Immuno-protective response of Asian seabass (Lates calcarifer) to inactivated vaccines against Streptococcus iniae and Vibrio harveyi.

BACKGROUND: In this study, the protective immunity and immunogenicity of the monovalent and bivalent Streptococcus iniae and Vibrio harveyi vaccine were evaluated in Asian seabass. To analyze immune responses, 1200 Asian seabass with an average weight of 132.6 ± 25.4 g were divided into eight treatments in triplicates (50 fish per tank) as follows: S. iniae immunized by injection (SI), V. harveyi immunized by injection (VI), bivalent S. iniae and V. harveyi (SVI) immunized by injection, S. iniae immunized by immersion (SIM), V. harveyi (VIM) immunized by immersion, bivalent S. iniae and V. harvei (SVIM) immunized by immersion, phosphate-buffered saline (PBS) by injection (PBSI) and control group without vaccine administration (CTRL). Blood and serum samples were taken at the end of the 30th and 60th days. Then the vaccinated groups were challenged with two bacteria (S. iniae) and (V. harveyi) separately and mortality was recorded for 14 days. RESULTS: This study reveals that there is no significant difference in the hematological parameters on the 30th and 60th days of the experiment in the vaccine-immunized groups compared to the CTRL group (P > 0.05). Meanwhile, there was no significant difference in the amount of serum albumin level, respiratory burst activity, and serum bactericidal activity in the vaccine-immunized groups compared to the CTRL group on the 30th and 60th days of the experiment (P > 0.05). Total protein on the 60th day (in the VI and SVI groups), globulin on the 30th day (in the VI and SVI groups) and the 60th day (in the VI group) compared to the CTRL and PBSI groups had a significant increase (P < 0.05). Complement activity (in the VI and SVI groups) and lysozyme (in the SI and SVI groups) increased significantly compared to the control group (P < 0.05). Serum antibody titer against S. iniae had a significant increase in the SI, VI, SVI and SVIM groups compared to the CTRL and PBSI groups (P < 0.05). Serum antibody titer against V. harveyi had a significant increase in the groups immunized with the vaccine compared to the CTRL and PBSI groups (P < 0.05). A significant increase in the relative percentage survival (RPS) following challenge with S. iniae in the SVI (86.6%), SI (83.3%,) and VI (73.3%) groups were observed compared to the CTRL (43.3%) and PBSI (40%) groups (P < 0.05). Also, a significant increase in the RPS after challenge with V. harveyi in the SVI group, VI 86.6%, SVI 83.3%, VIM 80% and SVIM 76.6% were observed compared to the CTRL (46.6%) and PBSI (50%) groups (P < 0.05). CONCLUSION: Overall, the results demonstrated that the bivalent vaccine of S. iniae and V. harveywas able to produce significant immunogenicity and RPS in Asian seabass.

Immunogenicity and protective efficacy of Yersinia ruckeri lipopolysaccharide (LPS), encapsulated by alginate-chitosan micro/nanoparticles in rainbow trout (Oncorhyncus mykiss).

Considering the many advantages of oral vaccines in aquaculture, several studies have been conducted in this area recently. In this study, immunization and protective power of the oral vaccine of Yersinia ruckeri encapsulated with Alginate-Chitosan micro/nanoparticles were evaluated in rainbow trout. For this purpose, 720 juvenile rainbow trout (9 ± 1.8 g) were divided into 8 groups in three replications (30 fish each) as follows: Groups A, B and C, were immunized with Yersinia ruckeri lipopolysaccharide (LPS), LPS+Formalin Killed Cells (FKC) and FKC alone, groups D, E, and F were immunized with encapsulated LPS, LPS+FKC and FKC, respectively. The G and H groups considered as encapsulated and non-encapsulated control, respectively. Micro/nanoencapsulation with alginate-chitosan was performed by internal emulsification method and vaccination were conductrd in the first and third weeks via oral route. Sampling was performed on days 0, 30, and 60 of experiment. Anti Y. ruckeri antibody titer in serum, intestine and skin mucus were measured via ELISA method. Non-specific immune response including: serum lysozyme, complement, bactericidal and respiratory burst activity, serum protein and globulin level, as well as white blood cell count were compared among the groups. The expression of IgT gene in the intestine and TCR gene in the anterior kidney were also investigated. At the end of the study, the fish were challenged with Y. ruckeri through immerssion and intraperitoneal routs and the relative survival rate was evaluated. Result showed that the antibody level in serum, skin and intestine was significantly higher in group E and F than control groups (P < 0.05), meanwhile serum, skin and intestine antibody level in all vaccinated groups were significantly (P < 0.01) higher in day 30 and 60 compare to zero day. Non-specific immunity factors including: serum lysozyme, complement, and respiratory burst activity as well as WBC, protein and Globulin level were significantly higher in E and F groups not only in day 30 but also in day 60 of experiment (P < 0.05). Cumulative mortality following injection and bath challenge were significantly (P = 0.004) lower (35%-45%) in groups E and F compare to control group (80%). The IgT and TCR gene expression in groups D, E and F were significantly higher (P < 0.05) than control group. Highest upregulation of IgT and TCR gene expression in vaccinated groups were seen at day 30 and 60 respectively which were significantly (P < 0.001) higher than day zero. Generally, it can be concluded that nano/micronanoencapsulation of Y. ruckeri FKC+LPS with chitosan-alginate, not only increases protective efficacy of oral vaccine, but improves specific and non-specific immune responses in rainbow trout.

