Dietary supplementation of Polygonum chinense improves the immunity of Asian seabass, Lates calcarifer (Bloch, 1790) against Vibrio harveyi infection

Vibriosis is a bacterial disease caused by Vibrio harveyi (V. harveyi; Vh ) that poses a substantial threat to Asian seabass (Lates calcarifer) aquaculture. In this study, we investigated alternative approaches to conventional injection vaccination for prevention of V. harveyi in Asian seabass. The first approach involved an immersion prime with heat-inactivated V. harveyi vaccine, followed by two oral booster doses (day 14-18 and day 28-32). Immunized fish demonstrated higher expression levels of immune genes (i.e. TNFα, IL1β, CD4, CD8, and IgM) following the immersion prime and the first oral booster dose, peaking at 14 and 28 days post vaccination (dpv). Specific antibodies (IgM) were detected in both systemic and mucosal compartments as early as 7 dpv and exhibited steady increases, reaching their peaks at 21 and 35 dpv. A laboratory challenge test at day 49 dpv showed relatively high protection, with a relative percent survival (RPS) of 72.22% ± 7.86%. The promising immersion prime-oral booster vaccination strategy warranted further improvement. In the second approach, we explored whether the integration of oxygen nanobubbles-enriched water (ONB) and positively charged chitosan (CS) can enhance antigen uptake to fish gills during immersion priming and improve immunological responses post oral booster. We compared antigen uptake in fish gills, antibody response and vaccine efficacy for four different combinations of vaccine with ONB and CS, and two control groups. Pre-exposure to ONB and CS prior to vaccine application resulted in superior antigen uptake and anti-V. harveyi antibody (IgM) production in both serum and mucus compared to other formulas. We further evaluated the integration of an oral booster (4.22 × 108 CFU/g, at day 21-25) in a vaccine trial experiment to improve the survival rates post exposure to V. harveyi. Antibody responses were measured weekly for over 42 days. Vaccine efficacy was assessed through an intraperitoneal challenge injection with 1.82 × 105 CFU/fish of virulent V. harveyi. The expression of immune-related genes (IL1β, TNFα, CD4, CD8, IgT) and antibody levels were assessed at 1, 3, and 7 day (s) post challenge (dpc). The results revealed that antibody level the group pre-exposed to ONB and CS were consistently higher than other groups post immersion immunization and oral booster. After challenge with V. harveyi, fish in this group exhibited the highest expression levels of immune-related genes in the gills, head kidney, and spleen on 1, 3, and 7 dpc compared to the other groups. Lastly, the group exposed to complex of ONB-CS and vaccine had a higher RPS (p < 0.05) of 63% ± 10.5% compared with the controls at 40.7% ± 10.5% and 25.9% ± 10.5%, respectively. In conclusion, pre-mixing of CS with ONB, followed by V. harveyi vaccine to form the ONB-CS-Vac complex, represents a promising immersion vaccination strategy. This novel approach can enhance antigen uptake, stimulate immunological responses, and improve survival of Asian seabass against vibriosis.

Molecular cloning and expression analysis of five heat shock protein 70 (HSP70) family members in Lateolabrax maculatus with Vibrio harveyi infection

A novel vaccination strategy against Vibrio harveyi infection in Asian seabass (Lates calcarifer) with the aid of oxygen nanobubbles and chitosan

