Probiotics from kefir: Evaluating their immunostimulant and antioxidant potential in the carpet shell clam (Ruditapesdecussatus).

This study aimed to investigate the impact of incorporating kefir into the diet on biometric parameters, as well as the immune and antioxidant responses of the carpet shell clam (Ruditapes decussatus) after an experimental infection by Vibrio alginolyticus. Clams were divided into a control group and a treated group. The control group was fed on spirulina (Arthrospira platensis) alone. While, the treated group was fed on spirulina supplemented with 10% dried kefir. After 21 days, clams were immersed in a suspension of V. alginolyticus 5 × 105 CFU mL -1 for 30 min. Seven days after experimental infection, survival was 100% in both groups. The obtained results showed a slight increase in weight and condition index in clams fed with kefir-supplemented diet for 21 days compared to control clams. Regarding antioxidant responses, the treated group showed higher superoxide dismutase activity compared to the control group. However, the malondialdehyde level was lower in the treated clams than in the control. In terms of immune parameters, the treated group showed slightly elevated activities of phenoloxidase, lysozyme and alkaline phosphatase, whereas a decreased lectin activity was observed compared to the control group. The obtained results suggest that kefir enhanced both the antioxidant and immune response of infected clams.

Transcriptomic analysis of Vibrio alginolyticus challenged by Rhizoma coptidis reveals mechanisms of virulence genes.

Rhizoma coptidis, a Chinese herbal medicine widely used to treat various bacterial infections, has the potential to develop antibiotic substitutes to overcome the drug resistance of Vibrio alginolyticus. To study the inhibitory effect of R. coptidis on V. alginolyticus, we sequenced the transcriptomes of three groups of samples of wild-type V. alginolyticus (CK) and V. alginolyticus, which were stressed by 5 mg/mL R. coptidis for 2 h (RC_2 h) and 4 h (RC_4 h). CK was compared with RC_2 h and RC_4 h, respectively, and a total of 1565 differentially expressed genes (DEGs) (988 up-regulated and 577 down-regulated) and 1737 DEGs (1152 up-regulated and 585 down-regulated) were identified. Comparing RC_2 h with RC_4 h, 156 DEGs (114 up-regulated and 42 down-regulated) were identified. The ability of biofilm formation and motility of V. alginolyticus altered upon with different concentrations of R. coptidis. Interestingly, relative expression patterns of virulence genes appeared statistically significantly varied, upon different concentrations of R. coptidis extract. DEGs were annotated to the Gene Ontology (GO) database for function enrichment analysis and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, the results showed that the main enriched pathways, was those related to the virulence of V. alginolyticus. This study provides a new perspective for understanding the complex pathogenic mechanism of V. alginolyticus. R. coptidis could potnetially be used as alternative or complimnetary to antibiotics to treat infections after further research.

Application of nZnO supported with nanoclay for improving shrimp immunity.

This study discloses the nanoscale silicate platelet-supported nZnO (ZnONSP) applied as novel feed additives in aquaculture. The preparation of the nanohybrid (ZnO/NSP = 15/85, w/w) was characterized by UV-visible spectroscopy, powder X-ray diffraction and transmission electron microscope. The effects of ZnONSP on growth, zinc accumulation, stress response, immunity and resistance to Vibrio alginolyticus in white shrimp (Penaeus vannamei) were \demonstrated. To evaluate the safety of ZnONSP, shrimps (2.0 ± 0.3 g) were fed with ZnONSP containing diets (200, 400 and 800 mg/kg) for 56 days. Dietary ZnONSP did not affect the weight gain, specific growth rate, feed conversion ratio, survival rate, zinc accumulation, and the expression of heat shock protein 70 in tested shrimps. To examine the immunomodulatory effect of ZnONSP, shrimps (16.6 ± 2.4 g) were fed with the same experimental diets for 28 days. Dietary ZnONSP improved the immune responses of haemocyte in tested shrimps, including phagocytic rate, phagocytic index, respiratory burst, and phenoloxidase activity, and upregulated the expression of several genes, including lipopolysaccharide, ß-1,3-glucan binding protein, peroxinectin, penaeidin 2/3/4, lysozyme, crustin, anti-lipopolysaccharide factor, superoxide dismutase, glutathione peroxidase, clotting protein and α-2-macroglobulin. In the challenge experiment, shrimps (17.2 ± 1.8 g) were fed with ZnONSP containing diets (400 and 800 mg/kg) for 7 days and then infected with Vibrio alginolyticus. Notably, white shrimps that received ZnONSP (800 mg/kg) showed significantly improved Vibrio resistance, with a survival rate of 71.4 % at the end of 7-day observation. In conclusion, this study discovers that ZnONSP is a new type of immunomodulatory supplement that are effective on enhancing innate cellular and humoral immunities, and disease resistance in white shrimp.

