Pseudomonas putida infection induces immune-antioxidant, hepato-renal, ethological, and histopathological/immunohistochemical disruptions in Oreochromis niloticus: the palliative role of titanium dioxide nanogel.

BACKGROUND: Pseudomonas putida is a pathogenic bacterium that induces great losses in fishes, including Nile tilapia (Oreochromis niloticus). Currently, the application of nanomaterials in aquaculture practices has gained more success as it endows promising results in therapies compared to traditional protocols. OBJECTIVE: Therefore, the current perspective is considered the first report to assess the anti-bacterial efficacy of titanium dioxide nanogel (TDNG) against Pseudomonas putida (P. putida) in Nile tilapia. METHODS: The fish (n = 200; average body weight: 47.50±1.32 g) were allocated into four random groups (control, TDNG, P. putida, and TDNG + P. putida), where 0.9 mg/L of TDNG was applied as bath treatment for ten days. RESULTS: Outcomes revealed that P. putida infection caused ethological alterations (surfacing, abnormal movement, and aggression) and depression of immune-antioxidant variables (complement 3, lysozyme activity, total antioxidant capacity, superoxide dismutase, and reduced glutathione content). Additionally, a substantial elevation in hepatorenal biomarkers (aspartate and alanine aminotransferases and creatinine) with clear histopathological changes and immuno-histochemical alterations (very weak BCL-2 and potent caspase-3 immuno-expressions) were seen. Surprisingly, treating P. putida-infected fish with TDNG improved these variables and obvious restoration of the tissue architectures. CONCLUSION: Overall, this report encompasses the key role of TDNG as an anti-bacterial agent for controlling P. putida infection and improving the health status of Nile tilapia.

Effects of supplementation with different zinc-based products on the growth and health of Nile tilapia.

Zinc is one of the essential microelements for the metabolism of animals. Zinc nanoparticles may have higher bioavailability due to their low specific surface area, facilitating absorption by fish. The present study aimed to evaluate the effects of supplementation with different zinc-based products on the growth and health of Nile tilapia Oreochromis niloticus. Zinc, in different sizes (nanoparticles or bulk) and forms (inorganic or organic), were used as a supplement in the tilapia diet at a dose of 15 mg kg feed-1 for 60 days. At the end of the feeding trial, production performance, hemato-immunological parameters, activity of antioxidant system enzymes, exposure to Streptococcus agalactiae and zinc concentration in the muscle were examined. After the bacterial challenge, the mean corpuscular hemoglobin concentration (MCHC) significantly increased in the fish treated with organic zinc, inorganic nano zinc, and organic nano zinc, while in the control group (inorganic zinc), MCHC remained unchanged. Regarding defense cells, dietary inorganic nano zinc increased the number of basophils (1.50 ± 1.10) compared to organic zinc (0.80 ± 0.90). Lymphocyte count increased after the challenge only in the organic zinc treatments (bulk and nanoparticles). Neutrophils decreased in the control (inorganic zinc) (2.20 ± 1.70) and inorganic nano zinc (2.60 ± 2.70) treatments after the challenge. When compared before and after the bacterial challenge, the plasma antimicrobial titer significantly increased after the bacterial challenge in all treatments. No significant differences were observed for total proteins, enzymes (SOD and CAT), cumulative survival and zinc deposition on fillet. In conclusion, organic zinc in nanoparticles or bulk size increased Nile tilapia innate defense during bacterial infection. However, the other parameters evaluated were not affected by zinc particle size or form (organic or inorganic), indicating that further evaluations should be conducted with organic zinc in nanoparticles or bulk size in the tilapia diet.

Acanthopanax senticosus cultures fermented by Lactobacillus rhamnosus enhanced immune response through improvement of antioxidant activity and inflammation in crucian carp (Carassius auratus).

Lactic acid bacteria (LAB) have been recognized as safe microorganism that improve micro-flora disturbances and enhance immune response. A well-know traditional herbal medicine, Acanthopanax senticosus (As) was extensively utilized in aquaculture to improve growth performance and disease resistance. Particularly, the septicemia, skin wound and gastroenteritis caused by Aeromonas hydrophila threaten the health of aquatic animals and human. However, the effects of probiotic fermented with A. senticosus product on the immune regulation and pathogen prevention in fish remain unclear. Here, the aim of the present study was to elucidate whether the A. senticosus fermentation by Lactobacillus rhamnosus improve immune barrier function. The crucian carp were fed with basal diet supplemented with L. rhamnosus fermented A. senticosus cultures at 2 %, 4 %, 6 % and 8 % bacterial inoculum for 8 weeks. After trials, the weight gain rate (WGR), specific growth rate (SGR) were significantly increased, especially in LGG-6 group. The results confirmed that the level of the CAT, GSH-PX, SOD, lysozyme, and MDA was enhanced in fish received with probiotic fermented product. Moreover, the L. rhamnosus fermented A. senticosus cultures could trigger innate and adaptive immunity, including the up-regulation of the C3, C4, and IgM concentration. The results of qRT-PCR revealed that stronger mRNA transcription of IL-1ß, IL-10, IFN-γ, TNF-α, and MyD88 genes in the liver, spleen, kidney, intestine and gills tissues of fish treated with probiotic fermented with A. senticosus product. After infected with A. hydrophila, the survival rate of the LGG-2 (40 %), LGG-4 (50 %), LGG-6 (60 %), LGG-8 (50 %) groups was higher than the control group. Meanwhile, the pathological damage of the liver, spleen, head-kidney, and intestine tissues of probiotic fermentation-fed fish could be alleviated after pathogen infection. Therefore, the present work indicated that L. rhamnosus fermented A. senticosus could be regard as a potential intestine-target therapy strategy to protecting fish from pathogenic bacteria infection.

