Relative Percent Survival Research Articles

Comparative study on antigen persistence and immunoprotective efficacy of intramuscular and intraperitoneal injections of squalene – aluminium hydroxide (Sq + Al) adjuvanted viral hemorrhagic septicaemia virus vaccine in olive flounder (Paralichthys olivaceus)

The profitability of the olive flounder (Paralichthys olivaceus) aquaculture industry in Korea depends on high production and maintenance of flesh quality, as consumers prefer to eat raw flounders from aquaria and relish the raw muscles as ‘sashimi’. For sustaining high production, easy-to-deliver and efficient vaccination strategies against serious pathogens, such as viral hemorrhagic septicemia virus (VHSV), is very important as it cause considerable losses to the industry. Whereas, a safe and non-invasive vaccine formulation that is free from unacceptable side-effects and does not devalue the fish is needed to maintain flesh quality. We previously developed a squalene–aluminium hydroxide (Sq + Al) adjuvanted VHSV vaccine that conferred moderate to high protection in flounder, without causing any side effects when administered through the intraperitoneal (IP) injection route. However, farmers often demand intramuscular (IM) injection vaccines as they are relatively easy to administer in small fishes. Therefore, we administered the developed vaccine via IP and IM routes and investigated the safety and persistency of the vaccine at the injection site. In addition, we conducted a comparative analysis of vaccine efficacy and serum antibody response. The clinical and histological observation of the IM and IP groups showed that our vaccine remained persistence at the injection sites for 10–17 weeks post vaccination (wpv), without causing any adverse effects to the fish. The relative percentage of survival were 100% and 71.4% for the IP group and 88.9% and 92.3% for the IM group at 3 and 17 wpv, respectively. Thus, considering the persistency period (24 wpv) and both short and long-term efficacy of our vaccine, the present study offers an option to flounder farmers in selecting either IM or IP delivery strategy according to their cultured fish size and harvesting schedule — IM vaccination for small-sized fish and IP vaccination for table-sized fish.

THE APPLICATION OF GUAJAVA LEAF EXTRACT (Psidium guajava Linnaeus) AS IMMUNOSTIMULANT OF COMMON CARP (Cyprinus carpio Linnaeus) INFECTED BY MOTILE AEROMONAS SEPTICEMIA


 This study aims to analyze the effect of the various concentration of guava leaf extract (Psidium guajava Linnaeus) on the prevention of Motile Aeromonas Septicemia disease in common carp (Cyprinus carpio Linnaeus). In a total of 150 common carp, length 9-11 cm with a density of 10 carps/aquarium. This study used a completely randomized design through 5 treatments, each treatment has 3 replications, namely (K+) consists of without guava Psidium guajava leaf extracts and without bacteria-infection Aeromonas hydrophila, (K-) consists of without guava Psidium guajava leaf extracts with bacteria-infection Aeromonas hydrophila, (A) the application of 125 ppm guava Psidium guajava leaf extracts, (B) the application of 250 ppm guava leaf extracts, (C) the application of 500 ppm guava Psidium guajava leaf extracts for 23 days. The parameters observed total of leukocytes, differensial leukocytes, hematocrit levels, erythrocyte, hemoglobin, phagocytic activity (AF), phagocytic index (IF), relative percent survival (RPS), survival rate (SR) and water quality. Data were analyzed with Anova 95% confidence interval and continued with Duncan test. The parameter also showed a significant result (P<0,05) with the application of guava Psidium guajava leaf extracts to the total leukocytes, differensial leukocytes, erythrocyte, hemoglobin, phagocytic activity, phagocytic index and relative percent survival. The results of this research indicate the concentration of 250 ppm guava Psidium guajava leaf extracts is the optimum application to increase common carp non-spesific immune responses.

Potential effects of dietary seaweeds mixture on the growth performance, antioxidant status, immunity response, and resistance of striped catfish (Pangasianodon hypophthalmus) against Aeromonas hydrophila infection

