Potential probiotic Lactobacillus plantarum VSG3 improves the growth,immunity, and disease resistance of tropical freshwater fish,Labeo rohita

The study evaluated the effects of dietary doses of Lactobacillus plantarum VSG3 on the growth performance, immunity, and disease resistance of Labeo rohita juveniles against Aeromonas hydrophila infection. Fish (mean body weight 60 g) were fed with diet containing 0 (control), 10⁶, 10⁸, 10¹⁰ cfu g^?1 L. plantarum VSG3 for 60 days. Various growth and immune parameters were examined at 30 and 60 days of post-feeding. Fish were challenged with A. hydrophila 60 days post-feeding and mortalities were recorded over 10 days post-infection. Results showed that administration of VSG3 for 60 days had significant effects (P < 0.05) on the specific growth rate (SGR) and feed utilization efficiency of L. rohita. Dietary administration of L. plantarum VSG3 significantly increased the serum lysozyme and alternative complement pathway (ACP) activities, phagocytosis and respiratory burst activity in L. rohita throughout the experimental period. The highest superoxide dismutase (SOD) activity (P < 0.05) was observed in the fish group fed diet containing VSG3 at 10⁸ cfu g^?1. The serum IgM levels were significantly higher in the experimental groups compared to the control group after 30 days of feeding; but, the result was opposite after 60 days of feeding. Further, fish fed the diet containing 10⁸ cfu g^?1 L. plantarum VSG3 had significantly higher (P < 0.05) post-challenge survival rate (77.7%). These results collectively suggest that dietary supplementation of L. plantarum VSG3 at 10⁸ cfu g^?1 to L. rohita is optimal for enhancing the growth, immunity, and disease resistance against A. hydrophila infection.     

Effect of dietary supplementation of probiotic and vitamin C on the immune response of Indian major carp, Labeo rohita (Ham.)

The immunostimulatory effect of probiotics and vitamin C has been established in many systems including fish. An investigation was carried out to study the effect of dietary supplementation of a probiotic bacterium "Bacillus subtilis", vitamin C in the form of ascorbyl polyphosphate and their combination on the immune response of Indian major carp, rohu, (Labeo rohita Ham.) fingerlings fed for a period of 60 days. The total serum protein and globulin content was significantly higher (p<0.05) in probiotic (B. subtilis @ 10(8) CFU/g of the feed) fed group while the respiratory burst activity of blood neutrophils was significantly high in vitamin C (ascorbyl polyphosphate @100 mg per kg diet) fed group. The antibody level was significantly high in Bacillus subtilis treated group followed by the probiotic (B. subtilis @ 10(8) CFU/g of the feed) and ascorbyl polyphosphate (ascorbyl polyphosphate @100 mg per kg diet) combined group. The least percentage of mortality was recorded in B. subtilis treated group (25%) followed by 35 and 40% in ascorbyl polyphosphate treated and B. subtilis and ascorbyl polyphosphate combined groups, respectively.     

Effects of polyherbal formulation 'ImmuPlus' on immunity and disease resistance of Indian major carp, Labeo rohita at different stages of growth

A series of experiments were performed to determine the impact of polyherbal immunomodulatory formulation 'ImmuPlus' (AquaImmu) on growth, immunity and disease resistance of rohu (Labeo rohita), one of the Indian major carp at different stages of growth. Rohu larvae were fed on plankton, ImmuPlus-mixed compound feed, and plankton plus ImmuPlus-mixed compound feed (ImmuPlus added at three dose levels of 0.25, 0.50, and 0.75 g/kg feed) from 4th day of hatching to 14th day. ImmuPlus-mixed diets enhanced growth of larvae, survival and disease resistance against Aeromonas hydrophila challenge, compared to only plankton-fed group. In two other experiments, advanced rohu larvae and fingerlings were fed with ImmuPlus-mixed compound feed (at 0, 0.5, 1.0 and 2.0 g/kg) for 15, 30 and 45 days. At the end of 45 days for advanced larvae and 30 days for fingerlings, the fish fed with ImmuPlus at 1.0 g/kg level showed significantly higher growth and disease resistance against A. hydrophila challenge. In a separate experiment, juveniles of rohu were fed with 1 g/kg of ImmuPlus incorporated feed for 15 and 30 days. At the end of the trial, the ImmuPlus fed fish showed enhanced non-specific immunity (as measured through nitroblue tetrazolium reduction assay, serum lysozyme activity, serum haemolysin titre and resistance against A. hydrophila challenge in non-vaccinated fish as well as specific immunity levels (as measured through bacterial agglutination titre against A. hydrophila in vaccinated fish). Incorporation of ImmuPlus at 1 g/kg level in the diet of rohu may be beneficial for enhancing disease resistance.     