An 8-week husbandry trial was carried out toexamine the impacts of graded levels of dietary fulvic acid (FA) on growth, stress, and disease resistance of Asian seabass (Lates calcarifer, 38.2 g) juveniles. Five levels of FA were included in a basal diet (470 g kg-1 crude protein, 150 g kg-1 crude lipid) to prepare experimental diets, including: 0 (control), 2.5, 5.0, 10, and 15 g kg-1 diet. Two hundred and twenty-five fish (38.2 ± 0.1 g) were stocked randomly into 15 1000-L circular polyethylene tanks (15 fish tank-1) filled with 800 L disinfected seawater and fed with the experimental feeds three times daily. Each dietary treatment had three replicates. Tanks were supplied with seawater (32.2 °C, 45.0 ppt) in a flow-throw system. At the end of the husbandry trial, fish were challenged with Vibrio harveyi and air exposure stress for one min. Fish fed FA-supplemented diets showed a positive quadratic growth response, and those fed with a 5 g FA kg-1 diet had higher final weight and weight gain percentage than the control. Feed intake in fish fed FA-supplemented diets were higher than in control, showing significant linear and quadratic trends (P < 0.05). The highest and lowest skin mucus lysozyme (LYZ) and serum alternative complement pathway (ACH50) activities were in fish fed 10 and 15 g FA kg-1 diets, respectively, exhibiting a quadratic response to dietary FA. Serum total protein level (TP) in fish fed FA-supplemented diets was higher than in the control group. Medium or high levels (10–15 g kg-1) of FA significantly enhanced the survival rate against Vibrio harveyi, which was associated with increased activities of serum lysozyme and ACH50 as well as liver glutathione peroxidase activity in these groups. Air exposure induced oxidative stress in all treatments and gradually reduced antioxidant enzyme activities associated with increased malondialdehyde content in the liver. Furthermore, air exposure suppressed some immune responses, including serum total immunoglobulin and TP levels. Still, LYZ activity was restored after 24 h, with the highest level found in fish fed 5 g FA kg-1 diet. Based on the broken-line regression analysis, the adequate level of FA in the diet for Asian seabass is 4.4 g kg-1.

The warm temperature acclimation related 65 kDa protein-2 (Wap65-2), a teleost plasma glycoprotein, plays an important role in immune regulation against bacterial infection. Here, for the first time we determined the full length cDNA sequence of the Japanese sea bass Wap65-2 gene (1 601 bp in length excluding the 3'-polyA tail). The sequence contains an open reading frame that encodes a protein of 436 amino acids with a molecular weight of 4.87×10(4). The predicted protein had a signal peptide in the N-terminal domain containing 19 residues. Sequence comparison and phylogenetic tree analysis showed that the Japanese sea bass Wap65-2 has a relatively high similarity to the Dicentrarchus labrax Wap65-2. In the healthy Japanese sea bass, Wap65-2 mRNA was expressed mainly in the liver and weakly in the heart and muscle. qRT-PCR results revealed that liver Wap65-2 transcripts were significantly increased after a Vibrio harveyi infection, and peaked 24 hour post injection (6.89 fold increase). The Japanese sea bass Wap65-2 protein was expressed in Escherichia coli and subsequently used for antiserum preparation. Western blot analysis showed that Wap65-2 was significantly increased in V. harveyi infected Japanese sea bass and reached a maximum of 5.33-fold increase at 36 h. In conclusion, the alteration of Japanese sea bass Wap65-2 expression was tightly associated with the progression of the V. harveyi bacterial infection.

Quorum quenching probiotics modulated digestive enzymes activity, growth performance, gut microflora, haemato-biochemical parameters and resistance against Vibrio harveyi in Asian seabass (Lates calcarifer)

