Higher Relative Percent Survival Research Articles

Mannose modified targeted immersion vaccine delivery system improves protective immunity against Infectious spleen and kidney necrosis virus in mandarin fish (Siniperca chuatsi)

Vaccination is an effective method to prevent viral diseases. However, the biological barrier prevents the immersion vaccine from achieving the best effect without adding adjuvants and carriers. Researches on the targeted presentation technology of vaccines with nanocarriers are helpful to develop immersion vaccines for fish that can break through biological barriers and play an effective role in fish defense. In our study, functionally modified single-walled carbon nanotubes (SWCNTs) were used as carriers to construct a targeted immersion vaccine (SWCNTs-M−MCP) with mannose modified major capsid protein (MCP) to target antigen-presenting cells (APCs), against iridovirus diseases. After bath immunization, our results showed that SWCNTs-M−MCP induced the presentation process and uptake of APCs, triggering a powerful immune response. Moreover, the highest relative percent survival (RPS) was 81.3% in SWCNTs-M−MCP group, which was only 41.5% in SWCNTs-MCP group. Altogether, this study indicates that the SWCNTs-based targeted immersion vaccine induces strong immune response and provided an effective protection against iridovirus diseases.

ADDITION OF Caulerpa racemosa EXTRACT ON RPS AND CLINICAL SIGNS OF VANAME SHRIMP AFTER Vibrio parahaemolyticus INFECTION

Prevention of AHPND in shrimp can be done by giving immunostimulants, one of which comes fromseaweed extract, explicitly Caulerpa racemosa. This study aimed to determine the effect of the administration of Caulerpa racemosa extract on the relative percent survival (RPS), clinical signs, and behavioral changes of white shrimp after being infected with Vibrio parahaemolyticus. This study used a completely random design with five treatments and three replications, namely K-, K+, and three treatments with the addition of Caulerpa racemosa extract, names P1 (3 μg/g), P2 (6 μg /g), and P3 (9 μg/g). The infection process with Vibrio parahaemolyticus bacteria was carried out 24 hours after being injected with Caulerpa racemosa extract. The results of this study indicate that the highest Relative Percent Survival (RPS) value was P3. Clinical signs of shrimp infected with Vibrio parahaemolyticus were characterized by empty intestines and a whitened hepatopancreas. In addition, there were behavioral changes in shrimp infected with Vibrio parahaemolyticus, namely decreased appetite, passive shrimp movement at the bottom of the aquarium, and erratic swimming movement of shrimp. Based on these results, it could be said that the best treatment is in P3 with a dose of 9 μg/g. This study can provide the best quantity of C. racemosa extract to treat AHPND in shrimp.

Correlation of Streptococcus agalactiae concentration on immune system and effective dose of inactivated vaccine for Chitralada 3 strain Nile tilapia (Oreochromis niloticus) in Thailand

The main pathogen in the Nile tilapia (Oreochromis niloticus) culture, Streptococcus agalactiae, causes economic harm. Infected fish’s immune systems worked to eliminate of the infection. This study demonstrated the effect of different bacterial concentrations on tilapia immunity and optimal vaccine concentration to induce immunity in Nile tilapia. The experiment was performed at 102, 104, 106, 108, and 1010 CFU/fish of S. agalactiae compared with the control (PBS) through intraperitoneal injection for 72 h. Fish that survived employed to gather blood, and immune responses were assessed through measures of the survival rate include blood smears, antibody titers, and immunoglobulin gene expression. The vaccine experiment investigated formalin-inactivated S. agalactiae vaccination and administered S. agalactiae injections for 14 days. The statistic revealed a significant difference (p < 0.05) in the 108 and 1010 CFU/fish injections with high survival rates (62.22% and 53.33%, respectively). Immunoglobulin gene expression was highly represented in the 1010 CFU/fish injection; antibody titers were significantly improved from the control group, and antibody levels were high in the 1010 CFU/fish injection. The analysis of blood cell types using the blood smear method revealed a progressive increase in leucocytes, particularly lymphocytes, neutrophils, and monocytes, in the treatment group compared to the control group. Moreover, the erythrocyte/leucocyte ratio decreased significantly in response to the high bacterial injection, indicating an increase in leucocytes. Conversely, the erythrocyte level stayed ed within at the 7.03–9.70 × 102 cell/ml and shown no significant difference (p > 0.05). The lymphocytes were almost two-fold in 1010 CFU/fish compared to 108 CFU/fish. As depicted in the lowest concentration of 106 CFU/fish, the vaccine performance had a high relative percent survival (RPS) at 86.67%. This research suggested that the tilapia infected with high S. agalactiae concentrations did not affect the mortality of the tilapia, and vaccine concentration was effective in 106 CFU/fish.