Effect of endotoxin on the immunity of Indian major carp, Labeo rohita

Endotoxin, a lipopolysaccharide component of outer cell wall membrane of the Gram-negative bacteria is a factor responsible for a number of biological effects including immunostimulatory activities in different animal species including fish. In this study, L. rohita yearlings of weight ranging from 80 to 100g were injected intraperitoneally with 0.5, 1, 2, 5, 10 and 20 EU/fish dose of endotoxin to find out its effect on the immunity. The L. rohita yearlings were found to resist the endotoxin dose up to 20 EU/fish and at the lower doses, i.e., at 1 and 2 EU/fish; it acted as an immune potentiator. Different serum and immune parameters like protein, globulin, lysozyme, respiratory burst activity, myeloperoxidase activity, natural agglutination titre were found to be significantly high (p<0.01) at a dose of 1 EU/fish. While at 10 and 20 EU/fish, most of these parameters were lower thereby indicating the immuno-suppressive nature of the endotoxin at these higher doses.     

Antigen adsorbed calcium phosphate nanoparticles stimulate both innate and adaptive immune response in fish, Labeo rohita H

Calcium phosphate nanoparticles as an antigen/protein delivery was explored in a fish model Labeo rohita H. S-layer protein (of Aeromonas hydrophila) adsorbed on nano sized calcium phosphate particles elicited both innate and adaptive immune parameters which persisted up to 63 days of post immunization through parenteral immunization and gave cross protections.     

Purification and properties of cytosolic alanine aminotransferase from the liver of two freshwater fish, Clarias batrachus and Labeo rohita

Cytosolic alanine aminotransferase (c-AAT) was purified up to 203- and 120-fold, from the liver of two freshwater teleosts Clarias batrachus (air-breathing, carnivorous) and Labeo rohita (water-breathing, herbivorous), respectively. The enzyme from both fish showed similar elution profiles on a DEAE-Sephacel ion exchange column. SDS-PAGE of purified enzymes revealed two subunits of 54 and 56 kDa, in both fish. The apparent Km values for l-alanine were 18.5+/-0.48 and 23.55+/-0.60 mM, whereas for 2-oxoglutarate the Km values were observed to be 0.29+/-0.023 and 0.33+/-0.028 mM for the enzyme from C. batrachus and L. rohita, respectively. With l-alanine as substrate, aminooxyacetic acid was found to act as a competitive inhibitor with KI values of 6.4 x 10(-4) and 3.4 x 10(-4) mM with c-AAT of C. batrachus and L. rohita, respectively. However, when 2-oxoglutarate was used as substrate, aminooxyacetic acid showed uncompetitive inhibition with similar KI values for purified c-AAT from both fish. Temperature and pH profiles of the enzyme did not show any marked differences between the two fish examined. These results suggest that liver c-AAT, isolated from these two fish species adapted to different modes of life, remain unaltered structurally. However, at the kinetic level, liver c-AAT from C. batrachus exhibits significantly higher affinity for the substrate l-alanine and decreased affinity for its metabolic inhibitor, in comparison to that of the enzyme purified from L. rohita. Such functional changes seem to be of physiological significance and also provide preliminary evidence for subtle changes in the enzyme as a mark of metabolic adaptation in the fish to different physiological demands.     

Immunostimulating effect of triiodothyronine: dietary administration of triiodothyronine in rohu (Labeo rohita) enhances immunity and resistance to Aeromonas hydrophila infection

The immunomodulatory effects of dietary administration of 3,3^′,5‐triiodo‐l ‐thyronine (T₃) at 0, 1, 5 and 10 mg  kg^?1 of diet for 60 days in rohu (Labeo rohita) fingerlings were studied. Oral administration of T₃ at all dose levels resulted in significantly (P<0.05) higher serum T₃ levels, total serum protein and globulin levels, and reduced albumin–globulin ratio (A : G) compared with the control group, whereas feeding of T₃ at 5 and 10 mg kg^?1 diet enhanced the growth and superoxide production by neutrophils. At the end of the 60‐day experimental period the optimum dosage of T₃ appeared to be the 5 mg  kg^?1 diet for rohu fingerlings, resulting in a significantly higher specific antibody titre against formalin‐killed Aeromonas hydrophila and lowering the mortality percentage against the A. hydrophila challenge.     