Striped catfish (Pangasianodon hypophthalmus) is one of the important fish species in the world, which is widely cultured in several countries worldwide including Egypt. Aeromonas hydrophila infection showed a high prevalence in the farmed striped catfish negatively affecting its production causing economic loss. Therefore, the finding of functional feed supplements with antimicrobial activity is urgently needed. The use of marine seaweeds as feed supplements has produced satisfactory results as antibacterial agents. Accordingly, the present study was designed to evaluate the dietary effects of seaweeds mixture (Ulva lactuca, Jania rubens, and Pterocladia capillacea) extract (SME) on the growth performance, haemato-chemical parameters, antioxidant capacity, immune indices, and resistance of striped catfish fry against A. hydrophila infection. Four isonitrogenous (30% crude protein) diets were prepared to contain 0.0% (control), 1%, 2%, and 3% SME and were fed to striped catfish fry (1.7 ± 0.1 g) up to apparent satiety three times a day for 60 days. After the feeding trial, fish of each treatment were intraperitoneally injected with a virulent A. hydrophila strain and the relative percentage of survival (RPS) was recorded. The growth-stimulating activity of dietary SME was observed especially at 2% and 3% SME treatments. Similarly, haematological parameters (red blood cells, haemoglobin, haematocrit, and white blood cells) and serum protein profile (total protein, albumin, and globulin) were significantly (P < 0.05) improved, meanwhile serum alanine and aspartate aminotransferases were significantly (P < 0.05) decreased in fish fed with 2 and 3% SME diets. Serum creatinine and uric acid levels revealed no significant (P > 0.05) differences among the experimental groups. Hepatic malondialdehyde and serum nitrous acid levels were significantly decreased; meanwhile hepatic superoxide dismutase, catalase, and glutathione peroxidase levels were significantly increased in all SME-treated groups than the control one. Also, SME supplementations significantly increased the serum lysozyme, total immunoglobulin, and complement C3 activities with highest values at 2% and 3% SME treatments. Interestingly, after bacterial challenge, fish fed SME-supplemented diet were more resistant with the lowest fish mortality (15%) at the 3% SME diet, which exhibit the highest RPS (81.9%). Meanwhile the control group showed highest mortality (85%). According to the regression fitting curve, the current study recommends using the dietary SME with optimum levels of 2.25–2.5% to improve the growth performance, welfare status, and resistance of striped catfish fry against A. hydrophila infection.

Evaluation of oil-based inactivated vaccine against Aeromonas hydrophila administered to Labeo rohita, Cirrhinus mrigala and Ctenopharyngodon idella at different concentrations: Immune response, immersion challenge, growth performance and histopathology

The present study aimed at developing Montanide oil based inactivated vaccine against Aeromonas hydrophila and assessing immunogenic potential of two Indian major carps (Rohu: Labeo rohita and Mori: Cirrhinus mrigala), and a Chinese carp (Grass carp: Ctenopharyngodon idella) commercially cultured in Pakistan. Fishes were randomly distributed in three vaccinated groups with three vaccine concentrations at 108, 109 and 1010 colony forming unit (CFU)/mL and an unvaccinated control group. Fixed dose of 0.1 mL was intraperitoneally injected to each fish on 1st day and a booster dose at 15 days post-vaccination (DPV). Blood samples were collected at 14, 28, 35, 48 and 60 DPV to determine antibody titers in blood serum using complement fixation test (CFT). Growth parameters were also measured on fortnightly basis. The immune response showed highest antibody Geometric Mean Titers (GMT) on 35 DPV in all vaccinated groups which remained significantly higher than control group till 60 DPV. Fishes were challenged at 60 DPV with infectious A. hydrophila with 108 CFU/mL through immersion bath. The relative percentage survival (RPS) was 89% for fishes vaccinated with 109 and 1010 CFU/mL potencies while 78% for 108 CFU/mL potency. Vaccination did not cause adverse effect on growth performance of fishes. Minor degenerations, with non-significant histopathological changes were observed in few vaccinated fishes as compared to control group in which majority of the fishes presented deteriorative changes in tissues histology.

Mitigating multiple stresses in Pangasianodon hypophthalmus with a novel dietary mixture of selenium nanoparticles and Omega-3-fatty acid

Effects of a novel dietary mixture of selenium nanoparticles (Se-NPs) and omega-3-fatty acids i.e., Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on mitigating arsenic pollution, high-temperature stress and bacterial infection were investigated in Pangasianodon hypophthalmus. To aim this, four isocaloric and iso-nitrogenous diets were prepared: control feed (no supplementation), Se-NPs at 0.2 mg kg−1 diet with EPA + DHA at 0.2, 0.4 and 0.6% as supplemented diets. Fish were reared under normal condition or concurrent exposure to arsenic (2.65 mg L−1), and temperature (34 °C) (As + T) stress for 105 days. The experiment was conducted with eight treatments in triplicates. Response to various stresses i.e., primary (cortisol), secondary (oxidative stress, immunity, and stress biomarkers) and tertiary stress response (growth performance, bioaccumulation and mortality due to bacterial infection) were determined. Supplementation of dietary Se-NPs at 0.2 mg kg−1 diet and EPA + DHA at 0.2 and 0.4% reduced the primary stress level. Exposure to arsenic and temperature (As + T) and fed with control diet and EPA + DHA at 0.6% aggravated the cortisol level. Anti-oxidative enzymes (Catalase, superoxide dismutase, glutathione peroxidase and glutathione-s-transferase) and immunity (Nitroblue tetrazolium, total protein, albumin, globulin, A:G ratio, total immunoglobulin and myeloperoxidase) of the fish were augmented by supplementation of Se-NPs and EPA + DHA at 0.2 and 0.4%. Neurotransmitter enzyme, HSP 70, Vitamin C were significantly enhanced (p < 0.01) with supplementation of Se-NPs at 0.2 mg kg−1 and EPA + DHA at 0.2 and 0.4%. Whereas total lipid, cholesterol, phospholipid, triglyceride and very low-density lipoprotein (VLDL) were reduced (p < 0.01) with the supplementation of Se-NPs at 0.2 mg kg−1 diet and EPA + DHA at 0.2 and 0.4%. Tertiary stress response viz. growth performance was also significantly enhanced with supplementation of Se-NPs at 0.2 mg kg−1 and EPA + DHA at 0.2 and 0.4% reared under As + T. Whereas arsenic bioaccumulation in fish tissues was significantly reduced with dietary supplementation of Se-NPs and EPA + DHA. Cumulative mortality and relative percentage survival were reduced with Se-NPs at 0.2 mg kg−1 and EPA + DHA at 0.2 and 0.4%. The investigation revealed that a novel combination of Se-NPs at 0.2 mg kg−1 and EPA + DHA at 0.4% followed by 0.2% has the potential to alleviate temperature stress, bacterial infection and arsenic pollution. Whereas diet containing Se-NPs at 0.2 mg kg−1 diet and EPA + DHA at 0.6% was noticeably enhanced the stress in P. hypophthalmus.