Humoral immune response in Asian seabass vaccinated with inactivated and recombinant viral nervous necrosis vaccine

Viral nervous necrosis is a serious viral disease of marine and brackishwater finfish hatcheries. The red-spotted grouper nervous necrosis virus (RGNNV) is the most prevalent genotype among fishes in tropical and temperate regions. RGNNV was propagated in SSN-1 cell line and inactivated using BEI. The capsid protein of RGNNV was cloned and expressed in Escherichia coli and purified. Inactivated and recombinant VNN vaccines were produced by emulsifying the antigen with commercial adjuvant and administered to Asian seabass, Lates calcarifer, fingerlings and broodstock intraperitoneally. The immune response was assessed at regular intervals by indirect ELISA. The maternal antibodies in one-day post-hatch larvae obtained from the vaccinated and control broodstock were assessed by indirect ELISA. The larvae were subjected to challenge with virulent RGNNV and the relative per cent survival (RPS) was estimated. The efficacy of the vaccine was also assessed under field conditions. The recombinant vaccine produced significantly higher specific antibody levels than the inactivated vaccine in fingerlings and broodstock and the immune response was dose-dependent. Recombinant vaccine at 50 and 100 μg/fish produced significantly higher antibody levels and immune response could be observed as early as one-week post vaccination. Vaccinated broodstock produced larvae with maternal antibodies and had higher RPS when challenged at 18 days post-hatch. The vaccine produced a significant immune response even under field conditions. The vaccine can be used to vaccinate broodstock to reduce loss due to VNN in early larval stages.

Asparagus racemosus improves immune-related parameters in Nile tilapia (Oreochromis niloticus) and mitigates deltamethrin-induced toxicity

Deltamethrin (DM) is one of the most toxic but widely used pyrethroid insecticides. Even though a non-target animal, fish are at high risk as they are deficient in the enzyme system that hydrolyses pyrethroids. Enhancing the immune system is a potential method in preventing fish diseases. The present investigation aims to study the modulations in the immune response-related parameters in Oreochromis niloticus that were exposed to DM, by dietary supplementation of aqueous root extract of Asparagus racemosus (ARE). The experiment compared fish in control, DM (1 μg/L) exposed (added to water), ARE (10 g, 20 g, and 30 g ARE/kg of feed) supplemented, and DM-ARE cotreated groups. After 21 days of experimental period, serological, histopathological, and immune response related-gene and protein analysis were carried out. The DM-ARE cotreated group showed significant increase in weight gain, specific growth rate, and decreased feed conversion ratio compared to the DM exposed group. The ARE cotreatment could significantly revert the alteration induced by DM in lysozyme, respiratory burst, myeloperoxidase, C-reactive protein, glucose, cortisol, total protein, albumin, and triglyceride levels. The liver histopathology showed membrane breakage, severe necrosis, infiltration of inflammatory cells, melano-macrophages, and nuclear atrophy, and the kidney showed tubular necrosis, hematopoietic necrosis, Bowman's capsule edema, and glomerulus degeneration in DM exposed group. In ARE cotreated group, the liver showed regenerative cellular changes and only mild to moderate cellular damages, and the kidney tubules and glomerulus had intact structure. ARE discernibly regulated the expression of immune-related genes and proteins (IgM, TNFα, IFN-γ, IL-1β, and IL-8) in fish. The DM-ARE cotreated groups showed reduced cumulative mortality and higher relative percent survival on experimental challenge with Aeromonas hydrophila compared to the DM group. Thus, ARE possess protective potential against DM-induced toxicity, and can be used as a cost-effective technique in aquafarming.