Antiparasitic efficacy of flavonoids identified from Psoralea corylifolia against Tetrahymena piriformis in guppy (Poecilia reticulate).

Tetrahymena piriformis belongs to the ciliated protists (ciliates), causing severe economic losses in aquaculture. Chemical drugs currently used usually have toxic side effects, and there is no specific drug against Tetrahymena. Therefore, it is an urgent need to identify new antiparasitic lead compounds. In the present study, the in vitro parasiticidal activity of ethyl acetate (EtOAc) extracts and water extracts from 22 selected traditional Chinese medicines (TCMs) were evaluated against T. piriformis. The EtOAc extract of P. corylifolia turned out to be the most active with the minimum parasiticidal concentration of 100 mg/L within 3 h. Thus, it was separated into 12 fractions by the first-dimensional (D1) normal phase liquid chromatography (NPLC), meanwhile combining with in vitro antiparasitic tests for activity tracking. Subsequently, 8 flavonoids were identified in the active fractions by the second-dimensional (D2) reverse phase liquid chromatography (RPLC) tandem high-resolution mass spectrometry. According to the results, 5 flavonoids were selected for in vitro antiparasitic test, of which isobavachalcone showed the minimum parasiticidal concentration of 3.125 mg/L in 2 h. Bathing treatment of infected guppies with isobavachalcone could significantly reduce the burden of T. piriformis, obtaining a 24-h median effective concentration (24-h EC50) value of 1.916 mg/L. And the concentration of isobavachalcone causing guppies to die within 24 h is 39 times than that of 24-h EC50. The results demonstrated that isobavachalcone has the potential to be developed into a novel commercial fish drug against T. piriformis.

Dietary effects of Saccharomyces cerevisiae and Allium sativum on growth, antioxidant status, hepatic and intestinal histoarchitecture, expression of growth- and immune-related genes, and resistance of Oreochromis niloticus to Aeromonas sobria.

This study investigated the benefits of yeast, Saccharomyces cerevisiae and/or garlic, Allium sativum supplementation in diets of Nile tilapia with regard to growth, antioxidant status, hepatic and intestinal histoarchitecture, expression of growth- and immune-related genes, and resistance to Aeromonas sobria infection. Fish (with an initial weight of 9.43 ± 0.08 g) were allocated to twelve hapas, organized into four triplicate treatment groups defined as control (no supplementation), yeast (4 g/kg diet), garlic (30 g/kg diet), and a mixture of both. This trial continued over a 60-day feeding period. Results revealed that combined treatment (yeast + garlic) demonstrated the most promising outcomes regarding growth, with significantly higher final body weights, weight gains, and specific growth rates compared to other groups. Moreover, this combination enhanced hepatic antioxidant status, as evidenced by elevated levels of reduced glutathione and activities of catalase and superoxide dismutase enzymes, reflecting improved defense against oxidative stress. Histological assessments of the livers and intestines demonstrated structural enhancements in yeast and garlic treatments, suggesting improvements in organ health. In comparison to the control, the gene expression analyses unveiled increased expression of growth-related (igf-1 and ghr1) and immune-related (il-10, lyz, and hep) genes in the test groups, indicating a possible reinforcement of the growth and immune responses. The combined treatment also showed the highest resistance to A. sobria infection, as evidenced by improved survival rates and lower mortality compared with the other groups. These findings highlight the benefits of a combination of both yeast and garlic as a dietary supplementation regimen. In conclusion, this study suggests that the combined treatment regimen could be considered an effective strategy to promote the health and productivity of Nile tilapia under production conditions.

From growth promotion to intestinal inflammation alleviation: Unraveling the potential role of Lactobacillus rhamnosus GCC-3 in juvenile grass carp (Ctenopharyngodon idella).