Characterization and protective effects of lytic bacteriophage pAh6.2TG against a pathogenic multidrug-resistant Aeromonas hydrophila in Nile tilapia (Oreochromis niloticus).

Bacteriophage (phage) is considered as one of the alternatives to antibiotics and an environmentally friendly approach to tackle antimicrobial resistance (AMR) in aquaculture. Here, we reported isolation, morphology and genomic characterizations of a newly isolated lytic phage, designated pAh6.2TG. Host range and stability of pAh6.2TG in different environmental conditions, and protective efficacy against a pathogenic multidrug-resistant (MDR) Aeromonas hydrophila in Nile tilapia were subsequently evaluated. The results showed that pAh6.2TG is a member of the new family Chaseviridae which has genome size of 51,780bp, encoding 65 putative open reading frames (ORFs) and is most closely related to Aeromonas phage PVN02 (99.33% nucleotide identity). The pAh6.2TG was highly specific to A. hydrophila and infected 83.3% tested strains of MDR A. hydrophila (10 out of 12) with relative stability at pH 7-9, temperature 0-40°C and salinity 0-40 ppt. In experimental challenge, pAh6.2TG treatments significantly improved survivability of Nile tilapia exposed to a lethal dose of the pathogenic MDR A. hydrophila, with relative per cent survival (RPS) of 73.3% and 50% for phage multiplicity of infection (MOI) 1.0 and 0.1, respectively. Phage treatment significantly reduced the concentration of A. hydrophila in both water and fish body. Interestingly, the surviving fish from A. hydrophila challenged groups provoked specific antibody (IgM) against this bacterium. In summary, the findings suggested that the lytic phage pAh6.2TG is an effective alternative to antibiotics to control MDR A. hydrophila in tilapia and possibly other freshwater fish.

Omega-3 fatty acids effectively modulate growth performance, immune response, and disease resistance in fish against multiple stresses

Arsenic pollution and climate change are two major global challenges that have threatened the aquatic environment and to a certain extent led to extinction of aquatic animals. Concerted and accelerated scientific efforts are in progress to restrict the ill- impacts of pollution and climate change in the aquatic ecosystem. In this context, we have conducted a study to delineate the effect of omega-3 fatty acids i.e., eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in mitigating arsenic pollution and temperature stress in Pangasianodon hypophthalmus. Four isocaloric and iso‑nitrogenous diets were prepared viz. control feed (no supplementation), and three treatment groups with combined level of EPA + DHA at 0.2, 0.4 and 0.6%. Fish were reared under arsenic (2.65 mg L−1) and elevated temperature (34 °C) (As+T) stress for 105 days with eight different treatment groups in triplicates. Growth performance, oxidative stress enzymes, stress biomarkers, immunity, lipid profile, bioaccumulation of the arsenic in fish tissues and bacterial infection in terms of cumulative mortality and relative percent survival were altered on exposure of arsenic and temperature stress. Supplementation of combinatorial mixture of EPA + DHA at the rate of 0.2 and 0.4% in the diet led to improved growth performance, anti-oxidative status, immunity of the fish and reduced the infection against Aeromonas hydrophila. Neurotransmitter enzyme, HSP 70 and Vitamin C were significantly enhanced (p < 0.01) with supplementation of EPA + DHA, whereas, total lipid, cholesterol, phospholipid, triglyceride and very low density lipoprotein (VLDL) were reduced (p < 0.01) at 0.2 and 0.4% of dietary incorporation of EPA + DHA. Overall, the present investigation clearly revealed that a novel combination of EPA + DHA at 0.4% has the potential to mitigate arsenic pollution, temperature stress and protect the fish against bacterial infection.