The recombinant subunit vaccine encapsulated by alginate-chitosan microsphere enhances the immune effect against Micropterus salmoides rhabdovirus.

The disease caused by Micropterus salmoides rhabdovirus (MSRV) has brought substantial economic losses to the largemouth bass aquaculture industry in China. Vaccination was considered as a potential way to prevent and control this disease. As a kind of sustained and controlled release system, alginate and chitosan microspheres (SA-CS) are widely used in the development of oral vaccination for fish. Here, we prepared a king of alginate-chitosan composite microsphere to encapsulate the second segment of MSRV glycoprotein (G2 protein) and then evaluated the immune effect of the microsphere vaccine on largemouth bass. Largemouth bass were vaccinated via intragastric immunization by different treatments (PBS, SA-CS, G2 and SA-CS-G2). The results showed that a stronger immune response including serum antibody levels, immune-related physiological indexes (acid phosphatase, alkaline phosphatase, superoxide dismutase and total antioxidant capacity) and the expression of immune-related gene (IgM、IL-8、IL-1β、CD4、TGF-β、TNF-α) can be induced obviously with SA-CS-G2 groups compared with G2 groups when fish were vaccinated. Furthermore, fish were injected with a lethal dose of MSRV after immunization for 28 days, and the highest relative percentage survival (54.8%) was observed in SA-CS-G2 group (40 μg per fish), which is significantly higher than that of G2 group (25.8%). This study showed that alginate-chitosan microspheres as the vaccine carrier can effectively improve the immune effect of oral vaccination and induce better immune protection effect against MSRV infection.

Dietary treatment of Nile tilapia (Oreochromis niloticus) with aquatic fern (Azolla caroliniana) improves growth performance, immunological response, and disease resistance against Streptococcus agalactiae cultured in bio-floc system

The present study was performed to examine the effects of aquatic fern (Azolla caroliniana) (AQF) on growth performance, skin mucus and serum immunities, and disease resistance of Nile tilapia reared in a biofloc system. Three hundred Nile tilapia fingerlings (average 10.48 ± 0.56 g fish-1) were distributed into 15 glass tanks (150 liters tank−1) at a concentration of 20 fish tank−1. A completely randomized design was used in triplicates, where the control group was fed 0 g kg−1 AQF (AQF1), while AQF2, AQF3, AQF4, and AQF5 were fed 25, 50, 100, and 200 g kg−1 AQF, respectively. Growth and immunological specimens were collected every 4 weeks, while disease resistance was conducted after 8 weeks post-feeding. The results showed that dietary administration of AQF significantly promoted the growth rate and feed efficiency of Nile tilapia, with the maximum level noticed in fish fed 100 g kg−1 AQF. However, no significant differences were observed between the AQF1 and AQF5 diets. Similarly, incorporation of AQF resulted in significantly higher skin mucus and serum immunities than the control, except for AQF5. The highest values were observed in fish fed the AQF5 diet. The challenge test showed that in contrast to the control (26.67%), the AQF administrated diets led to significantly higher survivability by 60% (AQF2), 66.67% (AQF3), 83.33% (AQF4), and 53.33 for AQF5. The relative percent survival (RPS) was 45.45%, 54.55%, 77.27%, and 36.36% in AQF2, AQF3, AQF4, and AQF5, respectively. The highest RPS against S. agalactiae was noted in the AQF4 diet. In conclusion, dietary supplementation of 100 g kg−1 AQF can be potentially used as a growth promoter and immunostimulant in Nile tilapia aquaculture.