Vibrio alginolyticus growth kinetics and the metabolic effects of iron.

IMPORTANCE: Transmission of V. alginolyticus occurs opportunistically through direct seawater exposure and is a function of its abundance in the environment. Like other Vibrio spp., V. alginolyticus are considered conditionally rare taxa in marine waters, with populations capable of forming large, short-lived blooms under specific environmental conditions, which remain poorly defined. Prior research has established the importance of temperature and salinity as the major determinants of Vibrio geographical and temporal range. However, bloom formation can be strongly influenced by other factors that may be more episodic and localized, such as changes in iron availability. Here we confirm the broad temperature and salinity tolerance of V. alginolyticus and demonstrate the importance of iron supplementation as a key factor for growth in the absence of thermal or osmotic stress. The results of this research highlight the importance of episodic iron input as a crucial metric to consider for the assessment of V. alginolyticus risk.

Effect of Clostridium butyricum on intestinal microbiota and resistance to Vibrio alginolyticus of Penaeus vannamei.

In order to evaluate the effect of Clostridium butyricum (C. butyricum) feeding on intestinal microorganisms and protection against infection by Vibrio alginolyticus (V. alginolyticus) in Penaeus vannamei (P. vannamei). We set up two groups, CG30 (fed normal feed) and CB30 (fed feed supplemented with C. butyricum), for the 30d C. butyricum feeding test, and four groups, CG (CG30 group injected with PBS), CB (CB30 group injected with PBS), VACG (CG30 group injected with V. alginolyticus), and VACB (CB30 group injected with V. alginolyticus), for the 24 h infection test. The protective effect of C. butyricum against acute V. alginolyticus infection in P. vannamei was explained in terms of survival, histopathology, changes in enzyme activity, transcriptome analysis, and immune-related genes. We found that feeding C. butyricum significantly altered intestinal microbial populations' abundance and significantly reduced Vibrio spp. In the V. alginolyticus stress test, C. butyricum improved the survival rate and alleviated pathological changes in hepatopancreatic tissues, alleviated the reduction of superoxide dismutase (SOD) and phenoloxidase (PO) activity caused by infection, and increased the lysozyme content in P. vannamei. VACB group compared with the VACG group, 1730 up-regulated differentially expressed genes (DEGs) and 2029 down-regulated DEGs were screened. Quantitative real-time PCR (qRT-PCR) showed that dietary supplementation with C. butyricum suppressed the upregulation of alkaline phosphatase (AKP) transcription factors and the downregulation of prophenoloxidase (proPO), alpha-2-macroglobulin (A2M), and anti-lipopolysaccharide factor (ALF) induced by V. alginolyticus infection. In conclusion, feed supplementation with C. butyricum changed P. vannamei's population ratio of intestinal microorganisms. Moreover, C. butyricum has the potential to act as an inhibitor of V. alginolyticus infection and enhance the resistance of P. vannamei to V. alginolyticus infection.

Effect of nanoclay supported nanosilver on the growth inhibition of aquatic pathogens and immunomodulatory effect in Penaeusvannamei.

