Relative Percent Survival Research Articles

Mannose functionalized biomimetic nanovaccine enhances immune responses against tilapia lake virus

Tilapia lake virus disease (TiLVD) caused by tilapia lake virus (TiLV) is a lethality truculence viral disease endangering the sustainable development of tilapia aquaculture. DNA vaccine present immense application potential in disease prevention and control, but the low gene transfection efficacy of naked DNA vaccine imposes restrictions on itself immune response that should have been induced. In this study, we constructed a biomimetic nanodelivery system (Cs-pS2@M-M) using the mannose modified erythrocyte membrane acted as a DNA vaccine carrier. The nanoparticle has a typical core-shell structure with a particle size of ~100 nm, and ensures sustained and efficient expression of plasmid DNA in muscle and spleen tissue. Intramuscular immunization of tilapia to evaluate the efficacy of the vaccine, the results showed that it has a high-effective induction of serum antibody production, enzyme activity, and immune-related gene expression compared to the same dose (10 μg/g) of non-mannosylated Cs-pS2@M nanoparticles and naked DNA vaccine. Notably, the TiLV challenge trial show that Cs-pS2@M-M provided 76.9% relative percentage survival (RPS), which was 26.9% higher than the 50.0% of the naked DNA vaccine (pS2), and not only that, it was 15.4% higher than that of Cs-pS2@M without mannose modification. These results showed that Cs-pS2@M-M biomimetic nanovaccine constructed in this study elicited robust immune responses to protect tilapia from TiLV challenge, and erythrocyte membrane-coated nanoparticles strategy show enormous promise potential as a vaccine delivery platform for aquatic animals.

Immunization with recombinant actin, enolase and 26S proteasome protects Epinephelus coioides against Cryptocaryon irritans

Cryptocaryon irritans (Brown, 1951) is a ciliated protozoan that infects various marine fish, causing severe economic losses in the aquaculture industry. We identified, expressed and purified three candidate antigens – actin, enolase and 26S proteasome subunit P45 protein family (26S) – from Cryptocaryon irritans and determined their roles in protecting grouper (Epinephelus coioides) against C. irritans infections as subunit vaccines. Serum immobilization titer was also carried out at 2 and 4 weeks post-immunization. Results indicated that all putative proteins highly corresponded with their orthologs from other ciliate species and contained multi-antigenic determinants (8–12). The CiActin, CiEnolase and Ci26S were optimized for successful expression in the Escherichia coli BL21 system. We found that all recombinant CiActin, CiEnolase, and Ci26S proteins conferred protection against C. irritans, with the relative percentage survival (RPS) of 36.7%, 23.3% and 46.7%, respectively. Additionally, the parasite abundance of infected groupers reduced by 70.1%, 15.4% and 67.5% in rCiActin, rCiEnolase and rCi26S groups compared to the PBS control. Conversely, the serum immobilization titers increased in all immunization groups, with the highest groups being rCiActin and rCi26S. These findings indicate that the rCiActin and rCi26S can provide a high level of immune protection against C. irritans infection in grouper and are therefore considered candidate C. irritans vaccines.

Development and Evaluation of a Bicistronic DNA Vaccine against Nervous Necrosis Virus in Pearl Gentian Grouper (Epinephelus lanceolatus × Epinephelus fuscoguttatus).

Nervous necrosis virus (NNV) can cause enormous economic losses in mariculture. Vaccines are promising ways to control the disease. In this study: the interferon regulatory factor 3 (IRF3) gene of pearl gentian grouper was cloned and functionally analyzed; then a bicistronic DNA vaccine encoding both capsid protein (CP) and IRF3 was constructed; then the cellular, humoral, and local immune responses in the grouper after immunization were investigated; and then the protective effects after the NNV challenge were investigated. The results showed that the vaccine successfully expressed CP and IRF3. After immunization, the lymphocytes were recruited at the injection site in the muscles. The percentage of sIgM+ lymphocytes in the head, kidney, and spleen significantly increased and peaked at 28.8 ± 3.1% and 42.6 ± 4.2% at the 3rd to 4th weeks. Six immune-related genes were significantly up-regulated. In the meantime, the total antibodies, anti-NNV specific antibodies, and neutralizing antibody titers in serum increased. After the challenge with 105, 106 or 107 TCID50/fish, the relative percent survival rate was 81.25%, 73.91%, and 66.67%, respectively. In 106 TCID50/fish groups, the percentages of sIgM+ lymphocytes, antibodies, and the viral load were investigated. In conclusion, the bicistronic vaccine significantly induced humoral and cellular responses in pearl gentian grouper and provided effective protection against NVV infection.

