Novel insights into immune stress markers associated with myxosporeans gill infection in Nile tilapia (molecular and immunohistochemical studies).

Nile tilapia (Oreochromis niloticus) is valued in aquaculture because of its quick development and ability to thrive in various environments. Myxosporeans are among the fish parasites that affect fish productivity, as they impact fish growth and reproduction, resulting in large fish deaths in farms and hatcheries. This study has been focused on morpho-molecular identification for the myxosporean parasites infecting Nile tilapia from three governorates in Egypt and assessment of gene expression of different cytokines (Interleukin-1ßeta (IL-1ß), major histocompatibility complex class II (MHC-II), and clusters of differentiation 4 (CD-4) and 8 (CD-8)) in tissues. Additionally, this work aimed to correlate the developed histopathological alterations and inflammatory reactions in gills with immunohistochemical expression of inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-α). Finally, the infected fish's cortisol levels and blood glucose were assessed. Results of BLAST sequence analysis of the 18S rRNA for the collected protozoans confirmed Myxobolus agolus, M. brachysporus, M. tilapiae, and Henneguya species. The molecular characterization of the immunological status of gills revealed marked upregulation of different inflammatory cytokines in the gills of infected fish. There was a significantly increased serum cortisol and glucose level in infected fish compared with control, non-infected ones. Severe histopathological alterations were observed in the infected fish gills, associated with increased expression of iNOS and TNF-α and related to myxosporean infection. The present study provides new insights into oxidative stress biomarkers in Nile tilapia infected with Myxosporeans and elucidates the gill's immune status changes as a portal of entry for protozoa that contribute to tissue damage.

The effects of dietary Spirulina platensis or curcumin nanoparticles on performance, body chemical composition, blood biochemical, digestive enzyme, antioxidant and immune activities of Oreochromis niloticus fingerlings.

CONTEXT: Recently, prioritize has been given to using natural phytogenic or nano compounds as growth promoters and immunostimulants in fish diets as an alternative to antibiotics. AIMS: The main propose of this trial was to determine the impact of supplementing diets with spirulina or curcumin nanoparticles on the performance and health indicators of Nile tilapia fingerlings. METHODS: In a 56-day feeding trial, 180 tilapia fingerlings were assigned into three main groups, as follows: 1st, control group, 2nd, Spirulina platensis (SP; 5 g kg-1 diet) and 3rd, curcumin nanoparticles (CUR-NPs; 30 mg kg-1 diet). KEY RESULTS: Incorporating tilapia diets with SP or CUR-NPs significantly improved performance, body chemical analysis, blood biochemical and hematological indices, digestive enzyme activities, and antioxidant and immunostimulant features compared to the control. CONCLUSION: Fortified tilapia diets with CUR-NPs or SP efficiently boost the productivity and health of Nile tilapia fingerlings. IMPLICATIONS: The research introduces new practical solutions for applying safe feed additives as alternatives to antibiotics in tilapia farming.

Immunomodulatory, antioxidant, and growth-promoting activities of dietary fermented Moringa oleifera in Nile tilapia (Oreochromus niloticus) with in-vivo protection against Aeromonas hydrophila.

