different species, including L. salmonis. Here we prove that the accepted life cycle of the salmon louse is wrong. By observations of chalimus larvae molting in incubators and by morphometric cluster analysis, we show that there are only two chalimus instars: chalimus 1 (comprising the former chalimus I and II stages which are not separated by a molt) and chalimus 2 (the former chalimus III and IV stages which are not separated by a molt). Consequently the salmon louse life cycle has only six post-nauplius instars, as in other genera of caligid sea lice and copepods in general. These findings are of fundamental importance in experimental studies as well as for interpretation of salmon louse biology and for control and management of this economically important parasite.

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-1ß (IL-1ß), 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.

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.

Molecular diagnosis of francisellosis, a systemic granulomatous inflammatory disease in Atlantic cod, Gadus morhua L.

A PCR-based assay for the detection of Francisella noatunensis causing francisellosis in Atlantic cod, Gadus morhua has been developed. Seven sets of primers targeting the flanking regions of the genes (rpoA, sdhA, atpA, rpoB, pgm, groEL and 16S rRNA) of the pathogen were designed. Among the primers, groEL was found to be the most suitable gene candidate for detecting the pathogen, due to its high sensitivity at various annealing temperatures and specificity in detection. The detection limit of the assay was 100 pg of bacterial DNA per milliliter or 100 fg bacterial DNA (approximately 50 genome equivalents) per PCR reaction, however, the sensitivity of the reaction decreased by 1 log dilution in the presence of 1 mg mL(-1) of serum and mucus samples as inhibitors. Nevertheless, the assay can potentially be used as a direct and non-lethal method to detect the pathogen in fish. Thus this PCR assay is a specific and sensitive molecular method to diagnose francisellosis in Atlantic cod, and will be helpful for controlling the infection through prompt detection of the disease in farms.

Influence of alginic acid and fucoidan on the immune responses of head kidney leukocytes in cod.

The present study investigated the immunomodulatory activities of alginic acid and fucoidan, both derived from brown seaweeds, on selected cellular immune responses and antibacterial activity of head kidney (HK) leukocytes of cod, Gadus morhua. Primary cultures of HK leukocytes were incubated with either 10 or 100 µg ml⁻¹ of the substances and the effects on respiratory burst, cellular proliferation, acid and alkaline phosphatase activity and cellular myeloperoxidase were measured at 3- and 24-h post-incubation. The antibacterial activity of the supernatants collected from the cell cultures incubated with 100 µg ml⁻¹ of the substances were tested against Vibrio anguillarum and Aeromonas salmonicida. Respiratory burst was significantly elevated in cells incubated with either alginic acid or fucoidan in a dose-dependent manner. Incubation with a higher dose of alginic acid and fucoidan resulted in lower cellular proliferation at 3- and 24-h, respectively. Both acid and alkaline phosphatase activities of HK leukocytes were not significantly modulated, except for a slight elevation of acid phosphatase in cells incubated with 100 µg ml⁻¹ of alginic acid for 24-h. Fucoidan, but not alginic acid significantly increased cellular myeloperoxidase activity at a concentration of 100 µg ml⁻¹. The growth of the bacteria in both the treated and control supernatants was significantly lower than what was observed in the bacterial culture medium. However, the supernatants from the treated cells had significantly higher bacterial growth compared with supernatants of the control cells. Taken together, these results showed that at the tested concentrations, both alginic acid and fucoidan are able to differentially stimulate some cellular immune responses of cod HK leukocytes in vitro and the respiratory burst activity was significantly stimulated by these brown algal derivatives. These substances could be tested as potential immunostimulants in future in vivo studies.

cDNA cloning of the immunoglobulin heavy chain genes in banded houndshark Triakis scyllium.

