Induction of trained immunity using ß-glucan and its protective responses in Nile tilapia, Oreochromis niloticus.

Emerging and re-emerging diseases in fish cause drastic economic losses in the aquaculture sector. To combat the impact of disease outbreaks and prevent the emergence of infections in culture systems, understanding the advanced strategies for protecting fish against infections is inevitable in fish health research. Therefore, the present study aimed to evaluate the induction of trained immunity and its protective efficacy against Streptococcus agalactiae in tilapia. For this, Nile tilapia and the Tilapia head kidney macrophage primary culture were primed using ß-glucan @200 µg/10 g body weight and 10 µg/mL respectively. Expression profiles of the markers of trained immunity and production of metabolites were monitored at different time points, post-priming and training, which depicted enhanced responsiveness. Higher lactate and lactate dehydrogenase (LDH) production in vitro suggests heightened glycolysis induced by priming of the cells using ß-glucan. A survival rate of 60% was observed in ß-glucan trained fish post challenge with virulent S. agalactiae at an LD50 of 2.6 × 107 cfu/ml, providing valuable insights into promising strategies of trained immunity for combating infections in fish.

Triclosan induces immunosuppression and reduces survivability of striped catfish Pangasianodon hypophthalmus during the challenge to a fish pathogenic bacterium Edwardsiella tarda.

Toxicological studies on the emergent pollutant, triclosan (TCS) have established the wide-ranging effects of the compound on fish and other aquatic organisms. Although the available literature describes the standalone effects of TCS on growth and metabolism of fish yet, reports about the combined effects of TCS with microbial pathogens are scarce. In a real environment, a combined exposure to TCS and pathogens is of common occurrence, therefore, such investigation facilitates in developing a better understanding about the gross effects of pollutants and microbial pathogens on aquatic organisms including fish. In this context, the experimental fish (striped catfish, Pangasianodon hypophthalmus) were exposed to three different concentrations of TCS viz. 10, 20 and 30% of 96 h LC50 (1177 µg L-1) for 45 days including two control group firstly solvent control (without TCS) group and another one (without solvent and TCS) group in triplicate. Sampling was performed fortnightly and blood, serum and tissues (liver, and gills) samples were collected for evaluating immunological and biochemical parameters. Following 45 days of the experiments, the experimental fish in each treatment group including controls were challenged with a fish pathogenic bacterium Edwardsiella tarda (LD50 dose) and fish mortality was daily monitored for calculating cumulative mortality till 7 days and further, relative per cent survivable was estimated. A significant reduction in cellular immune responses i.e. respiratory burst activity (RBA), myeloperoxidase activity (MPO), phagocytic activity (PA) and humoral immune components viz. serum lysozyme activity, total immunoglobulin in serum, ceruloplasmin level, serum total protein, albumin and globulin level was evident in TCS exposed groups in comparison to control during the experimental periods. Further, oxidative stress parameters viz. superoxide dismutase (SOD), catalase (CAT), glutathione-s-transferase (GST) activity in liver and gill tissue exhibited a dose-dependent increase in activity with related to TCS concentration during the experimental periods. A significant reduction in relative percentage survival was observed with increasing TCS concentration. The present study reveals that TCS can inhibit the cellular and humoral components of the innate immune system of the fish and can elevate the mortality due to TCS mediated immunosuppression in fish during the bacterial infection.

Duration of stunting impacts compensatory growth and carcass quality of farmed milkfish, Chanos chanos (Forsskal, 1775) under field conditions.

An 18-months field trial was performed to explore the effect of duration of stunting on growth, digestive enzymes and carcass quality in Chanos chanos. Milkfish fry (weight of 1.25 ± 0.03 g and length of 5.53 ± 0.03 cm) were stocked in earthen ponds of 0.02 ha, in triplicate, for different duration of stunting, viz., 4 months (Treatment-1; T4), 8 months (Treatment-2; T8) and 12 months (Treatment-3; T12) and a normal seed (Control; C) separately. In the stunting phase, fish were stocked at higher stocking density (0.2 million/ha) and fed de-oiled rice bran at sub-optimal level. Post-stunting or re-feeding phase commenced immediately after completion of respective stunting duration and fish were reared for the rest of the period to complete the total rearing period of 18 months. In post-stunting, fish stocking density was adjusted to (5000 pieces/ha) and fed at an optimum level (3%). At the end of stunting phase, the study found a significant reduction in growth, survival, digestive enzymes activity, except protease in the T4 group, and carcass nutrients composition of stunted fish. However, in the initial phase of post-stunting, T8 group exhibited an elevated specific growth rate (5.00 ± 0.092%/day), body weight gain (80.82 ± 1.28 g), amylase (0.585 ± 0.021 U/mg protein), protease (5.48 ± 0.13 U/mg protein), and lipase activity (7.92 ± 0.32 U/mg protein). All stunted fish groups displayed a compensatory growth response in post-stunting, but a complete growth compensation was observed in T8 group, which resulted in better feed conversion ratio (3.03 ± 0.04) feed efficiency ratio (0.33 ± 0.01), protein efficiency ratio (1.91 ± 0.03), survival (91.38 ± 0.07%) and digestive enzyme activities. Similarly, at the end of post-stunting, carcass analysis revealed a complete restoration of nutrients in stunted fish and significantly higher protein content in T8 group. Further, the study found lower meat and higher bone contents in normally reared fish than the post-stunted fish which revealed the carcass quality improvement in post-stunted fish thus indicates superiority of the stunting process over normal rearing. Overall, the study suggests that stunting of milkfish, for 8 months (T8), positively affects its growth, survival, digestive enzyme activities and carcass quality which in turn, shall help to overcome the contemporary challenges in milkfish culture.

