Comprehensive evaluation of immunomodulation by moderate hypoxia in S. agalactiae vaccinated Nile tilapia.

Streptococcus agalactiae is a major bacterial pathogen in tilapia aquaculture. Vaccines are known to provide protection but S. agalactiae clearance in tilapia can be reduced by marginal environmental conditions. Therefore, the purpose of this study is to examine S. agalactiae clearance in vaccinated Nile tilapia under moderate hypoxic (55± 5% DO) and normoxic (85 ± 5%DO) conditions. Fish were acclimatized to either moderate hypoxia or normoxia and immunized with formalin-inactivated S. agalactiae. Fish were experimentally challenged with S. agalactiae at 30 days post-vaccination. Serum antibody titer was significantly higher in vaccinated fish kept under normoxic condition compared to the moderate hypoxic condition at fifteen and thirty days post-vaccination. The cumulative mortality following challenge was significantly reduced in vaccinated fish kept under normoxic condition compared to those in moderate hypoxic condition reflecting that pre-challenge antibody titer may correlate with survival of fish. Blood and tissue pathogen burden detection of S. agalactiae studies revealed that culturable S. agalactiae cells could not be detected in the blood of normoxic vaccinated fish at all the sampling points. In contrast, fish vaccinated in moderate hypoxic condition had considerable number of culturable S. agalactiae cells in their blood up to 5 days following challenge. Phagocytosis and intracellular reactive oxygen species (ROS) production were lowered by moderate hypoxia in vitro. Furthermore, presence of specific antibodies and higher specific antibody level in the serum increased phagocytosis, ROS production and lowered intracellular survival of S. agalactiae in head kidney leukocytes. Overall this study has highlighted that S. agalactiae clearance in vaccinated Nile tilapia is modulated by moderate hypoxia. One of the possible explanations for this might be less efficient phagocytic activities due to low oxygen availability and lower specific antibody production in vaccinated fish.

Influence of moderate hypoxia on vaccine efficacy against Vibrio anguillarum in Oreochromis niloticus (Nile tilapia).

Hypoxia is known as a potential immunomodulator in fish. This study therefore assesses the impact of chronic, moderate hypoxia on vaccine efficacy in Oreochromis niloticus. Serum antibody titer was used as a surrogate marker to detect vaccine efficacy. The fish were acclimatized to either moderate hypoxia (55 ± 5% DO) or normoxia (85 ± 5%DO) and immunized with formalin inactivated Vibrio anguillarum. Significantly, a higher antibody titer was found in normoxic fish than in moderate hypoxia. The normoxic group titer peaked at 14th dpv (days post vaccination) while the moderate hypoxic group peaked at 21st or 28th dpv. The absolute blood lymphocyte counts and serum bactericidal activities against V. anguillarum were significantly higher in normoxic fish. Serum killing of V. anguillarum appeared to be mainly via antibody-dependent classical complement pathway. Furthermore, the first week following vaccination appears critical for antibody production. This view was further supported by results obtained from gene expression assay, where the transcription level of all the detected immune related genes (IgM, IL-1 ß, TCR-ß, MHC-II ß), except B cell activating factor, were significantly suppressed following exposure to moderate hypoxia. The overall results highlight that even though moderate hypoxia is not easily detectable in Oreochromis niloticus, it negatively affects antibody production by suppressing and delaying antibody response, ultimately affecting vaccine efficacy.

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.

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.