Immunity Of Olive Flounder Research Articles

Effects of dietary arachidonic acid supplementation in high plant protein diets on growth, feed utilization, and immunity of olive flounder, Paralichthys olivaceus

This study was designed to evaluate the effects of graded levels of arachidonic acid (ARA) in low fish meal diets (30% fish meal, 30% plant protein blends) on growth, feed utilization, and immunity of olive flounder (Paralichthys olivaceus). The control diet was formulated with 3% fish oil and 5.1% soybean oil (ARA0.0). Five other diets were formulated incorporating different levels of ARA into the ARA0.0 diet at the expense of soybean oil; that is, 0.3, 0.6, 0.9, 1.2, and 1.5% ARA (ARA0.3, ARA0.6, ARA0.9, ARA1.2, and ARA1.5), respectively. Three replicate groups of fish (88.24 ± 0.06 g) were placed into 18 polyvinyl circular tanks (215 L), with 24 fish each and fed an experimental diet twice a day for nine weeks. Overall, significantly improved growth performance was observed in the ARA0.6 group. Liver C18:2n-6 was significantly reduced, and liver ARA content was significantly increased with the increase in dietary ARA levels. Nitroblue tetrazolium activity was significantly higher in the ARA0.6 group, and significantly higher superoxide dismutase and myeloperoxidase activities were observed in the ARA0.9 group. Further, immunoglobulin level and lysozyme activity were significantly improved in the ARA0.3 group, antiprotease activity was increased in the ARA0.6 and ARA0.9 groups, and the ARA0.3 group showed lower liver lipid content than the ARA0.0 group. Serum prostaglandin E2 level was significantly increased with 1.5% ARA inclusion. Pro-inflammatory gene expressions of interleukin (IL)-6, IL-8, IL-10, perforin, and tumor necrosis factor in the intestine were significantly downregulated, and anti-inflammatory gene expression of transforming growth factor-β2 gene expression was significantly upregulated with higher dietary ARA supplementation (0.9–1.5%). These results indicate that 0.3–0.9% supplemented ARA may improve the growth, immune, and hematological parameters of olive flounder.

Saponin and chitosan-based oral vaccine against viral haemorrhagic septicaemia virus (VHSV) provides protective immunity in olive flounder (Paralichthys olivaceus)

Production losses of olive flounder (Paralichthys olivaceus) have increased owing to viral haemorrhagic septicaemia virus (VHSV) infection. In this study, we determined safe concentrations of orally administered saponin and chitosan by analysing serum enzyme (AST/ALT) levels as biochemical markers of hepatic injury. Furthermore, we demonstrated the efficacy, duration of protection, and safety of saponin and chitosan-based vaccines with inactivated VHSV (IV). Oral administration of saponin, chitosan, and their combination did not induce fish mortality at all tested concentrations (0.29, 1.45, and 2.9 mg/g of fish body weight/day) 10 days after administration. However, AST level was high at a dose >0.29 mg/g of fish body weight/day. Both saponin and chitosan were found to be safe and acceptable for vaccination studies at a dose of 0.29 mg/g of fish body weight/day. Administration of IV alone did not induce protection at 2 and 4 weeks post vaccination (wpv). Olive flounders administered saponin + IV and chitosan + IV vaccines had higher immunity against VHSV with relative percentage survival (RPS) of 12.5–7.5% and 0–20.1%, respectively; however, additional immunisation with combination of saponin + chitosan + IV clearly enhanced the protection with RPS values of 10–15%, 26.7%, 42.9%, and 37.5% at 4, 8, 12, and 20 wpv, respectively. Although the RPS value of oral immunisation was not comparable to that of injectable vaccines, the manufacturing process is simple and oral administration causes less stress to juvenile fish. To investigate the development of a protective immune response, olive flounder were re-challenged with VHSV (107.8 TCID50/fish) at 70 days postinfection; 100% of the previously unexposed fish died, whereas 80–100% of the previously immunised fish survived. Our results showed the possibility of developing preventive measures against VHSV using saponin and chitosan-based oral vaccines with inactivated virus.