High efficacy and economical procedure of oral vaccination against Lactococcus garvieae/Streptococcus iniae in rainbow trout (Oncorhynchus mykiss).

The present study was aimed to examine the efficacy of chitosan-alginate coated vaccines against pathogenicity of Lactococcus garvieae and Streptococcus iniae in rainbow trout. Fish were divided into four groups including: Group A: fish immunized by chitosan-alginate coated vaccine, Group B: fish immunized by non-coated vaccine, Group C: fish feed by chitosan-alginate coated pellets without vaccine and Group D: fish feed by basic diet (non-coated and without vaccine). In groups A and B, the vaccination was carried out for 14 days and after that supplemented with fundamental diet (control diet). Comparable to groups A and B, fish of group C were also fed 14 days with test diets and after that fed control food. On day 0, 20, 40 and 60 of the experiment, serum samples were given. Fish have been challenged with live L. garvieae and S. iniae after 60 days. The levels of bactericidal activity and complement activity among innate immunity components extended on day 20 of the research and after that decreased in group A and B (P < 0.05) all through the examination. The relative expression of IL-6 and IgM in groups A and B extended on examination day 20. The expression of these genes illustrated no advancements in different groups in during the examination (P > 0.05). In group A, the serum antibody titer against L. garvieae and S. iniae broadly raised on day 40 and 60 of examination, whereas in group B, the immune response titer against S. iniae and L. garvieae illustrated a significant elevation on day 60 of the trial (P < 0.05). After challenge with live bacteria, survival rate of 83 ± 9.1%(challenged with S. iniae) and 72.18 ± 9.8% (challenged with L. garvieae) were gotten independently in group A, which were higher than survival of other exploratory groups (P < 0.05). In conclusion, the results of the present examination appear that the orally vaccination of rainbow trout with chitosan-alginate covered vaccine stimulates immunity system and also efficiently protects rainbow trout against Lactococcus garvieae and Streptococcus iniae.

Phyco-linked vs chemogenic magnetite nanoparticles: Route selectivity in nano-synthesis, antibacterial and acute zooplanktonic responses.

Despite the fact that magnetic iron oxide nanoparticles (Fe3O4-MNPs) considered as the most promising nanoparticles (NPs) in biomedicine and environmental biotechnology, their safety and ecotoxicological impacts of biogenic and chemogenic routes of Fe3O4-MNPs in the marine aquatic system is scarcely studied. In this work, we report the optimized and suitable phyco-synthesis route for nano-Fe3O4 based on the six selected species of the Persian Gulf seaweeds: Ulva prolifera, U. flexuosa, U. linza, U. intestinalis, U. clathrata, and Sargassum boveanum. Moreover, antibacterial activities and acute zooplanktonic responses in Artemia salina and acorn barnacle Amphibalanus amphitrite to chemogenic and biogenic Fe3O4-MNPs, were evaluated. Although all the seaweeds extract showed reducing potential for Fe3O4-MNPs green synthesis - mainly on the basis of characterization results- the algal route selectivity has been demonstrated to be important for the biosynthesis of magnetite NPs. Herein, the cubo-spherical and polydisperse U. prolifera-derived Fe3O4-MNPs with particles sizes of 9.59 nm were the best ones. The comparative zooplanktonic cytotoxicity of chemo- and bio-route of Fe3O4-MNPs exhibited no acute toxicity in nauplii and adults of A. salina (96-h EC50 ≥ 1000 mg/L) and the potential of toxicity in A. amphitrite nauplii (48-h EC50 = 466.5 and 842.3 mg/L for chemo- and bio-route of Fe3O4-MNPs, respectively). The in vitro antimicrobial activity of both chemo- and bio-route of magnetite NPs to selective human pathogenic bacteria and fungi (i.e. n = 11) showed strong antagonistic activity against Staphylococcus epidermidis, Bacillus subtilis, B. pumulis, and Saccharomyces cerevisiae. In conclusion, these findings demonstrate the optimized phyco-fabrication of Fe3O4-MNPs as promising nontoxic approach in ecobiotechnology, the new insight about the potential adverse effects of chemosynthesized Fe3O4-MNPs to crustacean zoo-organisms after their possible entrance into the marine environments, and bio/chemo-route Fe3O4-MNPs as pivotal agent for nanoantimicrobials.