Evaluation of the efficacy of a novel Vibrio vulnificus vaccine based on antibacterial peptide inactivation in turbot, Scophthalmus maximus

Tongue sole tissue factor pathway inhibitor 2 (TFPI-2) C-terminus derived peptide, TC38, has previously been shown to kill Vibrio vulnificus cells without lysing the cell membrane; thus, the remaining bacterial shell has potential application as an inactivated vaccine. Therefore, this study aimed to evaluate the immune response induced by the novel V. vulnificus vaccine. The protective potential of TC38-killed V. vulnificus cells (TKC) was examined in a turbot model. Fish were intramuscularly vaccinated with TKC or FKC (formalin-killed V. vulnificus cells) and challenged with a lethal-dose of V. vulnificus. The results showed that compared with FKC, TKC was effective in protecting fish against V. vulnificus infection, with relative percent of survival (RPS) rates of 53.29% and 63.64%, respectively. The immunological analysis revealed that compared with the FKC and control groups, the TKC group exhibited: 1) significantly higher respiratory burst ability and bactericidal activity of macrophages at 7 d post-vaccination; 2) increased alkaline phosphatase, acid phosphatase, lysozyme, and total superoxide dismutase levels post-vaccination; 3) higher serum agglutinating antibody titer with corresponding higher serum bactericidal ability, and a more potent serum agglutination effect, as well as an increased IgM expression level; 4) higher expression of immune relevant genes, which were involved in both innate and adaptive immunity. Taken together, this is the first study to develop a novel V. vulnificus inactivated vaccine based on AMP inactivation, and TKC is an effective vaccine against V. vulnificus infection for aquaculture.

Efficacy of heat-killed and formalin-killed vaccines against Tilapia tilapinevirus in juvenile Nile tilapia (Oreochromis niloticus).

Tilapia tilapinevirus (also known as tilapia lake virus, TiLV) is considered to be a new threat to the global tilapia industry. The objective of this study was to develop simple cell culture‐based heat‐killed (HKV) and formalin‐killed (FKV) vaccines for the prevention of disease caused by TiLV. The fish were immunized with 100 µl of either HKV or FKV by intraperitoneal injection with each vaccine containing 1.8 × 106 TCID50‐inactivated virus. A booster vaccination was carried out at 21‐day post‐vaccination (dpv) using the same protocol. The fish were then challenged with a lethal dose of TiLV at 28 dpv. The expression of five immune genes (IgM, IgD, IgT, CD4 and CD8) in the head kidney and spleen of experimental fish was assessed at 14 and 21 dpv and again after the booster vaccination at 28 dpv. TiLV‐specific IgM responses were measured by ELISA at the same time points. The results showed that both vaccines conferred significant protection, with relative percentage survival of 71.3% and 79.6% for HKV and FKV, respectively. Significant up‐regulation of IgM and IgT was observed in the head kidney of fish vaccinated with HKV at 21 dpv, while IgM, IgD and CD4 expression increased in the head kidney of fish receiving FKV at the same time point. After booster vaccination, IgT and CD8 transcripts were significantly increased in the spleen of fish vaccinated with the HKV, but not with FKV. Both vaccines induced a specific IgM response in both serum and mucus. In summary, this study showed that both HKV and FKV are promising injectable vaccines for the prevention of disease caused by TiLV in Nile tilapia.

A multi‐epitope chimeric protein elicited a strong antibody response and partial protection against Edwardsiella ictaluri in Nile tilapia

Edwardsiella ictaluri infects several fish species and protection of the all the susceptible fish hosts from the pathogen using a monovalent vaccine is impossible because the species is composed of host-based genotypes that are genetic, serological and antigenic heterogenous. Here, immunoinformatic approach was employed to design a cross-immunogenic chimeric EiCh protein containing multi-epitopes. The chimeric EiCh protein is composed of 11 B-cell epitopes and 7 major histocompatibility complex class II epitopes identified from E.ictaluri immunogenic proteins previously reported. The 49.32kDa recombinant EiCh protein was expressed in vitro in Escherichia coli BL-21 (DE3) after which inclusion bodies were successfully solubilized and refolded. Ab initio protein modelling revealed secondary and tertiary structures. Secondary structure was confirmed by circular dichroism spectroscopy. Antigenicity of the chimeric EiCh protein was exhibited by strong reactivity with serum from striped catfish and Nile tilapia experimentally infected with E.ictaluri. Furthermore, immunogenicity of the chimeric EiCh protein was investigated in vivo in Nile tilapia juveniles and it was found that the protein could strongly induce production of specific antibodies conferring agglutination activity and partially protected Nile tilapia juveniles with a relative survival percentage (RPS) of 42%. This study explored immunoinformatics as reverse vaccinology approach in vaccine design for aquaculture to manage E.ictaluri infections.