In this study, cDNAs encoding the secreted forms of the immunoglobulin (Ig) heavy chains of IgM, IgNAR, and IgW were cloned from the banded houndshark Triakis scyllium. Two clones for the IgM heavy chains encoded 569 and 570 amino acids, whose conserved (C) region showed 47-70% amino acid identities to those reported in other cartilaginous fish. Four clones for the IgNAR encoded 673-670 amino acids with conserved Ig-superfamily domains. The IgNAR C region showed 56-69% amino acid identities to those so far reported. High-throughput sequencing revealed that in most of the IgNAR sequences, the two variable regions (CDR1 and CDR3) each possess a cysteine residue. Three types of IgW were identified; one contained Ig-superfamily domains that are in other cartilaginous fish, one lacks the 3rd domain in the constant region, and one lacks the 3rd to 5th domains. Despite these differences, the IgW isoforms clustered with IgWs of other cartilaginous fishes and the C regions showed 47-89% amino acid identities. mRNAs for IgM and IgNAR were detected in various tissues, while IgW mRNA was mainly detected in pancreas. The banded hounded shark also has IgM, IgW and IgNAR as well as the other cartilaginous fish with unique IgW isoform.

Rapid diagnosis of vibriosis and white spot syndrome (WSS) in the culture of shrimp, Penaeus monodon in Philippines.

Viral and bacterial pathogens have raised serious concerns in the sustainability of the shrimp culture industry in the Philippines. Heavy mortality associated with luminous vibriosis and white spot syndrome virus (WSSV) infection has been the major problem besetting the industry. Using published PCR protocols for the diagnosis of vibriosis and white spot syndrome virus (WSSV) disease in shrimp, we optimized these assays that could be suited to the shrimp aquaculture setting in the Philippines. Genomic DNAs of Vibrio spp. that exhibited luminescence as well as those that grew on thiosulfate citrate bile salt sucrose agar (TCBS) were used for the PCR amplification of the ribonuclease P (RNase P) gene. There was differential amplification of the RNase P gene based on the phenotypic characters of the Vibrio spp. Similar results were also obtained using direct colony PCR of the bacterial colonies. White spot syndrome virus was also detected in the infected shrimp and there were differences in the detection frequency in relation to the tissues used for PCR amplification. Duplex PCR was also optimized that could be used for simultaneous detection of these pathogens in shrimp.

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.

Unmethylated CpG oligodeoxynucleotides activate head kidney leukocytes of Atlantic cod, Gadus morhua.

Bacterial DNA and synthetic oligodeoxynucleotides (ODNs) that contain unmethylated CpG motifs are strong inducers of immune response in most mammalian organisms. The use of these synthetic CpG motifs in fish, particularly in salmonids and carp, resulted in the modulation of their immune system. However, much less is known in other species of fish such as gadoids including Atlantic cod, Gadus morhua. Using head kidney (HK) leukocytes of cod in an in vitro study, we determined the effects of some established CpG-ODNs on the cellular responses of the fish immunocytes. Incubation of the HK leukocytes with 2 µM concentration of the CpG-ODNs resulted in enhanced respiratory burst. There were differential effects on the activities of acid phosphatase and cellular myeloperoxidase. Only CpG-ODN 1826 triggered a significant increase in the level of both enzymes. On the other hand, the supernatants derived from the HK leukocytes after incubation with different CpG-ODNs did not possess bactericidal activity against Vibrio anguillarum and Aeromonas salmonicida. This study has shown that CpG-ODNs at low concentrations are able to stimulate respiratory burst in cod but have minimal effects on cellular enzymatic activities and antibacterial action.

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.

Characterization of GP21 and GP12: Two Potential Probiotic Bacteria Isolated from the Gastrointestinal Tract of Atlantic Cod.