The Anti-melanogenesis Activities of Some Selected Red Macroalgae from Northern Coasts of the Persian Gulf.

Tyrosinase is a key enzyme in melanin production. Therefore, tyrosinase inhibitors are used in cosmetic and medicinal industries to prevent or treat overproduction of melanin such as melasma, solar lentigo and post inflammatory melanoderma. Due to safety of natural whitening agents, in the present study, in-vitro anti-tyrosinase and in-vivo anti-melanogenesis activities of some selected red macroalgae of the Persian Gulf were investigated. The effects of various concentrations (100, 250 and 500 µg/mL) of methanolic extracts of three red macroalgae including Digenea simplex (D. simplex), Laurencia papillosa, and Laurencia paniculata on the activity of diphenolase of mushroom tyrosinase were studied by using L-Dopa as substrate. Subsequently, the activity of macroalgae with high inhibitory effect on hydroxylation of L-tyrosine was investigated by mushroom tyrosinase and zebrafish model. Anti-melanogenesis effects of algae extracts were studied on zebrafish as an alternative in-vivo model. Kojic acid was used as a positive control. All the tested macroalgae showed significantly a lower inhibitory effect on activities of diphenolase and monophenolase (of mushroom tyrosinase) compared to kojic acid. D. simplex showed the most anti-tyrosinase activity in zebrafish model among the samples. D. simplex extract and Kojic acid inhibited tyrosinase activity by 43.18% and 50.45%, and decreased total melanin content of zebrafish by 47.27% and 50.21%, respectively.

Valuable method for production of oral vaccine by using alginate and chitosan against Lactococcus garvieae/Streptococcus iniae in rainbow trout (Oncorhynchus mykiss).

The effectiveness of ionotropic gelation method (by combining alginate and chitosan) vaccine against Lactococcus garvieae and Streptococcus iniae was examined in rainbow trout. Fish were separated into four groups and fed the distinctive examined feeds. Our groups were included: A) fish immunized by chitosan-alginate coated vaccine, B) fish immunized by non-coated vaccine, C) fish feed by chitosan-alginate coated pellets without vaccine and D) fish feed by basic diet (non-coated and without vaccine). In groups A and B, the vaccination was carried out for 14 days. Fish of group C, like groups A and B were fed 14 days with pellets covered with chitosan-alginate without vaccine and a short time later they were fed with control diet. On day 0, 20, 40 and 60 of the trial, serum samples were extracted. Fish were challenged with L. garvieae and S. iniae after 60 days of research. Innate immunity components containing complement activity, total protein and IgM appeared no significant changes nearly in all groups during the 60 days that the examination finished. Although, bactericidal activity and lysozyme activity demonstrated a significant increase on days 20, 40 and 60 in group A compared to control groups (C and D) (P < 0.05) and similar results about the blood respiratory burst activity just on days 20 and 40 were obtained. Also, the relative expression of IL-6 of group A, was significantly higher compared to all of other groups (B, C and D) on days 20 and 60 of experiment (P < 0.05). The same results were obtained about the relative expression of IgM. The serum ELISA antibody titer against L. garvieae, increased significantly on days 20 and 40 of experiment in fish immunized by chitosan-alginate coated vaccine (Group A) compared to control groups (C and D)(P < 0.05) while the result of ELISA test against S. iniae was significantly higher on days 40 and 60 of experiment in group A compared to groups B, C and D (P < 0.05). After challenge with these two live bacteria (S. iniae and L. garvieae), a survival rates of 76.67 ±â€¯5.77% (challenged with S. iniae) and 66.67 ±â€¯5.77% (challenged with L. garvieae) were seen in group immunized with chitosan-alginate coated vaccine (Group A), which were higher than survival rates gotten in other trial groups (P < 0.05). The consequences of the present experiment show that the oral vaccination of rainbow trout with improved chitosan-alginate (via ionotropic procedure) (group A) properly secures this important fish against Lactococcus garvieae and Streptococcus iniae.