BACKGROUND: Moringa oleifera, a well-known medicinal plant, has been used in aquafeed as a dietary supplement. Based on previous studies, insufficient research is available on the dietary supplementation of Nile tilapia with M. oleifera leaf and seed mixtures, specifically the fermented form. Therefore, this study aimed to investigate the efficacy of fermented (FMO) versus non-fermented M. oleifera (MO) leaf and seed mixtures on immunological parameters, antioxidant activity, growth performance, and resistance to A. hydrophila infection after a 30-day feeding trial on Nile tilapia. METHODS: A total of 180 fingerlings were randomly divided into four groups in addition to the control group (36 fish each, in triplicate). Fish in the tested groups were fed on basal diet supplemented with MO5%, MO10%, FMO5%, and FMO10%, while those in control were fed on basal diet only. After the feeding trial, fish were challenged with A. hydrophila. The immunomodulatory activity of M. oleifera was evaluated in terms of phagocytic and lysozyme activities, immune-related cytokines and IgM gene expression. Antioxidants, and growth-promoting activities were also assessed. RESULTS: The results revealed that fish supplemented FMO markedly in FMO10% group followed by FMO5%, exhibited significant (P < 0.05) improvement in the tested immunological, hepatic antioxidants, and growth performance parameters. Furthermore, the highest survival rate post-challenge with mild clinical symptoms, and the lowest A. hydrophila bacterial count were reported in these groups. Meanwhile, MO10%-supplementation exhibited the opposite trend. CONCLUSIONS: The study' conclusion suggests that fermented M. oleifera leaf and seed mixture is a promising growth-promoting and immunostimulatory feed-additive candidate for Nile tilapia and could reduce the losses caused by A. hydrophila infection.

Effects of dietary fermented Saccharomyces cerevisiae extract (Hilyses) supplementation on growth, hematology, immunity, antioxidants, and intestinal health in Nile tilapia.

This study investigated the effects of dietary supplementation with the product Hilyses on growth performance, feed utilization, nutrient composition, hematological parameters, serum biochemistry, immune function, antioxidant status, and digestive enzyme activity in juvenile Nile tilapia (Oreochromis niloticus, initial body weight 4.24 ± 0.01 g). The fish were fed diets supplemented with Hilyses at concentrations of 0, 1, 2, or 3 g/kg for a period of 8 weeks. The results showed that supplementation with Hilyses at levels up to 2 g/kg diet significantly improved final body weight, weight gain, specific growth rate, feed efficiency ratio, protein efficiency ratio, apparent protein utilization, and energy utilization compared to the control diet without Hilyses. Carcass crude protein content and moisture were significantly higher in Hilyses-fed groups, while crude lipid content decreased at the 3 g/kg supplementation level. Hilyses supplementation enhanced various hematological parameters, including increased red blood cell count, total leukocyte count, hemoglobin concentration, hematocrit, and mean corpuscular volume. Serum biochemistry and immune function markers like total protein, albumin, complement component C3, IgM, and IgG were significantly elevated in the 2 and 3 g/kg Hilyses groups. Antioxidant enzyme activities (catalase, glutathione peroxidase, total superoxide dismutase) were enhanced, and lipid peroxidation was reduced, in the 2 g/kg Hilyses group. Digestive enzyme activities, particularly protease and lipase, were also improved with Hilyses supplementation. Histological examination showed reduced lipid deposition in the liver and increased branching of intestinal villi at the 2 g/kg Hilyses level. Overall, these results indicated that dietary Hilyses supplementation at 2 g/kg diet optimizes growth, feed utilization, nutrient composition, hematology, immunity, antioxidant status, and digestive function in juvenile Nile tilapia.

Novel C-type lectin mediated non-specific cytotoxic cells killing activity through NCCRP-1 in nile tilapia (Oreochromis niloticus).

Non-specific cytotoxic cells (NCCs) are vital immune cells involved in teleost's non-specific immunity. As a receptor molecule on the NCCs' surface, the non-specific cytotoxic cell receptor protein 1 (NCCRP-1) is known to play a crucial role in mediating their activity. Nevertheless, there have been limited studies on the signal molecule that transmits signals via NCCRP-1. In this study, a yeast two-hybrid (Y2H) library of tilapia liver and head kidney was constructed and subsequently screened with the bait vector NCCRP-1 of Oreochromis niloticus (On-NCCRP-1) to obtain a C-type lectin (On-CTL) with an interacting protein sequence. Consequently, the full-length sequence of On-CTL was cloned and analyzed. The expression analysis revealed that On-CTL is highly expressed in the liver and is widely distributed in other tissues. Furthermore, On-CTL expression was significantly up-regulated in the brain, intestine, and head kidney following a challenge with Streptococcus agalactiae. A point-to-point Y2H method was also used to confirm the binding between On-NCCRP-1 and On-CTL. The recombinant On-CTL (rOn-CTL) protein was purified. In vitro experiments demonstrated that rOn-CTL can up-regulate the expression of killer effector molecules in NCCs via its interaction with On-NCCRP-1. Moreover, activation of NCCs by rOn-CTL resulted in a remarkable enhancement in their ability to eliminate fathead minnow cells, indicating that rOn-CTL effectively modulates the killing activity of NCCs through the NCC receptor molecule On-NCCRP-1. These findings significantly contribute to our comprehension of the regulatory mechanisms governing NCC activity, paving the way for future research in this field.