Effects of dietary galactooligosaccharide on growth, antioxidants, immunity, intestinal morphology and disease resistance against Aeromons hydrophila in juvenile hybrid sturgeon (Acipenser baerii♀ × A. schrenckii♂)

The present study investigated the effects of dietary galactooligosaccharide (GOS) supplementation on growth, antioxidant capacity, immunity, intestinal morphology and resistance to Aeromons hydrophila of hybrid sturgeon (Acipenser baerii♀ × A. schrenckii♂). A total number of 360 fish were randomly distributed into 12 aquaria and fed diets supplemented with GOS at 0%, 1%, 3% and 5% concentrations (designated as control, GOS1, GOS3 and GOS5) for 8 weeks. Then 30 fish from each group were intraperitoneally challenged with A. hydrophila, and mortality was recorded for the next 15 days. The results showed that there were significant differences of final body weight (FBW), weight gain (WG), specific growth rate (SGR) and feed conversion ratio (FCR) in sturgeon fed GOS diets. The lysozyme, ACP, AKP and MPO activities in sturgeon serum fed GOS diets were higher than those with control diet. Significant increase of SOD, CAT and GSH-PX capacity and remarkably decrease of MDA content were observed in liver of fish fed GOS diets. Remarkable enhancement of muscular thickness, villus and microvilli height, and goblet cells in mid intestine was observed in GOS supplemented groups. In spiral valve, supplementation of any quantities of GOS promoted an evident increase in the muscular thickness, villus height and goblet cells, while the villus width and crypt depth were relatively invariant. The IL-1β and IL-8 mRNA levels were decreased and TNF-α gene expression was increased by dietary GOS. The challenge test with A. hydrophila resulted in lower mortalities by GOS administration in a dose-dependent manner and the highest relative percent survival (36.67 ± 1.92%) was observed in fish fed GOS5 diet. The study concludes that GOS inclusion at the rate of 3% is recommended to increase the growth performance, antioxidant capability and immunity as well as regulate intestinal morphology and promote resistance against A. hydrophila in hybrid sturgeon.

Impacts on the immune system of &lt;i&gt;Cyprinus carpio&lt;/i&gt; exposure with a mixed algal extract against &lt;i&gt;Aeromonas hydrophila&lt;/i&gt;

This study evaluates the influence of mixed algal extract (&lt;i&gt;Chlorella vulgaris&lt;/i&gt;, &lt;i&gt;Euglena viridis&lt;/i&gt; and &lt;i&gt;Spirulina platensis&lt;/i&gt;) on common carp &lt;i&gt;Cyprinus Carpio&lt;/i&gt;, which infected infect with bacterial pathogen &lt;i&gt;Aeromonas hydrophila&lt;/i&gt;. &lt;i&gt;C. carpio&lt;/i&gt; was administered intraperitoneally with various doses such as methanol extract (0, 0,1, 1, 10 and 100 mg/kg). The immunological parameters of fish blood and serum samples (Neutrophil activity, Lysozyme activity, Serum myeloperoxidase intensity, Serum bactericidal activity, and Serum antiprotease activity) were investigated at 7, 14, 21, and 28 days of post-immunization. Fish had been tested by virulent&lt;i&gt; A. hydrophila&lt;/i&gt; for 30 days after treatment and 14 days after infection were identified with mortalities. The findings showed that neutrophil levels, lysozyme activity, serum bactericidal activity, myeloperoxidase activity, and serum antiprotease activity significantly enhanced (p&lt;0.05) compared to untreated control. Mixed dietary algae at 1 and 10 mg/kg levels demonstrated slightly (p&lt;0.05) higher relative percentage survival (90 percent) than control against &lt;i&gt;A. hydrophila&lt;/i&gt; disease infection. Results indicated that mixed algal extract in &lt;i&gt;C. carpio&lt;/i&gt; positively impacts non-specific immune parameters and boosts disease tolerance to &lt;i&gt;A. hydrophila&lt;/i&gt; infections.

Enhancement of growth, antioxidative status, nonspecific immunity, and disease resistance in gibel carp (Carassius auratus) in response to dietary Flos populi extract