Aquaculture production activities have been experiencing a significant increase. Various economically valuable fish commodities have been developed, including Asian seabass, barramundi, and giant seaperch. Asian seabass farming offers advantages such as relatively fast growth, ease of maintenance, and high tolerance to environmental changes. However, the sustainability of Asian seabass farming faces challenges, including vibriosis infection. This disease can lead to economic losses due to mortality and has been reported in several Asian seabass-producing countries. This review discusses information on the causative agents of vibriosis, clinical symptoms, vibriosis control, and prevention through immunostimulants, prebiotics, and vaccination in Asian seabass. The primary causative agents of vibriosis in Asian seabass include Vibrio anguillarum, V. harveyi, and V. alginolyticus. Clinical symptoms include anorexia, darkening of body coloration, lesions around the anus and fin bases, scale loss and muscle degradation, hemorrhaging throughout the body, particularly at fin bases, ulcers on the operculum, kidney congestion, and liver necrosis. Infections caused by V. anguillarum, V. harveyi, and V. alginolyticus can be effectively managed using medicinal plants, probiotics, and vaccination, which have shown promising results in Asian seabass. Keywords: Lates calcarifer; Vibriosis; Vibrio alginolyticus; Vibrio anguillarum; Vibrio harveyi. Abstrak Produksi kegiatan akuakultur terus mengalami peningkatan yang signifikan. Berbagai komoditas ikan bernilai ekonomi telah dikembangkan salah satunya ikan kakap putih yang juga dikenal sebagai Barramundi, Asian seabass dan Giant seaperch. Budidaya kakap putih memiliki keunggulan, yaitu pertumbuhannya yang relatif cepat, mudah dipelihara dan mempunyai toleransi yang tinggi terhadap perubahan lingkungan. Namun, keberlanjutan budidaya kakap putih tidak terlepas dari berbagai kendala, salah satunya serangan penyakit vibriosis. Penyakit ini dapat mengakibatkan kerugian ekonomi akibat kematian dan telah dilaporkan pada pada beberapa negara produsen kakap putih. Ulasan ini membahas informasi mengenai agen penyebab vibriosis, gejala klinis, pengendalian vibriosis, serta pencegahan melalui pemanfaatan imunostimulan, prebiotik dan vaksinasi pada kakap putih. Agen utama penyebab vibriosis pada kakap putih adalah Vibrio anguillarum, V. harveyi, dan V. alginolyticus. Gejala klinis yang ditimbulkan adalah anoreksia, perubahan warna tubuh menjadi lebih gelap, lesi di sekitar anus dan pangkal sirip, kerontokan pada sisik dan degradasi otot, hemoragi di seluruh tubuh terutama di pangkal sirip, dan ulserasi di bagian operkulum, serta kongesti pada ginjal dan nekrosis pada hati. Infeksi V. anguillarum, V. harveyi, dan V. alginolyticus dapat dikendalikan dengan pemanfaatan tanaman obat, probiotik dan vaksinasi yang telah terbukti pada ikan kakap putih. Kata kunci: Lates calcarifer; Vibriosis; Vibrio alginolyticus; Vibrio anguillarum; Vibrio harveyi.

Molecular and functional characterisation of a mannose-binding lectin-like gene from Japanese sea bass (Lateolabrax japonicus)

Molecular characterization and chemotaxis assay of a CC motif chemokine ligand 25 from Japanese sea bass (Lateolabrax japonicus)

Immunostimulatory Effects of Polysaccharide Compound from Seaweed Kappaphycus alvarezii on Asian seabass (Lates calcarifer) and it’s Resistance against Vibrio parahaemolyticus Kappaphycus alvarezii is a red seaweed rich in polysaccharides. In the present study an attempt was made to isolate polysaccharide from K. alvarezii and also tested for immunostimulatory effects on Asian seabass (Lates calcarifer) using Vibrio parahaemolyticus as a test pathogen. Two sets of experiments were designed as mixed with fed diets and intraperitoneal (IP) injection of the crude extracts. Every five days of interval, the non specific immune responses were analysed by the parameters like WBCs count, Lysozyme activity and NBT assay. After 15 days of treatment, the animals were challenged against V. parahaemolytcius and cumulative survival rates were recorded. The total white blood cells were decreased in intraperitoneal injection of polysaccharides extract after 5th day. But in fed diet cells count were significantly increased from 1st to 15th day. Fish fed diet with polysaccharide extract improved the lysozyme level than IP injection, which reduced simultaneously over the period compared with control. In the present study, the respiratory burst activity significantly enhanced in the fed diet polysaccharide extract in Asian seabass (Lates calcarifer) by oral administration and the highest activity reached in 0.5% fed diet on 15th day. After challenge with V. parahaemolyticus, all treated groups showed a reduced mortality compared to the control group. The best survival rate was observed in the group polysaccharide extract treated with 0.5% by oral administration. Among the other groups, fish treated by intraperitoneal injection had a lower survival rate than the fish treated by oral administration. However, the differences between the two sets of experiment (oral administration or intraperitoneal injection) were only 10%. UV, FT-IR, 1H and 13C NMR spectra were carried out to characterize the polysaccharide extract. The UV spectrum of the crude extract compound was showed a major peak at 282 nm. In FT-IR spectrum, major peak was observed at 1000-1100 cm-1 supports the presence of ring vibrations C-O, C-O-C (polysaccharides region) and minor peak of 1647.93 cm-1, which supports amide group. And based on the 1H and 13C NMR spectra, the presence of carbonyl carbons in the purified compound was confirmed. Findings of the present study evidenced the excellent immunostimulatory activity of polysaccharide from the seaweed Kappaphycus alvarezii. Further complete purification and characterization of the active polysaccharide and its field evaluation is needed to bring this seaweed polysaccharide as a promising candidate for the development of good immunostimulatory agent in the field of aquaculture.