Immunological and molecular evaluation of zoonotic metacercarial infection in freshwater fish: A cross-sectional analysis.

Improperly cooked fish, carrying active metacercariae (MCs), can pose a significant risk for transmitting fish-borne zoonotic trematodes (FBZTs) to human consumers. This study aimed to enhance our understanding of FBZTs by conducting a comprehensive cross-sectional analysis involving various fish species, such as Nile tilapia (Oreochromis niloticus), African catfish (Clarias gariepinus), and red-belly tilapia (Tilapia zillii). These fish specimens were collected from distinct Egyptian governorates, specifically Giza, Kafr al-Shaykh, and Fayoum. The recovered flukes from experimentally infected domestic pigeons were identified as Prohemistomum vivax, Haplorchis pumilio, and Pygidiopsis genata based on morphological features. Furthermore, the identity of the retrieved adult flukes was confirmed using three species-specific primers for PCR amplification and sequencing analysis of the ITS rDNA region and have been deposited in GenBank with the following accession numbers: P. vivax (OR291421.1 and OR291422.1), P. genata (OP099561.1), and H. pumilio (OM439581.1-OP090510.1). Quantitative real-time PCR targeting the immunological genes Tumor Necrosis Factor-alpha (TNF-alpha) and Interleukin-1 (IL-1Β) was employed to compare the cellular immune response between infected with EMCs and uninfected O. niloticus. The results indicated a significant increase in TNF- and IL-1Β levels in FBZTs-infected vs un-infected fishes. Importantly, the presence of adult flukes and EMCs led to substantial histological alterations in both experimentally infected pigeons and naturally infected fish tissues. These changes included the necrosis of fish muscle bundles and a pronounced inflammatory reaction with muscular necrosis in the digestive tracts of experimentally infected pigeons.

Recombinant lactococcal-based oral vaccine for protection against Streptococcus agalactiae infections in tilapia (Oreochromis niloticus).

Streptococcosis outbreaks caused by Streptococcus agalactiae infection in tilapia aquaculture have been consistently reported and associated with high mortality and morbidity leading to significant economic losses. Existing vaccine candidates against Streptococcus spp. are designed for intraperitoneal injections that are not practical and labor-intensive which have prompted farmers to protect aquatic animals with antibiotics, thus encouraging the emergence of multidrug resistant bacteria. In this study, a live recombinant L. lactis vaccine expressing a 1403 bp surface immunogenic protein (SIP) and a 1100 bp truncated SIP (tSIP) gene was developed and evaluated against S. agalactiae infection in tilapia. Both SIP and tSIP sequences were cloned and transformed into L. lactis. The recombinant L.lactis vaccine was orally administered to juvenile tilapia for a month. Detection of SIP-specific serum IgM in vaccinated groups compared to control groups indicated that recombinant proteins expressed from L. lactis could elicit immunogenic reactions in tilapia. Fish immunized with the tSIP vaccine also showed the highest level of protection compared to other test groups, and the mortality rate was significantly reduced compared to both control groups. The relative percentage of survival (RPS) against S. agalactiae for both SIP and tSIP-vaccinated groups was 50 % and 89 %, respectively, at 14 days post-challenge. Significant up-regulation of IgM, IL-1ß, IL-10, TNF-α and IFN-γ were observed at day 34 between the vaccinated and control groups. These results indicated that the recombinant lactococcal tSIP vaccine can elicit both cell-mediated and humoral responses and is recommended as a potential oral vaccine against S. agalactiae infection. Future work will include further in vivo challenge assessments of this vaccine candidate fused with adjuvants to boost immunogenicity levels in tilapia.