The Anti-melanogenesis Activities of Some Selected Brown Macroalgae from Northern Coasts of the Persian Gulf

Abstract Melanogenesis is a biological process which led to the synthesis of melanin pigment. Abnormal melanin production results in melasma, solar lentigo, post inflammatory melanoderma, etc. In this study, we examined the potential inhibitory effects of 17 brown macroalgae from Persian Gulf on melanogenesis. The effects of various concentrations (100, 250 and 500 µg/mL) of methanolic extracts of macroalgae belonging to four genera (including: Padina, Colpomonia, Cystoseira and Sargassum) were studied on oxidation of L-Dopa by mushroom tyrosinase. Subsequently, the activity of macroalgae with high inhibitory effect on monophenolase activity of mushroom tyrosinase and zebrafish was investigated using L-tyrosine as a substrate. Anti-melanogenesis effects of algae extracts were studied on zebrafish as an alternative in vivo model. Kojic acid was used as a positive control. All the tested macroalgae showed inhibitory effect on activities of diphenolase and monophenolase (of mushroom tyrosinase). P. boergesinii exhibited the most in vivo anti-tyrosinase activity compared with other samples. P. boergesenii inhibited zebrafish tyrosinase more potent than kojic acid (83% vs 50% inhibition for kojic acid). Moreover, it reduced melanin synthesis in zebrafish 42% (kojic acid: 50%).

Efficacy of a Eudragit L30D-55 encapsulated oral vaccine containing inactivated bacteria (Lactococcus garvieae/Streptococcus iniae) in rainbow trout (Oncorhynchus mykiss).

The efficacy of a Eudragit L30D-55 encapsulated vaccine against Lactococcus garvieae and Streptococcus iniae was investigated in rainbow trout. Fish were divided into four groups and fed the different experimental feeds. Groups were: A) fish immunized by Eudragit-coated pellets containing vaccine, B) fish immunized by vaccine-coated pellets without Eudragit, C) fish fed Eudragit-coated pellets without vaccine and D) fish fed pellets without vaccine orEudragit (control group). In groups A and B, the vaccination was conducted for 14 days. Similar to groups A and B, fish of group C were fed 14 days with pellets coated with Eudragit and afterwards they were fed control diet. Serum samples were taken on day 0, 20, 40 and 60 of the experiment. After 60 days, fish were challenged with L. garvieae and S. iniae. In almost all groups, innate immunity components including alternative complement activity, lysozyme activity, bactericidal activity, IgM and total protein showed no significant changes during the 60 days that the experiment lasted. However, the blood respiratory burst activity and lysozyme activity showed a significant increase on day 20 of experiment in groups B and D respectively (P < 0.05). The relative expression of immune-related genes including IL-6 and IgM genes was higher in vaccinated fish, with the highest expression in those immunized by Eudragit-coated pellets (Group A). In addition, the relative expression of IL-6 and IgM peaked on day 20 but decreased on day 60 in vaccinated groups. The ELISA antibody titer against L. garvieae increased from day 20 and peaked on day 60 of experiment (P < 0.05). Also, the antibody titer against L. garvieae was higher in fish immunized by Eudragit-coated pellets (Group A) compared to fish of group C and control. After bacterial challenge, a survival percentages of % 85 ±â€¯7.07% (challenged with S. iniae) and % 72.21 ±â€¯7.8% (challenged with L. garvieae) were observed respectively in groups immunized with pellets coated with Eudragit L30D-55 (Group A), which were higher than survival percentages obtained in other experimental groups (P < 0.05). The results of the present study demonstrate that the oral administration of Eudragit L30D-55-encapsulated vaccine appropriately protects rainbow trout against Lactococcus garvieae and Streptococcus iniae.

Organic and inorganic nano-Fe3O4: Alga Ulva flexuosa-based synthesis, antimicrobial effects and acute toxicity to briny water rotifer Brachionus rotundiformis.