Lactobacillus rhamnosus is a probiotic, which not only promotes the growth of animals, but also has anti-inflammatory effects. However, the mechanism by which Lactobacillus rhamnosus regulates intestinal immunity is not well comprehended. Hence, the study aimed to research how Lactobacillus rhamnosus affects the intestinal immunity using juvenile grass carp (Ctenopharyngodon idella) as a model. We selected 1800 juvenile grass carp for testing. They were divided into six treatments and fed with six gradients of Lactobacillus rhamnosus GCC-3 (0.0, 0.5, 1.0, 1.5, 2.0, 2.5 g/kg) for 70 days. Enteritis was subsequently induced with dextroside sodium sulfate. Results indicated that dietary Lactobacillus rhamnosus GCC-3 addition improved growth performance. Meanwhile, appropriate levels of Lactobacillus rhamnosus GCC-3 alleviated excessive inflammatory response by down-regulating the expression of TLR4 and NOD receptors, up-regulating the expression of TOR, and then down-regulating the expression of NF-κB. Additionally, appropriate Lactobacillus rhamnosus GCC-3 improved intestinal immunity by reducing pyroptosis triggered by NLRP3 inflammasome and mediated by GSDME. Furthermore, 16 S rRNA sequencing showing appropriate levels of Lactobacillus rhamnosus GCC-3 increased Lactobacillus and Bifidobacterium abundance and decreased Aeromonas abundance. These results suggest that Lactobacillus rhamnosus GCC-3 can alleviate intestinal inflammation through down-regulating NF-κB and up-regulating TOR signaling pathways, as well as by inhibiting pyroptosis.

Roles of ß-catenin in innate immune process and regulating intestinal flora in Qi river crucian carp (Carassius auratus).

In mammals, ß-catenin participates in innate immune process through interaction with NF-κB signaling pathway. However, its role in teleost immune processes remains largely unknown. We aimed to clarify the function of ß-catenin in the natural defense mechanism of Qi river crucian carp (Carassius auratus). ß-catenin exhibited a ubiquitous expression pattern in adult fish, as indicated by real-time PCR analysis. Following lipopolysaccharide (LPS), Polyinosinic-polycytidylic acid (polyI: C) and Aeromonas hydrophila (A. hydrophila) challenges, ß-catenin increased in gill, intestine, liver and kidney, indicating that ß-catenin likely plays a pivotal role in the immune response against pathogen infiltration. Inhibition of the ß-catenin pathway using FH535, an inhibitor of Wnt/ß-catenin pathway, resulting in pathological damage of the gill, intestine, liver and kidney, significant decrease of innate immune factors (C3, defb3, LYZ-C, INF-γ), upregulation of inflammatory factors (NF-κB, TNF-α, IL-1, IL-8), and downregulation of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) activities, increase of Malondialdehyde (MDA) content. Following A. hydrophila invasion, the mortality rate in the FH535 treatment group exceeded that of the control group. In addition, the diversity of intestinal microflora decreased and the community structure was uneven after FH535 treatment. In summary, our findings strongly suggest that ß-catenin plays a vital role in combating pathogen invasion and regulating intestinal flora in Qi river crucian carp.

Acrylamide exposure induces growth retardation, neurotoxicity, stress, and immune/antioxidant disruption in Nile tilapia (Oreochromis niloticus): The alleviative effects of Chlorella vulgaris diets.

This study looked at the toxic impacts of water-born acrylamide (ACR) on Nile tilapia (Oreochromis niloticus) in terms of behaviors, growth, immune/antioxidant parameters and their regulating genes, biochemical indices, tissue architecture, and resistance to Aeromonas hydrophila. As well as the probable ameliorative effect of Chlorella vulgaris (CV) microalgae as a feed additive against ACR exposure was studied. The 96-h lethal concentration 50 of ACR was investigated and found to be 34.67 mg/L for O. niloticus. For the chronic exposure study, a total of 180 healthy O. niloticus (24.33 ± 0.03 g) were allocated into four groups in tri-replicates (15 fish/replicate), C (control) and ACR groups were fed a basal diet and exposed to 0 and 1/10 of 96-h LC50 of ACR (3.46 mg/L), respectively. ACR+ CV5 and ACR+ CV10 groups were fed basal diets with 5 % and 10 % CV supplements, respectively and exposed to 1/10 of 96-h LC50 of ACR for 60 days. After the exposure trial (60 days) the experimental groups were challenged with A. hydrophila. The findings demonstrated that ACR exposure induced growth retardation (P˂0.01) (lower final body weight, body weight gain, specific growth rate, feed intake, protein efficiency ratio, final body length, and condition factor as well as higher feed conversion ratio). A substantial decrease in the immune/antioxidant parameters (P˂0.05) (lysozyme, serum bactericidal activity %, superoxide dismutase, and reduced glutathione) and neurotransmitter (acetylcholine esterase) (P˂0.01) was noticed with ACR exposure. A substantial increase (P˂0.01) in the serum levels of hepato-renal indicators, lipid peroxidation biomarker, and cortisol was noticed as a result of ACR exposure. ACR exposure resulted in up-regulation (P˂0.05) of the pro-inflammatory cytokines and down-regulation (P˂0.05) of the antioxidant-related gene expression. Furthermore, the hepatic, renal, brain, and splenic tissues were badly affected by ACR exposure. ACR-exposed fish were more sensitive to A. hydrophila infection and recorded the lowest survival rate (P˂0.01). Feeding the ACR-exposed fish with CV diets significantly improved the growth and immune/antioxidant status, as well as modulating the hepatorenal functions, stress, and neurotransmitter level compared to the exposed-non fed fish. In addition, modulation of the pro-inflammatory and antioxidant-related gene expression was noticed by CV supplementation. Dietary CV improved the tissue architecture and increased the resistance to A. hydrophila challenge in the ACR-exposed fish. Noteworthy, the inclusion of 10 % CV produced better results than 5 %. Overall, CV diets could be added as a feed supplement in the O. niloticus diet to boost the fish's health, productivity, and resistance to A. hydrophila challenge during ACR exposure.