This study investigated the effects of dietary Flos populi extract (FPE) on the growth, antioxidation capability, innate immune response, and disease resistance in gibel carp. A total of 480 fish were fed with five different diets containing 0, 0.5, 1.0, 1.5, or 2.0 g kg−1 FPE (designated as control, D0.5, D1.0, D1.5, or D2.0 groups) for 45 days. The fish were challenged with A. hydrophila after the feeding trial. Compared with the control, the feed efficiency (FE), weight gain (WG), final body weight (FBW), and specific growth rate (SGR) were significantly improved in groups D1.0 and D1.5. Dietary FPE significantly increased serum superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) activities, as well as glutathione (GSH) content. The contents of protein carbonyl (PCC) and malondialdehyde (MDA) in serum decreased significantly. Additionally, FPE supplementation in diets resulted in significant improvement in serum lysozyme (LZM) and myeloperoxidase (MPO) activities, as well as immunoglobulin M (IgM) and complement 3 (C3) concentrations. The hepatic antioxidant enzymes (CAT and SOD) activities increased, whereas content of MDA decreased in fish treated with dietary FPE than those of control both pre- and post-challenged. After 12 h-challenge, an obvious downregulation of hepatic Kelch-like-ECH-associated protein 1 (Keap1), splenic tumor necrosis factor-α (TNF-α), interleukin (IL)-8, IL-1β, and toll-like receptor 2 (TLR2) mRNA levels was observed in fish treated with dietary FPE, whereas hepatic Nrf2 transcription level was upregulated compared to the control. Furthermore, compared to group D0.5, higher relative percent survival (RPS) was observed in gibel carp fed dietary 1.0–2.0 g/kg FPE. Our results reveal that FPE supplemented diet has a stimulatory effect on antioxidant capacity and nonspecific immune response, along with improved growth performance and enhanced resistance against A. hydrophila infection in juvenile gibel carp.

Impacts of Amla (Phyllanthus emblica) fruit extract on growth, skin mucosal and serum immunities, and disease resistance of Nile tilapia (Oreochromis niloticus) raised under biofloc system

This study aimed to investigate the effects of Amla (Phyllanthus emblica) fruit extract (AFE) on the growth, skin mucosal and serum immunity, and disease resistance of Nile tilapia (Oreochromis niloticus) against Streptococcus agalactiae. Three hundred fish (10.48 ± 0.56 g fish-1) were distributed in 15 tanks with 20 fish per tank and fed basal diet supplemented with 0, 5, 10, 20, and 40 mg kg-1 AFE for eight weeks. The completed randomized design with three replications was applied. Growth performance, peroxidase, alternative complement, phagocytosis, and respiratory burst activities were determined at weeks 4 and 8. The challenge study was conducted at the end of the experiment. Based on the results, Nile tilapia fed the AFE diets significantly enhanced skin mucus lysozyme and peroxidase activities (P ≤ 0.05) post-feeding. The highest levels were observed in the group that received 20 mg kg-1 of AFE (P ≤ 0.05), whereas the control group presented the lowest levels of lysosome and peroxidase activities. Similarly, incorporation of 20 mg kg-1 of AFE brought about the maximum levels of (P ≤ 0.05) serum lysozyme, peroxidase, alternative complement, phagocytosis, and respiratory burst activities in contrast to the other treatments; whereas fish fed the control diet generated the lowest immune parameter values (P ≤ 0.05). In the challenge study, the Nile tilapia’s relative percent survival (RPS) was 52.38%, 61.90%, 80.95%, and 47.62% in Diet 2, Diet 3, Diet 4, and Diet 5, respectively. Fish fed 20 mg kg-1 AFE showed significantly (P < 0.05) higher RPS, as well as the highest resistance towards S. agalactiae. The inclusion of AFE for 8 weeks resulted in a significant (P < 0.05) enhancement of growth parameters, with the highest growth rate observed in the 20 mg kg-1 AFE group. In conclusion, dietary supplementation with 20 mg AFE kg-1 may be used as a growth promoter and immunostimulant in Nile tilapia aquaculture.

Effects of dietary short‐chain fatty acid salts on the growth performance, digestive, antioxidant and immune enzyme activities, immune‐related gene expression and resistance to Vibro parahaemolytics infection in juvenile ridgetail white prawn ( Exopalaemon carinicauda )