Following the recent progress in magnetic nanotechnology, concern over the optimal benefits and potential risks of iron oxide nanoparticles (Fe NPs), has increased. Hence, to minimize the negative impacts of inorganic Fe NPs, we report the phyco-synthesis and characterization of superparamagnetic Fe3O4 NPs via reduction of ferric/ferrous chloride solution (2:1 M ratio; 88 °C) with green macroalga, Ulva flexuosa (wulfen) J.Agardh aqueous extract. The biogenic process is clean, eco-friendly, rapid, and facile to handle. These green fabricated magnetite NPs are characterized by Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), transmission electron microscopy (TEM), field emission scanning electron microscopy (FESEM), thermogravimetric analysis (TGA), particle size analysers (PSA), zeta potential (ZP) measurement, and vibrating sample magnetometry (VSM) techniques. The results confirmed that the cubo-spherical, polydisperse of biosynthesized Fe3O4 NPs with an average diameter of 12.3 nm was formed. The antagonistic effects of algal extract, chemo-route and U. flexuosa-derived Fe3O4 MgNPs on selective human pathogenic microbes (i.e. n = 11) resulted in strong antibacterial and moderate antifungal activity. The comparative toxic and reproductive effects of the chemo- and bio-routes of Fe3O4-MgNPs against rotifer B. rotundiformis exhibited low acute toxicity with a lower inducing effect of biogenic nano-magnetite on reduction rotifer reproductive rate than its chemogenic counterpart. In view of the nanoecotoxicity, though the current study covered a wide range of exposure concentrations (10-500 mg/L) of organic and inorganic nano sizes of Fe3O4 in brackish water rotifer, a biotoxicity assay at higher dosage or a comprehensive risk assessment in different aqua-organisms is recommended.

Changes in Immunity, Expression of some Immune-Related Genes of Shabot Fish, Tor grypus, Following Experimental Infection with Aeromonas hydrophila: Effects of Autochthonous Probiotics.

In this study, the effects of orally administrated two native probiotics (Lactobacillus plantarum and Lactobacillus delbrueckii ssp. bulguricus), isolated from the intestine of Shabot fish, Tor grypus, on some immune response parameters and immune-related genes expression against Aeromonas hydrophila in T. grypus were evaluated. Four hundred and eighty juveniles weighing 45 ± 10 g were randomly divided into four groups (with three replications) and fed with the experimental diet containing 5 × 107 cfu g-1 of L. plantarum (G1), Lactobacillus bulgaricus (G2), Lactobacillus casei (G3), and a control diet (without probiotics) for 60 continuous days. At the end of the dietary treatments, fish were challenged with a lethal concentration of A. hydrophila (5 × 108 CFU ml-1) via intra peritoneal (i.p) injection. Blood and head kidney samples were taken from six fish in each treatment before challenging and 6, 12, 24, and 48 h and also 7 days after injection. The results showed that lysozyme, complement, bactericidal, and NBT activity of probiotic-treated groups were significantly elevated (P < 0.05). The IL-8, IL-1ß, and TNF-α gene expressions were significantly higher in all probiotic-treated groups (P < 0.05). Meanwhile, a high direct correlation was observed between serum immune parameters and expression of immune-related genes (P < 0.0001); furthermore, the highest correlation (R 2 = 0.634, P < 0.0001) was recorded between IL-1ß expression and NBT activity. It can be concluded that not only two native probiotics strains stimulate serum immune responses parameters and immune-related gene expression in T. grypus, but also a high correlation was seen among these indices. The study suggests that gastrointestinal colonization is preferred for host specificity as the strain previously derived from shabot fish displayed better colonization than the non-indigenous bacteria strain such as L. casei. Therefore, these native probiotics bacteria can be accounted as suitable candidates to immune stimulation in fish.

Temperature-dependent protection against Ichthyophthirius multifiliis following immunisation of rainbow trout using live theronts.

Rainbow trout Oncorhynchus mykiss Walbaum, 1792 fingerlings were vaccinated by intraperitoneal (i.p.) injection using live theronts of the skin parasitic ciliate Ichthyophthirius multifiliis Fouquet, 1876 at 2 temperatures (12 and 20 degrees C), and protection against challenge infections was subsequently evaluated by bath exposure to live theronts. Vaccination conferred a relative protection (evaluated as the decrease in the number of established theronts) at 12 degrees C and almost complete immunity at 20 degrees C. Significantly increased immobilisation titers (using plasma immobilisation of live theronts) were found in immunised fish at Week 2 and 4 post-vaccination. Lysozyme activity of plasma from vaccinated fish increased from Week 1 to 4. Both immobilisation titers and lysozyme activity were significantly higher at 20 degrees C. This study demonstrated that live theronts are good candidates for an antigen source for development of effective vaccines against white spot disease in this fish host, and further indicated that the protection of rainbow trout against I. multifiliis infection is highly temperature dependent and may be associated with both adaptive and innate response mechanisms.