In vivo antibacterial activity of medicinal plant Sophora flavescens against Streptococcus agalactiae infection.

Streptococcosis disease caused by Streptococcus agalactiae (Group B Streptococcus, GBS) results in a huge economic loss of tilapia culture. It is urgent to find new antimicrobial agents against streptococcosis. In this study, 20 medicinal plants were evaluated in vitro and in vivo to obtain medicinal plants and potential bioactive compounds against GBS infection. The results showed that the ethanol extracts of 20 medicinal plants had low or no antibacterial properties in vitro, with a minimal inhibitory concentration ≥256 mg/L. Interestingly, in vivo tests showed that 7 medicinal plants could significantly inhibit GBS infection in tilapia, and Sophora flavescens (SF) had the strongest anti-GBS activity in tilapia, reaching 92.68%. SF could significantly reduce the bacterial loads of GBS in different tissues (liver, spleen and brain) of tilapia after treated with different tested concentrations (12.5, 25.0, 50.0 and 100.0 mg/kg) for 24 h. Moreover, 50 mg/kg SF could significantly improve the survival rate of GBS-infected tilapia by inhibiting GBS replication. Furthermore, the expression of antioxidant gene cat, immune-related gene c-type lysozyme and anti-inflammatory cytokine il-10 in liver tissue of GBS-infected tilapia significantly increased after treated with SF for 24 h. Meanwhile, SF significantly reduced the expression of immune-related gene myd88 and pro-inflammatory cytokines il-8 and il-1ß in liver tissue of GBS-infected tilapia. The negative and positive models of UPLC-QE-MS, respectively, identified 27 and 57 components of SF. The major components of SF extract in the negative model were α, α-trehalose, DL-malic acid, D- (-)-fructose and xanthohumol, while in the positive model were oxymatrine, formononetin, (-)-maackiain and xanthohumol. Interestingly, oxymatrine and xanthohumol could significantly inhibit GBS infection in tilapia. Taken together, these results suggest that SF can inhibit GBS infection in tilapia, and it has potential for the development of anti-GBS agents.

Isolation and Characterization of Aeromonas jandaei from Nile Tilapia in Lake Volta, Ghana, and Its Response to Antibiotics and Herbal Extracts.

Production of Nile Tilapia Oreochromis niloticus contributes to economic growth in many countries. However, there has been a decline in its production over the years due to the influx of bacterial infections, with Aeromonas jandaei as an emerging threat. In this study, we identified and characterized A. jandaei from cage-cultured Nile Tilapia in Akosombo Stratum II of Lake Volta in Ghana and evaluated its response to commonly used antibiotics using the disc diffusion and agar well diffusion methods for herbal extracts at various concentrations (10, 30, 50, 70, and 100 mg/mL). The herbs considered included guava Psidium guajava leaf, bitter leaf Vernonia amygdalina, neem Azadirachta indica leaf, and their cocktail (GBNL in the ratio of 1:1:1). The bacterium was isolated from swab samples from the head kidneys of 27 moribund Nile Tilapia collected from nine fish farms. Samples were screened for A. jandaei by culturing and identification using morphological and molecular techniques. The bacterium isolate from fish in the study, identified as A. jandaei GH-AS II, had 92-93% identity to A. jandaei reference strains. Infection of healthy Nile Tilapia (n = 210) with the bacterium isolate showed that 1.0 × 105 CFU/mL was the lethal dose causing 50% mortality. Antibiotic susceptibility testing showed that A. jandaei GH-AS II was resistant to tetracycline and ampicillin. Herbal extracts at the various concentrations inhibited the growth of the bacterium isolate, with a significant increment in the zones of inhibition with increasing concentrations of leaf extracts. However, GBNL showed prominence compared to the other extracts only at 100 mg/mL. Management of A. jandaei GH-AS II by using herbal extracts at Nile Tilapia farms in Lake Volta may be recommended since the use of antibiotics, such as tetracycline and ampicillin, may not yield the needed result.

The synergistic effects of plant polysaccharide and Pediococcus acidilactici as a synbiotic additive on growth, antioxidant status, immune response, and resistance of Nile tilapia (Oreochromis niloticus) against Aeromonas hydrophila.