Hybrid of nanosilver and nanoscale silicate platelet (AgNSP) is a safe, non-toxic nanomaterial which has been applied in medical use due to its strong antibacterial activity. The application of AgNSP in aquaculture was first proposed in the present study by evaluating the in vitro antibacterial activities against four aquatic pathogens, in vitro effects toward shrimp haemocytes as well as the immune responses and disease resistance in Penaeus vannamei fed with AgNSP for 7 days. For evaluating the antibacterial activities of AgNSP in culture medium, the minimum bactericidal concentration (MBC) values against Aeromonas hydrophila, Edwardsiella tarda, Vibrio alginolyticus and Vibrio parahaemolyticus were 100, 15, 625 and 625 mg/L, respectively. Moreover, the inhibition of pathogen growth over a period of 48 h could be achieved by the appropriate treatment of AgNSP in culturing water. In freshwater containing bacterial size of 103 and 106 CFU/mL, the effective doses of AgNSP against A. hydrophila were 12.5 and 450 mg/L, respectively while the effective doses against E. tarda were 0.2 and 50 mg/L, respectively. In seawater with same bacterial size, the effective doses against V. alginolyticus were 150 and 2000 mg/L, respectively while the effective doses against V. parahaemolyticus were 40 and 1500 mg/L, respectively. For the in vitro immune tests, the superoxide anion production and phenoloxidase activity in haemocytes were elevated after in vitro incubation with 0.5-10 mg/L of AgNSP. In the assessment of dietary supplemental effects of AgNSP (2 g/kg), no negative effect on the survival was found at the end of 7 day feeding trail. In addition, the gene expression of superoxide dismutase, lysozyme and glutathione peroxidase were up-regulated in haemocytes taken from shrimps received AgNSP. The following challenge test against Vibrio alginolyticus showed that the survival of shrimp fed with AgNSP was higher than that of shrimp fed with control diet (p = 0.083). Dietary AgNSP improved the Vibrio resistance of shrimp by increasing 22.7% of survival rate. Therefore, AgNSP could potentially be used as a feed additive in shrimp culture.

Andrographis paniculata improves growth and non-specific immunity of shrimp Litopenaeus vannamei, and protects it from Vibrio alginolyticus by reducing oxidative stress and apoptosis.

Andrographis paniculata (AP) is a traditional medicinal plant with many pharmacological activities, including anti-inflammatory, antimicrobial, immunity stimulation and so on. Several studies have reported that AP plays a strong role in promoting the immune system of aquatic animals to resist several pathogens. In the present study, we investigate the effects of a diet containing AP on the immune responses, growth, and the resistance to Vibrio alginolyticus (V. alginolyticus) in Litopenaeus vannamei (L. vannamei). Four diets were formulated by adding AP at the dosage of 0% (Control), 0.25%, 0.5%, and 1% in the basal diet, respectively. Each diet was randomly fed to one group with three replicates of shrimps in a 28-day feeding trial. The results showed that dietary AP improved the growth performance and non-specific immune function of shrimps. To investigate the effect of AP on disease resistance of L. vannamei, shrimps fed with diet containing AP were challenged with V. alginolyticus. Compared with the control group, the shrimps fed diet containing AP showed significantly higher survival. Furthermore, the hepatopancreas injury in the shrimp fed with AP was less than control group at 6 h after V. alginolyticus infection. However, no difference was observed in the degree of hepatopancreas injury between AP groups and control group at 12 h and 24 h after V. alginolyticus infection. Based on this result, the samples at 6 h after V. alginolyticus infection was selected for subsequent detection. Reactive oxygen species (ROS) accumulation in hemocytes and O2- production in hepatopancreas caused by V. alginolyticus infection was significantly reduced after feeding a diet containing 0.25% and 0.5% AP (p < 0.05). In addition, we found that feeding AP significantly up-regulated the expression of pro-apoptotic genes (Bax, Caspase 3, p53) and down-regulated the expression of anti-apoptotic genes (Bcl-2) in hepatopancreas after V. alginolyticus infection. In conclusion, AP promote the growth and immunity of L. vannamei, and protects shrimps against V. alginolyticus by regulating the oxidative damage and apoptosis. These results provide useful information regarding the effects of AP extracts as a shrimp feed additive for sustainable shrimp culture.

Sarcodia suae modulates the immunity and disease resistance of white shrimp Litopenaeus vannamei against Vibrio alginolyticus via the purine metabolism and phenylalanine metabolism.

