Higher Relative Percent Survival Research Articles

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.

Bicistronic DNA vaccine macromolecule complexed with poly lactic-co-glycolic acid-chitosan nanoparticles enhanced the mucosal immunity of Labeo rohita against Edwardsiella tarda infection

DNA vaccine is an important tool to elicit both humoral and cellular immunity. Present study investigates mucosal immune response of Labeo rohita (15 ± 04 g) to plasmid DNA (pDNA) vaccine macromolecule complexed with nanoparticles (NPs). Poly lactic-co-glycolic acid (PLGA), Chitosan (Chit) and PLGA-Chit-NPs were synthesized by double emulsion solvent evaporation method. Synthesized NPs were complexed with pDNA (pGPD + IFN) vaccine construct. Size and zeta potential of PLGA-NPs, Chit-NPs and PLGA-Chit-NPs-pDNA complex were recorded to be 120 nm and +0.5 mV, 117 nm and +32 mV, 189 nm and +11 mV, respectively. Immunization by immersion was carried out in two groups receiving PLGA-Chit-NPs-pDNA (T1) and PLGA-NPs-pDNA (T2) respectively. After immersion, samples were collected on 0, 2, 4, 7, 15 and 30 days from mucosa-associated lymphoid tissues (MALT) for mRNA expression studies of IgM, IgD and IgZ using qRT-PCR. Significant up-regulation of the mRNA expression of IgM, IgD, and IgT were observed in MALT in immunized fish compared to control. After 30 days post-immunization fish were infected with a virulent strain of Edwardsiella tarda. The highest relative percentage survival was observed in T1 (64.7%) compared to T2. The study showed better efficiency of pDNA-PLGA-Chit-NPs compared to pDNA-PLGA-NPs for inducing adaptive mucosal immunity in fish.

Benefits of nanotechnology: Dietary supplementation with nerolidol-loaded nanospheres increases survival rates, reduces bacterial loads and prevents oxidative damage in brains of Nile tilapia experimentally infected by Streptococcus agalactiae

Rampant and uncontrolled use of antibiotics is a major concern for aquaculture; the practice foments the emergence of resistant strains of Streptococcus agalactiae, among other negative impacts. Constituents of plant essential oils such as nerolidol are being considered as replacements for synthetic drugs to support fish nutrition and health. There is evidence to suggest that nanotechnology may enhance the efficacy of natural bioactive compounds; this is a substantial advance for the development and sustainability of aquaculture. Against the backdrop of this evidence, we aimed determine whether dietary supplementation with free nerolidol and nerolidol-loaded nanospheres would exert bactericidal effects against S. agalactiae, as well as prevent S. agalactiae-induced brain oxidative damage. In Experiment I, we measured the antimicrobial properties of dietary supplementation of nerolidol and nerolidol nanosphere in terms of mortality, longevity and relative percent survival. Fish infected with S. agalactiae fed 0.5 and 1.0mL nerolidol nanospheres kg/diet demonstrated lower mortality and higher relative percent survival than the control group, while longevity was higher in all infected plus supplementation groups. Experiment II showed significantly lower microbial loads in brains of fish infected with S. agalactiae that were fed 1.0mL nerolidol nanospheres kg/diet than in the control group. Brain nerolidol levels were significantly higher in uninfected as well as infected fish supplemented with nerolidol nanospheres than in fish supplemented with free nerolidol. Finally, brain reactive oxygen species and lipid peroxidation levels were higher in infected fish supplemented with basal diet compared to uninfected fish and supplemented with basal diet, and the supplementation with 1.0mL/kg nerolidol nanospheres prevented this augmentation caused by infection. These data suggest that dietary supplementation with nerolidol nanospheres (1.0mL/kg diet) has potent bactericidal effects in terms of augmentation of fish longevity and survival, and reduction of brain microbial loads. Also, S. agalactiae-induced brain oxidative damage that contributed to disease pathogenesis, and the dietary supplementation with nerolidol nanospheres (1.0mL/kg diet) prevented this alteration. In summary, nanotechnology is a compelling approach to enhancing the efficacy of nerolidol, giving rise to reduction of S. agalactiae loads in fish brains.