Introduction: Thiopurines and anti-tumor necrosis factor-alpha (anti-TNF) drugs are commonly used in treatment of inflammatory bowel diseases (IBD). Studies have established that thiopurines, when used in combination with anti-TNF drugs, affect the metabolism of anti-TNF drugs, resulting in higher serum drug concentrations. However, the data on the effect of anti-TNF drugs on thiopurine metabolism is limited. Methods: All adult IBD patients followed at our IBD center who had thiopurine metabolite concentrations measured between 2000-2016 were eligible for inclusion if they had at least 1 level measured while on thiopurine monotherapy, and 1 level measured while on combination therapy with an anti-TNF and thiopurine drug with the same thiopurine drug dose. Thiopurine metabolite concentration, white blood cell (WBC) count, mean corpuscular volume (MCV) and serum alanine aminotransferase (ALT) levels were compared on monotherapy and combination therapy. Paired t-test and Wilcoxon rank sum test were used for analysis of variables with normal and non-normal distribution, respectively. Results: 30 patients met the inclusion criteria (Table 1). Mean 6TGN level (in pmol/8 x 108 RBCs) on monotherapy and combination therapy were 201 and 267 respectively (% increase 33%; 95% CI 30.1-105.8; p 0.0004). There was a corresponding decrease in mean WBC count (in x1000/mL) from 7.6 to 6.1 (% decrease 20%; 95% CI 0.49-2.85; p 0.01) (Figure 1). There was no significant difference in 6-MMP levels, MCV and ALT levels on monotherapy and combination therapy (Table 2). On subgroup analysis, patients with therapeutic 6TGN levels >230 (n=13) had a significant increase in 6TGN levels (301 vs 369; p 0.03), and a corresponding decrease in WBC count (8.0 vs 5.7; p 0.07) on combination therapy. One patient developed leukopenia (WBC 2.8) after starting infliximab with an increase in 6TGN levels from 425 to 516.Figure: Thiopurine metabolite concentration and WBC count on monotherapy and combination therapy.Table: Table. Baseline CharacteristicsTable: Table. Lab parameters on monotherapy and combination therapyConclusion: In patients with IBD on thiopurine monotherapy, the addition of an anti-TNF agent can lead to an increase in 6-TGN concentration, thereby increasing the risk of leukopenia. Hence, elective thiopurine dose reduction should be considered prior to starting an anti-TNF agent, particularly in patients with therapeutic 6TGN concentration on thiopurine monotherapy. Our findings also suggest that benefits of combination therapy may be, in part, due to increase in thiopurine metabolite concentration with addition of an anti-TNF agent.