Red seaweeds have several biofunctional properties, including immunomodulatory, antitumor, antioxidant, and antibacterial activities. In this study, we examined the effects of diets containing Sarcodia suae on the immune response, immune-related gene expressions, and disease resistance against Vibrio alginolyticus in white shrimp Litopenaeus vannamei. In addition, 1H NMR metabolomics was applied to analyze the metabolites extracted from shrimp fed with S. suae and their functions in regulating immunity. A diet containing only fish meal was used as the control diet (S0), and three diets containing different concentrations of S. suae powder, 2.5% (S2.5), 5% (S5), and 7.5% (S7.5) were used as experimental diets. Shrimp were fed diets for 20 days. Compared to the control group (S0), results showed that (1) shrimp fed diets supplemented with 5-7.5% of S. suae powder significantly increased anti-V. alginolyticus activity; (2) phagocytic activity (PA) increased in all shrimp fed with S. suae, but total haemocyte count (THC) only increased in S7.5 group; and (3) the expression of glutathione peroxidase (GPx) in haemocyte were significantly higher in S7.5 groups. Results from the 1H NMR analysis revealed that 19 heapatopancreatic metabolites were matched and identified among groups. Based on the KEGG enrichment analysis, the up-regulated metabolites in the shrimp fed S5 and S7.5 diets were primarily due to the metabolism of purine and phenylalanine and their respective pathways. Results from these trials reveal that diets containing S. suae can increase immune response, thereby increasing shrimp resistance to V. alginolyticus. The purine and phenylalanine metabolic pathways may be considered as the relevant pathways for optimizing immunomodulatory responses.

Polyunsaturated fatty acids cause physiological and behavioral changes in Vibrio alginolyticus and Vibrio fischeri.

Vibrio alginolyticus and Vibrio (Aliivibrio) fischeri are Gram-negative bacteria found globally in marine environments. During the past decade, studies have shown that certain Gram-negative bacteria, including Vibrio species (cholerae, parahaemolyticus, and vulnificus) are capable of using exogenous polyunsaturated fatty acids (PUFAs) to modify the phospholipids of their membrane. Moreover, exposure to exogenous PUFAs has been shown to affect certain phenotypes that are important factors of virulence. The purpose of this study was to investigate whether V. alginolyticus and V. fischeri are capable of responding to exogenous PUFAs by remodeling their membrane phospholipids and/or altering behaviors associated with virulence. Thin-layer chromatography (TLC) analyses and ultra-performance liquid chromatography-electrospray ionization mass spectrometry (UPLC/ESI-MS) confirmed incorporation of all PUFAs into membrane phosphatidylglycerol and phosphatidylethanolamine. Several growth phenotypes were identified when individual fatty acids were supplied in minimal media and as sole carbon sources. Interestingly, several PUFAs acids inhibited growth of V. fischeri. Significant alterations to membrane permeability were observed depending on fatty acid supplemented. Strikingly, arachidonic acid (20:4) reduced membrane permeability by approximately 35% in both V. alginolyticus and V. fischeri. Biofilm assays indicated that fatty acid influence was dependent on media composition and temperature. All fatty acids caused decreased swimming motility in V. alginolyticus, while only linoleic acid (18:2) significantly increased swimming motility in V. fischeri. In summary, exogenous fatty acids cause a variety of changes in V. alginolyticus and V. fischeri, thus adding these bacteria to a growing list of Gram-negatives that exhibit versatility in fatty acid utilization and highlighting the potential for environmental PUFAs to influence phenotypes associated with planktonic, beneficial, and pathogenic associations.

Synbiotic combination of prebiotic, cacao pod husk pectin and probiotic, Lactobacillus plantarum, improve the immunocompetence and growth of Litopenaeus vannamei.