Construction and characterization of a DNA vaccine encoding the SagH against Streptococcus iniae

Streptococcus iniae is an important aquaculture pathogen that is associated with disease outbreaks in wild and cultured fish species. Streptolysin S has been identified as an important virulence factor of S. iniae. With an aim to develop effective vaccines against S. iniae for Japanese flounder (Paralichthys olivaceus), in this study, we constructed a DNA vaccine based on the sagH gene, which belongs to the streptolysin S-associated gene cluster. In fish vaccinated with pSagH, the transcription of sagH was detected in tissues and SagH protein was also detected in the muscles of pSagH-vaccinated fish by immunohistochemistry. The immunoprotective effect of SagH showed that fish vaccinated with pSagH at one and two months exhibited a high relative percent survival (RPS) of 92.62% and 90.58% against S. iniae serotype I, respectively. In addition, SagH conferred strong cross protection against S. iniae serotype II and resulted in an RPS of 83.01% and 80.65% at one and two months, respectively. Compared to the control group, fish vaccinated with pSagH were able to induce much stronger respiratory burst activity, and higher titer of specific antibodies. The results of quantitative real-time PCR demonstrated that pSagH upregulated the expression of several immune genes that are possibly involved in both innate and adaptive immune responses. These results indicate that pSagH is a candidate DNA vaccine candidate against S. iniae serotype I and II infection in Japanese flounder in aquaculture.

Efficacy of Montanide™ ISA 763 A VG as aquatic adjuvant administrated with an inactivated Vibrio harveyi vaccine in turbot (Scophthalmus maximus L.)

Turbot (Scophthalmus maximus L.) is a commercially important fish species in China. Despite of its great economic potential, fish farms often suffer severe economic losses due to certain fish diseases. Vaccination has become a common strategy to prevent diseases caused by pathogens in aquaculture industry. However, no inactivated vaccine against Vibrio harveyi of turbot has been reported so far. In this study, we developed an inactivated vaccine using formalin-killed cells of V. harveyi and the efficacy of a commercial adjuvant Montanide™ ISA 763 A VG on the inactivated vaccine was evaluated. We found that with an optimum vaccine dosage at 1.0 × 108 CFU/fish, a high relative percent survival (RPS) more than 75% was observed at 4 weeks post vaccination (w.p.v.). Moreover, enhanced antibody titer, lysozyme activity, total serum protein and antibacterial property in sera of vaccinated fish were observed at 4, 8, 12 and 16 w.p.v. In conclusion, we developed an efficient inactivated vaccine against V. harveyi in turbot, which not only induced humoral immunity, but also enhanced initial innate immune response for long-term protection.

Preparation, characterization and evaluation of the immune effect of alginate/chitosan composite microspheres encapsulating recombinant protein of Streptococcus iniae designed for fish oral vaccination

Streptococcus iniae has caused serious harm to the fish farming industry in recent years. Vaccination is a potential approach for preventing and controlling disease, being oral vaccination the most suitable vaccination route in fish. Alginate and chitosan microspheres have been widely used as controlled release systems for oral vaccination in fish. In this study, we prepared and characterized alginate/chitosan composite microspheres encapsulating the recombinant protein serine-rich repeat (rSrr) of S. iniae. We evaluated effect of these microspheres on the immune system of channel catfish. The microsphere preparation conditions were optimized by Response Surface Method and target microspheres were obtained under 1.68% alginate (w/v), the W/O ratio 3.6:7.4 (liquid paraffin with 4% Span 80, v/v) with stirring at 1000 rpm, 9.64% CaCl2 (w/v) and 0.95% chitosan (w/v) with an encapsulation efficiency of 92.38%. The stability and safety of rSrr-microspheres were evaluated in vitro and in vivo, respectively. Furthermore, compared with control group, oral vaccination with rSrr-microspheres induced higher serum antibody titers, higher lysozyme activity, higher total protein and higher expression of immune-related genes, and resulted in higher relative percent survival (RPS) with the value of 60% for channel catfish against S.iniae infection. Our results thus indicate that alginate/chitosan microspheres encapsulating rSrr can be used as oral vaccine for channel catfish, providing efficient immunoprotection against S. iniae infection.