This study evaluated the growth performance, immune responses, and disease resistance of Nile tilapia upon pistachio hulls derived polysaccharide (PHDP) and Pediococcus acidilactici (PA) separately or as synbiotic. Fish received four types of diets: T1, control; T2, PHDP (0.1%); T3, PA (0.2%); T4, PHDP (0.1%) +PA (0.2%) for 56 days. The results showed that final weight and weight gain were markedly higher in fish fed T4 diet than that given T1 and T2 diets (P ≤ 0.05). In addition, a significantly greater specific growth rate was obtained by the T4 diet compared to the control. Fish survival was significantly improved in all supplemented diets compared to the control. On the other hand, the activities of lipase, protease, and amylase showed significant increases in the T4 group compared with other feeding groups. The total leucocytes and lymphocytes proportion significantly elevated in T3 and T4 than remaining groups (P ≤ 0.05). Further, fish fed T3 diet presented significantly higher serum total protein, total immunoglobulin, lysozyme activity (LYZ), alternative complement activity (ACH50), and alkaline phosphatase activity compared to fish fed T1 and T2 diets, while the mentioned indices were found significantly highest in T4 group than others. Fish received T3 and T4 diets had higher skin mucus LYZ and ACH50 than those fed T1 and T2 diets (P ≤ 0.05). The malondialdehyde levels were significantly declined in T3 and T4 when compared to the control. Fish fed T3 and T4 diets demonstrated significantly enhanced superoxide dismutase, catalase, and glutathione peroxidase activities compared to the control. The intestinal propionic acid significantly increased by T2 and T4 diets, while the highest levels of acetic acid detected in fish given T4 diet. The expression levels of tumor necrosis factor α (TNF-α), interleukin 1ß (IL-1ß), and interleukin 10 (IL-10) were significantly affected by T3 and T4 supplements. The efficacy of T4 diet against Aeromonas hydrophila infection was documented by a significantly lower mortality rate. In conclusion, the combination of PHDP and PA presented promising results as a synbiotic feed additive for Nile tilapia.

Assessment the efficacy of some various treatment methods, in vitro and in vivo, against Aeromonas hydrophila infection in fish with regard to side effects and residues.

Aeromonas hydrophila is an opportunistic bacteria with an overwhelming impact on fish farming industry especially with upraising of drug resistant mutants. This study aimed to evaluate and compare the therapeutic and side effects of levofloxacin (LEV), chitosan-nanoparticles (CNPs), and fructooligosaccharides (FOS) in control of this infection in tilapia. A total of 160 Nile-tilapia divided into 8-groups; G1: negative-control, G2: infected-control, G3: non-infected-(levofloxacin (LEV) 10 mg/kg bwt), G4: non-infected-(chitosan-nanoparticles (CNPs) 1 g/kg ration), G5: non-infected-(fructooligosaccharides (FOS) 20 g/kg ration), G6: infected-LEV, G7: infected-CNPs and G8: infected-FOS for 7 days. MICs were (0.125 µg/ml and 1.25 mg/ml) for LEV and CNPs respectively. No mortalities or significant adverse effects were recorded in non-infected treated-groups while infected were (20%) LEV, (30%) CNPs, (40%) FOS and (70%) G2. Aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) decreased by LEV and CNPs than FOS while all increased total protein (TP) and albumin than G2. Malondialdehyde (MDA) significantly decreased and superoxide dismutase (SOD) and reduced glutathione (GSH) increased in all infected-treated groups than G2 in various degrees. Urea and creatinine descending order were FOS, LEV then CNPs decreased significantly than G2. LEV musculature residues, using HPLC, decreased gradually till the 5th day; 621.00 ± 0.66, 270.00 ± 0.48 then 64.00 ± 0.40, and 471.00 ± 0.79, 175.00 ± 0.52 ppb then not detected at 1st, 3rd, and 5th days of withdrawal in non-infected and infected groups respectively. Finally, LEV and CNPs were superior as bactericidal, decreasing mortalities and enzyme activities while CNPs and FOS increased performance, non-specific immunity, and antioxidant biomarkers.

Effects of sodium butyrate and Lippia origanoides essential oil blend on growth, intestinal microbiota, histology, and haemato-immunological response of Nile tilapia.

This study aimed to verify the effects of dietary supplementation with sodium butyrate and Lippia origanoides, combined and isolated, on the health and zootechnical performance of Nile tilapia juveniles Oreochromis niloticus. A total of 120 fish (5.38 ± 0.65 g) were randomly distributed in 12 experimental units and fed different experimental diets for 30 days, namely: commercial diet without supplementation (Unsupplemented); commercial diet supplemented with 0.5% sodium butyrate (Butyrate); commercial diet supplemented with 0.125% L. origanoides (Lippia) and commercial diet supplemented with a mixture of 0.5% sodium butyrate and 0.125% L. origanoides (Butyrate + Lippia). After preparing the experimental diets there was an increase in the pH of diet Butyrate when compared to the other diets. After 30 days the fish supplemented with Butyrate + Lippia showed reduction significate in the mean corpuscular haemoglobin, concentration of total heterotrophic bacteria in the intestine, and lymphocyte infiltrates in the liver. Besides that, the supplementation with Butyrate + Lippia promoted an increased number of intestinal villi compared to the fish Unsupplemented ones. Additionally, fish fed a diet containing only Lippia presented an increase in the villus perimeter in the posterior region of the gut and in the red blood cell number. Animals supplemented only with sodium butyrate demonstrated increased lactic acid bacterium in the gut and macrosteatosis in the liver, besides decreased melanomacrophages in the spleen. The use of sodium butyrate associated with essential oil had positive effects on the intestinal microbiota, intestinal structure, liver, and spleen integrity, suggesting a greater efficiency of the compounds when used together in the nutrition of Nile tilapia juveniles.