Effect of multiple injections of beta-glucan on non-specific immune response and disease resistance in Labeo rohita fingerlings

The purpose of this study was to determine if multiple injections of different dosages of beta-glucan derived from barley would enhance the immune response and disease resistance against infections due to opportunistic pathogens Aeromonas hydrophila and Edwardsiella tarda in Labeo rohita fingerlings. Hence, four different dosages of beta-glucan suspension in phosphate-buffered saline at the rate of 0, 5, 10, 15 mg kg(-1) body weight of fish were injected intraperitoneally to the fingerlings of Labeo rohita at two-week intervals for four times. After every two-week interval different serum biochemical, haematological and immunological parameters of fish were evaluated. At the end of immunostimulation trial of 56 days, fish were divided into four subgroups under each major treatment group for challenge through i.p injection and bath immersion with two pathogens, A. hydrophila and E. tarda. The mortality (%) and agglutinating antibody titre was recoded on 28th day post challenge. Most of the immune parameters such as leucocyte count, phagocytic ratio, phagocytic index, lysozyme activity, complement activity, serum bactericidal activity were significantly (P < 0.05) enhanced on 42 days after three i.p injections of 10 mg of beta-glucan kg(-1) body wt. Challenge study indicated least mortality in the group of fishes injected with medium dose of 10 mg of beta-glucan kg(-1) body wt. four times. Multiple injections of beta-glucan might have maintained the activation of phagocytic cells for a long period which in turn would lead to long-term protection in fishes. Thus, injections of 10 mg of beta-glucan kg(-1) body wt. for three times can be advocated to enhance the immune response of fish species under aquaculture.     

Effect of Achyranthes aspera on the immunity and survival of Labeo rohita infected with Aeromonas hydrophila

Achyranthes aspera seed was incorporated in the diets (at 0.01%, 0.1% and 0.5%) of Labeo rohita, rohu fingerlings (3.0+/-0.4 g). After 2 weeks, the fish were immunized with heat-killed Aeromonas hydrophila, and after a further 2 weeks the rohu were experimentally infected with Aeromonas hydrophila (ATCC 49140). After 7 days blood and serum were sampled to determine superoxide anion production, bactericidal activity, lysozyme, serum protein, albumin, globulin, serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT) and alkaline phosphatase (ALP). Superoxide anion production, serum bactericidal activity, lysozyme, ALP, serum protein, albumin:globulin ratio (A/G) were enhanced in Achyranthes treated groups compared to the control group. SGOT and SGPT levels were elevated in control group, but in Achyranthes treated groups the levels were similar to the uninfected-control group. Higher cumulative mortalities were observed in the control group (77%) up to day-9 after infection. This gradually decreased with increasing dose of Achyranthes, 66% mortality in 0.01% group, 57% mortality in 0.1% group and 28% mortality in 0.5% group. These results indicate that Achyranthes aspera stimulates immunity and increases resistance to infection in L. rohita.     

Effect of Mangifera indica kernel as a feed additive on immunity and resistance to Aeromonas hydrophila in Labeo rohita fingerlings

The study evaluated the efficacy of dietary doses of Mangifera indica (mango) kernel on the immune response and disease resistance of Labeo rohita fingerlings against the bacterial pathogen Aeromonas hydrophila infections in. L. rohita fingerlings fed diet containing 0 (Control), 1g, 5 g, 10 g mango kernel kg(-1) dry diet for 60 days. Biochemical (serum total protein, albumin, globulin, albumin:globulin ratio, blood glucose), haematological (WBC, RBC, haemoglobin content) and immunological (superoxide anion production, lysozyme, serum bactericidal activity) parameters of fish were examined at 20, 40 and 60 days of feeding. Fish were challenged with A. hydrophila 60 days post feeding and mortalities were recorded over 10 days post-infection. The results demonstrate that fish fed with mango kernel showed enhanced superoxide anion production, lysozyme, serum bactericidal, serum protein, albumin (P<0.05) compared with the control group. The mortality (%) was recorded up to 10 th day post-challenge. Less survivability was observed in control group (50%) up to day 10 after infection. The survivability was higher in experimental diets. The group fed 5 g kernel kg(-1) dry diet showed highest percentage survival (98%). These results indicate that mango kernel stimulates the immunity and makes L. rohita more resistant to A. hydrophila infection.     