Effects of dietary fructooligosaccharide on the growth, antioxidants, immunity and disease resistance of Chinese mitten crab

This study aimed to investigate the effects of dietary fructooligosaccharide (FOS) on the growth, antioxidant capability, immune response and disease resistance of Chinese mitten crab Eriocheir sinensis. Groups of crab (17.74±0.39g) were distributed into 18 cement pools (33 crabs per pool) with replicates and fed experimental diets supplemented with FOS at 0, 0.5, 1.0, 1.5, 2.0 and 2.5g/kg (designated as control, TF1, TF2, TF3, TF4 and TF5, respectively). After 60days of feeding trial, crabs were challenged by Aeromonas hydrophila and the survival was recorded for the next 96h. The results indicated that crabs fed TF4 had significantly higher weight gain (WG) than crabs fed control diet, whereas WG decreased with FOS level increasing to 2.5g/kg. There was no significant difference (P>0.05) in SGR among all experimental groups. Catalase (CAT) and superoxide dismutase (SOD) activities in crabs fed TF4 diet were significantly higher than those in control group, while malondialdehyde (MDA) concentrations in crabs fed TF4 diet were significantly lower than that in control group. Crabs fed TF4 diet showed significantly higher alkaline phosphatase (AKP) activity compared to the control group. There was a trend that acid phosphatase (ACP) activity was elevated with FOS levels increasing up to 2.0g/kg and decreased subsequently, and crabs fed TF4 diet showed significantly higher ACP activity compared to the control group. Supplementation of 2.0g/kg FOS up-regulated the mRNA expression of interleukin-2 enhancer binding factor 2 (ILF2) and peroxinectin (PX), and down-regulated the mRNA expression of lipopolysaccharide-induced TNF-α factor (LITAF). Bifidobacteriales abundance in the digestive tract increased significantly with dietary FOS levels increasing from 1.5 to 2.5g/kg. Crabs fed TF4 showed significantly higher Bacteroides abundance compare to the other groups. After Aeromonas hydrophila challenge, the highest relative percent survival (25.00%) was observed in crabs fed TF4 diet, which was significantly higher than that in control group. Taken together, 2.0g/kg FOS supplementation might be effective in improving growth performance, antioxidant capability, immune response and disease resistance of Chinese mitten crab.

GapA, a potential vaccine candidate antigen against Streptococcus agalactiae in Nile tilapia (Oreochromis niloticus)

Streptococcosis due to the bacterium Streptococcus agalactiae (S. agalactiae) has resulted in enormous economic losses in aquaculture worldwide, especially in the tilapia culture industry. Previously, there were limited vaccines that could be employed against streptococcosis in tilapia. This study aimed to develop a vaccine candidate using the glyceraldehyde-phosphate dehydrogenase protein (GapA) of S. agalactiae encoded by the gapA gene. Tilapia were intraperitoneally injected with PBS, PBS + Freund's adjuvant, PBS + Montanide's adjuvant, GapA + Freund's adjuvant, GapA + Montanide's adjuvant, killed S. agalactiae whole cells (WC)+Freund's adjuvant, or killed S. agalactiae whole cells (WC)+ Montanide's adjuvant. They were then challenged with S. agalactiae, and the relative percentage survival (RPS) was monitored 14 days after the challenge. The highest RPSs were observed in the WC groups, with 76.7% in WC + Freund's adjuvant and 74.4% in WC + Montanide's adjuvant groups; these were followed by the GapA groups, with 63.3% in GapA + Freund's adjuvant and 45.6% in GapA + Montanide's adjuvant groups. The RPS of the PBS group was 0%, and those of PBS + Freund's adjuvant and PBS + Montanide's adjuvant groups were 6.7% and 3.3%, respectively. Additionally, the IgM antibody responses elicited in GapA groups and WC groups were significantly higher than those in PBS groups. Furthermore, the expressions of cytokine (IL-1β and TNF-α) mRNAs in the GapA groups and WC groups were significantly higher than those in the PBS groups. Taken together, these results reveal that the GapA protein is a promising vaccine candidate that could be used to prevent streptococcosis in tilapia.