Transcriptome RNA-seq revealed lncRNAs activated by Edwardsiella anguillarum post the immunization of OmpA protecting European eel (Anguilla anguilla) from being infected

Long noncoding RNAs (lncRNAs) play important roles in various biological activities as vital regulators. However, no study has focused on the lncRNA regulation of Outer membrane protein (OMP) immunization against aquatic bacterial infection. In this study, we examined the genome-wide expression of lncRNAs in the liver of European eel (Anguilla anguilla, Aa) administrated by a recombinant OmpA (rOmpA) from Edwardsiella anguillarum (Ea) to elucidate the functions of lncRNAs in the process of Ea infection and Aa anti-Ea infection using strand specific RNA-seq. Eels were challenged by Ea at 28 d post the immunization (dpi) of OmpA, and the result showed, compared to uninfected livers in the PBS group (Con group), the infected livers in the PBS group (Con_inf group) showed severe bleeding, hepatocyte atrophy and thrombi formed in the hepatic vessels; livers in the OmpA group (OmpA_inf) also formed slight thrombi in the hepatic vessels. The relative percent survival of eels in OmpA_inf vs Con_inf was 78.6%. Using high-throughput transcriptomics, we found 13405 lncRNAs in 3 compares of Con_inf vs Con, OmpA_inf vs Con and OmpA_inf vs Con_inf, of which 111, 129 and 158 DE-lncRNAs were ascertained. GO analysis of the DE-lncRNAs revealed the targeting DEGs were mainly involved in single-organism process, signaling, biological process and response to stimulus in BP, component of membrane in CC and binding in MF; KEGG pathways showed that the targeting DEGs in co-expression and co-location enriched in cell adhesion molecules. Finally, 54 DE-lncRNAs targeting 1675 DEGs were involved in an interaction network of 21692 co-expression and 483 co-location related links, of which 18 DE-lncRNAs appear to play crucial roles in anti-Ea infection. Thus, the interaction networks revealed crucial DE-lncRNAs underlying the process of Ea infection and Aa anti-Ea infection pre and post the immunization of OmpA.

A live attenuated strain of HY9901ΔvscB provides protection against Vibrio alginolyticus in pearl gentian grouper (♀Epinephelus fuscoguttatus × ♂Epinephelus lanceolatu)

Vibrio alginolyticus, a bacterial pathogen in fish and humans, expresses a type III secretion system (T3SS) that is critical for pathogen virulence and disease development. In this study, the T3SS chaperone protein gene vscB was cloned from V. alginolyticus wild-type strain HY9901and the vscB gene has a length of 429 bp and encodes 142 amino acids. The amino acid sequence homology comparison results showed that VscB had the highest similarity with that of Vibrio parahaemolyticus VscB, reaching 91%. The mutant strain HY9901ΔvscB was constructed by the in-frame deletion method. The HY9901ΔvscB showed an attenuated swarming phenotype and a 23-fold decrease in virulence to pearl gentian grouper (♀Epinephelus fuscoguttatus × ♂Epinephelus lanceolatu). However, the HY9901ΔvscB showed no difference in morphology, growth, biofilm formation and extracellular protease (ECPase) activity. The results of antibiotic susceptibility testing showed that the wild-type HY9901 is more sensitive to Doxycycline, Minocyline and Kanamycin than in the HY9901ΔvscB. A total of 95 differentially expressed genes were screened by transcriptome sequencing analysis of HY9901 and strain ΔvscB, revealing 57 genes up-regulated and 38 genes were down-regulated in the ΔvscB compared to HY9901. qRT-PCR was employed to analyze the transcription levels of T3SS-related genes showing that ΔvscB had decreased expression of vopN and vscO and increased expression of hop, vscN, vscK, vscL and vopS compared to HY9901. Finally, grouper vaccinated via intraperitoneal (IP) injection with HY9901ΔvscB had a relative percent survival (RPS) of 77.6% following challenge with HY9901. Real-time qPCR assays showed that vaccination with HY9901ΔvscB enhanced the expression of immune-related genes, including MHC-I, IgM, and CD8α both in the liver and spleen, indicating that HY9901ΔvscB is able to induce humoral and cell-mediated immune responses in pearl gentian grouper. These results demonstrate that the HY9901ΔvscB mutant could be used as an effective live attenuated vaccine to combat V. alginolyticus infection in pearl gentian grouper.