Novel Perspective on Mechanism in Muscle Growth Inhibited by Ochratoxin A Associated with Ferroptosis: Model of Juvenile Grass Carp (Ctenopharyngodon idella) In Vivo and In Vitro Trials.

Ochratoxin A (OTA) is a common mycotoxin in food and feed that seriously harms human and animal health. This study investigated the effect of OTA on the muscle growth of juvenile grass carp (Ctenopharyngodon idella) and its possible mechanism in vitro. Our results have the following innovative findings: (1) Dietary OTA increased the expression of increasing phase I metabolic enzymes and absorbing transporters while reducing the expression of efflux transporters, thereby increasing their residue in muscles; (2) OTA inhibited the expressions of cell cycle and myogenic regulatory factors (MyoD, MyoG, and MyHC) and induced ferroptosis by decreasing the mRNA and protein expressions of FTH, TFR1, GPX4, and Nrf2 both in vivo and in vitro; and (3) the addition of DFO improved OTA-induced ferroptosis of grass carp primary myoblasts and promoted cell proliferation, while the addition of AKT improved OTA-inhibited myoblast differentiation and fusion, thus inhibiting muscle growth. Overall, this study provides a potential research target to further mitigate the myotoxicity of OTA.

Synergistic interaction of nanoparticles and probiotic delivery: A review.

Nanotechnology is an expanding and new technology that prompts production with nanoparticle-based (1-100 nm) organic and inorganic materials. Such a tool has an imperative function in different sectors like bioengineering, pharmaceuticals, electronics, energy, nuclear energy, and fuel, and its applications are helpful for human, animal, plant, and environmental health. In exacting, the nanoparticles are synthesized by top-down and bottom-up approaches through different techniques such as chemical, physical, and biological progress. The characterization is vital and the confirmation of nanoparticle traits is done by various instrumentation analyses like UV-Vis spectrophotometry (UV-Vis), Fourier transform infrared spectroscopy, scanning electron microscope, transmission electron microscopy, X-ray diffraction, atomic force microscopy, annular dark-field imaging, and intracranial pressure. In addition, probiotics are friendly microbes which while administered in sufficient quantity confer health advantages to the host. Characterization investigation is much more significant to the identification of good probiotics. Similarly, haemolytic activity, acid and bile salt tolerance, autoaggregation, antimicrobial compound production, inhibition of pathogens, enhance the immune system, and more health-beneficial effects on the host. The synergistic effects of nanoparticles and probiotics combined delivery applications are still limited to food, feed, and biomedical applications. However, the mechanisms by which they interact with the immune system and gut microbiota in humans and animals are largely unclear. This review discusses current research advancements to fulfil research gaps and promote the successful improvement of human and animal health.

Modulatory effects of dietary prickly pear (Opuntia ficus-indica) peel on high salinity tolerance, growth rate, immunity and antioxidant capacity of Nile tilapia (Oreochromis niloticus).

This study evaluated the effects of prickly pear (Opuntia ficus-indica) peel (PPP) on salinity tolerance, growth, feed utilization, digestive enzymes, antioxidant capacity, and immunity of Nile tilapia (Oreochromis niloticus). PPP was incorporated into four iso-nitrogenous (280 g kg-1 protein) and iso-energetic (18.62 MJ kg-1) diets at 0 (PPP0), 1 (PPP1), 2 (PPP2), and 4 (PPP4) g kg-1. Fish (9.69 ± 0.2 g) (mean ± SD) were fed the diets for 75 days. Following the feeding experiment, fish were exposed to a salinity challenge (25‰) for 24 h. Fish survival was not affected by the dietary PPP inclusion either before or after the salinity challenge. Fish fed the PPP-supplemented diets showed lower aspartate aminotransferase, alanine aminotransferase, cortisol, and glucose levels compared to PPP0, with the lowest values being observed in PPP1. Fish fed dietary PPP had higher growth rates and feed utilization than PPP0. Quadratic regression analysis revealed that the best weight gain was obtained at 2.13 g PPP kg-1 diet. The highest activities of protease and lipase enzymes were recorded in PPP1, while the best value of amylase was recorded in PPP2, and all PPP values were higher than PPP0. Similarly, PPP1 showed higher activities of lysozyme, alternative complement, phagocytic cells, respiratory burst, superoxide dismutase, glutathione peroxidase and catalase, and lower activity of malondialdehyde than in PPP0. Further increases in PPP levels above 2 g kg-1 diet led to significant retardation in the immune and antioxidant parameters. Thus, the inclusion of PPP at about 1 to or 2 g kg-1 diet can improve stress tolerance, immunity, and antioxidant capacity in Nile tilapia.

Traditional Chinese medicine bufalin inhibits infectious hematopoietic necrosis virus infection in vitro and in vivo.