ADDITION OF ADJUVANTS IN RECOMBINANT SUBUNIT VACCINES FOR THE PREVENTION OF GROUPER SLEEPY DISEASE IRIDOVIRUS (GSDIV) INFECTION IN HUMPBACK GROUPER, Cromileptes altivelis

Infection of grouper sleepy disease iridovirus (GSDIV) which is a member of Megalocytivirus causes mass mortalities in marine fish in Indonesia. This study was conducted to know the effectiveness of recombinant subunit vaccine of GSDIV with an addition of adjuvants against GSDIV infection. Inactive bacteria Eschericia coli containing recombinant MCP-GSDIV protein was added with montanide ISA adjuvant at a ratio of 3:7. The vaccine was administered to humpback grouper, Cromileptes altivelis by intramuscular and intraperitoneal injection at a dose of 0.1 mL/fish. Test of the vaccine in humpback grouper was performed in four replicates (four trials). Results of the vaccination showed that the recombinant protein vaccine added with the adjuvant increased immunity of humpback grouper, indicated by higher relative percent survival (RPS= 77.78%) compared to negative control (PBS) and 50% higher compared to protein control (pET Sumo CAT) at two weeks post vaccination. The RPS values of the recombinant protein vaccine were still higher (53.57%-72.73%) than those of the control vaccine and 25%-53.33% of the protein control in the 4th week post vaccination. GSDIV detection by PCR showed that MCP-GSDIV-DNA and pET Sumo CAT-DNA were not detected in the vaccinated fish after one, two, three, and four weeks post vaccination. The fish died in both of vaccinated and control groups after experimental challenge with GSDIV were found to be infected with GSDIV. It can be stated that recombinant subunit vaccine of GSDIV with the addition of montanide ISA adjuvant could be used to prevent and diminish mortalities of grouper against GSDIV infection.

Benih Keturunan Induk Ikan Nila yang Divaksinasi pada Tingkat Kematangan Gonad-2 Lebih Tahan Terhadap Infeksi Streptococcus agalactiae (RESISTANCE OF TILAPIA (OREOCHRIMIS NILOTICUS) FRY VACCINATED AT DIFFERENT GONADAL DEVELOPMENTAL STAGES TOWARD STREPTOCOCCUS AGALACTIAE INFECTION)

The aim of this study was to evaluate the effectiveness of vaccination based on gonad maturationstages on tilapia brood stocks in which the released antibodies was able to be transferred to the seed.Vaccine composed with whole cells and extracellular product (ECP) was injected at stage 2 and stage 3 ofthe gonad development stages at concentration of 109 CFU mL1 as much as 4 mL to 1 kg of brood fish.Control fish was unvaccinated treatment. Challenge study at seed was conducted by immersing S. agalactiaefor 30 minutes at 7, 14, 21, and 28 days post hatching (DPH) in 107 CFU/mL. Antibody levels on broodstocks, eggs, and body fluids of seed, and relative percentage survival (RPS) of seed post challenge studywere evaluated. The results showed that stage 2 of gonad developmental stages was found on 7 days postinitial spawning and stage 3 found on 14 days post initial spawning of brood fish. Vaccinated done in stage 2 of gonad developmental stages gave immunoglobulin serum in brood (0,166±0,001), egg (0,165±0,002),and seed aged 7, 14, 21, and 28 days post hatching (0,164±0,002, 0,162±0,005, 0,155±0,006, and 0,14±0,008respectively) were significantly higher (P<0,05) compared to other treatment. Challenged test that doneby immersing with S. agalactiae suspension on larval aged 7, 14, 21, and 28 days had highest RPS(95,24%, 83,33%, 72,22%, and 56,02% respectively) formed on seed from brood stock vaccination in gonaddevelopment stage 2. Vaccination in tilapia brood stocks at stage 2 of gonad developmental stages gavehighest protection by maternal immunity to the seed against S. agalactiae.

Feeding pyridoxine prevents Saprolegnia parasitica infection in fish Labeo rohita

A 60-day experiment was carried out to delineate the role of dietary pyridoxine (DP) in Labeo rohita fingerlings in modulating immunity and prevention of fungal infection. Two hundred and seventy fingerlings were randomly distributed into three treatments in triplicates. Three iso-caloric and iso-nitrogenous purified diets were prepared with graded levels of pyridoxine. Three experimental groups were C (0.0% DP), T1 (0.01% DP) and T2 (0.02% DP). The role of dietary pyridoxine in modulating immunity and prevention of fungal infection was assessed by haemato-immunological parameters like erythrocyte counts (EC), leucocyte counts (LC), haemoglobulin (Hb), packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), nitro-blue tetrazolium (NBT), phagocytic activity, albumin, globulin, total plasma protein, albumin/globulin and by challenge study with Saprolegnia parasitica, where relative percentage survival (RPS) were recorded. Hb, PCV, MCV, MCH, NBT, total plasma protein, albumin, globulin contents, lysozyme and phagocytic activity was significantly (P < 0.05) higher in DP fed group. Significantly (P < 0.05) higher RPS was recorded from T2 group fed with 0.02% DP for 45 days. Hence, DP has the capacity to stimulate nonspecific immunity and increase resistance to S. parasitica infection in L. rohita fingerlings.