An 8-week feeding trial was conducted to study the effects of dietary short-chain fatty acid salts, sodium acetate (SA), sodium propionate (SP) and sodium butyrate (SB), on the growth performance, digestive ability, immune parameters and disease resistance of juvenile Exopalaemon carinicauda. Shrimps (14.60 ± 1.48 mg) were fed with 10 experimental diets containing 0.0 (control), 0.25%, 0.5% and 1% of SA, SB and SP in triplicate. The highest survival rate was observed in the SP (0.5%) group (p < 0.05). The final weight, specific growth rate and feed efficiency was highest in the SP (0.25%) group (p < 0.05). Shrimps that were fed SP (0.25%) had greater intestinal digestive enzyme (chymotrypsin) activity. Shrimps in the SA (0.5%) group had the highest activities of glutathione peroxidase, catalase, lysozyme, acid phosphatase and alkaline phosphatase, which was accompanied by the highest relative percent survival after a Vibro parahaemolyticus challenge (p < 0.05). The expression of immune-related genes (prophenoloxidase, C-type lectin and mannose binding lectin) in the hepatopancreas of shrimps fed SA (0.5%) and SP (0.25%) was higher than that of the control group (p < 0.05). These data suggest that SA (0.5%) and SP (0.25%) are beneficial to the growth performance and health of E. carinicauda.

Dietary supplementation with miswak (Salvadora persica) improves the health status of Nile tilapia and protects against Aeromonas hydrophila infection

The potential effect of Salvadora persica dietary supplementation on Nile tilapia (Oreochromis niloticus) health was investigated. Four diets were formulated to contain 0 (control), 0.5, 1 and 2 % (w/w) S. persica powder. Fish were fed for eight weeks (pre-challenge period), then challenged with A. hydrophila for seven days (post-challenge period). Results showed that the immune and biochemical parameters besides, white blood cell counts (WBCs), increased significantly at 1 % and 2 % or both levels of S. persica in the pre- and post-challenge period. Most of the hematological indices were elevated pre- and post-challenge at 1 % and 2 % or both S. persica groups compared to other groups. Malondialdehyde (MDA) exhibited a significant decrease with 2 % S. persica diet in pre-challenge and at 1 and 2 % in the post-challenge period compared to the control. Additionally, antioxidant enzyme activities revealed a significant increase in pre- and post-challenge groups, principally in fish fed 2 % S. persica. Furthermore, the highest relative percentage survival, 90 % was recorded in 2 % S. persica. The findings suggested that dietary supplementation of miswak improved hematoimmunological parameters and enhanced antioxidant responses of cultured Nile tilapia and confer protection against A. hydrophila infection.

Octominin: An antibacterial and anti-biofilm peptide for controlling the multidrug resistance and pathogenic Streptococcus parauberis

Streptococcus parauberis is a pathogenic gram-positive bacterium that causes streptococcosis infection in fish. Since S. parauberis is becoming resistant to multiple antibiotics, the development of alternatives, such as antimicrobial peptides, has gained great attention. Octominin, derived from the defense protein of Octopus minor, showed a significant antimicrobial activity against multidrug resistance S. parauberis, with a minimum inhibitory concentration (MIC) and a minimum bactericidal concentration (MBC) of 50 and 100 μg/mL, respectively. Furthermore, time-kill kinetics, agar diffusion, and bacterial viability assays confirmed the concentration-dependent antibacterial activity of Octominin against S. parauberis. Field emission scanning electron microscopy analysis showed morphological and ultra-structural changes in S. parauberis upon Octominin treatment. Moreover, Octominin treatment demonstrated changes in membrane permeability, induced reactive oxygen species (ROS), and its binding ability to genomic DNA, suggesting its strong bactericidal activity with multiple modes of action. We confirmed the inhibition of biofilm formation and the eradication of existing biofilms in a concentration-dependent manner. Additionally, Octominin on S. parauberis at transcriptional level exhibited downregulation of membrane formation (pgsA and cds1), DNA repairing (recF), biofilm formation (pgaB and epsF) genes, while upregulation of ROS detoxification (sodA) and DNA protecting (ahpF) related genes. An in vivo study confirmed a significantly (P < 0.05) higher relative percentage survival in Octominin-treated larval zebrafish exposed to S. parauberis (93.3%) compared to the control group (20.0%). Collectively, our results confirm that Octominin could be a potential antibacterial and anti-biofilm agent against S. parauberis.