Immersion immunization of common carp with bacterial ghost‐based DNA vaccine inducing prophylactic protective immunity against spring viraemia of carp virus

The interactive applications of immunization route, vaccine type and delivery vectors are emerging as a key area of research within the field of mass immunization in fishery production. In an effort to improve DNA vaccine's immune efficiency in large-scale immunization, a promising bacterial ghost-loaded DNA vaccine was constructed based on Escherichia coli DH5α. In common carp was investigated the immune response to immersion immunization via related indicator analysis, and the challenge test of spring viraemia of carp virus (SVCV) was carried out. The result indicated that BG-loaded DNA vaccine induced higher serum antibody level than naked pEG-G. Simultaneously, the immunophysiological indicators and genes change at the more advanced levels in the BG/pEG-G immune group. At the treatment concentration of 20mg/L of the BG/pEG-G group, IgM and IgZ expressions in vivo were markedly increased by 21.62 times and 6.91 times, respectively, and the relative percentage survival reached the peak of 59.57%. This study paves the way for future aquatic animal vaccine research, which aimed to develop the highly effective immersion vaccine system by delivery vectors, with the ultimate aim to prevent and restrict SVCV in actual production.

Effects of dietary short‐chain fatty acid salts on the growth performance, digestive, antioxidant and immune enzyme activities, immune‐related gene expression and resistance to Vibro parahaemolytics infection in juvenile ridgetail white prawn ( Exopalaemon carinicauda )

An 8-week feeding trial was conducted to study the effects of dietary short-chain fatty acid salts, sodium acetate (SA), sodium propionate (SP) and sodium butyrate (SB), on the growth performance, digestive ability, immune parameters and disease resistance of juvenile Exopalaemon carinicauda. Shrimps (14.60 ± 1.48 mg) were fed with 10 experimental diets containing 0.0 (control), 0.25%, 0.5% and 1% of SA, SB and SP in triplicate. The highest survival rate was observed in the SP (0.5%) group (p < 0.05). The final weight, specific growth rate and feed efficiency was highest in the SP (0.25%) group (p < 0.05). Shrimps that were fed SP (0.25%) had greater intestinal digestive enzyme (chymotrypsin) activity. Shrimps in the SA (0.5%) group had the highest activities of glutathione peroxidase, catalase, lysozyme, acid phosphatase and alkaline phosphatase, which was accompanied by the highest relative percent survival after a Vibro parahaemolyticus challenge (p < 0.05). The expression of immune-related genes (prophenoloxidase, C-type lectin and mannose binding lectin) in the hepatopancreas of shrimps fed SA (0.5%) and SP (0.25%) was higher than that of the control group (p < 0.05). These data suggest that SA (0.5%) and SP (0.25%) are beneficial to the growth performance and health of E. carinicauda.

Efficacy of encapsulated biogenic silver nanoparticles and its disease resistance against Vibrio harveyi through oral administration in Macrobrachium rosenbergii.

Biological synthesis of silver nanoparticles (AgNPs) by Cheatomorpha antennia and its in vitro and in vivo antibacterial activity against Vibrio harveyi in Macrobrachium rosenbergii was demonstrated in the study. In vitro growth curve analysis, cell viability and bacterial inhibitory assays were performed to test the efficacy of synthesised AgNPs against bacteria. Sodium caseinate was used as an encapsulating agent to deliver the antibacterial drugs and the commercial process of microencapsulation comprises the antibacterial bioelements for oral administration to improve the disease resistance of AgNPs against V. harveyi due to the eco-friendly for non-toxic behaviour of nanoparticle and their treatment. Characterisation of antibacterial silver was performed by UV spectroscopy, X-ray diffraction, Fourier Transform Infrared spectroscopy and Scanning Electron Microscopy. The peak at 420 nm showed the presence of nanoparticles in the solution and the crystal nature of the particle was identified by the XRD. FTIR characterised the functional harveyi biomolecules and further SEM confirmed the size of the nanoparticles around 24 ± 2.4 nm. Experimental pathogenicity of V. harveyi showed 100% mortality at the 120th hour. Treatment of encapsulated AgNPs was administered orally for the relative percentage of survival which acquired almost 90% of survival till 30 days of exposure. In conclusion, the microencapsulation of AgNPs in the biopolymer matrices promotes the health, growth responses, immunity and disease resistance of encapsulated AgNPs with an improved relative percentage of survival.

The Efficacy of Moina micrura Enriched with Probiotic Bacillus pocheonensis in Enhancing Survival and Disease Resistance of Red Hybrid Tilapia (Oreochromis spp.) Larvae.