Molecular cloning, expression and the adjuvant effects of interleukin-8 of channel catfish (Ictalurus Punctatus) against Streptococcus iniae.

Interleukin-8 (IL-8) as an important cytokine involving in inflammatory and immune response, has been studied as effective adjuvants for vaccines in mammals. However, there are fewer reports about the characterization and adjuvant effects of IL-8 in fish. In this study, cloning and sequence analysis of IL-8 coding region of channel catfish (Ictalurus punctatus) were conducted, mature IL-8(rtIL-8) was expressed and evaluated for its adjuvant effects on the immunoprotection of subunit vaccine encoding α-enolase (rENO) of Streptococcus iniae from several aspects in channel catfish. The results showed co-vaccination of rENO with rtIL-8 enhanced immune responses including humoral and cellular immunity, with higher relative percent survival(RPS,71.4%) compared with the moderate RPS of rENO alone(50%) against S. iniae infection at 4 week post vaccination. While rtIL-8 failed to maintain long-lasting immune protection, only with RPS of 26.67% in rENO + rtIL-8-vaccinated fish compared with that of rENO alone(20%) at 8 week, signifying that IL-8 hold promise for use as potential immunopotentiator in vaccines against bacterial infections in fish, whereas it is insufficient to extend the immunoprotection for long time, and further studies are required to understand the mechanisms of IL-8 used as an adjuvant and seek for more effective way to strengthen the adjuvanticity of IL-8.

Development of three-valent vaccine against streptococcal infections in olive flounder, Paralichthys olivaceus

Streptococcus iniae and S. parauberis types I and II are major bacterial pathogens affecting the olive flounder, Paralichthys olivaceus. With the introduction of formalin-killed vaccine for S. iniae, recent epidemiological studies have indicated that S. parauberis infections are becoming increasingly severe and frequent in the olive flounder farming industry. Here, the formalin-killed bacterins were used to develop a three-valent vaccine against three streptococcal infections, and its efficacy was assessed in laboratory- and field-challenge trials. In the laboratory-challenge test, it obtained 75%, 75% and 90% relative percent survival (RPS) values in vaccinated olive flounders challenged with S. iniae, S. parauberis type I, and S. parauberis type II, respectively. For the field-challenge trial, the three-valent vaccine was administered to over 100,000 olive flounders, which were compared to non-injected control fish. An RPS of 74% was observed when S. parauberis infection naturally occurred at 11 fish farms. Serum antibody measurements using olive flounder immunoglobulin-specific antibodies indicated that immunized fish had significantly higher serum antibody levels than control fish up to 6months post-vaccination. These results demonstrate that the three-valent vaccine using formalin-killed bacterins could effectively protect olive flounders against three major streptococcal infections. Statement of relevanceThe olive flounder, P. olivaceus, is an economically important fish species in Northeast Asia and the most valuable fish species in South Korea, corresponding to 56.5% of the total mariculture production in 2010 (Baeck et al., 2006). However, streptococcal infections often break out among farmed olive flounders subjected to certain conditions (e.g., high stocking density, poor water quality and high water temperature), leading to mass mortality and severe economic losses (Baeck et al., 2006; Cho et al., 2008) Antibiotic treatments are usually recommended to address such outbreaks, but the prevalence of multi-drug-resistant bacteria means that antibiotic compounds are not always able to eradicate the infections (Creeper and Buller, 2006).The Gram-positive bacteria, Streptococcus iniae and S. parauberis, cause streptococcosis and have severe effects on aquaculture of the olive flounder, barramundi, channel catfish, European sea bass, rainbow trout, striped bass, tilapia, turbot and yellow tail (Gudmundsdóttir and Björnsdóttir, 2007; Han et al., 2011). In the olive flounder, fish infected with S. iniae usually show severe pathological changes, such as darkening, exophthalmia, rectal hernia, abdominal distension, ascites and congestion of intestinal organs, whereas fish infected with S. parauberis rarely exhibited any remarkable pathological finding except for darkening of the skin (Kim et al., 2006).Some vaccines have been reported to prevent S. iniae infection, showing high relative percent survival (RPS) among olive flounders in laboratory trials; such vaccines are now commercially available (Cheng et al., 2010a). However, recent epidemiological studies have suggested that S. parauberis infection has increased and become more severe and frequent as much as infections of S. iniae, which was previously considered the major bacterial pathogen among olive flounder (Park et al., 2012; Park, 2009; Perera et al., 1998). S. parauberis can be divided into two serotypes (types I and II) based on serological testing of a distinctive capsular polysaccharide layer (Bromage and Owens, 2002; Plant and Lapatra, 2011). Since the two serotypes possess specific antigenic characteristics, a vaccine should be able to address both types in order to effectively prevent S. parauberis infection.The present study was undertaken to develop a three-valent vaccine against both serotypes of S. parauberis plus S. iniae using formalin-killed vaccine preparation method. The efficacy of this vaccine against the major streptococcal infections of olive flounder was calculated as RPS and conducted in both laboratory trials and large-scale field trial. This vaccine will be highly useful and more advantageous since the use of this does not require several antibiotics which means safer food production and more sustainable environment.

