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Background Aeromonas hydrophila is an opportunistic pathogen. Thus, it has received significant attention mainly in the fish sectors with high production scales. Nile tilapia broodstock confined in the environment of fish hatcheries can be stressed. Hence, they are vulnerable to A. hydrophila. Results Sequencing of the gyr B gene revealed the presence of 18 different A. hydrophila strains (kdy 10,620–10,637), which were deposited in the NCBI under accession numbers ON745861–ON745878. The median lethal doses of the isolates ranged from 2.62 × 10 4 to 3.02 × 10 6  CFU/mL. Antibiotic resistant genes, sulfonamide (sul1) and tetracycline (tetA) were found in the eighteen isolates . Approximately 83.3% of A. hydrophila strains were sensitive to ciprofloxacin and florfenicol. Further, eight A. hydrophila strains had high MDR indices at 0.27–0.45. All isolates presented with hemolysin activity. However, only 72.22% of them had proteolytic activity, and only 61.11% could form biofilms. Bacterial isolates harbored different pattern virulence genes, the heat-stable cytotonic enterotoxin (ast), cytotoxic enterotoxin (act), and hemolysin (hly) genes were the most prevalent. Also, a trial to inhibit bacterial growth was conducted using titanium dioxide nanoparticles (TiO 2 NPs) with three sizes (13, 32, and 123 nm). If A. hydrophila strains with a high MDR index were tested against TiO 2 NPs (20 µg/mL) for 1, 12, and 24 h, those with a small size had a greater bactericidal action than large ones. Bacterial strains were inhibited at different percentages in response to TiO 2 NP treatment. Conclusions Nile tilapia broodstock, mortality is associated with different A. hydrophila strains , which harbored virulent and MDR genes. Furthermore, TiO 2 NPs had bactericidal activity, thereby resulting in a considerable reduction in bacterial load.

Aflatoxins (AFB) are adventitious plant toxins that act as immunosuppressants. Experimental Nile tilapia were divided into eight groups: AFB0 (control, uncontaminated diet), AFB50 (50 ppbAFB 1 ), AFB100 (100 ppbAFB 1 ), AFB150 (150 ppbAFB 1 ), AFB0 + Sil (0 ppbAFB 1 +Sil), AFB50 + Sil (50 ppbAFB 1 +Sil), AFB100 + Sil (100 ppbAFB 1 +Sil), and AFB150 + Sil (150 ppbAFB 1 +Sil). The AFB 1 -residues in fish liver (AFB0, AFB50, AFB100, and AFB150) were 0.34, 0.54, and 0.82 ppb, respectively, while traces were recorded in muscles. Sil-supplemented fish had lower residues than other contaminated groups, which were still higher than AFB0. The gene expression of GPx , SOD , IL-1β , and TNF-α was significantly and linearly decreased with increasing aflatoxin levels, whereas Sil-supplementation (AFB50 + Sil) resulted in a high and significant increase of 3.86, 5.37, and 7.85 fold-change, respectively, compared with the fish groups and the control. While, CAT expression increased in response to a low aflatoxin level (AFB50; 2.17-fold-change), it decreased linearly with increasing aflatoxin level. Moreover, IL-10 expression was significantly higher in fish of AFB100 and AFB150, with 8.7- and 8.85-fold changes, respectively, compared to the control. Significant declines in immunoglobulin (Ig)M-2 and insulin-like growth factor (IGF) gene expression were observed in fish receiving AFB 1 -contaminated diets and Sil could enhance the expression. Innate immunity of the experimental fish was assessed by measuring the following parameters; serum antibacterial activity (SAB), oxidative burst activity (OBA), phagocytic activity (PA), phagocytic index (PI), and lysozyme (LYZ) as well as by challenge with Streptococcus agalactiae . Nile tilapia fed AFB 1 -contaminated diets showed immune-antioxidant suppressive status, indicating compromised health and a lowered survival rate (SR%) during bacterial challenge and antibiotic treatment Sil-supplementation could improve SR%.