Evaluation of the efficacy of MONTANIDE™ GR01, a new adjuvant for feed-based vaccines, on the immune response and protection against Streptococcus agalactiae in oral vaccinated Nile tilapia (Oreochromis niloticus) under laboratory and on-farm conditions.

Streptococcosis, an emerging infectious disease caused by Streptococcus agalactiae, has had adverse effects on farmed tilapia. Several vaccines have been developed to prevent this disease and induce a specific immune response against S. agalactiae infection. In this study the use of MONTANIDE™ GR01, a new adjuvant for oral vaccination, was optimized for use in tilapia under laboratory and field studies. In the laboratory trial the immune response and protective efficacy of two doses of MONTANIDE™ GR01, 20 % (w/w) and 2 % (w/w), included into the feed-based adjuvanted vaccines were assessed comparatively. Following immunization, the innate immune parameters studied in serum, including lysozyme, myeloperoxidase, catalase and glutathione peroxidase activity, were all increased significantly. Furthermore, specific IgM antibodies against S. agalactiae were induced significantly in serum post-vaccination, with higher levels observed in both groups that received the feed-based adjuvanted vaccine. Under both injection and immersion challenge conditions, the relative percent survival for the feed-based adjuvanted vaccine groups ranged from 78 % to 84 %. Following use of the low dose concentration of MONTANIDE™ GR01 for oral vaccination of tilapia in cage culture systems, several innate immune parameters were effectively enhanced in the immunized fish. Similarly, the levels of specific IgM antibodies in the serum of feed-based vaccinated fish were significantly enhanced, reaching their highest levels 2-5 months post-vaccination. Cytokines associated with innate and adaptive immunity were also examined, and the expression levels of several genes showed significant up-regulation. This indicates that both cellular and humoral immune responses were induced by the feed-based adjuvanted vaccine. The economic impact of a feed-based adjuvanted vaccine was examined following vaccination, considering the growth performance and feed utilization of the fish. It was found that the Economic Performance Index and Economic Conversion Ratio were unaffected by vaccination, further demonstrating that there are no negative impacts associated with administering a feed-based vaccine to fish. In conclusion, the data from this study indicate that MONTANIDE™ GR01 is a highly valuable adjuvant for oral vaccination, as demonstrated by its ability to induce a strong immune response and effectively prevent streptococcal disease in Nile tilapia.

Infection dynamics of Shewanella spp. in Nile tilapia under varied water temperatures: A hematological, biochemical, antioxidant-immune analysis, and histopathological alterations.

In aquaculture, fluctuating water temperatures can act as a potent stressor, influencing the virulence and transmission dynamics of pathogenic bacteria, potentially triggering outbreaks and impacting fish health. The purpose of this work was to examine the impact of Shewanella spp. infection on hematological, biochemical, and antioxidant-immune parameters of Nile tilapia (Oreochromis niloticus) under different water temperatures. For this purpose, 180 fish were divided into 6 groups in triplicate (30 fish per group; 10 fish per replicate). Group 1 (G1), G2, and G3 were reared at varying water temperatures (22 °C, 28 °C, and 31 °C, respectively) without infection. While G4, G5, and G6 were IP-injected with 0.2 mL of Shewanella spp. (0.14 × 105) and reared at 22 °C, 28 °C, and 31 °C, respectively. Shewanella spp. infection induced significant lowering (p < 0.05) in hematological parameters (red and white blood cells, hemoglobin, and packed cell volume%) and immune-antioxidant responses (phagocytic activity%, phagocytic index, lysozyme, nitric oxide), total antioxidant capacity, catalase, and reduced glutathione, especially at 22 °C. Moreover, a significant increase (p < 0.05) in the hepato-renal function indicators (alanine aminotransferase, aspartate aminotransferase, urea, and creatinine), stress biomarkers (glucose and cortisol), malondialdehyde, and pro-inflammatory cytokines (interleukin-1ß and tumor necrosis factor-α) were the consequences of the Shewanella spp. infection, especially at 22 °C. The Shewanella spp. infection exhibited marked histopathological changes in the hepatic and renal tissues. Worthily, Shewanella spp. can cause detrimental alterations in Nile tilapia's hematological, biochemical, and antioxidant-immune parameters at various water temperatures, but the major detrimental changes were observed at a water temperature of 22 °C. Consequently, we can conclude that the infection dynamics of Shewanella spp. are exaggerated at 22 °C. These outcomes could help in understanding the nature of such an infection in Nile tilapia.