Growth, non-specific immunity and disease resistance of Labeo rohita against Aeromonas hydrophila in biofloc systems using different carbon sources

A 60day growth trial was carried out to evaluate biofloc based rearing system for Labeo rohita fingerlings. The C/N ratio of 15 was maintained in four biofloc treatments (T1, T2, T3 and T4) using indirect and long-lasting carbon sources (tapioca, wheat, corn and sugar bagasse) along with one control (C). Each group consisted of triplicate tanks and each tank was stocked with 50 fingerlings (4.80g±0.12) of L. rohita. Fishes were challenged with A. hydrophila 60days post rearing and relative percentage survival (RPS) was recorded over 14days post challenge. Serum samples were collected on 20, 40 and 60days interval of sampling for the evaluation of non-specific immune and stress parameters. The results revealed that fishes reared in tapioca based biofloc system showed significantly (p<0.05) higher serum protein, serum albumin, total immunoglobulin, respiratory burst activity, myeloperoxidase activity and lower serum glucose and serum cortisol when compared to wheat, corn, sugar bagasse based biofloc system and control. Growth, feed conversion ratio (FCR), feed efficiency ratio (FER), and specific growth rate (SGR) of fishes in the biofloc treatments were significantly better (p<0.05) than control. Tapioca based biofloc treatment showed significantly (p<0.05) higher RPS (66.66%) than other treatments. The study suggests that the fishes reared using in-situ biofloc produced by tapioca can enhance growth and non-specific immune responses under zero water exchange system and hence ensures sustainability. Statement of relevance•Zero water exchange reduces water scarcity and pollution problems•Improves welfare of the animals by stimulating immunity and protection against diseases•In-situ production of valuable microbial protein thus reducing dependence on fishmeal.

Immune response of flounder (Paralichthys olivaceus) was associated with the concentration of inactivated Edwardsiella tarda and immersion time