Effect of total replacement of fish oil with tallow and emulsifier in diet on growth, feed utilization, and immunity of olive flounder (Paralichthys olivaceus)

AbstractThe supplementary effects of an emulsifier mixture (polyoxyethylene (20) sorbitan monolaurate and distilled monoglycerides) were investigated on feed utilization, growth performance, and immunity of olive flounder, Paralichthys olivaceus, replacing fish oil with beef tallow. A fish oil containing diet was considered as positive control (PC) and a diet containing beef tallow instead of fish oil was considered as negative control (TW). Two other diets (EM01 and EM02) were prepared by adding 0.01 and 0.02% of the emulsifier into the TW diet. After 8 weeks of a feeding trial, growth performance and feed utilization were significantly higher in fish fed EM01, EM02, or PC diets compared to those fed the TW diet. Dry matter digestibility was higher in EM01 and EM02 diets than the TW diet and comparable to PC diet. Lipid digestibility was significantly higher in PC diet compared to TW diet. Liver EPA and DHA levels were lower, and oleic acid level was higher in fish fed diets containing tallow compared to PC group. Significantly increased aspartate aminotransferase and triglyceride levels were observed in TW group. Dietary supplemented emulsifier restored the reduced performance of olive flounder fed diets containing tallow instead of fish oil. However, the optimum inclusion level of the emulsifier should be elucidated in future studies.

Dietary Supplementation of Bacillus sp. SJ-10 and Lactobacillus plantarum KCCM 11322 Combinations Enhance Growth and Cellular and Humoral Immunity in Olive Flounder (Paralichthys olivaceus)

Experiments were conducted to identify different ratios of Bacillus sp. SJ-10 and Lactobacillus plantarum KCCM 11322 mixtures at a concentration of 1 × 108CFU/g diet; the effects on growth and cellular and humoral immune responses and the characteristics of disease protection in olive flounder (Paralichthys olivaceus). Flounder were divided into six groups and fed control diet D-1 (without Bacillus sp. SJ-10 and L. plantarum KCCM 11322), positive control diets D-2 (Bacillus sp. SJ-10 at 1 × 108CFU/g feed) and D-3 (L. plantarum KCCM 11322 at 1 × 108CFU/g feed); or treatment diets D-4 (3:1 Bacillus sp. SJ-10 and L. plantarum KCCM 11322 at 0.75 + 0.25 × 108CFU/g feed), D-5 (1:1 Bacillus sp. SJ-10 and L. plantarum KCCM 11322 at 0.50 + 0.50 × 108CFU/g feed), or D-6 (1:3 Bacillus sp. SJ-10 and L. plantarum KCCM 11322 at 0.25 + 0.75 × 108CFU/g feed) for 8 weeks. Group D-4 demonstrated better growth and feed utilization (P < 0.05) compared with the controls and positive controls. Similar modulation was also observed in respiratory burst for all treatments and in the expression levels of TNF-α, IL-1β, IL-6, and IL-10 in different organs in D-4. D-4 and D-5 increased respiratory burst, superoxide dismutase, lysozyme, and myeloperoxidase activities compared with the controls, and only D-4 increased microvilli length. When challenged with 1 × 108CFU/mL Streptococcus iniae, the fish in the D-4 and D-5 groups survived up to 14days, whereas the fish in the other groups reached 100% mortality at 11.50days. Collectively, a ratio-specific Bacillus sp. SJ-10 and L. plantarum KCCM 11322 mixture (3:1) was associated with elevated growth, innate immunity, and streptococcosis resistance (3:1 and 1:1) compared with the control and single probiotic diets.