To reach the sustainable development goals on waste recycling, cacao pod husk (CPH), produced as an agricultural waste byproduct during the cacao bean processing was applied to manufacture CPH pectin for developing the potential for diverse application in aquaculture, minimizing CPH impact to the environment and bringing benefits to the agriculture and aquaculture industries. In this study, CPH pectin (5 g/kg diet) and Lactobacillus plantarum (LP; 1010 cfu/kg diet) were separately introduced to the diets of Litopenaeus vannamei for a 56-day feeding trial, and two synbiotic combinations of CPH pectin and LP (CPH pectin at 5 g/kg diet + LP at 107 cfu/kg diet or at 1010 cfu/kg diet) were also conducted. After the 56-day feeding trial, significantly elevated percent weight gain, percent length gains and feeding efficiency in L. vannamei were only observed in synbiotic combination of CPH pectin at 5 g/kg diet and LP at 107 cfu/kg diet treatment, and the remainder of the treatments remained consistently similar to the control. Significantly increases in total haemocyte count, granular cells, phenoloxidase activity, and respiratory bursts were observed in L. vannamei fed with synbiotics at 7-28 days of feeding, accompanied by significant promotion of phagocytic activity and clearance efficiency in response to V. alginolyticus challenge during 56 days of feeding trial. Furthermore, at the end of the 56 days of feeding trial, shrimp receiving CPH pectin and/or LP treatments showed a significantly higher survival ratio against V. alginolyticus infection and hypothermal stress. It was therefore concluded that CPH pectin or LP was confirmed as an immunostimulant for L. vannamei to trigger immunocompetence through oral administration without negative effects within 56 days of feeding trial, and the synbiotic combination of CPH pectin and LP exhibited complementary and synergistic effects on growth performance and immunocompetence in L. vannamei.

Dietary astaxanthin augments disease resistance of Asian seabass, Lates calcarifer (Bloch, 1790), against Vibrio alginolyticus infection.

This investigation describes the impacts of dietary provisioning with astaxanthin on hemato-biochemistry, non-specific immunity, and disease resistance of the Asian seabass, Lates calcarifer, against the virulent Vibrio alginolyticus; with specific reference to dose-response associations and variations over different post-infection periods (0-, 7-, and 14-day). Triplicate groups of fish weighing 28 g, on average, were fed various diets (C, the control or astaxanthin-free; AXT50, 50 mg astaxanthin kg-1 diet; AXT100, 100 mg astaxanthin kg-1 diet; and AXT150, 150 mg astaxanthin kg-1 diet) for 90 days and subsequently challenged with V. alginolyticus at the end of the feeding period. Experimental infection unveiled that supplemented fish demonstrated significant improvements (P < 0.05) of hematological parameters (white blood cell [WBC] and red blood cell [RBC] counts, and hemoglobin and hematocrit levels) when fed diets with elevating supplemental doses of astaxanthin through distinct post-infection periods (0-, 7-, and 14-day). Furthermore, the administration of dietary astaxanthin at escalating levels markedly enhanced (P < 0.05) the serum biochemical profile (aspartate aminotransferase [AST], alanine aminotransferase [ALT], glucose, cortisol, cholesterol, and triglyceride contents) of challenged fish, resulting in better welfare. Significantly higher (P < 0.05) contents of serum total protein were observed in supplemented fish, as opposed to the control. Additionally, immunological defense mechanisms (lysozyme activity, phagocytic activity, respiratory burst activity, and total serum immunoglobulin) of challenged fish were pronouncedly elicited (P < 0.05) following the ingestion of astaxanthin. Besides, the supplementation with dietary astaxanthin significantly augmented (P < 0.05) the post-challenge survival rate of fish. Collectively, the results manifest that supplementary feeding of astaxanthin is effective in reinforcing fish immunocompetence and disease resistance against V. alginolyticus infection.

Early immune response in large yellow croaker (Larimichthys crocea) after immunization with oral vaccine.

Mesoporous silica nanoparticles (MSNs) have been used in the field of biomedicine as antigen carriers and adjuvants for protective antigens. In the present study, an oral nanovaccine against Vibrio alginolyticus was prepared employing MSNs as carriers. The uptake of the dihydrolipoamide dehydrogenase (DLDH) antigens in the intestine of large yellow croaker was evaluated using an immunohistochemistry assay. Additionally, the effects of the nanovaccine on the early immune response in large yellow croaker were investigated via oral vaccination. The presence of the antigens was detected in the mucosa and lamina propria of the foregut, midgut, and hindgut of large yellow croaker at 3 h following oral immunization. The expression levels of cytokines (i.e., lysozyme, IFN-γ, IFITM, TNF-α, IL-1ß, IL-2, IL-4, IL-10, and IL-13) in the intestine, spleen, and head kidney tissues of large yellow croaker before and after the immune challenge were determined via RT-qPCR assay. The obtained results revealed that the expression levels of lysozyme, IFN-γ, IFITM, TNF-α, IL-1ß, IL-2, IL-4, IL-10, and IL-13 in the intestine and head kidney of the vaccinated large yellow croaker, as well as the expression of lysozyme, IL-1ß, and IL-10 in the spleen, exhibited time-dependent oscillation regulation patterns. Notably, the nanovaccine immunization could induce early (6 h) and high expression of IFN-γ in the spleen and kidney tissues after the bacterial infection. The current study supplements the available data on the early immune response to fish nanovaccines. It also provides a valuable theoretical basis for the future development of large yellow croaker oral vaccines.