Exogenous enzymes and probiotics alter digestion kinetics, volatile fatty acid content and microbial interactions in the gut of Nile tilapia.

Sustainable aquafeed production requires fishmeal replacement, leading to an increasing use of plant-derived ingredients. As a consequence, higher levels of antinutritional substances, such as non-starch polysaccharides and phytate, are present in aquafeeds, with negative effects on fish performance, nutrient digestibility and overall gut health. To alleviate these negative effects, providing exogenous digestive enzymes and/or probiotics can be an effective solution. In this study, we tested the effect of dietary supplementation of enzymes (phytase and xylanase) and probiotics (three strains of Bacillus amyloliquefaciens) on nutrient digestion kinetics and volatile fatty acid content along the gut, and the distal gut microbiome diversity in Nile tilapia. Chyme volatile fatty content was increased with probiotic supplementation in the proximal gut, while lactate content, measured for the first time in vivo in fish, decreased with enzymes along the gut. Enzyme supplementation enhanced crude protein, Ca and P digestibility in proximal and middle gut. Enzymes and probiotics supplementation enhanced microbial interactions as shown by network analysis, while increased the abundance of lactic acid bacteria and Bacillus species. Such results suggest that supplementation with exogenous enzymes and probiotics increases nutrient availability, while at the same time benefits gut health and contributes to a more stable microbiome environment.

Tinospora cordifolia ameliorated titanium dioxide nanoparticle-induced toxicity via regulating oxidative stress-activated MAPK and NRF2/Keap1 signaling pathways in Nile tilapia (Oreochromis niloticus).

Titanium dioxide nanoparticle (TNP) has been suggested for use in fish farms to prevent or alleviate bacterial diseases owing to its bactericidal property. Unfortunately, the interaction of TNP with cells impaired the host defenses of fish resulting in increased mortality during bacterial challenges. The present study evaluated the efficacy of the ethanolic extract of Tinospora cordifolia (TCE) as a dietary supplement in ameliorating TNP induced toxicity in Nile tilapia (Oreochromis niloticus). The fishes were exposed to environmentally relevant concentration (10 mg/L) of TNP for 14 days and the effect of TCE supplemented feed at 3 different doses (5, 10, and 15 g/kg) was studied. TCE signally increased the weight gain, specific growth rate, and decreased feed conversion ratio in fish. TCE significantly (P < 0.05) ameliorated the toxic effects caused by TNP by increasing the antioxidant (CAT, SOD, GPx) activity and decreasing the levels of serum enzymes (ALT, AST, ALP, ACP), macromolecular oxidation, excessive ROS production, and pro-inflammatory cytokines (IL-1ß, IL-6, IL-8, INF-γ, TNF-α, PGE-2). TNP bioaccumulation and histopathological alterations in gill, liver, and kidney were also significantly alleviated by TCE supplementation. TCE perceptibly regulated the expression of heat shock proteins (HSP60, -70), MAPKs (pERK1/2, pp38), antioxidant (NRF2, Keap1, HO-1), apoptotic (p53, PDRG1), and anti-apoptotic (AKT, Bcl2) proteins in fish. Regarding disease resistance, the TCE co-treated groups showed reduced cumulative mortality and higher relative percent survival with A. hydrophila. Our results suggest that TNP-induced apoptosis is mediated by the MAPK/NRF2/Keap1 pathway and underlines the therapeutic potential of TCE in aqua-farming.

Treatment Trial of Nile Tilapia (Oreochromis niloticus) Experimentally Infected with Vibrio alginolyticus Isolated from Sea bass (Dicentrarchus labrax).