Effective protection against Ranavirus-type grouper iridovirus and Vibrio harveyi by priming a bivalent GrouperVAC-Irido-R-Vh followed by boosting a monovalent V. harveyi vaccine

Natural outbreaks of Ranavirus-type grouper iridovirus (GIV-R) and/or Vibrio harveyi result in massive mortalities and severe economic losses in grouper industry. However, effective vaccines against both pathogens have not yet been applied commercially. In this study, a water-in-oil formation of formalin-killed bivalent vaccine (GrouperVAC-Irido-R-Vh) against both GIV-R and V. harveyi and a monovalent vaccine (GrouperVAC-Vh) against V. harveyi were developed and evaluated both under laboratory and field trial tests. The result showed that the single intraperitoneally-injected grouper juveniles (N = 35) with bivalent GrouperVAC-Irido-R-Vh under laboratory condition were protected against GIV-R and V. harveyi at 21 days post vaccination (dpv) with relative percent survivals (RPS) of >90% and 80%, respectively. In a field trial test (N = 7500), the effective protections of single intraperitoneally-injected fish were also observed in vaccinated grouper juveniles at 90 dpv, with RPSs of 87.5% and 70.4% against GIV-R and V. harveyi challenge, respectively, indicating that the bivalent GrouperVAC-Irido-R-Vh conferred effective protections against both GIV-R and V. harveyi infection for at least 3 months. Moreover, based on primary injection of GrouperVAC-Irido-R-Vh, a booster immunization of the single component of GrouperVAC-Vh (N = 4500) can prolong conferring effective protection against vibriosis caused by V. harveyi from 3 months to 9 months with an RPS of 88.2%. Given that the large-sized grouper is much less sensitive to GIV-R but remains highly susceptible to V. harveyi, we propose that the optimal immunization programs against GIV-R and V. harveyi are primary immunization with the bivalent GrouperVAC-Irido-R-Vh vaccine followed by boosting a monovalent GrouperVAC-Vh vaccine.

Protective immunity induced by ankyrin repeat-containing protein-based DNA vaccine against rock bream iridovirus (RBIV) in rock bream (Oplegnathus fasciatus)

Rock bream iridovirus (RBIV) causes severe mass mortalities in rock bream (Oplegnathus fasciatus) and remains an unsolved problem in Korea aquaculture industry. In this study, we assessed the potential of ankyrin repeat (ANK)-containing proteins to induce protective immunity in RBIV-infected rock bream. Rock bream administered with ankyrin repeat-containing protein-based DNA vaccine (200 ng/fish) exhibited significant protection against at 4 and 8 weeks post vaccination to infected with 6.7 × 105 RBIV at 23°C; relative percent survival (RPS) of 60.04% and 40.1%, respectively. Furthermore, survivors from the first infection were strongly protected from RBIV (1.1 × 107) re-infection at 70 days post infection, as 100% RPS was observed and without clinical signs of RBIV diseases. Moreover, TLR3 (9.5-fold), TLR9 (5.2-fold), MyD88 (15.9-fold), Mx (55.5-fold), ISG15 (19.0-fold), PKR (24.2-fold), MHC class I (5.1-fold), perforin (6.5-fold), Fas (6.4-fold), Fas ligand (7.1-fold), caspase8 (5.0-fold), caspase9 (12.5-fold), and caspase3 (6.3-fold) responses were significantly elevated in the muscle (vaccine injection site) of ANK-based DNA vaccinated fish at 7 days post vaccination. However, inflammatory cytokines (IL1β, IL8, and TNFα) were not enhanced in the vaccinated rock bream. Moreover, ANK gene may be a good candidate to detect RBIV infection or in revealing specific information to elucidate the pathogenic mechanisms underlying RBIV infection. In summary, ANK-based DNA vaccination in rock bream induced TLR- and IFN-mediated or apoptosis-related immune responses and suggest efficient preventive measures against RBIV.