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

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

Effects of Shrimp Shell-Derived Chitosan on Growth, Immunity, Intestinal Morphology, and Gene Expression of Nile Tilapia (Oreochromis niloticus) Reared in a Biofloc System.

Chitosan (CH) shows great potential as an immunostimulatory feed additive in aquaculture. This study evaluates the effects of varying dietary CH levels on the growth, immunity, intestinal morphology, and antioxidant status of Nile tilapia (Oreochromis niloticus) reared in a biofloc system. Tilapia fingerlings (mean weight 13.54 ± 0.05 g) were fed diets supplemented with 0 (CH0), 5 (CH5), 10 (CH10), 20 (CH20), and 40 (CH40) mL·kg-1 of CH for 8 weeks. Parameters were assessed after 4 and 8 weeks. Their final weight was not affected by CH supplementation, but CH at 10 mL·kg-1 significantly improved weight gain (WG) and specific growth rate (SGR) compared to the control (p < 0.05) at 8 weeks. Skin mucus lysozyme and peroxidase activities were lower in the chitosan-treated groups at weeks 4 and 8. Intestinal villi length and width were enhanced by 10 and 20 mL·kg-1 CH compared to the control. However, 40 mL·kg-1 CH caused detrimental impacts on the villi and muscular layer. CH supplementation, especially 5-10 mL·kg-1, increased liver and intestinal expressions of interleukin 1 (IL-1), interleukin 8 (IL-8), LPS-binding protein (LBP), glutathione reductase (GSR), glutathione peroxidase (GPX), and glutathione S-transferase (GST-α) compared to the control group. Overall, dietary CH at 10 mL·kg-1 can effectively promote growth, intestinal morphology, innate immunity, and antioxidant capacity in Nile tilapia fingerlings reared in biofloc systems.

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.

Potential effects of probiotics (immunobacteryne; IMB) on growth performance, feed efficacy, blood biochemical, redox balance, nonspecific immunity and heat-shock protein expression of Nile tilapia (Oreochromis niloticus) fingerlings.

The supplementation of aquafeed with probiotics is recommended for feasible aquaculture activities. Therefore, the aim of current study was to investigate the potential effects of probiotics on growth performance, feed utilization, biochemical attributes, redox status and immunity response as well as the transcription of heat-shock protein 70 (HSP70) and insulin-like growth factor-1 (IGF-1) genes of Nile tilapia (Oreochromis niloticus; n = 120). Fish with an initial weight of 8.17 ± 0.02 g/fish were randomly divided into four treatment groups and were fed diets containing 0, 0.5, 1 and 1.5 mg immunobacteryne (IMB)/kg diet respectively. Dietary IMB at 1.5 g/kg diet significantly improved the growth performance, feed consumption and growth hormone secretion of the experimental fish (p < 0.05). The 1 or 1.5 g IMB/kg diet boosted phagocytic activities and innate immune response. Serum total protein, total cholesterol, triglycerides and glucose were significantly increased in the groups that were fed 1 and 1.5 mg IMB/kg diet compared to the control (p < 0.05). Meanwhile, the levels of uric acid, creatinine, liver enzymes (aspartate transaminase and alanine transaminase) and cortisol hormone were significantly reduced in the aforementioned treated groups compared to the control (p < 0.05). All fish fed IMB-supplemented diet showed a significant increase in the expression of IGF-1 gene, while the transcription of HSP70 was significantly decreased (p < 0.05). In conclusion, the dietary inclusion of IMB (1 g/kg diet) enhanced growth promoters, feed efficacy, blood biochemical, redox balance and nonspecific immune responses in Nile tilapia fingerlings.