BACKGROUND AND OBJECTIVE: In Egypt, Nile tilapia represents the main cultured type due to its economical price, palatability and easy culturing. This study was aimed to elucidate the pathogenicity of V. alginolyticus isolated from diseased sea bass and experimentally infected healthy Nile tilapia fish. MATERIALS AND METHODS: Healthy Nile tilapia fish were injected I/P with V. alginolyticus isolated from diseased sea bass. Symptoms and mortality rates of infected Nile tilapia fish were recorded during the experimental period. Re-isolation of V. alginolyticus was done from infected tilapia fish by bacteriological methods. For confirmation the pathogenicity of Vibrio isolated either from marine fish or tilapia fish, PCR test was done using tdh and bla gens. Liver and kidney function tests with histopathological examinations of some organs were performed. Treatment trial was done according to the antibiotic sensitivity test. RESULTS: The isolated Vibrio is highly pathogenic to Nile tilapia fish causing deterioration in all parameters which finished by severe mortalities. Treatment with florfenicol, enrofloxacin, or oxytetracycline reduced the mortality rate and improved liver and kidney function parameters of infected Nile tilapia fish. CONCLUSION: V. alginolyticus can infect both marine and fresh water fish inducing a high mortality rate. Treatment of infected fish with florfenicol, enrofloxacin, or oxytetracycline reduces the mortality rate.

Effects of oligochitosan on the growth, immune responses and gut microbes of tilapia (Oreochromis niloticus).

The immunomodulatory effects of oligochitosan have been demonstrated in several fish. However, the underlying mechanisms are not well characterized. The profound interplay between gut microbes and aquaculture has received much scientific attention but understanding the alternations of microbes populating in gut of tilapia (Oreochromis niloticus) fed with oligochitosan remains enigmatic. In this study, the effects of oligochitosan on the growth, immune responses and gut microbes of tilapia were investigated. The feeding trial was conducted in triplicates with the control diet supplemented with oligochitosan at different concentrations (0, 100, 200, 400 or 800 mg/kg). Following a six-week feeding trial, body weights of the fish supplemented with 200 mg/kg and 400 mg/kg oligochitosan were significantly higher than that of the control group. To address the immune responses stimulated by oligochitosan, by the quantitative real time PCR (qRT-PCR), the mRNA expression levels of CSF, IL-1ß, IgM, TLR2 and TLR3 genes from head kidney were all significantly up-regulated in the 400 mg/kg group compared to the control. To characterize the gut microbes, bacterial samples were collected from the foregut, midgut, and hindgut, respectively and were subjected to high-throughput sequencing of 16S rDNA. The results showed that significantly lower abundance of Fusobacterium was detected in the hindgut of 400 mg/kg group compared to the control. Additionally, beta-diversity revealed that both gut habitat and oligochitosan had effects on the gut bacterial assembly. To further elucidate the mechanism underlying the effects of oligochitosan on bacterial assembly, the results showed that difference dosages of dietary oligochitosan could alter the specific metabolic pathways and functions of the discriminatory bacterial taxa, resulting in the different bacterial assemblies. To test the antibacterial ability of tilapia fed with oligochitosan, when the tilapias were challenged with Aeromonas hydrophila, the mortality of groups fed with dietary oligochitosan was significantly lower than that of the control. Taken together, appropriate dietary oligochitosan could improve growth, immune responses and alter the bacterial flora in the intestine of tilapia, so as to play a role in fighting against the bacterial infection.

Diet type influences the gut microbiome and nutrient assimilation of Genetically Improved Farmed Tilapia (Oreochromis niloticus).

Nile tilapia, Oreochromis niloticus is the third most commonly farmed finfish species in the world, accounting for nearly 5% of global aquaculture production. In the past few decades much of the success of this species has been attributed to the development and distribution of Genetically Improved Farmed Tilapia (GIFT). Despite the increasing availability of GIFT, the productivity of small-scale farming remains highly variable, particularly in developing nations. Commercial fish-feed pellets can increase fish farm productivity; however, many small-scale farmers rely on other means of feeding fish due to the high cost and limited availability of commercial fish feed pellets. Therefore, understanding how locally-sourced feeds affect the production of GIFT is an important step towards improving feeding practices, particularly for farmers with low financial capital. This study used stable isotope analysis (SIA) and 16S rRNA gene sequencing to compare the effects of a locally-sourced vegetable-based diet and commercial pellet-based diets on the relative condition, nutrient assimilation patterns and gastrointestinal microbiota of GIFT. GIFT fed a locally-sourced diet were smaller, and in a significantly poorer condition than those fed with commercial fish feeds. SIA showed no differences in dietary carbon between the two diets; however, δ13C, poor fish condition and the abundance of specific bacterial taxa (of such as Fusobacteria) were correlated. SIA revealed that GIFT fed locally-sourced diets that predominantly consisted of vegetables were significantly enriched in δ15N despite a perceived lack of dietary protein. This enrichment suggests that GIFT fed a locally-sourced diet may be supplementing their diet via cannibalism, a behaviour representative of poor farming practice. Overall this study highlights the need to increase the availability of suitable GIFT feeds in developing nations. The development a low-cost feed alternative could improve the success of small-scale GIFT farmers in PNG, increasing both food and income security within the region.