Infectious hematopoietic necrosis virus (IHNV) causes infectious hematopoietic necrosis and severe economic losses to salmon and trout aquaculture worldwide. Currently, the only commercial vaccine against IHNV is a DNA vaccine with some biosafety concerns. Hence, more effective vaccines and antiviral drugs are needed to prevent IHNV infection. In this study, 1,483 compounds were screened from a traditional Chinese medicine monomer library, and bufalin showed potential antiviral activity against IHNV. The 50% cytotoxic concentration of bufalin was >20 µM, and the 50% inhibitory concentration was 0.1223 µΜ against IHNV. Bufalin showed the inhibition of diverse IHNV strains in vitro, which confirmed that it had an inhibitory effect against all IHNV strains, rather than random activity against a single strain. The bufalin-mediated block of IHNV infection occurred at the viral attachment and RNA replication stages, but not internalization. Bufalin also inhibited IHNV infection in vivo and significantly increased the survival of rainbow trout compared with the mock drug-treated group, and this was confirmed by in vivo viral load monitoring. Our data showed that the anti-IHNV activity of bufalin was proportional to extracellular Na+ concentration and inversely proportional to extracellular K+ concentration, and bufalin may inhibit IHNV infection by targeting Na+/K+-ATPase. The in vitro and in vivo studies showed that bufalin significantly inhibited IHNV infection and may be a promising candidate drug against the disease in rainbow trout. IMPORTANCE: Infectious hematopoietic necrosis virus (IHNV) is the pathogen of infectious hematopoietic necrosis (IHN) which outbreak often causes huge economic losses and hampers the healthy development of salmon and trout farming. Currently, there is only one approved DNA vaccine for IHN worldwide, but it faces some biosafety problems. Hence, more effective vaccines and antiviral drugs are needed to prevent IHNV infection. In this study, we report that bufalin, a traditional Chinese medicine, shows potential antiviral activity against IHNV both in vitro and in vivo. The bufalin-mediated block of IHNV infection occurred at the viral attachment and RNA replication stages, but not internalization, and bufalin inhibited IHNV infection by targeting Na+/K+-ATPase. The in vitro and in vivo studies showed that bufalin significantly inhibited IHNV infection and may be a promising candidate drug against the disease in rainbow trout.

In vivo and ex vivo studies support the immunostimulant and immunoprotective effect of Damiana (Turnera diffusa Willd) in Almaco Jack (Seriola rivoliana).

Damiana (Turnera diffusa Willd) was evaluated in vitro for antioxidant and antibacterial activities against Staphylococcus aureus and Streptococcus pyogenes (as a preliminary screening assessment) by high-performance thin-layer chromatography (HPTLC)-Direct bioautography. A study was performed in vivo to evaluate the effects of Damiana enriched diets at 0.5 % on immune parameters in mucus and serum and gene expression in Almaco Jack (Seriola rivoliana) intestine after two and four weeks; an infection with Aeromonas hydrophila at 1x107 colony forming units (CFU) followed and an ex vivo study was carried out using head-kidney leukocytes. Ferric reducing ability of plasma (FRAP) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assays showed high antioxidant activities in Damiana leaves; even in the ABTS assay, Damiana at 300 µg/mL showed similar activity to ascorbic acid - the standard control. Damiana exhibited strong in vitro antimicrobial activity against S. aureus and S. pyogenes. In vivo studies showed a strong enhancement of myeloperoxidase, nitric oxide, superoxide dismutase, and catalase activities in mucus and serum of S. rivoliana supplemented with Damiana; their immunological response enhanced after infection with A. hydrophila. IL-1ß, TNF-α, and IL-10 gene expressions upregulated in the fish intestine challenged with the bacterium. Piscidin and macrophage (MARCO) receptor gene expression up-regulated at week 4 and down-regulated after infection. Intestinal histology results confirm that Damiana not cause inflammation or damage. Finally, the ex vivo study confirmed the immunostimulant and protective effects of Damiana through increased phagocytic, respiratory burst, myeloperoxidase activities and nitric oxide generation before and upon the bacterial encounter. These results support the idea that Damiana has the potential as an immunostimulant additive for diets in aquaculture by enhancing immune parameters and protecting Almaco Jack against A. hydrophila infections upon four weeks of supplementation.

Chicken egg lysozyme enhanced the growth performance, feed utilization, upregulated immune-related genes, and mitigated the impacts of Aeromonas hydrophila infection in Nile tilapia (Oreochromisniloticus).