Optimizing the immunization procedure of single-walled carbon nanotubes based vaccine against grass carp reovirus for grass carp

Hemorrhagic disease caused by grass carp reovirus (GCRV) is considered to be one of the most serious threats to grass carp, resulting in significant economic loss in grass carp culture industry. This study was aimed at optimizing the immunization program of SWCNTs-vaccine (SWCNTs-M-VP4-3) controlling infectious GCRV, determining the best immunization program for grass carp, and then providing reference for the commercial promotion and use of vaccines in aquaculture. We designed an compete experiment to optimize different parameters affecting vaccination such as immunization time, antigen concentration, and fish density when grass carp was vaccinated by bath treatment. Our results showed that the highest relative percent survival (88.33%) was found in 12 h of immunization time, 10 mg L−1 of antigen concentration, and 15 fish per liter of fish density compared to 100% mortality for control groups. And other immune responses (serum antibody production, enzyme activities, and immune genes expression) also demonstrated similar results. These results indicated that the administration protocol which induce highest immune response of the host, had the highest vaccine effect against the disease. This study lays the foundation for providing reference for the commercial promotion and use of vaccines in aquaculture.

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

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

Expression of LamB Vaccine Antigen in Wolffia globosa (Duck Weed) Against Fish Vibriosis.

Vibriosis is a commonly found bacterial disease identified among fish and shellfish cultured in saline waters. A multitude of Vibrio species have been identified as the causative agents. LamB, a member of outer membrane protein (OMPs) family of these bacteria is conserved among all Vibrio species and has been identified as an efficient vaccine candidate against vibriosis. Rootless duckweed (Wolffia) is a tiny, edible aquatic plant possessing characteristics suitable for the utilization as a bioreactor. Thus, we attempted to express a protective edible vaccine antigen against fish vibriosis in nuclear-transformed Wolffia. We amplified LamB gene from virulent Vibrio alginolyticus and it was modified to maximize the protein expression level and translocate the protein to the endoplasmic reticulum (ER) in plants. It was cloned into binary vector pMYC under the control of CaMV 35S promoter and introduced into Wolffia globosa by Agrobacterium-mediated transformation. Integration and expression of the LamB gene was confirmed by genomic PCR and RT-PCR. Western blot analysis revealed accumulation of the LamB protein in 8 transgenic lines. The cross-protective property of transgenic Wolffia was evaluated by orally vaccinating zebrafish through feeding fresh transgenic Wolffia and subsequently challenging with virulent V. alginolyticus. High relative percent survival (RPS) of the vaccinated fish (63.3%) confirmed that fish immunized with transgenic Wolffia were well-protected from Vibrio infection. These findings suggest that Wolffia expressed LamB could serve as an edible plant-based candidate vaccine model for fish vibriosis and feasibility of utilizing Wolffia as bioreactor to produce edible vaccines.

Optimization of the efficacy of a SWCNTs-based subunit vaccine against infectious spleen and kidney necrosis virus in mandarin fish

Infectious spleen and kidney necrosis virus (ISKNV) cause a high mortality disease which brings substantial economic losses to the mandarin fish culture industry in China. This study was aimed at optimizing the efficacy of a SWCNTs-based immersion subunit vaccine (SWCNTs-M-MCP) which as a promising vaccine against ISKNV. Mandarin fish were vaccinated by immersion, then we designed an orthogonal experiment to optimize different parameters affecting vaccination such as immune duration of bath immunization, immune dose, and fish density when immunized. Our results showed that the highest relative percent survival (86.7%) was found in the group 6 with 8 h of immune duration, 20 mg/L of immune dose, and 8 fish per liter of fish density. And other immune responses (serum antibody production, enzyme activities, and immune-related genes expression) also demonstrated similar results. In addition, the expression of IRF-I in group 6 (8 h, 20 mg/L, 8 fish per liter) was significant extents, and about 16-folds increases were obtained than the control group at 21 d post-vaccination. And the highest specific antibody response was significantly increased (more than 4-folds) than control group which was found in group 6. The optimum immune duration, immune dose, and fish density of SWCNTs-M-MCP were 8 h, 20 mg/L, 8 fish per liter, respectively. Importantly, our results also showed that immune duration had the greatest effect on the immune response of our vaccine, followed by immune dose. The study reported herein provides a helpful reference for the effective use of vaccine in fish farming industry.