Nowadays, nanomaterials enter high numbers of daily used products and drug manufacture. A nanocomposite of vitamins C (VC) and vitamin E (VE) with chitosan as a vehicle and protector was used in a comparative eight-week feeding study, Nile tilapia weighing 31.2 ± 0.36 g distributed in seven groups and fed (G1) basal diet, (G2) bulk VC, (G3) VC- nanoparticles (NPs), (G4) bulk VE, (G5) VE-NPs, bulk VCE (G6), and (G7) VC plus VE (VCE)-NPs, respectively. The Nile tilapia-fed nanocomposite vitamins had significantly higher growth performance compared to the control; VCE-NPs had the superiority among tested supplementations where total weight gain (63.6 g), daily weight gain (1.13 g), relative growth rate (206.1%) with lower feed conversion rate (1.6) and insignificant feed intake (101.5 g). Overall, the level of liver enzymes was significantly decreased in fish serum after eight-week nanocomposite supplementation, and dietary VCE-NPs caused a significant reduction of serum AST (18.45 IU/L) and ALT (14.77 IU/L) compared to the control 25.5 IU/L and 17.6 IU/L, respectively. Fish fed dietary VCE-NPs, VC-NPs, and VE-NPs had significant enhancement of RBCs 4.2 × 10 6 /μL, 3.8 × 10 6 /μL, and 3.55 × 10 6 /μL; WBCs 46.15 × 10 3 , 42.9 × 10 3 , and 44 × 10 3 /μL, respectively, Also TP was significantly higher 6.38 g/dL in VCE-NPs group compared to the control and the other treatments. Over all, the dietary nanocomposite vitamins boost the innate immunity of the experimental Nile tilapia, the oxidative burst activity (OBA), phagocytic activity (PA), phagocytic index (PI), and serum antibacterial (SAA) were significantly increased compared to those received bulk vitamins and the control. The activity of antioxidant biomarkers in fish serum including glutathione peroxidase (GPx), catalase (CAT), superoxide dismutase (SOD), total antioxidant capacity (TAC), glutathione reductase (GR), and myeloperoxidase (MPO) showed a rise in the serum of Nile tilapia received nano- and bulk-form of VC and VCE compared to the control and both forms of VE. Furthermore, the level of malondialdehyde (MDA), reduced glutathione (GSH), and oxidized glutathione (GSSG) were significantly increased in the fish serum following the trend of antioxidants enzymes. In conclusion, a dietary nanocomposite of vitamin C and vitamin E enhanced Nile tilapia's growth performance and feed utilization. It could also improve health status and immune response. The values of antioxidant biomarkers indicated that the nanocomposite could help the fish body scavenge the generated reactive oxidative species (ROS).

Phyllanthus niruri L. is a widespread tropical plant which is used in Ayurvedic system for liver and kidney ailments. The present study aims at specifying the most active hepatoprotective extract of P. niruri and applying a bio-guided protocol to identify the active compounds responsible for this effect. P. niruri aerial parts were extracted separately with water, 50%, 70% and 80% ethanol. The cytoprotective activity of the extracts was evaluated against CCl4-induced hepatotoxicity in clone-9 and Hepg2 cells. Bioassay-guided fractionation of the aqueous extract (AE) was accomplished for the isolation of the active compounds. Antioxidant activity was assessed using DPPH (1, 1-diphenyl-2-picrylhydrazyl) radical scavenging method and ferric reducing antioxidant power (FRAP). The in vivo hepatoprotective activity of AE was evaluated in CCl4-induced hepatotoxicity in rats at different doses after determination of its LD50. Pretreatment of clone-9 and Hepg2 with different concentrations of AE (1, 0.1, 0.01 mg/ml) had significantly reduced the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) against CCl4 injures, and restored the activity of the natural antioxidants; glutathione (GSH) and superoxide dismutase (SOD) towards normalization. Fractionation of AE gave four fractions (I-IV). Fractions I, II, and IV showed a significant in vitro hepatoprotective activity. Purification of I, II and IV yielded seven compounds; corilagin C1, isocorilagin C2, brevifolin C3, quercetin C4, kaempferol rhamnoside C5, gallic acid C6, and brevifolin carboxylic acid C7. Compounds C1, C2, C5, and C7 showed the highest (p< 0.001) hepatoprotective potency, while C3, C4, and C6 exhibited a moderate (p< 0.001) activity. The AE exhibited strong antioxidant DPPH (IC50 11.6 ± 2 μg/ml) and FRAP (79.352 ± 2.88 mM Ferrous equivalents) activity. In vivo administration of AE in rats (25, 50, 100 and 200 mg/kg) caused normalization of AST, ALT, alkaline phosphatase (ALP), lactate dehydrogenase (LDH), total cholesterol (TC), triglycyrides (TG), total bilirubin (TB), glucose, total proteins (TP), urea and creatinine levels which were elevated by CCl4. AE also decreased TNF-α, NF-KB, IL-6, IL-8, IL10 and COX-2 expression, and significantly antagonizes the effect of CCl4 on the antioxidant enzymes SOD, catalase (CAT), glutathione reductase (GR), and glutathione peroxidase (GSP). The histopathological study also supported the hepatoprotective effect of AE. P. niruri isolates exhibited a potent hepatoprotective activity against CCl4-induced hepatotoxicity in clone-9 and Hepg2 cell lines through reduction of lipid peroxidation and maintaining glutathione in its reduced form. This is attributable to their phenolic nature and hence antioxidative potential.