Effects of amino acid supplementation on the nutritive quality of fermented linseed meal protein in the diets for rohu, Labeo rohita, fingerlings

A feeding trial was conducted for 8 weeks to examine the effects of partial substitution of fish meal (FM) protein (crude protein content: 58.5%) with linseed meal protein with and without supplemental amino acids in diets for rohu Labeo rohita (Hamilton), fingerlings (mean weight: 1.50 ± 0.3 g). Prior to incorporation into the diets, linseed meal was fermented with lactic acid bacteria ( Lactobacillus acidophilus ) to reduce/eliminate the antinutritional tannin and phytic acid factors. Twelve experimental diets (diets D1–D12) were formulated to replace the FM protein from a reference diet (RD) with linseed meal protein at different levels (four sets of diets, of which each set of three diets contained 25%, 50% and 75% replacement of FM protein by linseed meal protein, respectively). Diets D1–D3 were not supplemented with any amino acid. Lysine was supplemented in diets D4–D6. Diets D7–D9 were supplemented with methionine + cystine (together), and diets D10–D12 contained lysine and methionine + cystine (together). Lysine and methionine + cystine (together) were added to the diets at 5.7% and 3.1% of dietary protein, respectively. The groups of fish fed diets without amino acid supplementation had significantly lower percentages of weight gain, specific growth rate and high feed : gain ratio than the fish groups fed other experimental diets. The addition of lysine and methionine + cystine to the diet in which 50% of the FM protein was replaced by linseed meal protein (diet D11) significantly improved fish performance. The results of the present study suggest that rohu fingerlings can effectively utilize the supplemented amino acids and that linseed meal protein can replace up to 50% of the FM protein in rohu diets if the linseed meal is properly processed (fermented) and supplemented with the lacking amino acids.     

Parenteral immunization of fish,Labeo rohita with Poly d,l-lactide-co-glycolic acid (PLGA) encapsulated antigen microparticles promotes innate and adaptive immune responses

Immunogenicity of different antigen preparations of outer membrane proteins (OMP) of Aeromonas hydrophila such as Poly d, l-lactide-co-glycolic acid (PLGA) microparticles, oil emulsion, neat OMP and bacterial whole cells were compared through intra-peritoneal injection in fish, Labeo rohita. Among these preparations, PLGA encapsulated antigen stimulated both innate and adaptive immune parameters and the immunogenicity exhibited by PLGA microparticles was significantly higher (p < 0.05) at both 21 and 42 days post-immunization suggesting that the above delivery system would be a novel antigen carrier for parenteral immunization in fish, Labeo rohita     

Effect of Allium sativum on the immunity and survival of Labeo rohita infected with Aeromonas hydrophila

The aim of this study was to evaluate dietary dosages of garlic on the immune response and disease resistance against infections due to the opportunistic pathogen Aeromonas hydrophila in Labeo rohita fingerlings. Garlic, Allium sativum, was incorporated into the diets (0%, 0.1%, 0.5% and 1.0%) of rohu, L. rohita fingerlings (10 ± 2 g). Every 20 days, different biochemical (serum total protein, albumin, globulin, albumin : globulin ratio, blood glucose), haematological (WBC, RBC and haemoglobin content) and immunological (superoxide anion production, lysozyme activity and serum bactericidal activity) parameters were evaluated. Superoxide anion production, lysozyme, serum bactericidal, serum protein and albumin were enhanced in garlic treated groups compared with the control group. After 60 days, fish were challenged with A. hydrophila and mortality (%) was recorded up to day 10 post‐challenge. Survival decreased in control group (57%) up to day 10 after infection. However, this was increased in the garlic treatment group, i.e. 85% survivability in the 1 g garlic kg^?1 (B group) and 5 g garlic kg^?1 (C group), and 71% survivability in the 10 g garlic kg^?1 (D group), respectively. These results indicate that Allium sativum stimulates the immunity and makes L. rohita more resistant to infection by A. hydrophila.     