Effects of butaphosphan and cyanocobalamin mixture on immunity and stress in olive flounder, Paralichthys olivaceus

BackgroundThe study evaluated the effects of a butaphosphan and cyanocobalamin mixture on the immune system and stress in olive flounders, Paralichthys olivaceus.MethodsThe mixture was intramuscularly injected into olive flounders at the current recommended dose. Furthermore, to determine the toxicity of overdose, a histological examination was performed after injection of 1-, 2-, and 4-fold higher than the recommended dose.ResultsImmunity parameters were altered during the first 2 weeks after a single intramuscular injection of the mixture in olive flounders (average weight 20.5 ± 1.1 g). The levels of all tested items, except glutathione and anti-protease, were higher in the treated group than in the control group in the first week; the levels of all tested items were even higher in the second week in the treated group than in the control group. The level of nitro-blue tetrazolium, myeloperoxidase, and superoxide dismutase between the two groups differed significantly. Changes in the stress response to different seawater temperatures (increase or decrease in seawater temperature by 3–5 °C using 50 L heated or cooled seawater tanks) were studied by determining the changes in cortisol and glucose levels on days 1 and 7. Both cortisol and glucose levels were significantly lower in the treated group than in the control group. Histological analysis did not reveal any abnormalities after intramuscular injection of the mixture at doses that were 1-, 2-, and 4-fold higher than the recommended dose.ConclusionsIntramuscular injection of a butaphosphan and cyanocobalamin mixture is safe and effective in reducing stress and improving immunity in olive flounders.

Enhancing immunogenicity of a reporter protein by fusion to glycoprotein and nucleoprotein of viral hemorrhagic septicemia virus (VHSV) particles

The introduction of reverse genetic technology to generate recombinant VHSVs (rVHSVs) has contributed to the uncovering of functional roles of viral genes and to the development of attenuated prophylactic vaccines. In this study, to assess the possible use of rVHSVs as a tool of combined vaccines, we newly rescued rVHSVs that harbor viral envelop-studded eGFP (rVHSV-A-SGT) or nucleoprotein-fused eGFP (rVHSV-A-NLG), and the ability of these rVHSVs to induce adaptive humoral immunity in olive flounder (Paralichthys olivaceus) was compared with that of rVHSV-A-eGFP that expresses eGFP as a soluble form in the cytoplasm of infected cells. The results showed that antibodies against eGFP were efficiently induced by the immunization of olive flounder with rVHSV-A-SGT and rVHSV-A-NLG, while rVHSV-A-eGFP was poor in the ability to induce antibody response against eGFP. These results suggest that the display of heterologous antigens on VHSV envelop is a good way to develop efficient combined vaccines and the fusion of foreign antigen with N protein can also be a way to enhance immunogenicity of a foreign antigen. The present recombinant VHSVs - rVHSV-A-SGT and rVHSV-A-NLG – not only express foreign antigens in host cell cytoplasm but also display antigens in or on the virus particles. Further researches on the availability of recombinant VHSVs as combined vaccines against multiple fish pathogens are needed.

Effects of probiotic supplementation of a plant-based protein diet on intestinal microbial diversity, digestive enzyme activity, intestinal structure, and immunity in olive flounder (Paralichthys olivaceus)

The aim of this study was to investigate the effects of intestinal microbial manipulation by dietary probiotic supplementation on digestive enzyme activity, immune-related gene transcription, intestinal structure alteration, and viability against pathogenic challenge in olive flounder. Similar-sized flounders (14.92 ± 0.21 g) were divided into three groups and supplemented with a control (without probiotic) or 1 × 108 CFU/g diet of each of Bacillus sp. SJ-10 (ProB) and Lactobacillus plantarum (ProL) for eight weeks. At the end of the feeding trial, the estimated intestinal microbial richness (Chao1) and diversity (Shannon) demonstrated a significant (P < 0.05) abundance in the ProB group (484.80 ± 88.75, 5.08 ± 0.17) compared to the ProL (285.32 ± 17.78, 4.54 ± 0.09) and control groups (263.23 ± 20.20, 4.30 ± 0.20). A similar alteration phenomenon was also found at the phylum level, with a higher abundance of Proteobacteria, Actinobacteria, and Acidobacteria. Trypsin and lipase activities were elevated in both the ProB and ProL groups compared to the control, but amylase was only higher in the ProB group. The expression levels of pro-inflammatory cytokines, such as tumor necrosis factor-α, interleukin (IL)-1β, and IL-6 were significantly higher in the ProB group than in the other two groups. There was a significant increase in transcription of IL-10 in both the ProB and ProL groups compared to the control. The length of villi and microvilli of probiotic-fed olive flounder was increased but was not significantly different from the control group. In an in vivo challenge experiment with Streptococcus iniae (1 × 108 CFU/mL), the survival rates of the ProB and ProL groups were 29.17% and 12.50%, respectively, when control mortality reached 100%. Therefore, intestinal microbiota manipulation by probiotic supplementation increased the richness of the bacterial population, digestive enzyme activity, intestinal immune gene transcription, and infectious disease protection in olive flounder.