Functional supplements, including lysozyme, are highly approved as immunostimulant and antibacterial agents with a high potential for use in aquaculture. In this regard, Nile tilapia was treated with lysozyme at 0, 0.5, 1, 1.5, and 3 g/kg for 60 days, then challenged with Aeromonas hydrophila. Fish were stocked in 15 glass aquaria (70 L each) with an equal initial weight of 10.72 ± 0.71 g per fish and 15 fish per aquarium. The regression analysis revealed that dietary lysozyme supplementation at 1.83-2 g/kg enhanced the growth performance, protein efficiency ratio, and protein productive value while reducing the feed conversion ratio of tilapia. Markedly, tilapia treated with lysozyme had a low mortality rate (30-50 %) compared to the control, which recorded a 70 % mortality rate after 15 days of challenge with A. hydrophila. The regression analysis also revealed that the highest lysozyme activity of tilapia-fed lysozyme for 60 days is achieved by 2.05 g/kg lysozyme. The expression of Nf-κb, IL-1ß, and IL-8 genes is upregulated in tilapia-fed lysozyme at 0.5, 1, 1.5, and 3 g/kg for 60 days before and after A. hydrophila infection. The expression of GPX and CAT genes was higher in tilapia-fed lysozyme at 0.5, 1, 1.5, and 3 g/kg for 60 days before and after A. hydrophila infection. Before infection, the relative transcription of the lysozyme and C3 was upregulated in tilapia-fed lysozyme at 0.5, 1, 1.5, and 3 g/kg. However, lysozyme gene expression in tilapia treated with 0.5 g/kg lysozyme had no significant differences from those fed 0 g/kg lysozyme. After infection, the relative transcription of the lysozyme gene was upregulated in tilapia fed 1 and 1.5 g/kg, while tilapia fed 1 g/kg lysozyme had the highest C3 gene transcription. After infection, the hepatocytes in the livers of fish fed 0 g/kg lysozyme exhibited a noticeable fatty alteration, along with congestion, a light infiltration of inflammatory cells, and the start of necrosed cell regeneration. However, the livers of fish that received lysozyme were normal except for infiltrations of perivascular and interstitial mononuclear cells, depending on the supplementation dose. In conclusion, dietary lysozyme is recommended at 1.83-2.05 g/kg to gain high growth performance, immune response, and high resistance to A. hydrophila in Nile tilapia.

Chlorella vulgaris extract conjugated magnetic iron nanoparticles in nile tilapia (Oreochromis niloticus): Growth promoting, immunostimulant and antioxidant role and combating against the synergistic infection with Ichthyophthirius multifiliis and Aeromonashydrophila.

Nile tilapia reared under intensive conditions was more susceptible for Ichthyophthirius multifilii (I. multifiliis) infection eliciting higher mortality, lower productive rate and further bacterial coinfection with Aeromonas hydrophila (A. hydrophila). The higher potency of magnetic field of iron oxide nanoparticles (NPs) can kill pathogens through inhibiting their viability. Herein, coating of Chlorella vulgaris extract (ChVE) with magnetic iron oxide NPs (Mag iron NPs) can create an external magnetic field that facilitates their release inside the targeted tissues. Thus, the current study is focused on application of new functionalized properties of Mag iron NPs in combination with ChVE and their efficacy to alleviate I. multifiliis and subsequent infection with A. hydrophila in Nile tilapia. Four hundred fingerlings were divided into: control group (with no additives), three groups fed control diet supplemented with ChVE, Mag iron NPs and ChVE@Mag iron NPs for 90 days. At the end of feeding trial fish were challenged with I. multifiliis and at 9 days post challenge was coinfected by A. hydrophila. A remarkable higher growth rate and an improved feed conversion ratio were detected in group fed ChVE@Mag iron-NPs. The maximum expression of antioxidant enzymes in skin and gills tissues (GSH-Px, CAT, and SOD) which came in parallel with higher serum activities of these enzymes was identified in groups received ChVE@Mag iron-NPs. Furthermore, group fed a combination of ChVE and Mag iron-NPs showed a boosted immune response (higher lysozyme, IgM, ACH50, and MPO) prior to challenge with I. multifiliis. In contrast, fish fed ChVE@Mag iron-NPs supplemented diet had lower infection (decreased by 62%) and mortality rates (decreased by 84%), as well as less visible white spots (decreased by 92 % at 12 dpi) on the body surfaces and mucous score. Interestingly, post I. multifiliis the excessive inflammatory response in gill and skin tissues was subsided by feeding on ChVE@Mag iron-NPs as proved by down regulation of IL-1ß, TNFα, COX-2 and iNOS and upregulation of IL-10, and IgM, IgT and Muc-2 genes. Notably, group exposed to I. multifiliis-showed higher mortality when exposed to Aeromonas hydrophilia (increased by 43 %) while group fed ChVE@Mag iron-NPs exhibited lower morality (2%). Moreover, the bacterial loads of A. hydrophilia in fish infected by I. multifiliis and fed control diet were higher than those received dietary supplement of ChVE, Mag iron-NPs and the most reduced load was obtained in group fed ChVE@Mag iron-NPs at 7 dpi. In conclusion, ChVE@Mag iron-NPs fed fish had stronger immune barrier and antioxidant functions of skin and gills, and better survival following I. multifiliis and A. hydrophilia infection.

Molecular characterization, expression and functional analysis of TAK1, TAB1 and TAB2 in Nile tilapia (Oreochromis niloticus).