Inorganic mercury and dietary safe feed additives enriched diet impacts on growth, immunity, tissue bioaccumulation, and disease resistance in Nile tilapia (Oreochromis niloticus).

Little is known about the impacts of dietary exposure to inorganic mercury (Hg) for a long duration on the health indicators, growth, and disease resistance in Oreochromis niloticus. Accordingly, the current study was designed to assess the effects of Hg contaminated diets on blood biochemistry, growth, chemical composition, Hg bioaccumulation in the tissues, histopathology of liver and head kidneys, and disease resistance to Aeromonas hydrophila of O. niloticus. Also, the efficiency of citronella oil, geranium oil (GO), curcumin (CUR), Bacillus toyonensis (BT), and Bacillus subtilis (BS) as dietary supplements on reversing the negative impacts of Hg were assessed. A total of 240 tilapia fingerlings were assigned to eight dietary treatments fed on the basal diet (G1), G1 diet contaminated with 50 ppm Hg (G2), whereas the other groups fed the G2 diet and enriched with 400 mg CO (G3), 400 mg GO (G4), 200 mg CUR (G5), 7 × 107 cells BT (G6), 7 × 107 cells BS (G7), and 7 × 107 BT + BS/ kg diet (G8) for 16 weeks. The obtained results showed that fish fed on the G2 diet had significantly impaired growth performance indicators, blood parameters, and resistance to bacterial infection compared with fish in the control group. Additionally, distinct pathological perturbations in liver and head kidneys were observed. In contrast, fish groups G3 to G8 had a significant enhancement in the growth performance, Hg bioaccumulation in fish tissues, blood biochemistry, and resistance against A. hydrophila infection compared with fish in the G2 group. Maximum improvement was recorded in G5, G6, and G8. Conclusively, from both health and an economic point of view, these results suggested that several benefits might be gained by adding these additives, especially CUR, BT, and BT + BS, on growth enhancement and ameliorating Hg negative impacts in O. niloticus.

Ziziphus mauritiana supplementation of Nile tilapia (Oreochromis niloticus) diet for improvement of immune response to Aeromonas hydrophila infection.

This study evaluated the effectiveness of dietary Ziziphus mauritiana leaf powder (ZLP) to control Aeromonas hydrophila infection in Nile tilapia and reduce damage to vital immune organs. Four experimental groups were fed a diet supplemented with ZLP at concentrations of 0, 5, 10, and 20 g/kg (w/w) for 6 weeks. At the end of the feeding trial, all groups were intraperitoneally injected with pathogenic A. hydrophila. It was found that Z. mauritiana significantly (P < 0.05) upregulated (lysozyme, interleukin 1 beta) and superoxide dismutase gene expressions as well as improved the activity of serum lysozyme and liver antioxidant enzymes. The fish that were fed a ZLP-supplemented diet also exhibited significantly higher survival rates after A. hydrophila challenge than those that were fed a ZLP-free diet (P < 0.05). Supplementation of 10 g/kg ZLP most effectively reduced the histopathological alterations caused by A. hydrophila challenge in the liver, spleen, kidney, and muscle of the fish. In conclusion, ZLP can be effective in controlling A. hydrophila infection in Nile tilapia (particularly at a concentration of 10 g/kg) through enhancement of its immune and antioxidant status.

Benefits of nanotechnology: Dietary supplementation with nerolidol-loaded nanospheres increases survival rates, reduces bacterial loads and prevents oxidative damage in brains of Nile tilapia experimentally infected by Streptococcus agalactiae.