The growth performance, antioxidant capacity, immunological responses, and the resistance against Aeromonas hydrophila in Nile tilapia (Oreochromis niloticus) fed Pistacia vera hulls derived polysaccharide

The present study aimed at evaluating the supplementation of dietary Pistacia vera hulls derived polysaccharide (PHDP) at varying levels (0, 2.5, 5, and 10 g/kg diet) on the growth rate, digestive enzyme activity, immune response, and antioxidative capacity of Nile tilapia. After the feeding trial (60 days), fish were injected intraperitoneally with Aeromonas hydrophila, and the cumulative mortality was calculated for 10 days. The final body weight, weight gain, specific growth rate, survival rate, amylase activity, red blood cells, hemoglobin, serum total protein, and serum lysozyme activity (LZM) were significantly higher in fish fed PHDP at 5 and 10 g/kg than fish fed 0 and 2.5 g/kg diet (P ≤ 0.05). However, the feed conversion ratio and glucose levels were significantly decreased by 5 and 10 g PHDP/kg diet. Further, the protease digestive activity, serum alkaline phosphatase, and serum alternative complement (ACH50) had higher values in fish fed 5 g PHDP/kg diet than tilapia fed 0 g PHDP/kg diet (P ≤ 0.05). The serum protease and immunoglobulin (Ig) presented the highest values in fish fed 10 g PHDP/kg diet followed by fish fed 5 g PHDP/kg diet (P ≤ 0.05). The LZM, ACH50, and Ig in the mucus samples were significantly higher in fish fed 5 or 10 g PHDP/kg diet than fish fed 0 g PHDP/kg diet (P ≤ 0.05). The levels of superoxide dismutase and catalase in the liver tissue was significantly higher in fish fed 5 or 10 g PHDP/kg diet than fish fed 2.5 g PHDP/kg diet (P ≤ 0.05). On the other hand, malondialdehyde concentration was significantly lower in tilapia fed 5 and 10 g PHDP/kg diet than tilapia fed 0 and 2.5 g PHDP/kg diet (P ≤ 0.05). The lowest mortality rate and the highest relative percentage survival were in fish fed 5 g/kg followed by 10 g level after 10 days of A. hydrophila injection. Accordingly, dietary PHDP at 5–10 g/kg is recommended to improve the growth performance, antioxidative capacity, immune response, and resistance against A. hydrophila in Nile tilapia.

Development of IglC and GroEL recombinant vaccines for francisellosis in Nile tilapia, Oreochromis niloticus

Warm-water piscine francisellosis is a granulomatous bacterial disease caused by Francisella orientalis (Fo). The disease has been detected in a wide range of fish species globally, causing mortalities as high as 90% and significant economic losses. Currently there are no commercially available vaccines and few treatment options exist. In the current study, two novel recombinant vaccines were prepared using diatom-expressed IglC or bacterial-expressed GroEL proteins. The vaccine antigens were emulsified with either nanoparticles or a commercially available oil-based adjuvant. Nile tilapia, Oreochromis niloticus, fingerlings were immunized intracoelomically with the recombinant IglC or GroEL vaccines, diatoms alone or phosphate buffer saline. Approximately 840-degree days post-vaccination, fish were challenged via immersion with 106 CFU/mL of wild-type Fo. Twenty-one days post challenge (dpc), the highest relative percent survival was recorded in the IglC-Montanide group (75%), compared to 53%, 50%, 22%, 19% and 16% in the IglC-nanoparticles, GroEL-Montanide, GroEL-nanoparticles, diatoms-Montanide and diatoms-nanoparticles groups, respectively. Protection correlated with significantly higher specific antibody responses in the IglC-Montanide group. Moreover, a significantly lower bacterial load was detected in spleen samples from the IglC-Montanide survivor tilapia compared to the other experimental groups. This is the first report of recombinant vaccines against piscine francisellosis in tilapia. The Fo vaccines described in our study may facilitate development of a safe, cost-effective and highly protective vaccine against francisellosis in farmed tilapia.