TGF-ß1 suppresses the T-cell response in teleost fish by initiating Smad3- and Foxp3-mediated transcriptional networks.

Transforming growth factor-ß1 (TGF-ß1) can suppress the activation, proliferation, and function of many T-cell subsets, protecting organisms from inflammatory and autoimmune disease caused by an overexuberant immune response. However, whether and how TGF-ß1 regulates T-cell immunity in early vertebrates remain unknown. Here, using a Nile tilapia (Oreochromis niloticus) model, we investigated suppression of the T-cell response by TGF-ß1 in teleost species. Tilapia encodes an evolutionarily conserved TGF-ß1, the expression of which in lymphocytes is significantly induced during the immune response following Edwardsiella piscicida infection. Once activated, tilapia T cells increase TGF-ß1 production, which in turn suppresses proinflammatory cytokine expression and inhibits T-cell activation. Notably, we found administration of TGF-ß1 cripples the proliferation of tilapia T cells, reduces the potential capacity of Th1/2 differentiation, and impairs the cytotoxic function, rendering the fish more vulnerable to bacterial infection. Mechanistically, TGF-ß1 initiates the TGF-ßR/Smad signaling pathway and triggers the phosphorylation and nuclear translocation of Smad2/3. Smad3 subsequently interacts with several transcriptional partners to repress transcription of cytokines IL-2 and IFN-γ but promote transcription of immune checkpoint regulator CTLA4 and transcription factor Foxp3. Furthermore, TGF-ß1/Smad signaling further utilizes Foxp3 to achieve the cascade regulation of these T-cell genes. Taken together, our findings reveal a detailed mechanism by which TGF-ß1 suppresses the T cell-based immunity in Nile tilapia and support the notion that TGF-ß1 had already been employed to inhibit the T-cell response early in vertebrate evolution, thus providing novel insights into the evolution of the adaptive immune system.

α-MSH is partially involved in the immunomodulation of Nile tilapia (Oreochromis niloticus) antibacterial immunity.

α-Melanocyte-stimulating hormone (α-MSH) is a well-studied neuropeptide controlling skin and hair color. Besides, numerous immunomodulation roles of α-MSH were recorded in humans and mice. However, the regulatory effects of α-MSH in teleost immunity haven't been well elucidated. In this study, several precursor molecules of α-MSH (POMCs) and its receptors (MCRs) in Nile tilapia (Oreochromis niloticus) were characterized, and their expression characteristics and specific functions on antibacterial immunity were determined. Overall, POMCs and MCRs were principally detected in the brain, skin, and liver, and were remarkably promoted post Streptococcus agalactiae infection. However, tiny POMCs and MCRs were observed in tilapia immune organs (head kidney and spleen) or lymphocytes, and no evident immunomodulation effect was detected in vitro. Moreover, the in vivo challenge experiments revealed that α-MSH protects tilapia from bacterial infection by regulating responses in the brain and intestine. This study lays theoretical data for a deeper comprehension of the immunomodulation mechanisms of teleost α-MSH and the evolutional process of the vertebrate melanocortin system.

ß-glucan mitigation on toxicological effects in monocytes/macrophages of Nile tilapia (Oreochromis niloticus) following copper exposure.