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.

A high efficacy DNA vaccine against Tilapia lake virus in Nile tilapia (Oreochromis niloticus)

Tilapia lake virus (TiLV) is the main tilapia-infecting virus worldwide, causing serious economic losses to the tilapia aquaculture. However, there is no vaccine for this viral disease. Here, TiLV ORF10 (TiLV-ORF10) encoding a protein with abundant epitopes was cloned into the eukaryotic expression vector pcDNA3.1 as a vaccine candidate, and then used to evaluate the protective effects against TiLV in Nile tilapia (Oreochromis niloticus). RT-PCR and Western blot analyses confirmed pcDNA3.1–ORF10 expression in tilapia. The transcription levels of immunity-related genes such as immunoglobulin M, Toll-like receptor 2, myeloid differentiation factor 88, interleukin 8, tumor necrosis factor alpha, gamma-IFN, and nuclear factor κB were significantly upregulated in the spleen, liver, and kidney of the vaccinated tilapias (P < 0.05). TiLV challenge experiments showed that relative percent survival (RPS) was significantly enhanced in fish vaccinated with the DNA vaccine. Moreover, the RPS was significantly higher in fish vaccinated with a high dose of the DNA vaccine (85.72% RPS at a dosage of 45 μg DNA vaccine plasmid). Meanwhile, vaccination with pcDNA3.1–ORF10 significantly reduced virus replication, as evidenced by the lower amount of virus in the spleen, liver, and kidney of vaccinated tilapia than tilapias vaccinated with the empty pcDNA3.1 plasmid. Thus, pcDNA3.1–ORF10 could induce protective immunity in tilapia and may be a potential vaccine candidate for controlling disease caused by TiLV.

Development of a hyper-adhesive and attenuated Edwardsiella ictaluri strain as a novel immersion vaccine candidate in yellow catfish (Pelteobagrus fulvidraco)

Edwardsiella ictaluri, a Gram-negative intracellular pathogen, is the causative agent of enteric septicemia in channel catfish, and catfish aquaculture in China suffers heavy economic losses due to E. ictaluri infection. Vaccination is an effective control measure for this disease. In this study, an attenuated E. ictaluri strain was acquired through deletion mutation of the T3SS protein eseJei, and the ΔeseJei strain fails to replicate in the epithelioma papillosum of carp cells. The type 1 fimbria plays a pivotal role in the adhesion of E. ictaluri, and it was found in this study that deletion of −245 to −50 nt upstream of fimA increases its adhesion to around five times that of the WT strain. A hyper-adhesive and highly attenuated double mutant (ΔeseJeiΔfimA-245--50 strain) was constructed, and it was used as a vaccine candidate in yellow catfish via bath immersion at a dosage of 1 × 105 CFU/mL. It was found that this vaccine candidate can stimulate protection when challenged with E. ictaluri HSN-1 at 5 × 107 CFU/mL (∼20 × LD50). The survival rate was 83.61% for the vaccinated group and 33.33% for the sham-vaccinated group. The RPS (relative percent of survival) of the vaccination trial reached 75.41%. In conclusion, the ΔeseJeiΔfimA-245--50 strain developed in this study can be used as a vaccine candidate. It excels in terms of ease of delivery (via bath immersion) and is highly efficient in stimulating protection against E. ictaluri infection.