The MAPK pathway is the common intersection of signal transduction pathways such as inflammation, differentiation and proliferation and plays an important role in the process of antiviral immunity. Streptococcus agalactiae will have a great impact on tilapia aquaculture, so it is necessary to study the immune response mechanism of tilapia to S. agalactiae. In this study, we isolated the cDNA sequences of TAK1, TAB1 and TAB2 from Nile tilapia (Oreochromis niloticus). The TAK1 gene was 3492 bp in length, contained an open reading frame (ORF) of 1809 bp and encoded a polypeptide of 602 amino acids. The cDNA sequence of the TAB1 gene was 4001 bp, and its ORF was 1491 bp, which encoded 497 amino acids. The cDNA sequence of the TAB2 gene was 4792 bp, and its ORF was 2217 bp, encoding 738 amino acids. TAK1 has an S_TKc domain and a coiled coil structure; the TAB1 protein structure contains a PP2C_SIG domain and a conserved PYVDXA/TXF sequence model; and TAB2 contains a CUE domain, a coiled coil domain and a Znf_RBZ domain. Homology analysis showed that TAK1 and TAB1 had the highest homology with Neolamprologus brichardi, and TAB2 had the highest homology with Simochromis diagramma (98.28 %). In the phylogenetic tree, TAK1, TAB1 and TAB2 formed a large branch with other scleractinian fishes. The tissue expression analysis showed that the expression of TAK1, TAB1 and TAB2 was highest in the muscle. The expression of TAK1, TAB1 and TAB2 was significantly induced in most of the tested tissues after stimulation with LPS, Poly I:C and S. agalactiae. The subcellular localization results showed that TAK1 was located in the cytoplasm, and TAB1 and TAB2 had certain distributions in the cytoplasm and nucleus. Coimmunoprecipitation (Co-IP) results showed that TRAF6 did not interact with the TAK1 protein but interacted with TAB2, while TAB1 did not interact with P38γ but interacted with TAK1. There was also an interaction between TAK1 and TAB2.

Dietary effects of microalga Tetraselmis suecica on growth, antioxidant-immune activity, inflammation cytokines, and resistance of Nile tilapia fingerlings to Aeromonas sobria infection.

The dietary effects of the green microalga Tetraselmis suecica (TS) on the growth, digestive enzymes, immune and antioxidant responses, genes expression, and disease resistance of Nile tilapia (Oreochromis niloticus) fingerlings were investigated. This microalga was mixed with the diet' ingredients at doses of 0.0 (the control), 5, 10, 15, and 20 g/kg diet and then fed to fish daily for 84 days. After the feeding trial, fish were experimentally challenged with Aeromonas sobria, infection and fish mortalities were recorded for another 10 days. Dietary TS significantly (p < 0.05) enhanced growth, digestive enzymes activities, and blood proteins, particularly at the level of 15 g/kg diet. Feeding the fish on 15 TS/kg feed exhibited highest mRNA expressions of GH and IGF-1 genes as well as SOD, CAT, and GPx genes compared to other TS groups. Moreover, highest levels of hepatic antioxidant and immune indices were found in the treatment of 15 g TS/kg feed. Significant downregulation of IL-1ß and IL-8 genes expression and significant upregulation of IL-10 gene expression were observed in TS-fed fish, principally in fish groups fed on 15-20 g TS/kg feed. Conversely, hepatic malondialdehyde levels, blood glucose, and the activities of transaminases (ALT and AST) were significantly (p < 0.05) decreased in fish fed with 15-20 g TS/kg diet. Serum bactericidal activity against A. sobria was significantly higher in TS-fed fish groups, and its highest levels were found in treatments of 15-20 g/kg diet. Of interest, the survival rates of fish groups fed diets with 10-20 g TS/kg feed were higher after the challenge with A. sobria infection than the control group. Accordingly, we can conclude that supplementing fish diets with a 15 g TS/kg diet enhanced the growth, antioxidant and immune activities, and resistance of Nile tilapia fingerlings to possible A. sobria infection.

Effect of dietary intervention with Capsicum annuum extract on growth performance, physiological status, innate immune response, and related gene expression in Nile tilapia.

The red pepper (Capsicum annuum) has gained great attention recently because of its biological and pharmacological characteristics. The present approach aimed to evaluate the effects of C. annuum alcoholic extract (CAE) supplementation on Nile tilapia (Oreochromis niloticus) growth performance, physiological status, some metabolic, immune, and regulatory genes expression, and resistance against Streptococcus agalactiae infection. Fish (22.26 ± 0.19 g) were assigned to four treatments (five replicates, each with 10 fish replicate-1) and fed tested diets for 60 days. The experimental diets were supplemented with CAE at 0, 0.4, 0.8, and 1.6 g kg-1, expressed as CAE0, CAE0.4, CAE0.8, and CAE1.6, respectively. The findings exhibited that CAE dietary supplementation improved growth performance, feed utilization, elevated growth hormone level, and digestive enzyme activities (amylase and protease), and lowered leptin hormone in a level-dependent manner. Boosting the mRNA expression of the transporter proteins (solute carrier family 15 member 2 and solute carrier family 26 member 6) and insulin-like growth factor-1 genes with a decrease in the myostatin gene expression was noticed in the CAE-fed groups. The innate immune (serum bactericidal activity %, complement 3, and phagocytic activity %) and antioxidant (glutathione peroxidase and total antioxidant capacity) parameters were significantly (p < 0.05) improved, and the serum malondialdehyde level was significantly decreased by CAE dietary inclusion. A marked upregulation in the mRNA expression of interleukins (il-1ß, il-6, il-8, and il-10), transforming growth factor-ß, glutathione peroxidase, and glutathione synthetase genes were observed in CAE-fed groups. Dietary CAE decreased the cumulative mortalities after the challenge with S. agalactiae by 20, 13.33, and 10% in CAE0.4, CAE0.8, and CAE1.6, respectively, compared to the control (40%). Overall, dietary supplementation with CAE could improve growth performance and physiological status, and modulate the expression of several regulatory genes in Nile tilapia. The recommended level of CAE is 1.6 g kg-1 to augment growth and health status.