Effects of potential probiotic Bacillus subtilis T13 on growth, immunity and disease resistance against Vibrio splendidus infection in juvenile sea cucumber Apostichopus japonicus

A feeding experiment was conducted to determine influences of potential probiotic Bacillus subtilis T13 (isolated from intestine of healthy sea cucumbers) on growth, immunity and disease resistance against Vibrio splendidus infection in juvenile sea cucumbers Apostichopus japonicus. Animals were fed with diets containing B. subtilis T13 at 0, 10(5), 10(7) and 10(9) CFU/g for 30 days, respectively. At the end of the growth trial, fifteen sea cucumbers from each aquarium were sampled for immune indices measurement. Then twenty sea cucumbers from each replicate were challenged with V. splendidus. Results showed that administration of B. subtilis T13 had significant effect on the specific growth rates (SGR) of sea cucumbers (P < 0.05). Phagocytosis, respiratory burst activity and total nitric oxide synthase (T-NOS) activity were significantly improved in coelomocytes of sea cucumbers fed with T13 at 10(9) CFU/g diet (P < 0.05). The highest values of the total coelomocytes counts (TCC) and superoxide dismutase (SOD) activity were found in sea cucumbers fed diet containing T13 at 10(9) CFU/g. The cumulative mortality after V. splendidus challenge decreased significantly in sea cucumbers fed with T13 at dose of 10(9) CFU/g (P < 0.05). The present study confirmed the potential beneficial effects of B. subtilis T13 as dietary probiotic in juvenile A. japonicus.     

Effect of dietary beta-1,3 glucan on immune responses and disease resistance of healthy and aflatoxin B1-induced immunocompromised rohu (Labeo rohita Hamilton).

The immunostimulant beta-1,3 glucan was fed at 0.1% in feed for 7 days to healthy and aflatoxin B1 (AFB1)-induced immunocompromised fish, Labeo rohita (one of the major tropical carp species), in a 60 day trial. The effects of AFB1, glucan and their interactions on non-specific and specific immunity levels and disease resistance of fish were studied. A single intraperitoneal injection of AFB1 at 1.25 mg kg(-1) body weight) caused a significant (P < 0.05) reduction in non-specific immunity as measured through neutrophil phagocytic indices, serum bactericidal activity, and specific immunity as measured through bacterial agglutination titre against Edwardsiella tarda, as well as reduced protection against Aeromonas hydrophila challenge in comparison to control fish which were exposed neither to aflatoxin nor to glucan. Feeding of glucan to healthy fish raised the non-specific and specific immunity level and protection against bacterial infection compared with the control. Feeding of glucan to AFB1-induced immunocompromised fish for 7 days significantly raised the degree of resistance against A. hydrophila challenge and the non-specific immunity level in comparison to non-treated AFB1 exposed fish. Although feeding of glucan was able to increase specific immunity, all measured through haemagglutination titre against sheep red blood cells, and bacterial (E. tarda) agglutination titre in healthy fish in comparison to all other groups, no significant increase in specific immunity to the aflatoxin-exposed group was seen.     

Perturbations in nitrogen metabolic profiles in the tissues of fish, Labeo rohita exposed to fenvalerate.

Changes in the levels of nitrogen metabolic profiles in gill, brain, liver and muscle tissues of fish, Labeo rohita exposed to sublethal concentration (10 micrograms/L) of fenvalerate for 6, 12, 24 and 48 hours were studied. Ammonia content was decreased, whereas urea and glutamine levels were increased in the tissues of fenvalerate exposed fish. Changes were more pronounced with an increase in the period of exposure. Within the tissues, the alterations in excretory products were in the following order: liver greater than muscle greater than brain greater than gill. The survivability of fish in the polluted habitats might be due to the operation of compensatory mechanism in the metabolic profiles such as detoxification and transamination of more toxic ammonia by conversion to less toxic compounds like urea and glutamine.     

Protease IV,a quorum sensing‐dependent protease of Pseudomonas aeruginosa modulates insect innate immunity

In Pseudomonas aeruginosa, quorum sensing (QS) plays an essential role in pathogenesis and the QS response controls many virulence factors. Using a mealworm, Tenebrio molitor as a host model, we found that Protease IV, a QS‐regulated exoprotease of P. aeruginosa functions as a key virulence effector causing the melanization and death of T. molitor larvae. Protease IV was able to degrade zymogens of spätzle processing enzyme (SPE) and SPE‐activating enzyme (SAE) without the activation of the antimicrobial peptide (AMP) production. Since SPE and SAE function to activate spätzle, a ligand of Toll receptor in the innate immune system of T. molitor, we suggest that Protease IV may interfere with the activation of the Toll signaling. Independently of the Toll pathway, the melanization response, another innate immunity was still generated, since Protease IV directly converted Tenebrio prophenoloxidase into active phenoloxidase. Protease IV also worked as an important factor in the virulence to brine shrimp and nematode. These results suggest that Protease IV provides P. aeruginosa with a sophisticated way to escape the immune attack of host by interfering with the production of AMPs.