Rampant and uncontrolled use of antibiotics is a major concern for aquaculture; the practice foments the emergence of resistant strains of Streptococcus agalactiae, among other negative impacts. Constituents of plant essential oils such as nerolidol are being considered as replacements for synthetic drugs to support fish nutrition and health. There is evidence to suggest that nanotechnology may enhance the efficacy of natural bioactive compounds; this is a substantial advance for the development and sustainability of aquaculture. Against the backdrop of this evidence, we aimed determine whether dietary supplementation with free nerolidol and nerolidol-loaded nanospheres would exert bactericidal effects against S. agalactiae, as well as prevent S. agalactiae-induced brain oxidative damage. In Experiment I, we measured the antimicrobial properties of dietary supplementation of nerolidol and nerolidol nanosphere in terms of mortality, longevity and relative percent survival. Fish infected with S. agalactiae fed 0.5 and 1.0 mL nerolidol nanospheres kg/diet demonstrated lower mortality and higher relative percent survival than the control group, while longevity was higher in all infected plus supplementation groups. Experiment II showed significantly lower microbial loads in brains of fish infected with S. agalactiae that were fed 1.0 mL nerolidol nanospheres kg/diet than in the control group. Brain nerolidol levels were significantly higher in uninfected as well as infected fish supplemented with nerolidol nanospheres than in fish supplemented with free nerolidol. Finally, brain reactive oxygen species and lipid peroxidation levels were higher in infected fish supplemented with basal diet compared to uninfected fish and supplemented with basal diet, and the supplementation with 1.0 mL/kg nerolidol nanospheres prevented this augmentation caused by infection. These data suggest that dietary supplementation with nerolidol nanospheres (1.0 mL/kg diet) has potent bactericidal effects in terms of augmentation of fish longevity and survival, and reduction of brain microbial loads. Also, S. agalactiae-induced brain oxidative damage that contributed to disease pathogenesis, and the dietary supplementation with nerolidol nanospheres (1.0 mL/kg diet) prevented this alteration. In summary, nanotechnology is a compelling approach to enhancing the efficacy of nerolidol, giving rise to reduction of S. agalactiae loads in fish brains.

Effects of dietary supplementation with a microalga (Schizochytrium sp.) on the hemato-immunological, and intestinal histological parameters and gut microbiota of Nile tilapia in net cages.

Nutritional improvements in intensive aquaculture production systems is necessary for the reduction of stress, maximum utilization of nutritional components, and expression of the genetic potential of fish. The objective of this study was to evaluate the hemato-immunological, and histological parameters and gut microbiota of Nile tilapia fed with the microalga Schizochytrium sp. Males of Nile tilapia were distributed among eight net cages (6 m3), and fed for 105 days with two diets: control (CON), without Schizochytrium sp., and supplemented (SUP), with 1.2% Schizochytrium sp. in the diet. The final weight, mortality, hematocrit, total erythrocyte count (RBC), hemoglobin, hematimetric indices, white blood cell count (WBC), total protein, and serum lysozyme were measured. Alterations in intestinal morphology were evaluated. The gut microbiota was evaluated with next-generation sequencing. No significant differences (p>0.05) were found in the final weight and mortality between diets. Regarding the hematological parameters, a difference (p<0.05) was detected only in RBC, with there being lower values in the SUP, although this group also showed a tendency toward having an increased mean corpuscular hemoglobin level. There were no differences (p>0.05) in total protein and serum lysozyme concentrations or in WBCs between diets, except for lymphocytes, which presented lower values (p<0.05) in the SUP, suggesting immunomodulation by the polyunsaturated fatty acids present in the microalga. There was no difference (p>0.05) in the intestinal morphology between diets. Metagenomic data indicated greater richness (represented by the Chao index) and a higher abundance of the bacterial phylum Firmicutes in the gut microbiota of the tilapia fed with the SUP diet, demonstrating that the digestion and use of the components of the microalga could influence the microbial community. The results indicated that the microalga had modulatory effects on blood cells and the intestinal microbiota, without affecting the structure and integrity of the intestinal villi.

Time-dependent gut microbiota analysis of juvenile Oreochromis niloticus by dietary supplementation of resveratrol.

To evaluate the changes in bacterial diversity at various time points under resveratrol supplementation, we aimed to investigate the diversification of gut microbiota and the changes in total genetic diversity. We performed 16S rDNA gene sequencing at different time points (15, 30, and 45 days) to analyze the gut microbiota of tilapia. Fusobacteria, Proteobacteria, and Bacteroidetes (15 days) or Cyanobacteria (30 and 45 days) were found to be the three most abundant phyla. Cyanobacteria (15 and 30 days), Proteobacteria (15 days), Firmicutes and Chlamydiae (30 and 45 days), Planctomycetes (30 days), Bacteroidetes, Actinobacteria, and Fusobacteria (45 days) in the 0.05 g/kg RES group increased as compared to that in the controls. Proteobacteria and Cyanobacteria significantly decreased and increased at 30 and 45 days, respectively, while the reverse pattern was observed at 15 days. The Bacteroidetes:Firmicutes and Proteobacteria:Cyanobacteria ratios were significantly increased (15 and 45 days, P < 0.05) and decreased (30 days, P < 0.05). RES supplementation did not affect the richness and diversity of the gut microbiota in tilapia. Our findings may contribute to the development of strategies for the management of diseases.