The protective effect of ß-glucan against toxicological effects caused by copper oxide nanoparticles (Cu NPs) and copper ions (Cu ions) were studied in monocytes/macrophages (MO/MФ) of Nile tilapia (Oreochromis niloticus). Our results demonstrated that CuO NPs and Cu ions exposure aroused strong oxidative lesion in MO/MФ by detection of cellular reactive oxygen species (ROS) and reduced glutathione (GSH), as well as identification of several antioxidant-related cytokines. Meanwhile, the serious pro-inflammatory responses were accompanied during the processes of oxidative lesion by TNFα, IL-1ß, and IL-6 genes validation. Copper induced MO/MФ underwent apoptosis through mitochondrial signaling pathway by mitochondrial membrane potential (ΔΨm) detection and Bax, Bcl-2, Cyt-c, Apaf-1, Caspase 9, Caspase 3 genes validation. Furthermore, the phagocytic abilities were inhibition in MO/MФ by evaluation of microspheres (0.5 and 1.0 µm beads) and bioparticles (S. agalactiae and A. hydrophila) uptake and LPS-induced NO production. However, ß-glucan might participate in immunomodulation through C-type lectin receptor (CLR) and complement receptor 3 (CR3) to suppress pro-inflammatory responses, thereby revered all the copper induced aforementioned adverse effects in MO/MΦ. Taken together, our results provide insights on the mechanisms through ß-glucan administration to mitigate toxicological effects of CuO NPs and Cu ions exposure to the MO/MΦ, which will benefit aspects related to fish farming and aquaculture production.

DDX43 recruits TRIF or IPS-1 as an adaptor and activates the IFN-ß pathway in Nile tilapia (Oreochromis niloticus).

DDX43 is one of the members of the DExD/H-box protein family, and emerging data suggest that it may play an important role in antiviral immunity across mammals. However, little is known about DDX43 in the fish immune response. In this study, we isolated the cDNA sequence of ddx43 in Nile tilapia (Oreochromis niloticus). The ddx43 gene was 2338 bp in length, contained an open reading frame (ORF) of 2064 bp and encoded a polypeptide of 687 amino acids. The predicted protein of OnDDX43 has three conserved domains, including the RNA binding domain KH, DEAD-like helicase superfamily DEXDc and C-terminal HELICc domain. In healthy Nile tilapia, the Onddx43 transcript was broadly expressed in all examined tissues, with the highest expression levels in the muscle and brain and the lowest in the liver. After challenge with Streptococcus agalactiae, lipopolysaccharides (LPS) and polyinosinic polycytidylic acid (Poly I:C), the expression level of Onddx43 mRNA was upregulated or downregulated in all of the tissues tested. Overexpression of OnDDX43 in 293 T cells showed that it has a positive regulatory effect on IFN-ß. The subcellular localization showed that OnDDX43 was expressed in the cytoplasm. We performed further pull-down assays and found that OnDDX43 interacted with both interferon-ß promoter stimulator1 (IPS-1) and TIR domain-containing adaptor inducing interferon-ß (TRIF).

Development and efficacy of Streptococcus iniae live-attenuated vaccines in Nile tilapia, Oreochromis niloticus.

Streptococcus iniae is a re-emerging bacterial pathogen in freshwater and marine aquaculture worldwide. There are no commercial vaccines available for S. iniae in the United States, and autogenous vaccines are restricted to inactivated whole-cell preparations with limited protection against heterogenous strains. Live-attenuated vaccines (LAV) represent an advantageous alternative to these bacterins, as they induce robust cellular and humoral immunity, and may provide longer lasting protection through less stressful routes of administration. We investigated whether accumulation of mutations in S. iniae by serial passage in the presence of rifampin can generate immunogenic LAV conferring protection against challenge with heterologous wild-type (WT) S. iniae strains in Nile tilapia (Oreochromis niloticus). Three lineages of rifampin-resistant S. iniae strains were generated from three genetically distinct parent strains (n = 9) by multiple passages in increments of Rifamycin SV sodium salt. Growth in liquid media, extent of capsulation, antimicrobial susceptibility, survival in Nile tilapia whole blood, and cytotoxicity in an O. mossambicus endothelial cell line were compared between the passaged and WT strains. Nile tilapia challenges were used to assess strain virulence, generation of anti-S. iniae IgM, and the protection conferred by LAV candidates against virulent S. iniae. Rifampin-resistant strains demonstrated changes in growth rate and cytotoxicity in endothelial cells, as well as significant reductions in whole blood survival (p < 0.05). Selected strains also showed attenuated virulence in the Nile tilapia challenge model, and anti-S. iniae IgM generated against these strains demonstrated cross-reactivity against heterologous bacteria. Immunization by intracoelomic injection induced protection against a virulent WT strain of S. iniae, with relative percent survival up to 95.05%.