Heat-killed Bacillus sp. SJ-10 probiotic acts as a growth and humoral innate immunity response enhancer in olive flounder (Paralichthys olivaceus)

Investigations were carried out to evaluate and quantify the effects of dietary supplementation with heat-killed (HK) Bacillus sp. SJ-10 (BSJ-10) probiotic (1 × 108 CFU g−1) on the growth and immunity of olive flounder (Paralichthys olivaceus). Flounder (averagely 9.64 g) were divided into two groups, and fed control and HK BSJ-10 (HKBSJ-10)-inoculated diets for 8 weeks. Investigations were carried out on growth and feed utilizations, innate immunity, serum biochemical parameters, microvilli length, and pro- and anti-inflammatory cytokine gene (tumor necrosis factor [TNF]-α, interleukin [IL]-1β, IL-6, and IL-10) transcriptions. Compared to control, HKBSJ-10 diet significantly (P < 0.05) enhanced weight gain and protein efficiency ratio, 1.17 and 1.11 folds respectively. Humoral innate immune parameters, lysozyme and superoxide dismutase in treatment group were also elevated by 1.34 and 1.16 folds. Similarly, an increased (P < 0.05) relative expressions of TNF-α, IL-1β, IL-6 were recorded in liver (2.71, 3.38, and 4.12 folds respectively), and gill (2.08, 1.98, and 1.81 folds respectively) than that of controls. Moreover, after challenge with Streptococcus iniae (1 × 108 CFU mL−1), the HKBSJ-10-fed group exhibited significantly higher protection (P < 0.05) against streptococcosis compared to controls, validating the observed changes in immune parameters and induction on the cytokine-encoding genes. Therefore, HKBSJ-10 increases growth, modulates innate immune parameters, and protects olive flounders against streptococcosis.

Generation of VHSV replicon particles carrying transmembrane and C-terminal cytoplasmic region-deleted G gene (rVHSV-GΔTM) and comparison of vaccine efficacy with G gene-deleted VHSV (rVHSV-ΔG)

Vaccines based on viral replicon particles would be advantageous to induce immune responses compared to inactivated viruses in that they can infect host cells (only once) and can produce viral proteins in the infected cells like live viruses. Furthermore, as viral replicon particles are replication-defective, they are safer than live attenuated viruses. Previously, we had rescued viral hemorrhagic septicemia virus (VHSV) replicon particles lacking full ORF of G gene (rVHSV-ΔG). In the present study, to enhance the immunogenicity of VHSV replicon particles, we newly generated another form of VHSV replicon particles that can produce the transmembrane and C-terminal cytoplasmic region-deleted G protein in host cells (rVHSV-GΔTM), and compared the protective efficacy of rVHSV-GΔTM with that of rVHSV-ΔG through immunization of olive flounder (Paralichthys olivaceus). In addition, we evaluated the safety of rVHSV-GΔTM by the analysis of effects on wild-type VHSV replication. In the vaccine experiment, olive flounder immunized with rVHSV-GΔTM showed significantly higher titers of serum neutralization activity than fish immunized with rVHSV-ΔG suggesting that the G protein that is not only spiked on the viral envelop but also secreted extracellularly can contribute to the enhancement of adaptive humoral immunity. Moreover, fish immunized with rVHSV-GΔTM showed higher survival rates than fish immunized with rVHSV-ΔG, suggesting that the amount of G protein provided to hosts is an important factor for the enhancement of vaccine efficacy against VHSV disease. In a safety aspect, rVHSV-GΔTM could not replicate in infected cells, and significantly inhibited the replication of wild-type VHSV when co-infected, suggesting that rVHSV-GΔTM would not worsen disease progression caused by wild-type VHSV infection.