Black soldier fly (Hermetia illucens) larvae meal in diets of European seabass: Effects on antioxidative capacity, non-specific immunity, transcriptomic responses, and resistance to the challenge with Vibrio alginolyticus.

Black soldier fly larvae meal (BSFLM) has been successfully demonstrated as a promising fish meal (FM) replacer in diets of several fish species including European seabass (Dicentrarchus labrax). However, its impacts on antioxidant capacity, and immune responses of treated fish are still poorly understood. A 60-day feeding trial was carried out to evaluate the effects of partial substitution of FM with different levels of dry BSFLM on the antioxidative status, non-specific immunity, transcriptomic responses, and resistance of European seabass to the challenge with Vibrio alginolyticus. Four isoproteic (45%) and isolipidic diets were formulated by replacing 0.0%, 25%, 35%, and 50% of the dietary FM. Each diet was randomly assigned to four fish groups (in triplicates) (initial mean body weight, 12.1 ± 0.21 g) (20 fish per aquarium) (n = 240). Fish were fed three times daily to the apparent satiation. At the end of the feeding trial, serum antioxidant biomarkers such as malondialdehyde levels, and catalase, superoxide dismutase, and glutathione peroxidase enzyme activities were significantly increased in all BSFLM groups in a dose-dependent manner compared to the control group (P < 0.05). The non-specific immune indices, including phagocytic activity, phagocytic index, serum lysozyme and respiratory burst activities were significantly elevated in BSFLM groups compared to those in the control group (P < 0.05). Significant upregulation of the mRNA expression levels of hepatic heat shock protein 70, interleukin-1beta and interleukin-10 genes were observed in all BSFLM groups compared to the control group (P < 0.05). Additionally, after the challenge with V. alginolyticus, the relative percent of survival was significantly elevated in fish groups fed on diets containing graded levels of BSFLM over the control group (P < 0.05). Conclusively, the present study suggests the potential efficacy of partial replacement of dietary FM protein for up to 50% by BSFLM without negative effects on fish health with possible potentiation of the antioxidative status, and the immune responses of the European seabass.

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

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

Pectin of cacao pod husk, an efficient immunostimulant for white shrimp, Litopenaeus vannamei.

The disposal of cacao pod husk, a byproduct of cacao bean processing, can cause serious adverse environmental impacts, motivating scientist to explore and develop potential beneficial applications of this resource. Dried cacao pod husk was extracted with ethanol to obtain a 10.6% pectin of cacao pod husks (pCPH), and its effects on the immunocompetence of Litopenaeus vannamei were estimated. Measured variables included total haemocyte count, differential haemocyte count, phenoloxidase activity, respiratory bursts, as well as phagocytic activity and clearance efficiency against Vibrio alginolyticus after receiving pCPH at 0, 1.5, 3, and 6 µg shrimp-1 for 0, 1, 3 and 7 days via injection, and their resistance to thermal stress and V. alginolyticus infection were further evaluated. No significant differences were observed in total haemocyte count, differential haemocyte count, and respiratory bursts in shrimp receiving pCPH at 1.5 µg shrimp-1 for 1 day; however, these variables were significantly elevated after 3 days of injection, compared to the control group. The significantly increased phenoloxidase activity was assessed in shrimp receiving pCPH at 1.5, 3 and 6 µg shrimp-1 within 3 days, and activity returned to the baseline after 7 days. Furthermore, the reduced phenoloxidase activity per granulocytes or respiratory bursts per haemocytes maintained homeostasis following the variation of haemogram. For gene expression assessments in haemocytes, the immune-related genes of the lipopolysaccharide and ß-1,3-glucan binding protein, prophenoloxidase II and anti-lipopolysaccharide factor as well as innate immune signaling pathway-related genes of toll-like receptors 1 and 3 significantly increased after shrimp received pCPH for 1 day. The increases in phagocytic activity and clearance efficiency were only detected in shrimp receiving pCPH at 6 µg shrimp-1 within 7 days, compared to the control. There was no significant difference in the mortality ratio of shrimp against hyperthermal stress when they received pCPH for 1 day, and the significant higher resistance to hypothermal stress and V. alginolyticus infection were found in shrimp received pCPH at 6 µg shrimp-1 for 1 days than those in the other treatments. It is therefore found that pCPH triggers immune responses serving as an immunostimulant capable of enhancing resistance against V. alginolyticus and hypothermal stress.