Edwardsiellosis is a bacterial fish disease that mostly occurs in freshwater farms and is characterized by a high mortality rate. Edwardsiella tarda strain was recovered from 17 fish out of 50 Nile tilapia, which were harboring clinical signs of systemic septicemia. The level of un-ionized ammonia (NH 3 ) in the fish farm’s water was 0.11–0.15 mg/L, which was stressful for the Nile tilapia. Sequencing of the gyrB1 gene confirmed that the isolate was E. tarda JALO4, and it was submitted to NCBI under the accession number PP449014. The isolated E. tarda harbored the virulence gene edw1 AHL-synthase (quorum sensing). In addition, the isolate was sensitive to trimethoprim and sulfamethoxazole mean while it was intermediate to florfenicol. The median lethal dose (LD 50 ) of E. tarda JALO4 was determined to be 1.7 × 10 5 CFU/mL in Nile tilapia. In the indoor experiment, Nile tilapia (45.05 ± 0.4 g), which received dietary Spirulina platensis (5 and 10 g/kg fish feed), showed optimum growth and feed utilization. Meanwhile, after receiving dietary S . platensis , the fish’s feed conversion ratio (FCR) was significantly enhanced compared to the control, which was 1.94, 1.99, and 2.88, respectively. The expression of immune-related genes interleukin ( IL)-1β and tumor necrosis factor (TNF)-α were upsurged in E. tarda- challenged fish with higher intensity in S. platensis groups. Dietary S. platensis at a dose of 10 g/kg fish feed could provide a relative protection level (RPL) of 22.2% Nile tilapia challenged against E. tarda . Nile tilapia experimentally infected E. tarda , drastically altering their behavior: higher operculum movement, low food apprehension, and abnormal swimming dietary S. platensis (10 g/kg fish feed) could rapidly restore normal status. It was concluded that Edwardsiellosis could alter Nile tilapia behavior with a high loss in fish population. Fish received dietary- S. platensis could rapidly restore normal behavior after E. tarda infection. It is recommended the incorporation of S. platensis at doses of 10 g/kg into the Nile tilapia diet to boost their immunity and counteract E. tarda infection.

In aquaculture, fish are exposed to many stressors, such as climate changes and infectious diseases that affect their performance, immunity, and welfare. Freshwater fish subjected to salt bath become exhausted and stressed. In this experiment, Nile tilapia were exposed to a salt bath at a dose of 30 ppt for 30 min a day. Vitamin C and vitamin E are well-known antioxidants that are used in aquaculture. Fish received dietary nanoparticles of chitosan-vitamin C and chitosan-vitamin E (CCE-NPs) for different periods (7 and 14 days) pre- (G2) and post-salt treatment (G3). In the control fish (G1), cortisol 5.44 µg/dL and glucose 91.67 mg/dL were significantly up-regulated post-salt treatment by 1 h and 24 h, respectively, whereas those (G2) fed CCE-NPs diet had significantly lower values of 4.72 and 3.25 µg/dL; 86.3 and 84.3 mg/dL, respectively. A rapid decrease of glucose 68.3 and 66.3 mg/dL was noticed in those (G2) fed CCE-NPs diet compared to the control 84.67 mg/dL at 48 h post-stress. Regardless of the supplementation period, fish (G2) could partially restore normal food reflex at 48 h (post-salt bath) and fully restored at 72 h compared to 7 days in the control (G1). After 48 h, fish that received dietary CCE-NPs (G2 and G3) restored normal mucus lysozyme levels, whereas the control did not restore pre-treatment values till the seventh day. Mucus antibacterial activity, fish received rapid dietary CCE-NPs (G2) and partially restored average values (pre-salt bath) at 96 h. The salt treatment could provoke gene expression of pro-inflammatory cytokines interleukin (IL-1β) and tumor necrosis (TNF)-α in the head kidney of fish at 24 h post-salt bath to 5.9–8.35 fold-change, respectively, with a rapid decline in fish (G2) the gene expression. Post-salt bath (24 h), the gene expression of glutathione peroxidase (GPx) , superoxide dismutase (SOD) , and catalase (CAT) was higher in fish (G2) than in the control group (G1) regardless of the supplementation period (7 and 14 days). Bacterial infection S. agalactiae (OL471408), a significantly lower MR was recorded in G2 at 40% and 33.3% compared to the control G1 MR (53.3%), with an RPL of 24.95% and 37.5%. In conclusion, Nile tilapia treated with a 30 ppt salt became more vulnerable to S. agalactiae. Adding CCE-NPs to the Nile tilapia diet for 7- and 14-day pre-salt bath could increase immune and antioxidant-related gene expression to counteract S. agalactiae infection.