PLGA encapsulated inactivated-viral vaccine: Formulation and evaluation of its protective efficacy against viral haemorrhagic septicaemia virus (VHSV) infection in olive flounder (Paralichthys olivaceus) vaccinated by mucosal delivery routes

Viral haemorrhagic septicaemia virus (VHSV), an OIE listed viral pathogen, is the etiological agent of a contagious disease, causing huge economic losses in farmed olive flounder (Paralichthys olivaceus) and significant mortalities among several other marine fish species in Korea, Japan, and China. In continuation with our previous work, where injection vaccination with inactivated VHSV mixed with squalene (as adjuvant) conferred higher protective immunity to olive flounder, the present study focused on replacing the injection route of vaccine delivery by immersion/oral route to overcome the limitations of the parenteral immunization method. Here, we encapsulated the inactivated VHSV vaccine with PLGA (poly lactic-co-glycolic acid) nanoparticles (PNPs-IV) and evaluated its ability to induce protective immunity in olive flounder (12.5 ± 1.5 g) by initially immunizing the fishes by immersion route followed by a booster with the same dose two weeks later with half of the fish through immersion route and other half through oral route (incorporated into fish feed). Cumulative mortalities post-challenge (1 × 106 TCID50 virus/fish) with virulent VHSV-isolate, were lower in vaccinated fish and RPS of 60% and 73.3% were obtained for PNPs-IV (immersion/immersion) and PNPs-IV (immersion/oral) groups, respectively. In addition, specific (anti-VHSV) antibody titre in the fish sera, skin mucus and intestinal mucus of the immunized groups were significantly (p < 0.05) enhanced following vaccination. Furthermore, PNPs-IV immunized fish showed significant (p < 0.05) upregulation of different immune gene transcripts (IgM, IgT, pIgR, MHC-I, MHC-II, IFN-γ, and Caspase3) compared to controls, in both the systemic (kidney) and mucosal (skin and intestine) immune compartment of the host post immunization as well as post challenge. Thus it can be inferred that the adopted immunization strategy efficiently protected and transported the inactivated viral antigen to target immune organs and positively stimulated the protective immune response against VHSV in olive flounder.

Effects of LEDs of different spectra on immunity-related responses in olive flounder Paralichthys olivaceus during short starvation

In this study, we investigated the regulation of innate immune responses in olive flounder Paralichthys olivaceus under irradiation with various light-emitting diodes (LEDs), blue (460 nm), green (520 nm), and red (630 nm), at two intensities (0.3 and 0.6 W/m2) for 9 days. To evaluate the effects, we analyzed the levels of melatonin receptor 1 (MTR-1), melatonin, IgM, and lysozyme, which are related to the immunity index. The results showed that melatonin, IgM, and lysozyme significantly decreased during starvation compared to their levels in a normally fed control group. However, the decreases were less pronounced in the green and blue LED-exposed groups than those in the red LED-exposed group during the experimental period. These results confirmed that short-wavelength light (green and blue) is effective in maintaining enhanced immunity in olive flounder.

Effects of Lactococcus lactis subsp. lactis I2 with β-Glucooligosaccharides on Growth, Innate Immunity and Streptococcosis Resistance in Olive Flounder (Paralichthys olivaceus).