Effects of Phyllanthus amarus extract on nonspecific immune responses, growth, and resistance to Vibrio alginolyticus in white shrimp Litopenaeus vannamei.

This study investigates the effects of Phyllanthus amarus extract (PAE) on immune responses, growth, and resistance to Vibrio alginolyticus in white shrimp (Litopenaeus vannamei). In vitro PAE treatment did not alter the cell viability of haemocytes and significantly enhanced immune parameters such as phenoloxidase (PO) activity, phagocytic activity, and superoxide anion (O2-) production. We conducted two feeding trials to examine the effects of PAE on the growth, disease resistance, and innate immune parameters of white shrimp. In the first in vivo trial, shrimps (4.01 ± 0.03 g) were fed a diet containing 0 g (control), 10 g (PAE10), 20 g (PAE20), or 40 g (PAE40) of PAE per kilogram of feed for 56 days. After the feeding period, the PAE20 group showed a significantly higher weight gain and specific growth rate than shrimp fed the control diet. Furthermore, after challenge with V. alginolyticus, shrimp fed a diet containing PAE showed significantly higher survival than those fed the control diet. The second in vivo trial (28 days) was performed to identify the mechanisms of enhanced immunity in PAE-fed shrimp. Shrimp fed the PAE20 diet generally had the highest total haemocyte count, PO activity, phagocytic activity, and O2- production, followed by the PAE40 and PAE10 groups. Thus, our results suggest that administration of 20 g of PAE per kilogram of feed can enhance immunity, growth, and resistance to V. alginolyticus in white shrimp.

ß-glucan alleviates the immunosuppressive effects of oxytetracycline on the non-specific immune responses and resistance against Vibrio alginolyticus infection in Epinephelus fuscoguttatus × Epinephelus lanceolatus hybrids.

This study was conducted to examine the combinatory effects of ß-glucan and oxytetracycline (OTC) on hybrid giant tiger groupers (Epinephelus fuscoguttatus × Epinephelus lanceolatus). In vitro tests, OTC significantly reduced superoxide anion production and phagocytic activity in primary head kidney leukocytes. However, this suppressive effect was alleviated by co-treatment with ß-glucan. Subsequently, feeding trials were performed to investigate the potential immunomodulatory effects of dietary ß-glucan alone or in combination with OTC on groupers. A total of 210 healthy groupers (368.00 ± 51.03 g) were divided into six groups. Group 1 was the control group, group 2 (BG) received 5 g ß-glucan per kg feed weight, groups 3-5 received 5 g/kg ß-glucan in combination with 10, 30, or 50 mg OTC/kg fish weight/day (groups M1, M2, and M3, respectively), and group 6 (O) received 50 mg OTC/kg fish weight/day. Fish were sampled to determine the innate immunity parameters and residual OTC levels in the muscle tissue during a 28-day feeding regimen. Residual OTC levels were considerably higher in groups M3 and O compared with the other groups, and peaked on day 14. This was followed by a slight decrease on day 28, despite a continuous supply of OTC. Notably, fish fed with OTC alone had significantly decreased phagocytic rates and superoxide anion production observed in head kidney leukocytes, as well as poorer protection against Vibrio alginolyticus infection. These immunosuppressive effects were not observed in the fish fed with ß-glucan in combination with a lower dose of OTC (group M2). Thus, these data suggest that the combination of dietary ß-glucan and OTC exerts synergistic immunostimulating effects that protect groupers from bacterial infection.