Interleukin-6 mediates lipopolysaccharide-inhibited irisin secretion in Nile tilapia (Oreochromis niloticus).

Irisin is a novel immunomodulatory adipomyokine released upon cleavage of the fibronectin type III domain-containing protein 5 (FNDC5). We aimed to examine interleukin-6 (IL-6) role in mediating irisin secretion in immunologically challenged animal and primary head kidney leukocytes cultured from tilapia. Intraperitoneal injection of lipopolysaccharide (LPS) increased plasma IL-6 levels and decreased irisin secretion, suggesting a causal relationship between the induction of IL-6 and irisin. To address this relationship, we further produced recombinant tilapia IL-6 and the anti-tilapia IL-6 polyclonal antiserum. Intraperitoneal injection of recombinant tilapia IL-6 inhibited plasma irisin levels. Consistent with this observation, LPS-induced inhibition of plasma irisin was significantly attenuated by neutralizing circulating IL-6 using an IL-6 antiserum. Besides, IL-6 treatment could inhibit irisin secretion and FNDC5 gene expression in primary cultures of tilapia head kidney leukocytes. In parallel experiments, both LPS and IL-6 blockade of irisin secretion could be reverted by IL-6 receptor antagonism. At the level of the leukocyte, IL-6 treatment also triggered rapid phosphorylation of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3), whereas IL-6-reduced irisin secretion could be negated by inhibiting the JAK2 and STAT3 signaling pathways. These results, as a whole, provide the first evidence that IL-6 is the mediator of LPS-inhibited irisin secretion via activation of the JAK2/STAT3 signaling pathway.

Pistachio hull polysaccharide protects Nile tilapia against LPS-induced excessive inflammatory responses and oxidative stress, possibly via TLR2 and Nrf2 signaling pathways.

Polysaccharides are polymeric carbohydrates found in living organisms, which have several physiological functions. In the present study, Nile tilapia (Oreochromis niloticus) were fed diets containing three levels (0%, 0.2%, and 0.6%) of Pistacia vera hull polysaccharide (PHP) for 45 days and then injected with PBS or bacterial lipopolysaccharide (LPS). Before the LPS challenge, Nile tilapia fed 0.2% and 0.6% PHP showed significantly increased mean final weight and weight gain compared to those received 0% PHP. The specific growth rate and feed conversion ratio were significantly improved in the treatment fed 0.6% PHP compared to the remaining groups. After LPS challenge, the activities of liver antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase exhibited the highest values in the 0.6% PHP group. Malondialdehyde (MDA) levels were significantly augmented in the model (fed 0% PHP diet and injected with LPS) and 0.2% PHP groups compared to the control. However, MDA showed decreased levels in the 0.6% PHP group. LPS induced higher mRNA and/or protein levels of Toll-like receptor 2 (TLR2), nuclear factor kappa B (NF-κB), myeloid differentiation primary response protein 88 (Myd88), tumor necrosis factor α (TNF-α), interleukin 1ß (IL-1ß), and interferon γ (IFN-γ) in Nile tilapia liver. However, PHP administration significantly upregulated the expression of interleukin 10 (IL-10), nuclear erythroid 2-related factor 2 (Nrf2), SOD, and CAT, but markedly suppressed TLR2, NF-κB, Myd88, and pro-inflammatory cytokine expression and/or production in the liver. The findings of the current study indicated that PHP has positive effects on growth performance, immune gene-related expression, and antioxidative activities. We can conclude that PHP can attenuate LPS-induced oxidative stress and inflammatory responses in vivo, possibly via induction of Nrf2 and blockade of TLR2/Myd88/NF-κB pathways in Nile tilapia.