Growth Promotion and Immune Stimulation in Nile Tilapia, Oreochromis niloticus, Fingerlings Following Dietary Administration of a Novel Marine Probiotic, Psychrobacter maritimus S.

A 50-day feeding trial was conducted to evaluate the effects of dietary supplementation of a novel marine psychrotrophic bacterium, Psychrobacter maritimus S, on growth performance and immune responses of Nile tilapia (Oreochromis niloticus) fingerlings. Triplicate groups of Nile tilapia (10 fish each) were fed for 50 days with three different diets: T0 (without probiotic, negative control), T0.5 (supplemented with 3.3 × 108 CFU g-1P. maritimus S), and T1 (supplemented with 6.6 × 108 CFU g-1P. maritimus S). The test diets were fed to fish at a daily rate of 3% of their live weights, three times per day. On the 50th day, fish were weighed and blood samples were collected to determine the main nonspecific humoral and cellular immune responses and digestive enzymes (protease, amylase, and lipase). Fish performance (weight gain, specific growth rate, feed conversion ratio, protein efficiency ratio, and protein productive value) was also calculated. Growth rates and digestive enzyme activity were significantly increased (P < 0.05) with increasing dietary P. maritimus S up to 0.5%, and leveled off with further increase in bacterial concentrations. Phagocytic activity, lysozyme activity, alternative complement hemolysis, and hematological parameters were also significantly increased (P < 0.05) with increasing P. maritimus S concentration to 0.5%, followed by a slight decrease (P > 0.05) at 1% level. The expression of interleukin-4 and interleukin-12 genes was significantly upregulated, while heat shock protein gene was downregulated, with dietary supplementation of P. maritimus S up to 0.5% level. These results suggest that 0.5% of dietary Psychrobacter maritimus S supplementation could be considered as a novel probiont for optimum growth performance and immune response of Nile tilapia fingerlings.

Effects of three host-associated Bacillus species on mucosal immunity and gut health of Nile tilapia, Oreochromis niloticus and its resistance against Aeromonas hydrophila infection.

Skin and intestinal mucosa lymphoid tissues are known to be the fish's first line of defence since they serve as the first point of contact for pathogens. Only few studies have investigated the influence of host-associated Bacillus on mucosal immunity. In this study, the effects of three host-associated Bacillus species on mucosal immunity, intestinal morphology, intestinal digestive enzymes activity, intestinal microbiome and resistance of Nile tilapia against Aeromonas hydrophila infection was evaluated. The fish were divided into five treatment groups and fed with diets containing no bacteria denoted as Control, Bacillus velezensis TPS3N denoted as group V, Bacillus subtilis TPS4 denoted as group S, Bacillus amyloliquefaciens TPS17 denoted as group A and a 5th group containing the three Bacillus species at a ratio 1:1:1 denoted as group CB. At the end of the feeding trial, significant enhancement of both skin mucus and intestinal immune titres were recorded in terms of nitric oxide (NO) (except in the mucus of V and S groups), immunoglobulin M (IgM) (except in the intestine of group V), lysozyme (LZM), and alkaline phosphatase (AKP) in all fish fed the Bacillus supplemented groups relative to the untreated group. Intestinal antioxidant enzymes (catalase (CAT) (except in the intestine of group S) and superoxide dismutase (SOD)) capacity of Nile tilapia were higher in the Bacillus groups. Intestinal lipase activity was elevated in the Bacillus supplemented groups. The intestinal morphological parameters (villus height, villus width, goblet cells count (except in group S and A), and intestinal muscle thickness) were significantly enhanced in the Bacillus supplemented groups relative to the Control group. Dietary probiotic supplementation also influenced the intestinal microflora composition of Nile tilapia. Proteobacteria recorded the highest abundance followed by Firmicutes, Fusobacteria, and Bacteroidetes at the phylum level in this study. At the genus level, the abundance of pathogenic bacteria viz Staphylococcus and Aeromonas were reduced in the Bacillus supplemented groups in comparison to the Control group. A challenge test with A. hydrophila resulted in lower mortalities (%) in the Bacillus treated groups thus 86.67%, 50.00%, 43.33%, 63.33%, and 30.00% for Nile tilapia fed Control, V, S, A, and CB diets respectively. In conclusion, the inclusion of B. velezensis TPS3N, B. subtilis TPS4, and B. amyloliquefaciens TPS17 in the diet of Nile tilapia singularly or in combination, could enhance the mucosal immunity, intestinal health, and resistance of Nile tilapia against A. hydrophila infection.

Modulation of mucosal parameters, innate immunity, growth and resistance against Streptococcus agalactiae by enrichment of Nile tilapia (Oreochromis niloticus) diet with Leucas aspera.