The administration of probiotics via live feeds, such as Artemia and rotifers, has gained significant attention. Moreover, indiscriminate use of antibiotics in conventional aquaculture practices in order to prevent or control disease outbreaks has resulted in the occurrence of residues and antimicrobial resistance. Thus, the application of eco-friendly feed additives, such as probiotics, as a safer alternative has received increasing attention in recent years. However, only minimal information on the administration of probiotics via freshwater cladoceran Moina micrura is available despite being commonly used for larval and post-larval feeding of freshwater crustaceans and fish. Thus, this study aimed to evaluate the application of Bacillus pocheonensis strain S2 administered via M. micrura to red hybrid tilapia (Oreochromis spp.) larvae. Bacillus pocheonensis that has been previously isolated from Spirulina sp. was subjected to preliminary in vitro evaluation of antagonistic properties. The agar well-diffusion assay revealed that this probiont could inhibit the growth of Streptococcus agalactiae and Aeromonas hydrophila. The size of inhibition zones ranged from 8.8 ± 0.2 to 18.2 ± 0.4 mm. Moina micrura was later used as a biological model in preliminary in vivo bacterial challenge assays to evaluate the efficacy of B. pocheonensis in protecting the host from diseases. Moina micrura was pre-enriched with B. pocheonensis at and before S. agalactiae and A. hydrophila were introduced into the culture. The study revealed that B. pocheonensis at was able to significantly enhance the survival of M. micrura after being challenged with both pathogens (63 ± 3%) in comparison to the control group. The relative percentage survival (RPS) of M. micrura was highest (p < 0.05) when treated with B. pocheonensis at both concentrations and (38.33) after being challenged against S. agalactiae. To assess the efficacy of B. pocheonensis in protecting red hybrid tilapia against streptococcosis, the larvae were fed with either unenriched (control) Moina or probiont-enriched Moina daily for 10 days. A significantly (p < 0.05) higher survival rate (77 ± 3%) was observed in larvae fed with probiont-enriched M. micrura compared to other treatments, and the RPS was recorded at 62.90. In addition, the S. agalactiae load was suppressed in larvae fed probiont-enriched M. micrura () in comparison to the control group (), indicating that the probiont might have contributed to the improvement of tilapia health and survival. This study illustrated that M. micrura was suitable to be used as a vector for probiotics in freshwater fish larvae as an alternative to hazardous antibiotics for disease control.

Optimization of immunization procedure for SWCNTs-based subunit vaccine with mannose modification against spring viraemia of carp virus in common carp.

Immersion vaccination of single-walled carbon nanotubes loaded with mannose-modified glycoprotein (SWCNTs-MG) vaccine has been proved to be effective in preventing spring viraemia of carp virus (SVCV). Immunization procedure has immense consequence on the immune effect of the immersion vaccine. However, immunization procedure optimization for SWCNTs-MG vaccine against SVCV has not been reported. In this study, accordingly, a full-factor experiment was designed to optimize the immunization procedure of SWCNTs-MG vaccine by three aspects of vaccine dose (30mg/L, 40mg/L and 50mg/L), immunization density (8 fish L-1 , 24 fish L-1 and 48 fish L-1 ) and immunization time (6, 12 and 24hr). Furthermore, we used the immunization group (A1B2C1, 30mg/L, 24 fish L-1 and 6hr) in the previous study as a positive control (PC) to evaluate the immunization effect optimized conditions from the expression of immune-related genes and relative percentage survival (RPS). At 28days post-vaccination (DPV), common carps were intraperitoneal injected SVCV challenged test indicated that the A1B2C2 group (30mg/L, 24 fish L-1 , 12hr) displayed superiority of protective efficacy compare with other groups and the RPS with 77.9%, which was 15.6% higher than the PC group of RPS with 62.3%. Moreover, the expression of immune-related genes such as IL-10, CD4 and MHC-II was also significantly higher than PC group. The specific experimental flow chart is shown in Figure1. Conclusively, these results demonstrated that vaccine dose, immunization density and immunization time are 30mg/L, 24 fish L-1 and 12hr, which is the more appropriate immunization programme with juvenile carp for SWCNTs-MG vaccine. This study provides a profitable reference for improving the immune efficiency of aquatic immersion vaccine. [Figure: see text].

Effect of dietary Andrographis paniculata leaf extract on growth, immunity, and disease resistance against Aeromonas hydrophila in Pangasianodon hypopthalmus

ABSTRACT Aeromonas hydrophila severely hampers the farmed catfish production. A 60-day feeding trial was conducted to study the effect of Andrographis paniculata leaf extract on growth, immunomodulation, and disease outbreak against A. hydrophila in Pangasianodon hypophthalmus. A total of 180 juveniles of P. hypophthalmus (avg. wt. 123.33 ± 1.8 g) were distributed randomly in four groups following a completely randomized design in triplicate. Four experimental diets—C (no extract), T1 (1%), T2 (2%), and T3 (4%)—were prepared and fed twice daily. Growth metrics were calculated after 45 days and challenged with A. hydrophila. Relative percentage survival (RPS) was recorded over 15 days postchallenge. Results indicated that A. paniculata extract has a positive effect on hematobiochemical parameters and immune response. The highest (P < 0.05) pre- and postchallenge serum lysozyme activity, respiratory burst activity, and globulin level were found in the T2 group and lowest in the T0 group. Significantly (P < 0.05) higher percentage weight gain and SGR and lowest FCR were found in the T2 group than in the other groups. RPS was highest (P < 0.05) in the T2 group, followed by the T1 and T3 groups, compared to the control. Results suggest that A. paniculata leaf extract at a level of 2% could improve growth, immunity, and disease resistance against A. hydrophila by enhancing innate immunity in P. hypophthalmus.