Recombinant Lactococcus lactis Expressing Grass Carp Reovirus VP6 Induces Mucosal Immunity Against Grass Carp Reovirus Infection.

Grass carp haemorrhagic disease caused by grass carp reovirus II is a serious disease of the aquaculture industry and vaccination is the only effective method of GCRV protection. In this study, Lactococcus lactis was used as oral vaccine delivery to express the GCRV II VP6 protein. We evaluated the protective efficacy of the live vaccine strain to induce mucosal immune protection. After oral administration, the recombinant strains remained in the hindgut for antigen presentation and increased the survival rate 46.7% and the relative percent survival 42.9%, respectively versus control vaccination. Though L. lactis alone can induce the inflammatory response by stimulating the mucosal immune system, the recombinant L. lactis expressing VP6 greatly enhanced nonspecific immune responses via expression of immune related genes of the fish. Furthermore, both systemic and mucosal immunity was elicited following oral immunization with the recombinant strain and this strain also elicited an inflammatory response and cellular immunity to enhance the protective effect. L. lactis can therefore be utilized as a mucosal immune vector to trigger high levels of immune protection in fish at both the systemic and mucosal levels. L. lactis is a promising candidate for oral vaccine delivery.

Characterization of Co-Stimulatory Ligand CD80/86 and Its Effect as a Molecular Adjuvant on DNA Vaccine Against Vibrio anguillarum in Flounder (Paralichthys olivaceus).

The CD80/86 molecule is one of the important co-stimulatory ligands and involves antigen-specific immune responses by ligating with CD28 and then delivering the required second signal to T-cell activation. In this study, a CD80/86 homolog was identified, and its expression characteristics were studied in flounder (Paralichthys olivaceus). The open reading frame (ORF) of CD80/86 is 906 bp, encoding 301 aa, and the extracellular amino acid sequence encoded two IgV- and IgC-like structural domains; fCD80/86 is highly expressed in head kidney, peripheral blood leukocytes (PBLs), and spleen, and has relatively high expression in muscle. Antibodies specific for CD80/86 were produced, and CD80/86 was colocalized with MHCII+, CD40+, and CD83+ leukocytes but not with IgM+, CD3+, or CD4+ lymphocytes. The cloned CD80/86 in flounder shares conserved structural features with its mammalian counterparts and is mainly distributed on antigen-presenting cells. Based on these data, CD80/86 as an adjuvant to enhance the immune response of DNA vaccine was investigated. A bicistronic DNA vaccine expressing both CD80/86 and the outer membrane protein (OmpK) of Vibrio anguillarum (p-OmpK-CD80/86) was successfully constructed. After immunization, p-OmpK-CD80/86 could induce the upregulation of the proportion of IgM+ and CD4+ cells in flounder, compared to the p-OmpK- or p-CD80/86-immunized group; CD28 genes were significantly induced in the p-CD80/86 and p-OmpK-CD80/86 groups. Compared to the p-OmpK group, the higher expression of CD83, MHCI, CD4, CD8, and IL-2 was detected at the injection site. The relative percent survival (RPS) produced by p-OmpK-CD80/86 is 66.11% following the V. anguillarum challenge, while the RPS of p-OmpK or p-CD80/86 is 46.30% and 5.56%, respectively. The results revealed that CD80/86 is mainly found in antigen-presenting cells, and could help elicit humoral immune responses in teleost through the CD80/86-CD28 signaling pathway involving CD4+ lymphocytes.

Cross-protective efficacy of a live-attenuated Edwardsiella ictaluri vaccine against heterologous Edwardsiella piscicida isolates in channel and channel × blue catfish hybrids.