In the optimization of immersion strategy, vaccine concentration and immersion time are two key factors needed to be considered, which largely determined the immune efficacy. In this work, the healthy flounder were vaccinated in formalin-killed Edwardsiella tarda at four concentrations (106, 107, 108, 109CFUml−1) for three immersion times (30, 60, 90min), respectively. At the 6th week post vaccination, the flounders were challenged with live E. tarda, and the relative percent survival (RPS) of flounder in 109–30, 108–60, 108–90 and 107–90min groups were 70%, 78%, 74% and 65%, respectively, which were much higher than the other vaccination groups. Meanwhile, the sIg+ cells in the leucocytes of peripheral blood (PBL), spleen (SL), head kidney (HKL) were monitored by flow cytometry, and the specific sera and mucosal antibodies were measured by indirect ELISA for 6 weeks. The results showed that the proportions of sIg+ cells in PBL, SL and HKL of vaccinated fish were significantly higher than the untreated fish since the 2nd week (P<0.05), and the fish in 109–30, 108–60, 108–90 and 107–90min groups exhibited stronger responses than other groups, especially the 108–60min group displayed the strongest response, which reached the peaks (54.63% in PBL, 37.21% in SL, 36.51% in HKL) at the 5th week. ELISA assay showed that the kinetics of specific antibodies in sera were similar to the variation of sIg+ cells, and the higher antibody levels were also detected in the four groups with higher RPS and stronger sIg+ response, whereas the mucosal antibody showed a faster response, which were significantly higher than the control since the 1st week (P>0.05). These results demonstrated that the higher RPS was closely associated with stronger immune response, and immersion with formalin-inactivated E. tarda under 108CFUml−1 for 60min induced the highest immune response of flounder against E. tarda bacterin, which might be applied for the control of edwardsiellosis in flounder.

Microencapsulation of a putative probiotic Enterobacter species, C6-6, to protect rainbow trout, Oncorhynchus mykiss (Walbaum), against bacterial coldwater disease.

Flavobacterium psychrophilum is the causative agent of bacterial coldwater disease (BCWD), which has a major impact on salmonid aquaculture globally. An Enterobacter species, C6-6, isolated from the gut of rainbow trout, Oncorhynchus mykiss (Walbaum), has been identified as a potential probiotic species providing protection against BCWD. This study examined the effects of alginate microencapsulation on the protective efficacy of C6-6 against BCWD in vivo when administered to rainbow trout fry orally or by intraperitoneal (IP) injection. Viable C6-6 bacteria were microencapsulated successfully, and this process (microencapsulation) did not significantly deteriorate its protective properties as compared to the administration of non-microencapsulated C6-6 bacteria. Both oral and IP delivery of C6-6 achieved significantly better protection than control treatments that did not contain C6-6 bacteria. The highest relative percent survival (RPS) resulted from IP delivery (71.4%) and was significantly greater than the highest oral RPS (38.6%). Successful intestinal colonization was not critical to protective effects of C6-6. The study showed that C6-6 administration, with or without encapsulation, was a viable choice for protecting fry from BCWD especially when administered intraperitoneally.

Effect of dietary supplemented andrographolide on growth, non-specific immune parameters and resistance against Aeromonas hydrophila in Labeo rohita (Hamilton)

The present study evaluated the effect of dietary andrographolide (EC 50%) on growth, non-specific immune parameters and disease resistance against Aeromonas hydrophila infection in Indian major carp, Labeo rohita fingerlings. Fishes were fed with formulated diet containing andrographolide as T0 (0.00%), T1 (0.05%), T2 (0.10%), T3 (0.20%), T4 (0.40%) and T5 (0.80%) for 42 days. Fishes were challenged with A. hydrophila 42 days post feeding and relative percentage survival (RPS) was recorded over 14 days post challenge. Blood and serum samples were collected for nonspecific immune parameters on 14, 28 and 42 days of feeding and growth performance was evaluated at the end of experiment. The results revealed that fishes fed with andrographolide showed significant (p < 0.05) increase in NBT levels, myeloperoxidase activity, phagocytic activity, serum lysozyme activity, and serum antiprotease activity when compared to the control group. The weight gain, specific growth rate, feed conversion ratio and protein efficiency ratio of fishes fed with andrographolide were found to be significantly (p < 0.05) differed compared with control. Dietary andrographolide at the level of 0.10% showed significantly (P < 0.05) higher RPS (74.06%) against A. hydrophila infection than control. The results revealed that andrographolide supplemented diet has a stimulatory effect on non-specific immune parameters along with improved growth performance and increased disease resistance against A. hydrophila infection in L. rohita fingerlings.