The production of striped catfish (Pangasianodon hypothalamus) has increased worldwide; recently, it was farmed with Nile tilapia in polyculture farms. Polyculture systems and water temperature (25℃ and 33℃) could affect Edwardsiella tarda infection, antibiotic efficacy, and residues. Moribund fishes were collected from three Farms 1–3: Farm 1 (monoculture, Nile tilapia), Farm 2 (monoculture, striped catfish), and Farm 3 (polyculture). Four E. tarda , LAMSH1, and LAMAH2-4 were isolated, whereas LAMAH3 was isolated from both fish spp., where striped catfish were highly susceptible to infection. The obtained E. tarda , which was isolated from striped catfish, has a significantly lower LD 50 than those retrieved from Nile tilapia, and co-infection occurred only in striped catfish on Farm 3. The infection was screened and confirmed by gyrB1 gene presence while detecting the cds1 , pvsA , and qseC genes indicated virulence. All isolates were sensitive to ciprofloxacin and florfenicol but showed resistance to a high number of other antibiotics, resulting in high multi-drug resistant (MDR) indices exceeding 0.2, except for strain LAMAH4, which had an index of 0.18. Analyses of farms water revealed high ammonia compounds total ammonia nitrogen (TAN), unionized ammonia (NH 3 ), nitrite (NO 2 ), and nitrate (NO 3 ) in Farm 2 (monoculture, striped catfish), and the recorded significantly higher concentrations were 2.75, 0.29, 0.24, and 2.01 mg/L, respectively, which were compared with Farm 1 and Farm 3. In the indoor experiment, at high water temperatures (33 °C), Nile tilapia and striped catfish had a high mortality rate and re-isolation of E. tarda (10–20%) compared to those exposed to low water temperatures (25 °C). These observations were concurrent with low antibiotic residues in their hepatic tissues. Despite water temperature, Nile tilapia showed higher ciprofloxacin residues than striped catfish. The study concluded that striped catfish are more susceptible to the bacteria E. tarda compared to Nile tilapia, particularly in polyculture farms, which resulted in a higher infection rate. Both Nile tilapia and striped catfish exposed to elevated water temperatures exhibited increased vulnerability to bacterial infections. Additionally, these fish showed a high re-isolation rate of E. tarda while having low ciprofloxacin residues in their hepatic tissues.