Edwardsiella piscicida is a growing problem for catfish aquaculture in the southeastern United States, particularly in channel (Ictalurus punctatus) x blue (I. furcatus) catfish hybrids. Research has shown E.piscicida isolates recovered from farmed catfish in Mississippi form at least five discrete phyletic groups, with no apparent differences in virulence in channel and hybrid catfish. Laboratory trials have shown a live-attenuated E.ictaluri vaccine (340X2) cross-protects against at least one E.piscicida isolate (S11-285) in channel and hybrid catfish, although it is unknown if this protection exists for other E.piscicida variants. To this end, channel and hybrid catfish were immunized by immersion with E.ictaluri 340X2. Thirty days later, fish were challenged by intracoelomic injection with representative E.piscicida variants from each phyletic group. Relative percent survival (RPS) for hybrids ranged from 54.7% to 77.8%, while RPS in channels ranged from 80.5% to 100%. A second study investigated whether channel and hybrid catfish exposed to heterologous E.piscicida isolates were similarly protected against wild-type E.ictaluri. Fish were exposed by bath immersion to representative E.piscicida isolates from each phyletic group. Thirty days post-immunization, fish were challenged by immersion with wild-type E.ictaluri isolate S97-773. Regardless of variant, previous exposure to heterologous E.piscicida isolates significantly improved survival following E.ictaluri challenge. These findings suggest the presence of shared and conserved antigens among E.piscicida and E.ictaluri that could be exploited by application of polyvalent or cross-protective vaccines.

Dietary astaxanthin modulated the performance, gastrointestinal histology, and antioxidant and immune responses and enhanced the resistance of Litopenaeus vannamei against Vibrio harveyi infection

Astaxanthin (AX) is one of the commonly used feed supplements to enhance the growth performance and provide antioxidant and immune functions of several aquatic animals. In the current study, juveniles of white-leg shrimp (Litopenaeus vannamei) with mean initial weight of 0.340 ± 0.041 g were fed with diets supplemented with 0 (control), 25, 50, 100, and 200 mg/kg feed for 8 weeks. At the end of the feeding trial, shrimps were exposed to Vibrio harveyi, and their mortality rates were observed for additional 10 days. The growth indices in the AX-fed groups were significantly (P < 0.05) higher than what were observed in shrimps in the control group. Dietary AX stimulated the final weight, weight gain, and specific growth rate and optimum growth levels were achieved at 100–200 mg AX/kg feed. Furthermore, the AX-enriched diets significantly enhanced feed intake more than the control diet, and the amount of AX had no effects on feed conversion ratios. In comparison to the control group, the AX-fed animals had significantly (P < 0.05) higher villi length, villi width, and absorption area and their optimum values were observed at 100–200 mg AX/kg feed treatments. Moreover, the intestinal morphometry especially villi and its crypt, both internal and external tunica muscularis, and submucosal tissues did not show any inflammatory and/or degenerative changes in AX-fed shrimp. Furthermore, the dietary AX at escalating levels linearly and quadratically enhanced (P < 0.05) the activities of serum superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and increased levels of total antioxidant capacity and reduced glutathione. In addition, malondialdehyde levels decreased significantly in AX-fed animals, and the highest levels were observed in the control group (without AX). The expression levels of cMn-SOD, CAT, and GPx genes were significantly upregulated in the hepatopancreas of L. vannamei fed with AX-enriched diets (especially in the 200-mg/kg feed treatment) as compared with the control diet. The immunity indices of the AX treatments (hematocyte count, total protein, lysozyme, phagocytic activity, and phenoloxidase) of L. vannamei were linearly (P < 0.05) and quadratically (P < 0.05) increased. This study revealed the antibacterial activity of AX against V. harveyi abundance. After the bacterial challenge, feeding L. vannamei with dietary AX significantly increased (P < 0.05) the relative percentage of survival, especially in the treatment of 200-mg/kg diet (82.7%). The findings of the current study demonstrate that dietary AX (100–200 mg/kg feed) was effective in enhancing the growth, antioxidant status, immune response, and increasing the resistance of L. vannamei against V. harveyi infection.