This study evaluated the probiotic potential of GP21 (Pseudomonas sp.) and GP12 (Psychrobacter sp.), two bacteria isolated from the intestinal tract of a cold-water fish, Atlantic cod. The antagonistic activity of the two intestinal bacteria against two fish pathogens (Vibrio anguillarum and Aeromonas salmonicida subsp. salmonicida) was studied under different physical conditions. Further, their resistance to physiological barriers and their ability to form biofilms were examined. In addition, a test was conducted to confirm that the isolates were not pathogenic to the host fish. The two bacteria exhibited differences in their antagonism to the pathogens. Both were active against V. anguillarum at mildly acidic conditions over a 5-day period. The activity of GP21 against A. salmonicida was greater at pH 7-8. The maximum antagonistic activity was observed at a temperature of 15°C and at a salt concentration of 15 ppt for both the isolates. They did not produce acids, could release siderophores and tolerated both the acidic environment and the bile salts. Their ability to form biofilms was high around 15°C and when iron was supplemented in the medium at 5 µmol l(-1). There was no mortality of fish during the pathogenicity experiment, confirming the safety of both isolates for further applications. Considering the favorable characteristics identified here, it could be concluded that GP21 and GP12 isolated from the gastrointestinal tract of Atlantic cod are potential probiotic candidates.

Responses of Atlantic cod Gadus morhua head kidney leukocytes to phytase produced by gastrointestinal-derived bacteria.

This study identified phytase-producing bacteria that were previously isolated from the gastrointestinal tract of Atlantic cod, Gadus morhua and determined its effect on head kidney leukocytes. Out of the 216 bacterial strains tested, the two phytase producers were identified as Pseudomonas sp. and Psychrobacter sp. based on their 16S rDNA sequence. Crude phytase from these two bacterial strains was produced employing the shake flask method. Even though the total protein of the crude phytase was not significantly different for the two bacteria, the phytase activity of the crude enzyme produced by Pseudomonas sp. (97.1±16.7 U) was significantly higher than that of the enzyme from Psychrobacter sp. (75.9±2.4 U). When cod head kidney leukocytes were incubated with the crude phytase (50 µg ml(-1)), it resulted in enhanced cell proliferation, higher myeloperoxidase, and acid phosphatase activities. Extracellular responses-respiratory burst activity and hydrogen peroxide production were not enhanced by the crude enzyme. As a consequence, the growth of two pathogenic bacteria Aeromonas salmonicida and Vibrio anguillarum was not suppressed by the supernatants obtained from head kidney leukocytes incubated with the crude bacterial phytase. Thus, the enzyme from phytase-producing intestinal bacteria of Atlantic cod can stimulate intracellular head kidney leukocyte activities but not the production of extracellular substances that are involved in antibacterial response. These have implications on the potential use of bacterial phytase as feed supplement to boost cellular immune response of the fish and could be employed as a health management strategy in culture systems.

In vivo modulation of immune response and antioxidant defense in Atlantic cod, Gadus morhua following oral administration of oxolinic acid and florfenicol.

Oxolinic acid and florfenicol are the commonly used antibiotics for the treatment of bacterial diseases in Atlantic cod, Gadus morhua. The changes in selected innate humoral immune response of the fish, bacterial proliferation in serum and transcriptional activity of selected immune and antioxidant defense-related genes following oral administration of these antimicrobial compounds were evaluated. Juvenile cod (75-100 g) were fed commercial feed coated with either florfenicol (10 mg kg(-1) fish, active ingredient) or oxolinic acid (20 mg kg(-1) fish, active ingredient) at a ration of 0.5% body weight for 10 days. Whole blood and serum samples were collected on the 10th day of feeding the antibiotics and at 3, 5 and 10 days after their withdrawal. Serum protein was significantly higher in fish at the 10th day post-withdrawal of both antibiotics. Florfenicol-fed fish had lower myeloperoxidase activity at 3 days post-withdrawal, while there were differential effects on alkaline phosphatase activity. Vibrio anguillarum and Aeromonas salmonicida were significantly reduced in the sera of antibiotics-fed fish until the 5th day post-withdrawal. Florfenicol could inhibit V. anguillarum better than oxolinic acid, while A. salmonicida was more susceptible than V. anguillarum upon treatment with both antibiotics. Furthermore, transcriptional profiles of selected genes related to bacterial defense, inflammation and antioxidant defense were dependent on the type of antibiotics that was administered and the time of sampling. These results indicate that oral administration of antibiotics modulates the immune response and antioxidant defense in Atlantic cod and these may, in turn, affect their ability to resist bacterial pathogens.