Many governments have approved the use of oxytetracycline as an antibiotic additive to food fish, with oxytetracycline now routinely used in many nations. However, oxytetracycline is known to have immunosuppression impacts. We, therefore, evaluated the immunological, antioxidative, and histopathological status of Nile tilapia fed a diet containing silymarin (100 mg/kg fish feed) for 0, 2, 4, 6, and 8 weeks. The protective effects of silymarin against  Aeromonas hydrophila  ( A. hydrophila ) infection and oxytetracycline treatment were evaluated. Blood parameters (erythrocyte count, white blood cell count, hemoglobin, and packed cell volume) improved over time in fish fed on dietary silymarin. Serum levels of alanine aminotransferase (ALT) were lower in fish fed on dietary silymarin, whereas serum levels of aspartate transferase (AST)and alkaline phosphatase (ALK) were unchanged. Dietary silymarin affected serum lipid profiles as decreases in serum triglyceride and low-density lipoprotein cholesterol levels and a trend toward lower cholesterol levels, whereas serum high-density lipoprotein cholesterol levels were increased compared to fish fed on the control diet. Dietary silymarin resulted in an increase of serum total protein levels and globulin fractions. Significant and progressive increases in catalase and glutathione peroxidase levels were observed after six weeks of feeding on a dietary silymarin before decreasing to control levels at the end of the experimental period. Fish fed on dietary silymarin, interleukin-1 and fish tumor necrosis factor-alpha were upregulated in hepatic tissues; however, interleukin-10 levels decreased to comparable levels to controls after eight weeks. Fish infected with A. hydrophila displayed septicemia (opaque eye, hemorrhagic ulcers, dentated fins, hepatomegaly, and splenomegaly). Reduced mortality was observed in Nile tilapia infected with A. hydrophila and fed a diet containing silymarin, indicating that silymarin improves fish responses to oxytetracycline with a 37% reduction in mortality.

Vitamin E (VE) is an essential vitamin liposoluble antioxidant in aquatic animals that is usually lost during feed processing and digestion, whereas nano-chitosan, a polysaccharide, could protect VE. In this study, Nile tilapia (70.85 ± 0.2 g) was fed VE (100 mg/kg dry diet) and a chitosan protected-VE nanoparticle (NPs) with gradual percentages of recommended dose 25%, 50%, 75%, and 100% for 4, 6, and 8 weeks. Growth parameters total weight gain (TG), daily weight gain (DWG), and relative growth rate (RGR) were significantly and positively correlated with VENPs additions. Regardless of the addition level, the feed conversion ratio (FCR) was significantly lower in the VENP groups. Lysozyme, serum antibacterial activity, and oxidative burst activity indicated the superiority of VENPs (VENPs75 and VENPs100) in enhancing the fish’s innate immunity compared to bulk VE and the control groups. Fish were experimentally challenged with pathogenic Aeromonas hydrophila; those received dietary showed a low mortality rate (MR%), about 40% compared with 70% in the control with lower re-isolation compared to the control and VE groups. VENPs could provide ascending relative protection level during the period of 4 to 8 weeks; RPL ranged from 33.3 to 42.86% (VENPs100), 16.67–42.86% (VENPs75), 0 to 28.57% (VENPs50), and 0 to 14.29% (VENPs25 and VE), respectively. Finally, this study recommended incorporating VENPs into the Nile tilapia diet at 50, 75, and 100 mg/ kg fish feed. Fish in the VENPs75 and VENPs100 groups were immune boosted, becoming less vulnerable to A. hydrophila infection.

Aflatoxin B1 (AFB1) is a plant-origin toxin that could induce oxidative stress in fish. The micromineral selenium (Se) possesses well-documented antioxidant properties. To assess the ameliorative effects of SeNPs (1 mg/kg fish feed) on oxidative stress induced by AFB1 (500 μg/kg fish feed), Nile tilapia (32.2±1.7 g body weight) were distributed randomly and even in six groups for 8-week feeding trial. Live enzymes, AST, ALT, and ALP levels were increased in the serum of fish fed AFB1-contaminated diet, and the addition of SeNPs could restore normal values compared to the control. The gene expression of antioxidant enzymes, superoxide dismutase (SOD) enzyme and catalase (CAT) enzyme, and DNA fragmentation were significantly increased in response to aflatoxin exposure, while dietary SeNPs could mitigate the generated oxidative stress. The innate immunity, serum antibacterial activity (SAA), oxidative burst activity (OBA), phagocytic activities (PA and PI), and gene expression of cytokines (interleukin ( IL ) -1β , heat shock protein70 ( Hsp ), and tumor necrosis factor ( TNF )- α ) revealed a status of immunosuppression in Nile tilapia fed on AFB1-contaminated diet. These findings showed that fish became more vulnerable to Streptococcus agalactiae infection with a high mortality rate while dietary SeNPs provided a high relative protection level (RPL). From the obtained findings, SeNPs could mitigate the oxidative stress induced by feeding the AFB1 diet and could boost the immunity of stressed Nile tilapia.