Preliminary investigations on the role of Drp-1 dependent mitochondrial fission in attenuating RLR downstream signaling during nervous necrosis virus infection.

Member of the dynamin family of large GTPases, dynamin-related protein 1 (Drp1) dependent mitochondrial fission is an intricate process regulating both cellular and organ dynamics. Present study shows that NNV perturbs mitochondrial dynamics by promoting Drp-1 dependent mitochondrial fission, which attenuates MAVS mediated downstream signaling. NNV infected SISS cells revealed induction in Drp1 expression and subsequent translocation into mitochondria. The level of MAVS expression was up-regulated over a period of 24 hpi and declined with the progression of NNV infection at 48 and 72 hpi confirmed by western blot and mRNA transcript analysis. Drp-1 displayed its association with fragmented mitochondria and the transcript abundance was significant post infection along with Mff. Expression levels of IRF-3 IFN-1 and Mx followed a similar pattern with abundant expression at 48 hpi and diminished expression during the further period. Importantly, silencing of Drp1 caused significant elevation in the RLR downstream molecules and reduction in viral RNA expression. These results suggest that NNV-induced mitochondrial fission serve to attenuate host RLR signaling. This provides an illustration of host-pathogen interaction in which the virus evades innate immunity by enhancing mitochondrial fission and perturbs MAVS, and the downstream molecules.

Molecular cloning, characterization and expression profiling of galectin-9 gene from Labeo rohita (Hamilton, 1822).

Galectin-9 is a b-galactoside-binding tandem repeat galectin that regulates many cellular functions, ranging from cell adhesion to pathogen recognition. In spite of extensive study of mammalian galectin importance in immune system, little is known about that of fish. To study the normal expression and immune response of Labeo rohita to pathogens, a tandem-repeat galectin-9 from Labeo rohita was identified and named LrGal-9. Its full-length cDNA was 1534 bp encoded 291 amino acids (35.12 KDa), shared the highest 81% identity with the galectin-9 of Danio rerio. LrGal-9 identified in this study lacked signal peptide and a transmembrane domain like galectin-9 members reported in other fishes. Quantitative PCR showed that LrGal-9 was lowly expressed in gill, muscle, heart, highly expressed in tested immune tissues (intestine, kidney, liver, spleen) in normal body. After Aeromonas hydrophila challenge, LrGal-9 was remarkably increased in all tested immune tissues in a time-dependent manner. These results suggest that LrGal-9 plays a role in innate immunity in Labeo rohita.

Effect of feeding Lipopolysaccharide as an immunostimulant on immune response and immune gene expression of Labeo bata.

This study investigates the effects of dietary lipopolysaccharide (LPS) as an immunostimulant on hematology, innate immunity, immune gene expression and protection against Edwardsiella tarda on Labeo bata. A basal diet supplemented with 0, 50, 100 and 150mg LPS kg-1diet was fed to the four different groups for 30days. The haematological (total erythrocyte count, total leukocyte count, total serum protein, albumin and globulin), innate immune parameters (respiratory burst, serum lysozyme, myeloperoxidase and serum bactericidal activity), immune gene expression (C3, ß-2 microglobulin, lysozyme g, transferrin, IFN-1, IFN-γ) were monitored at 7th, 15th, 30th day and one day post challenge (DPC) with E. tarda. All the studied haematological, innate immune parameters and expression of immune gene increased significantly (p≤0.05) in LPS fed group in comparison with control. However the group fed 100mgkg-1 LPS in feed showed highest activity on 7th day and 1DPC. The group fed 100mgkg-1 LPS also recorded highest relative percent survivability after challenge with E. tarda. Therefore this study suggests that LPS at 100mgkg-1 could be used as an immunostimulant in feed to enhance the protection of bata during periods of increased disease risk.

Effect of dietary supplemented andrographolide on growth, non-specific immune parameters and resistance against Aeromonas hydrophila in Labeo rohita (Hamilton).

The present study evaluated the effect of dietary andrographolide (EC 50%) on growth, non-specific immune parameters and disease resistance against Aeromonas hydrophila infection in Indian major carp, Labeo rohita fingerlings. Fishes were fed with formulated diet containing andrographolide as T0 (0.00%), T1 (0.05%), T2 (0.10%), T3 (0.20%), T4 (0.40%) and T5 (0.80%) for 42 days. Fishes were challenged with A. hydrophila 42 days post feeding and relative percentage survival (RPS) was recorded over 14 days post challenge. Blood and serum samples were collected for nonspecific immune parameters on 14, 28 and 42 days of feeding and growth performance was evaluated at the end of experiment. The results revealed that fishes fed with andrographolide showed significant (p < 0.05) increase in NBT levels, myeloperoxidase activity, phagocytic activity, serum lysozyme activity, and serum antiprotease activity when compared to the control group. The weight gain, specific growth rate, feed conversion ratio and protein efficiency ratio of fishes fed with andrographolide were found to be significantly (p < 0.05) differed compared with control. Dietary andrographolide at the level of 0.10% showed significantly (P < 0.05) higher RPS (74.06%) against A. hydrophila infection than control. The results revealed that andrographolide supplemented diet has a stimulatory effect on non-specific immune parameters along with improved growth performance and increased disease resistance against A. hydrophila infection in L. rohita fingerlings.