To identify and quantify the effects of a combination of dietary 1 × 108 CFU/g Lactococcus lactis subsp. lactis I2 (LI2) and 0.1% β-glucooligosaccharides (BGO) on the growth and immunity of olive flounder (Paralichthys olivaceus), a feeding experiment was conducted. Flounder (14 ± 0.5 g) were divided into two groups and fed control and synbiotic feeds for 8 weeks. Investigations were carried out on growth and feed utilization, innate immunity, serum biochemical parameters, intestinal lactic acid bacterial (LAB) viability, microvillus length, and changes in the expression levels of genes encoding pro-inflammatory cytokines (tumor necrosis factor [TNF]-α, interleukin [IL]-1β, and IL-6). Results demonstrated the synbiotic diet had significantly better (p < 0.05) responses in terms of weight gain and specific growth rate, three innate immune parameters (respiratory burst, serum lysozyme, and superoxide dismutase), intestinal LAB viability, and the relative TNF-α expression level (p < 0.05). Moreover, after challenge with Streptococcus iniae (1 × 108 CFU/ml), the synbiotically fed group exhibited significantly higher (p < 0.05) protection against streptococcosis, validating the observed changes in immune parameters and induction of the cytokine-encoding gene. Therefore, according to the results of the present study, synbiotic feed (LI2 + BGO) increased growth, modulated innate immune parameters and protected olive flounder against streptococcosis.

Montanide IMS 1312 VG adjuvant enhances the efficacy of immersion vaccine of inactivated viral hemorrhagic septicemia virus (VHSV) in olive flounder, Paralichthys olivaceus

Vaccination by immersion is suitable for mass vaccination of small size fish. However, no viral vaccine has been developed for immersion applications, because of low efficacy. In this study, we evaluated the efficacy and safety of immersion vaccine against viral hemorrhagic septicemia (VHS) containing Montanide IMS 1312 VG adjuvant in olive flounder (Paralichthys olivaceus). Healthy fish were vaccinated by an immersion method with a heat-inactivated FP-VHS2010-1 strain of VHS virus (VHSV) in combination with Montanide IMS 1312 VG for 5 min at 20 ± 2 °C. The control group was vaccinated with sterile PBS. No toxicity of immersion vaccine with Montanide IMS 1312 VG adjuvant was observed by hematological and histopathological analysis. Immersion vaccine with adjuvant enhanced gene expression of immune-associated genes, i.e., genes encoding interleukin (IL)-1β, IL-6, IL-8, and Toll-like receptor (TLR) 3. Relative percent survival (RPS) of fish was measured on weeks 4 and 8 post vaccination. In fish vaccinated with adjuvant, RPS was significantly higher than that of fish vaccinated without adjuvant. The results of the present study provide evidence that the VHSV immersion vaccine with Montanide IMS 1312 VG induces protective immunity in olive flounder against VHS.

Effect of G gene-deleted recombinant viral hemorrhagic septicemia virus (rVHSV-ΔG) on the replication of wild type VHSV in a fish cell line and in olive flounder (Paralichthys olivaceus).