Azadirachta indica (neem) leaf dietary effects on the immunity response and disease resistance of Asian seabass, Lates calcarifer challenged with Vibrio harveyi

Asian sea bass (Lates calcarifer), commonly known as barramundi, is an economically important food fish inhabiting coastal and brackish waters. In Sri Lanka, demand for commercial and recreational fishing has increased due to its mild flavor and culinary versatility. However, disease outbreaks pose significant challenges to aquaculture production, particularly in cage culture systems. This study investigated the causative agent of mass mortality events in Asian sea bass farms in Trincomalee, Sri Lanka. Fish exhibiting clinical signs, including skin ulcers, fin rot, exophthalmia, and lethargy, were collected for examination. Samples from the kidney, liver, and spleen were cultured on Tryptic Soy Agar (TSA) and Thiosulfate Citrate Bile Salts Sucrose (TCBS) agar at 37 °C for 24-48 hr. Isolates produced small to medium, round, convex colonies on TSA and green colonies on TCBS. Biochemical characterization revealed Gram-negative, oxidase-positive, catalase-positive, motile rods that fermented glucose without gas production. Molecular identification using 16S rRNA polymerase chain reaction confirmed the pathogen as Vibrio harveyi with 99.44% similarity to strain CP014038.2. The sequence was deposited in GenBank (accession number OR351233). Histopathological examination revealed tubular injury, necrotic epithelial cells, and macrophage-lymphocyte infiltration in liver and kidney tissues. This study confirms that Vibrio harveyi is strongly associated with Vibriosis, causing mass mortality in Asian sea bass cage culture in Trincomalee. These findings provide essential baseline data for developing effective disease prevention and control strategies in Sri Lankan aquaculture systems.

Evaluation of immune responses and protection in Asian seabass (Lates calcarifer Bloch, 1790) against Vibrio vulnificus using immersion and oral nanoemulsion vaccines.

A feeding trial was done for 60 days to examine the influence of supplementing low-fish meal (FM) diet with a mixture of acidifiers on the performance of Asian seabass (Lates calcarifer) juveniles (initial weight: 54.2 ± 0.5 g, mean ± standard deviation). Dietary FM was replaced (35% and 70%) with a mixture of alternative protein sources, including soybean meal, corn gluten, wheat gluten, and poultry meal then supplemented with two levels (0.5% and 1.0%) of an acidifier mixture (butyric acid, sodium diformate and fulvic acid, 1:1:1 ratio). In this regard, seven isonitrogenous (45%) and isolipidic (15%) diets were formulated including: FM70 (FM-based diet, control), FMR35 (35% FM replacement), FMR35+0.5% (35% FM replacement + 0.5% acidifier mixture), FMR35+1.0% (35% FM replacement + 1.0% acidifier mixture), FMR70 (70% FM replacement), FMR70+0.5% (70% FM replacement + 0.5% acidifier mixture), and FMR35+1.0% (70% FM replacement + 1.0% acidifier mixture). Fish were stocked into twenty-one 2000-L rectangular concrete tanks (53 fish/tank) that were filled with running seawater in a flow-through system (26.5 ± 1.5 °C and 46.0 ± 0.2 ppt). Fish were fed with the diets twice every day up to visual satiation. Before the beginning (day 0), middle (day 30), and after finishing the feeding trial (day 60), fish were individually weighed, and samples were collected from their blood and gut for evaluating hematological, antioxidant and digestive enzymes, respectively. After finishing the feeding trial, fish fed FMR70 had lower weight gain (89.1% lower than FM70) than other groups (P = 0.001) that associated with the lowest feed intake in this group. The trypsin, protease, and α-amylase activities were decreased by increasing FM replacement level, but it enhanced alkaline phosphatase activity (P < 0.05). Serum catalase activity increased by the interactive effects of dietary FM replacement and acidifier level (P = 0.029). The blood hemoglobin, mean cell volume, mean cell hemoglobin, lysozyme activity, serum protein, albumin, globulin, cholesterol, and triglycerides increased by including a 0.5% acidifier mixture in the low FM content diets (P < 0.05). In addition, serum calcium content increased with increasing acidifier mixture level in the diet (P = 0.006). In summary, the inclusion of 0.5% acidifier mixture in a low FM-diet not only improved growth performance and feed utilization but also promoted humoral immunocompetance and hematological indices in L. calcarifer juveniles.