Immune-antioxidant trait, Aeromonas veronii resistance, growth, intestinal architecture, and splenic cytokines expression of Cyprinus carpio fed Prunus armeniaca kernel-enriched diets

Currently, the intervention of plant by-products in the fish diet has gained tremendous attention owing to the economic and high nutritious value. The current study is a pioneer attempt to incorporate the apricot, Prunus armeniaca kernel powder (PAKP) into the Common carp, Cyprinus carpio diets, and assess its efficacy on growth, digestion, intestinal morphology, immunity, antioxidant capacity, and splenic cytokines expression, besides the antibacterial role against Aeromonas veronii infection. Apparently healthy fish (N = 120) with an initial body weight of 24.76 ± 0.03g were allotted in 12 glass aquaria (60 L) and randomly distributed into four groups (triplicates, 10 fish/aquarium). The control group (PAKP0) was fed a basal diet without additives. The second, third, and fourth groups were provided PAKP diets with various concentrations (2.5 (PAKP2.5), 5 (PAKP5), and 10 g kg−1 (PAKP10)) respectively. After 60 days (feeding trial), sub-samples of the fish (12 fish/group) were intraperitoneally injected with 1 × 107 CFU mL−1 of A. veronii. Results revealed that body weight gain, feed conversion ratio, and specific growth rates were significantly augmented in the PAKP10 group in comparison to the other groups. The dietary inclusion of PAKP at all concentrations boosted the digestive capacity and maintained the intestinal morphology (average villus length, villus width, and goblet cells count) with a marked improvement in PAKP10. Moreover, fish fed on PAKP10 followed by PAKP5 then PAKP2.5 diets had noticeably elevated values of immunological biomarkers (IgM, antiprotease, and lysozyme activity) and antioxidant capabilities (the total antioxidant capacity, superoxide dismutase, and reduced glutathione) as well as significant up-regulation of immune and antioxidant-related genes (TGF-β2, TLR-2, TNF-α, IL-10, SOD, GPx, and GSS). Fourteen days post-infection with A. veronii, the highest relative percentage survival of fish was observed in PAKP10 (83.33%), followed by PAKP5 (66.67%), and PAKP2.5 (50%). Our results indicated that a dietary intervention with PAKP could promise growth, digestion, immunity, and protect C. carpio against A. veronii infection in a dose-dependent manner. This offers a framework for future application of such seeds as a growth promotor, immune-stimulant, and antioxidant, besides an alternative cheap therapeutic antibacterial agent for sustaining the aquaculture industry.

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

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

Antioxidant and innate immunity of Danio rerio against Edwardsiella tarda in response to diets including three kinds of marine microalgae

Disease is one of the most critical factors restricting the green and sustainable development of aquaculture. Microalgae, serving as excellent aquatic bait with high nutritional value, can strengthen the immune protection of aquatic organisms. The purpose of this study is to select superior marine microalgae to as feed additives that can defend against pathogens to a certain extent. In this study, the nutrient compositions of three marine microalga species were analyzed, and their antioxidant and innate-immunity protection effects as feed additions on Danio rerio were further investigated. Results demonstrated that Dunaliella salina contained 36.72% carbohydrates, 0.51% total chlorophyll, and 1.20% total carotenoids. Isochrysis galbana contained 1.33% total chlorophyll and 0.91% total carotenoids. The total fatty acid content of Phaeodactylum tricornutum and I. galbana was 60.77 μg mg−1 dry cell weight (DCW) and 76.40 μg mg−1 DCW, respectively. The docosahexaenoic acid content of I. galbana was 8.57 μg mg−1 DCW. After 30 days of feeding with different diets on D. rerio, the relative percent survival of the D. salina and I. galbana group challenged with Edwardsiella tarda EIB 202 were 17.39% and 26.09%, respectively. In the I. galbana group, the relative expression levels of the antioxidant factor catalase and glutathione peroxidase increased 1.5–2.5 times. Furthermore, the Toll-like receptors 2 signaling pathway was activated, and the signaling transcription-factor nuclear factor kappa-B was activated depending on the myeloid differentiation factor 88 pathway to promote cytokine expression. The relative expression of defensins increased 1.26 times, which helped enhance the innate-immunity defense system. Therefore, I. galbana can be developed as feed additives in the freshwater-fish field and be an excellent carrier for the development of recombinant microalga vaccine in aquaculture.