Nelumbo nucifera synthesized selenium nanoparticles modulate the immune-antioxidants, biochemical indices, and pro/anti-inflammatory cytokines pathways in Oreochromis niloticus infected with Aeromonas veronii.

Bacterial infection is considered one of the major issues in fish culturing that results in economic losses. Metal nanoparticles are a cutting-edge and effective disease management and preventive strategy because of their antibacterial ability. In this investigation, the selenium nanoparticles were prepared by a biological method using Nelumbo nucifera leaves extract. The in-vitro antibacterial activity of N. nucifera synthesized selenium nanoparticles (NN-SeNPs) was tested against Aeromonas veronii. A treatment assay was conducted on 210 Oreochromis niloticus (average body weight: 27 ± 2.00 g). A preliminary approach was conducted on 90 fish for determination of the therapeutic concentration of NN-SeNPs which was found to be 4 mg/L. Fish (n = 120) were categorized into four groups for 10 days; G1 (control) and G2 (NN-SeNPs) were non-challenged and treated with 0 and 4 mg/L NN-SeNPs, respectively. While, G3 and G4 were infected with 2 × 106 CFU/mL of A. veronii and treated with 0 and 4 mg/L NN-SeNPs, respectively. NN-SeNPs exhibited an inhibition zone against A. veronii with a diameter of 16 ± 1.25 mm. The A. veronii infection increased the hepato-renal biomarkers (alanine and aspartate aminotransferases and creatinine) than the control group. An oxidative stress was the consequence of A. veronii infection (higher malondialdehyde and hydrogen peroxide levels with lower glutathione peroxidase superoxide, dismutase, and catalase activity). A. veronii infection resulted in lower immunological biomarker values (immunoglobulin M, lysozyme, and complement 3) with higher expression of the inflammatory cytokines (interleukin-1ß and tumor necrosis factor-ɑ) as well as lower expression of the anti-inflammatory cytokines (interleukin-10 and transforming growth factor-ß). Therapeutic application with 4 mg/L NN-SeNPs prevented the disease progression; and modulated the hepato-renal function disruptions, oxidant-immune dysfunction, as well as the pro/anti-inflammatory cytokines pathway in the A. veronii-infected fish. These findings suggest that NN-SeNPs, employed as a water therapy, can safeguard fish from the harmful effects of A. veronii and serve as a promising antibacterial agent for sustainable aquaculture.

Rhein: A potent immunomodulator empowering largemouth bass against MSRV infection.

Micropterus salmoides rhabdovirus (MSRV) is a significant viral pathogen in largemouth bass aquaculture, causing substantial annual economic losses. However, effective prevention methods remain elusive for various reasons. Medicinal plant extracts have emerged as valuable tools in preventing and managing aquatic animal diseases. Thus, the search for immunomodulators with straightforward, safe structures in plant extracts is imperative to ensure the continued health and growth of the largemouth bass industry. In our research, we employed epithelioma papulosum cyprinid (EPC) cells and largemouth bass as models to assess the anti-MSRV properties and immunomodulatory effects of ten plant-derived bioactive compounds. Among them, rhein demonstrated noteworthy potential, exhibiting a 75 % reduction in viral replication in vitro at a concentration of 50 mg/L. Furthermore, rhein pre-treatment significantly inhibited MSRV genome replication in EPC cells, with the highest inhibition rate reaching 64.8 % after 24 h, underscoring rhein's preventive impact against MSRV. Likewise, rhein displayed remarkable therapeutic effects on EPC cells during the early stages of MSRV infection, achieving a maximum inhibition rate of 85.6 % in viral replication. Subsequent investigations unveiled that rhein, with its consistent activity, effectively mitigated cytopathic effects (CPE) and nuclear damage induced by MSRV infection. Moreover, it restrained mitochondrial membrane depolarization and reduced the apoptosis rate by 38.8 %. In vivo experiments reinforced these findings, demonstrating that intraperitoneal injection of rhein enhanced the expression levels of immune related genes in multiple organs, hindered virus replication, and curtailed the mortality rate of MSRV-infected largemouth bass by 29 %. Collectively, our study endorses the utility of rhein as an immunomodulator to combat MSRV infections in largemouth bass. This not only underscores the potential of rhein as a broad-spectrum antiviral and means to bolster the immune response but also highlights the role of apoptosis as an immunological marker, making it an invaluable addition to the armamentarium against aquatic viral pathogens.