Impact of aqueous exposure to silver nanoparticles on growth performance, redox status, non-specific immunity, and histopathological changes of Nile Tilapia, Oreochromis niloticus, challenged with Aeromonas hydrophila

Silver nanoparticles (AgNP) are one of the most synthesised nanomaterials due to their widespread applications, which in turn increases the potential of release and accumulation in nature. Moreover, they are known as antibacterial and recommended to be used in different commercial drugs. However, the evaluation of their effect on living organisms, especially fish, at low levels is still not completely understood. Therefore, the present study was conducted to investigate the effects of aqueous exposure of AgNPs on growth performance, oxidative status, non-specific immunity, and histopathological changes of epidermis, gills, liver, and intestine of Nile Tilapia, Oreochromis niloticus. The fish were exposed to four concentrations of AgNP (0, 10, 20, and 30 μg L-1) for 8 weeks. At the end of the experiment, fish were intraperitoneally injected with pathogenic bacteria Aeromonas hydrophila and fish mortalities were recorded up to 14 days post-challenge. The results revealed that growth performance and feed utilisation of fish exposed to the lower AgNPs concentration improved significantly compared to other treatments. The increasing AgNP exposure levels decreased different growth indicators in a dose-dependent manner. The highest catalase, superoxide dismutase, glutathione peroxidase, lysozyme, and respiratory burst activities were recorded with 10 μg AgNPs L−1 and the enzyme activities were retarded with increasing AgNP concentrations. The total bacteria count in the water decreased with increasing AgNP concentrations. Meanwhile, the relative percentage of survival improved with low AgNP concentration and decreased with increasing exposure levels. The histopathological investigation of different studied tissues (gills, skin, liver, and intestine) revealed an increase of histological lesions’ frequency and severity with increasing AgNP concentrations. It can be concluded that the exposure of Nile Tilapia to AgNP at levels exceeding 10 μg L−1 could interrupt growth performance, immunity, and antioxidant status and induced histopathological changes.

Evaluation of an oral DNA nanovaccine against photobacteriosis in Solea senegalensis

Infectious diseases are one of the main causes of social and economical losses in world aquaculture. Senegalese sole (Solea senegalensis) is an important species for aquaculture in southern Europe, whose production is affected by the appearance of bacterial diseases such as photobacteriosis, a septicemia caused by Photobacterium damselae subsp. piscicida (Phdp). The aim of this study was to obtain an oral DNA nanovaccine and to evaluate its efficacy against Phdp in S. senegalensis juveniles. For this purpose, the amplified product corresponding to the protein inosine-5′-monophophate dehydrogenase (IMPDH) from Phdp, was cloned into the expression vector pcDNA™6.2/C-EmGFP-GW obtaining the DNA vaccine named as pPDPimpdh. The correct transcription and protein expression was verified at 48 h post tansfection in HEK293 cells. Chitosan nanoparticles (CS-TPP NPs) were prepared by ionotropic gelation and their features were appropriate for use as oral delivery system. Therefore, pPDPimpdh was protected with chitosan CS-TPP NPs throughout complex coacervation method giving as a result a DNA nanovaccine referred as CS-TPP+pPDPimpdh NPs. Sole juveniles were vaccinated orally with CS-TPP NPs, pPDPimpdh and CS-TPP+pPDPimpdh NPs followed by a challenge with Phdp at 30 days post vaccination (dpv). The relative percentage survival (RPS) for pPDPimpdh vaccinated groups was 6.25%, probably due to its degradation in the digestive tract. RPS value obtained for CS-TPP NPs and CS-TPP+pPDPimpdh NPs was 40% and antibodies were observed in both cases. However, a delay in mortality was observed in sole juveniles vaccinated orally with CS-TPP+pPDPimpdh NPs. In fact, an upregulation of tf, mhcII, cd8a and igm in the posterior gut and c3, hamp1, tf and cd4 in spleen was observed in juveniles vaccinated with CS-TPP+pPDPimpdh NPs. After challenge, a modulation of cd8a and cd4 expression levels in the posterior gut and c3, tf, lyg, cd4, igm and igt expression levels in spleen was observed. Moreover, the concentration of lysozyme in skin mucus significantly increased in fish vaccinated orally with CS-TPP+pPDPimpdh NPs at 11 dpc. These data indicate that oral vaccination with CS-TPP+pPDPimpdh NPs could be acting through the non-specific immune responses as well as the specific humoral and cell mediated immunity and provide the first step toward a development of an oral DNA nanovaccine against Phdp in sole.