Characterization of a Diguanylate Cyclase VAGM001033 of Vibrio alginolyticus and Protective Efficacy as a Live Attenuated Vaccine Candidate in Pearl Gentian Grouper

Vibrio alginolyticus (V. alginolyticus) is one of the important epizootic pathogens in marine animals. VAGM001033 belongs to a diguanylate cyclase, responsible for the synthesis of dimeric guanosine monophosphate (c-di-GMP), a ubiquitous second messenger involved in the function of biofilm formation, motility, and virulence. This study confirmed that VAGM001033 was an active diguanylate cyclase by Congo red assay. The red-stained, dry, and rough form of colonies were observed with the increasing concentration of the L-arabinose on Congo red plates. Also, an in-frame deleted ΔVAGM001033 mutant was constructed and changes of ΔVAGM001033 mutant in physiology and pathogenicity were detected. The ΔVAGM001033 mutant displayed similar morphology and growth curve with the wild-type strain showing no significant differences. The swarming ability of the ΔVAGM001033 mutant was significantly enhanced showing bigger swarming circles, while the biofilm formation, extracellular proteases, and virulence were significantly attenuated. The results of the test for antibiotic susceptibility showed that the wild type and ΔVAGM001033 mutant had similar sensitivity or resistance to most antibiotics used in this study, except cefotaxime and nitrofurantoin. The mutant was sensitive to cefotaxime and nitrofurantoin, while the wild type was intermediate. A total of 756 differentially expressed genes (DEGs) were identified by RNA-seq, of which 109 were upregulated and 647 were downregulated. Flagellar assembly, two-component system, ATP-binding cassette (ABC) transporters, and peptidoglycan biosynthesis were significantly enriched in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. Moreover, the ΔVAGM001033 mutant induced high antibody titers and provided immune protectivity with a relative percent survival (RPS) of 82%. Immune-related genes of pearl gentian grouper (♀Epinephelus fuscoguttatus × ♂ Epinephelus lanceolatus), namely, IgM, MHC-Iα, interleukin-1β (IL-1β), interleukin-16 (IL-16), and tumor necrosis factor-α (TNF-α) were upregulated after vaccination. Overall, the results suggested that VAGM001033 plays a crucial role in V. alginolyticus. The ΔVAGM001033 mutant might be applied as an effective live vaccine candidate against V. alginolyticus.

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

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

Immunoproteomic identification of OmpA with potential stimulation of serum‐specific antibody in Nile tilapia ( Oreochromis niloticus ) and its ability to protect against Edwardsiella ictaluri infection

Edwardsiella ictaluri is a Gram-negative bacterium that has been reported to be disease causing in several economic fish including Nile tilapia. Here, immunoproteomic identification found that outer membrane protein A (OmpA) could react with serum from fish naturally infected with E. ictaluri. The expression and production of recombinant OmpA protein could be successfully conducted in Escherichia coli strain BL21(DE3) with the molecular mass of 38 kDa after inclusion bodies were successfully solubilized and refolded. Immunization of fish with the recombinant OmpA protein showed that the recombinant proteins could induce the production of specific antibodies in serum of immunized fish (p < 0.05) at 1-week postimmunization (7 dpi) and reached the highest antibody titre at 3 weeks postimmunization (21 dpi). The serum-specific antibody from fish immunized with the recombinant OmpA exhibited bacterial agglutination property. Furthermore, the partial protection of Nile tilapia was observed in OmpA immunized fish with the relative per cent survival (RPS) of 39.81%. In summary, this study explored the immunoproteomic method as a starting point to investigate OmpA of E. ictaluri and might be considered as a candidate protein for recombinant vaccine development in Nile tilapia against this bacterial infection.