In an earlier study, we generated a replicon viral hemorrhagic septicemia virus (VHSV) particle that was lacking the G gene in the genome (rVHSV-ΔG), and proved the potential of it as a protective vaccine through the immunization of olive flounder (Paralichthys olivaceus) fingerlings. Safety is the most important preconsideration for the development of recombinant live vaccines, and a major concern of propagation-incompetent viral particles would be the possible harmful effect to hosts through the interaction with wild-type viruses. Thus, in the present study, we analyzed the replication of rVHSV-ΔG in the presence of wild-type VHSV and the effect of rVHSV-ΔG on the replication of wild-type VHSV in Epithelioma papulosum cyprini (EPC) cells and in olive flounder fingerlings. The replication of wild-type VHSV in EPC cells was severely suppressed when the MOI of rVHSV-ΔG was 0.1 or 0.01, on the other hand, the titers of rVHSV-ΔG were not increased and stayed in a relatively constant according to time lapse. Furthermore, the replication of other novirhabdoviruses, IHNV and HIRRV, was also inhibited by co-infection with high titers of rVHSV-ΔG. There were no big differences in mortalities between groups infected with wild-type VHSV plus rVHSV-ΔG and groups infected with wild-type VHSV alone, when the challenged wild-type VHSV was more than 10(2)PFU/fish. However, a group of fish infected with 10PFU/fish of wild-type VHSV plus rVHSV-ΔG showed significantly lower and slowly progressing cumulative mortality than a group of fish infected with 10PFU/fish of wild-type VHSV alone. This result suggests that rVHSV-ΔG has an ability to attenuate the disease progression caused by wild-type VHSV when co-infected with relatively low titers of wild-type VHSV. These results indicate that the propagation-incompetent rVHSV-ΔG would not worsen but attenuate the progression of a disease caused by wild-type VHSV infection. Therefore, rVHSV-ΔG-based vaccines can provide a safe and effective way to control VHSV.

Potential of auxotrophic Edwardsiella tarda double-knockout mutant as a delivery vector for DNA vaccine in olive flounder (Paralichthys olivaceus)

To evaluate potential of an auxotrophic Edwardsiella tarda mutant (Δalr Δasd E. tarda) as a delivery vehicle for DNA vaccine in fish, olive flounder (Paralichthys olivaceus) were immunized with the E. tarda mutant harboring plasmids (pG02-ASD-CMV-eGFP) for eukaryotic expression of the enhanced green fluorescent protein (eGFP) gene through either intraperitoneal (i.p.) or oral route, and the expression of eGFP in the internal organs and generation of antibody against eGFP in fish were analyzed. In fish i.p. injected with 2×107CFU/fish of Δalr Δasd E. tarda harboring pG02-ASD-CMV-eGFP, expression of eGFP was detected in liver, kidney, and spleen from 1 day to 28 days post-injection. In fish orally administered with 1×109CFU/fish of the bacteria, the eGFP band was detected in liver, kidney, and spleen from 1 day to 14 days post-administration, whereas, in intestine, the band was detected only at 1 day post-administration. Either oral or i.p. immunization of olive flounder with recombinant E. tarda that carried eGFP-expressing eukaryotic plasmids was successful to induce humoral adaptive immunity against not only E. tarda that was used as a delivery vehicle but also eGFP that was used as the reporter protein of DNA vaccine, suggesting attenuated E. tarda-vectored DNA vaccine has a potential to be used as a combined vaccine against infectious diseases in fish.

Probiotics and herbal mixtures enhance the growth, blood constituents, and nonspecific immune response in Paralichthys olivaceus against Streptococcus parauberis

The present study was reported the effect of probiotics and herbals mixture supplementation diet on growth, blood constituents, and nonspecific immune response in olive flounder Paralichthys olivaceus against Streptococcus parauberis on weeks 1, 2, 4, 6, 8, 10, and 12 after injected intraperitoneally (i.p.) with 50 μl of PBS (phosphate buffer saline) containing S. parauberis (2.1 × 10 7 CFU ml -1). The initial weight did not significantly increased in supplementation diet group from 1 to 4 weeks, whereas it was significantly increased from weeks 6 to 12 as compared to fish fed without supplementation diet. The serum aspartate aminotransferase (SGOT) and alanine aminotransferase (SGPT) activities significantly increased from weeks 4 to 12 in infected fish fed with supplementation diet compared to fish fed without supplementation diet. However, the total protein (TP) and glucose (GLU) levels were significantly increased in infected fish fed with supplementation diet after 6 weeks. The phagocytic, respiratory burst, complement, and lysozyme activities significantly enhanced in infected fish fed with supplementation diet from weeks 4 to 12 as compared to fish fed without supplementation diet. These results suggested that different probiotics and herbals mixture supplementation diet enhanced the growth, blood biochemical constituents, and nonspecific immunity in olive flounder against S. parauberis.