Bio-inspired antibacterial cellulose paper-poly(amidoxime) composite hydrogel for highly efficient uranium(vi) capture from seawater.

A bio-inspired cellulose paper-poly(amidoxime) composite hydrogel is explored via UV-polymerization. This hydrogel has a highly efficient uranium capture capacity of up to 6.21 mg g-1 for WU/Wdry gel and 12.9 mg g-1 for WU/Wpoly(amidoxime) in seawater for 6 weeks, due to its enhanced hydrophilicity, good hydraulic/ionic conductivity and broad-spectrum antibacterial performance.

Antibacterial activity in secondary metabolite extracts of heterotrophic bacteria against Vibrio alginolyticus, Aeromonas hydrophila, and Pseudomonas aeruginosa.

Background: Disease causing bacteria such as Vibrio alginolyticus, Aeromonas hydrophila, and Pseudomonas aeruginosa present a problem for fish farming. Treatment to remove them are generally carried out using antibiotics which have side effects on fish, the environment and humans. However, the use of antibacterial compounds derived from heterotrophic bacteria serve as a good alternative for antibiotics. Therefore, this study aimed to explore antibacterial activity in the secondary metabolite extracts of heterotrophic bacteria against Vibrio alginolyticus, Aeromonas hydrophila, and Pseudomonas aeruginosa. Methods: Heterotrophic bacteria namely Bacillus sp. JS04 MT102913.1, Bacillus toyonensis JS08 MT102920.1, Bacillus cereus JS10 MT102922.1, Bacillus sp. JS11 MT102923.1, Pseudoalteromonas sp. JS19 MT102924.1, Bacillus cereus JS22 MT102926.1, and Bacillus sp. strain JS25 MT102927.1 were used in this study. The sequences of these bacteria have been deposited and are available from NCBI GenBank. Each heterotrophic bacterium was cultured on 6L nutrient broth for 8 days, and extracts produced using ethyl acetate to obtain their secondary metabolites. These extracts were tested for their phytochemical contents using FT-IR and also tested for their inhibitory property in pathogenic bacteria by agar diffusion method. Results: Phytochemical test results showed that the seven heterotrophic bacterial isolates produced terpenoid compounds. Based on the inhibitory test, the secondary metabolite extracts from Bacillus sp strain JS04 had the highest inhibitory effect on the growth of pathogenic bacteria namely, V. alginolyticus (17.5 mm), A. hydrophila (16.8 mm), and P. aeruginosa (17.3 mm). Conclusion: It was concluded that the secondary metabolite extracts of heterotrophic bacteria inhibit the growth of V. alginolyticus, A. hydrophila, and P. aeruginosa.

Highly oxygenated polyketides produced by Trichoderma koningiopsis QA-3, an endophytic fungus obtained from the fresh roots of the medicinal plant Artemisia argyi.

Eight new highly oxygenated fungal polyketides, namely, 15-hydroxy-1,4,5,6-tetra-epi-koninginin G (1), 14-hydroxykoninginin E (2), koninginin U (3), 4'-hydroxykoninginin U (4), koninginin V (5), 14-ketokoninginin B (6), 14-hydroxykoninginin B (7), and 7-O-methylkoninginin B (8), together with six known related analogues (9-14), were isolated from Trichoderma koningiopsis QA-3, a fungus obtained from the inner root tissue of the well known medicinal plant Artemisia argyi. All these compounds are bicyclic polyketides, with compound 1 contains unusual hemiketal moiety at C-5 and compounds 2-14 having ketone group at C-1 and double bond at C-5(6). The structures and absolute configurations of the new compounds were established by spectroscopic analysis, X-ray crystal diffraction, modified Mosher's method, and ECD calculation. The absolute configurations of the known compounds 9, 10, and 12 were determined by X-ray crystal diffractions for the first time. The antimicrobial activities against human pathogen, marine-derived aquatic bacteria, and plant-pathogenic fungi of compounds 1-14 were evaluated, and compound 1 showed remarkable activity against aquatic pathogen Vibrio alginolyticus with MIC value 1 µg/mL, which is as active as that of the positive control.