The present study aimed at evaluating the possible effects of Leucas aspera as immunostimulant on mucosal and serum immunity, as well as on growth and resistance against Streptococcus agalactiae infection in Nile tilapia (Oreochromis niloticus) fingerlings. In a 45 days trial, fish were fed experimental diets containing L. aspera 0 g kg-1 (Diet 1- control), 1 g kg-1 (Diet 2), 2 g kg-1(Diet 3), 4 g kg-1 (Diet 4) and 8 g kg-1 (Diet 5). The results revealed a significant increase in the specific growth rate (SGR), weight gain (WG), and final weight (FW) in fish fed diet 3 (2 g kg-1) of L. aspera compared to the control and other supplemented groups (P < 0.05). Also, feeding on diet 3 (2 g kg-1) of L. aspera enriched diet significantly (P < 0.05) increased lysozyme activities in the serum and mucus, serum peroxidase and phagocytosis activity. However, significant (P < 0.05) increase in mucus peroxidase activity was reported in fish fed diet 4 (4 g kg-1) and diet 5 (8 g kg-1) of L. aspera, whereas significantly higher (P < 0.05) alternative complement activity was reported in fish fed diet 2 (1 g kg-1) of L. aspera. At the end of the experiment, nine fish per replication were selected for a challenge test against S. agalactiae. The dietary supplementation of L. aspera significantly reduced the mortality rate and increased the resistance of Nile tilapia following by challenge with S. agalactiae. The highest post challenge survival of 100% was observed in tilapia fed diet 5 (8 g kg-1) following by 92.6% of RPS in fish fed diet 4 (4 g kg-1) and 88.9% in diet 3 (2 g kg-1), 77.8% in diet 2 (1 g kg-1) and 74.1% in diet 1(0 g kg-1).

Varieties of immunity activities and gut contents in tilapia with seasonal changes.

We performed 16S rDNA sequencing of tilapia fecal samples to analyze changes in tilapia gut contents after cultivation of the fish in the presence of sandwich-like floating beds of Chinese medicinal herbs (5 and 10% planting-areas; 5% Polygonum cuspidatum). The interactive effects between water quality and blood and hepatic pro- and anti-inflammatory concentrations were also assessed. Our results showed that the water quality (i.e., NO3--N, NO2--N, TP removal rates) improved, and the abundance of Chloroflexi and Cyanobacteria increased. The abundance of Bacteroidetes, Verrucomicrobia, Saccharibacteria, and Actinobacteria showed both significant seasonal decreases and increases in the presence of P. cuspidatum (increases in August and decreases in July). Fish blood and hepatic IL-10 and IFN-γ levels (together with fish sampled in September) significantly increased in the P. cuspidatum group sampled in August, while those of TNF-α (10% sandwich-like, P. cuspidatum), IL-1ß (P. cuspidatum), IL-8 (5% sandwich-like in September, S905S) significantly decreased. Heat shock proteins 60 and 70 levels significantly increased in the P. cuspidatum group, and complement C3 and C4 concentrations significantly increased in S905S. This study demonstrated that enhanced immunity through the regulation of pro- and anti-inflammatory proteins was sustained throughout development until harvest, particularly in fish grown with P. cuspidatum.

Effects of a phytogenic, alone and associated with potassium diformate, on tilapia growth, immunity, gut microbiome and resistance against francisellosis.

This work evaluated the effects of dietary supplementation of A-Live (phytogenic) either individually or in combination with Aquaform (potassium diformate, acidifier) on juvenile Nile tilapia (Oreochromis niloticus) growth performance, innate immune parameters, gut microbiome, and resistance against Francisella noatunensis subsp. orientalis challenge. Each experimental group contained 140 fishes (34.3 ± 0.33) in two 150L tanks. The experimental design consisted of five groups: a negative control; treated groups (G1, G2, G3) supplemented with different concentrations of A-Live and Aquaform in the feed; and a positive control (PC) for pathogen infection. Groups G1, G2, G3, and PC were challenged with Francisella spp. after 15 days. After infection, the mortality was significantly lower in groups G1, G2, and G3 (p < 0.01). Furthermore, these groups showed significant increase (p < 0.05) in daily weight gain, feed conversion rate, and specific growth rate. The PC group presented increase (p < 0.05) in the leukocytes and neutrophils number. Innate immunity parameters showed no difference between treatments after infection. Microbiome analysis revealed an increased number of bacteria belonging to the Vibrionaceae family after pathogen infection suggesting a secondary pathogen function of these bacteria. These results validate the beneficial effects of these products in tilapia farming.