Effect of orally administered azadirachtin on non-specific immune parameters of goldfish Carassius auratus (Linn. 1758) and resistance against Aeromonas hydrophila

Modulation of the immune responses using active bio-ingredients as a possible prophylaxis measure has been novel prospect for aquaculture. The present study evaluated the effects of azadirachtin EC 25% on non-specific immune responses in goldfish Carassius auratus and resistance against pathogenic bacteria Aeromonas hydrophila. The experimental trial for effects of azadirachtin on immuno-haematoloical parameters in goldfish was conducted by feeding the various levels of azadirachtin as control T0 (without azadirachtin), T1 (0.1%), T2 (0.2%), T3 (0.4%), T4 (0.8%) and T5 (1.6%) for a period of 28 days. Fishes were challenged with A. hydrophila 28 days post feeding and relative percentage survival (%) was recorded over 14 days post infection. Immuno-haematoloical (total erythrocyte count, total leukocyte count, haemoglobin, packed cell volume, NBT activity, phagocytic activity, serum lysozyme activity, myeloperoxidase activity, total immunoglobulin) and serum biochemical parameters (serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT) and blood glucose) of fishes were examined at 14 and 28 days of feedings. Fish fed with azadirachtin, showed significantly (p < 0.05) enhanced TEC, TLC, Total Ig, total protein, NBT activity, serum lysozyme activity and myeloperoxidase level in different treatment groups in comparison with control group. Similarly, SGOT, SGPT and blood glucose level were found to be significantly (p < 0.05) high but PCV and Hb did not differ significantly (p > 0.05) in the treatment groups compared to control groups. Azadirachtin at the concentration of 4 g kg−1 showed significantly (p < 0.05) higher relative percentage survival (42.60%) when compared with the control against A. hydrophila infection. This study indicated that azadirachtin EC 25% (4 g kg−1) showed higher NBT activity, serum lysozyme, protein profiles, leukocyte counts and resistance against A. hydrophila infection and thus, can be used as a potential immunostimulant in aquaculture.

Construction and evaluation of an Edwardsiella ictaluri fhuC mutant

Edwardsiella ictaluri is a Gram-negative facultative intracellular pathogen causing enteric septicemia in channel catfish. Iron is an essential micronutrient needed for bacterial virulence, and to acquire iron, many Gram-negative bacteria secrete ferric iron chelating siderophores. The ferric hydroxamate uptake (Fhu) system consists of four genes (fhuC, fhuD, fhuB, and fhuA), and is involved in the uptake of hydroxamate type siderophores across bacterial membranes. However, the Fhu system and its importance in E. ictaluri virulence have been uninvestigated. Here, we present construction and evaluation of an E. ictaluri ΔfhuC mutant. The E. ictaluri fhuC gene was deleted in-frame by allelic exchange, and the mutant's growth in media and virulence in catfish were determined. Our results indicated that deletion of the E. ictaluri fhuC gene did not affect the growth of E. ictaluri largely in both iron-replete and iron-depleted media. Addition of ferric iron sources into the iron-depleted medium improved the growth of both E. ictaluri ΔfhuC and wild type (WT). Catfish mortalities indicated that E. ictaluri ΔfhuC mutant was attenuated 2.05-fold compared with the parent strain. The catfish immunized with the E. ictaluri ΔfhuC mutant showed a high relative percent survival rate (97.50%) after re-challenge with the WT E. ictaluri strain. Taken together, our data indicates that the fhuC gene contributes to E. ictaluri virulence.

Development and preliminary validation of an antibody filtration-assisted single-dilution chemiluminometric immunoassay for potency testing of Piscirickettsia salmonis vaccines

Challenge with live pathogens could be substituted by serology for many veterinary diseases, however little progress has been made in the development of alternative batch vaccine potency tests for fish. This study reports the development and preliminary validation of a single-dilution filtration-assisted chemiluminometric immunoassay (SD FAL-ELISA) applied to measure anti Piscirickettsia salmonis IgM in individual or pooled serum and mucus samples. The assay was set up to test a single-dilution of the sample. Serum SD FAL-ELISA yielded a sensitivity of 90% and a specificity of 96%. SD FAL-ELISA was applied to evaluate pooled and individual samples from P. salmonis challenge assessments. Relative-light units values (RLU) obtained by SD FAL-ELISA were proportional to antibody levels in serum. RLU values obtained from pooled and individual serum samples increased with the observed relative percent survival (RPS) values, indicating a correlation between protection and specific IgM levels. Results obtained for specific IgM in mucus samples was not related to the RPS, but discriminated the vaccine that yielded high RPS (86.4%) from the others (40.9 and 54.5%). This is the first report on the development of an indirect high-throughput serological assessment for P. salmonis vaccine potency testing using both pooled or individual serum and cutaneous mucus samples.