Pre-treatment of Nile tilapia (Oreochromis niloticus) with ozone nanobubbles improve efficacy of heat-killed Streptococcus agalactiae immersion vaccine

Nanobubble technology has shown appealing technical benefits and potential applications in aquaculture. We recently found that treatment with ozone nanobubbles (NB–O3) activated expression of several immune-related genes leading to effective response to subsequent exposure to fish pathogens. In this study, we investigated whether pre-treatment of Nile tilapia (Oreochromis niloticus) with NB-O3 can enhance specific immune responses and improve efficacy of immersion vaccination against Streptococcus agalactiae. Spleen and head kidney of fish in the vaccinated groups showed a substantial upregulation in expression levels of pro-inflammatory cytokine genes (IL-1β, TNF-α, IL-6) and immunoglobulin classes (IgM, IgD, IgT) compared with the unvaccinated control groups. The mRNA transcript of pro-inflammatory cytokine genes was greatest (approx. 2.8–3.3 folds) on day 7 post-vaccination, whereas the relative expression of immunoglobulin genes was greatest (approx. 3.2–4.1 folds) on day 21 post-immunization. Both systemic and mucosal IgM antibodies were elicited in vaccinated groups. As the result, the cumulative survival rate of the vaccinated groups was found to be higher than that of the unvaccinated groups, with a relative percent survival (RPS) ranging from 52.9 to 70.5%. However, fish in the vaccinated groups that received pre-treatment with NB-O3, bacterial antigen uptakes, expression levels of IL-1β, TNF-α, IL-6,IgM, IgD, and IgT, as well as the specific-IgM antibody levels and percent survival, were all slightly or significantly higher than that of the vaccinated group without pre-treatment with NB-O3. Taken together, our findings suggest that utilizing pre-treatment with NB-O3 may improve the immune response and efficacy of immersion vaccination in Nile tilapia.

The efficacy of Aeromonas hydrophila GPl-04 feed-based vaccine on African catfish (Clarias gariepinus)

Abstract. Mulia DS, Utomo T, Isnansetyo A. 2022. The efficacy of Aeromonas hydrophila GPl-04 feed-based vaccine on African catfish (Clarias gariepinus). Biodiversitas 23: 1505-1510. Aeromonas hydrophila is a pathogenic bacterium to African catfish (Clarias gariepinus). Vaccination is one of the strategic efforts to control this type of bacterial attack. The objective of this study was to determine the effectiveness of A. hydrophila GPl-04 feed-based vaccine for African catfish. This study used A. hydrophila GPl-04 strain isolate as vaccine material. Sample used was African catfish measuring 10-13 cm length, weighing 16-25 g, taken from aquaculture ponds in the Purbalingga area, Central Java, Indonesia. This study applied experiments under the completely randomized design (CRD) method with 5 treatments and 3 replications. The treatments consisted of P0: feed without vaccine (control); P1: feed with 10 mL/100 g dose of vaccine given for 10 days; P2: feed with 10 mL/100 g dose of vaccine given for 15 days; P3: feed with 15 mL/ 100 g dose of vaccine given for 10 days; and P4: feed with 15 mL/ 100 g dose of vaccine given for 15 days. The challenge test was carried out on week 3, by injecting 0.1 mL of A. hydrophila suspension at 105CFU/mL dose per fish. The main parameters included the fish antibody titer, survival rate (SR), relative percent survival (RPS), mean time to death (MTD), and growth rate. Supporting parameters included water quality such as water temperature, water pH, and dissolved O2 levels. The main parameter data were analyzed using analysis of variance (ANOVA) and Duncan multiple range test (DMRT) at 5% test level, and the supporting parameter was descriptively quantitative analyzed. The results showed A. hydrophila GPl-04 feed-based vaccine could increase antibody titer, SR, RPS, and MTD of African catfish (P&lt;0.05). In addition, vaccination did not adversely affect the growth. The A. hydrophila GPl-04 feed-based vaccine was effective in protecting African catfish from A. hydrophila attack where 10 mL/100 g dose, given for 10 days (P1) was indicated as the most effective compared to other vaccination treatments.