Evaluation of the efficacy of MONTANIDE™ GR01, a new adjuvant for feed-based vaccines, on the immune response and protection against Streptococcus agalactiae in oral vaccinated Nile tilapia (Oreochromis niloticus) under laboratory and on-farm conditions.

Streptococcosis, an emerging infectious disease caused by Streptococcus agalactiae, has had adverse effects on farmed tilapia. Several vaccines have been developed to prevent this disease and induce a specific immune response against S. agalactiae infection. In this study the use of MONTANIDE™ GR01, a new adjuvant for oral vaccination, was optimized for use in tilapia under laboratory and field studies. In the laboratory trial the immune response and protective efficacy of two doses of MONTANIDE™ GR01, 20 % (w/w) and 2 % (w/w), included into the feed-based adjuvanted vaccines were assessed comparatively. Following immunization, the innate immune parameters studied in serum, including lysozyme, myeloperoxidase, catalase and glutathione peroxidase activity, were all increased significantly. Furthermore, specific IgM antibodies against S. agalactiae were induced significantly in serum post-vaccination, with higher levels observed in both groups that received the feed-based adjuvanted vaccine. Under both injection and immersion challenge conditions, the relative percent survival for the feed-based adjuvanted vaccine groups ranged from 78 % to 84 %. Following use of the low dose concentration of MONTANIDE™ GR01 for oral vaccination of tilapia in cage culture systems, several innate immune parameters were effectively enhanced in the immunized fish. Similarly, the levels of specific IgM antibodies in the serum of feed-based vaccinated fish were significantly enhanced, reaching their highest levels 2-5 months post-vaccination. Cytokines associated with innate and adaptive immunity were also examined, and the expression levels of several genes showed significant up-regulation. This indicates that both cellular and humoral immune responses were induced by the feed-based adjuvanted vaccine. The economic impact of a feed-based adjuvanted vaccine was examined following vaccination, considering the growth performance and feed utilization of the fish. It was found that the Economic Performance Index and Economic Conversion Ratio were unaffected by vaccination, further demonstrating that there are no negative impacts associated with administering a feed-based vaccine to fish. In conclusion, the data from this study indicate that MONTANIDE™ GR01 is a highly valuable adjuvant for oral vaccination, as demonstrated by its ability to induce a strong immune response and effectively prevent streptococcal disease in Nile tilapia.

Pathogenicity of Streptococcus iniae causing mass mortalities of yellow catfish (Tachysurus fulvidraco) and its induced host immune response.

The outbreak of mass mortality occurred in Tachysurus fulvidraco farm in Hubei province of China. The pathogenic strain of Streptococcus iniae (termed 2022SI08) was isolated and identified from diseased T. fulvidraco, based on morphological, physiological, and biochemical characteristics, as well as 16S rRNA gene sequence and phylogenetic analysis. Further, the whole genome of isolate S. iniae was sequenced and predicted to contain one single circular chromosome of 1,776,777 bp with a GC content of 37.14%. The genomic sequence analysis showed that 2022SI08 was positive for 204 virulent and 127 antibiotic resistant genes. The experimental challenge demonstrated the high pathogenicity of the retrieved isolate of S. iniae, with a median lethal dosage (LD50) 9.53 × 105 CFU/g. Histopathological examination indicated that the 2022SI08 strain could induce extensive tissue cell degeneration, necrosis, hemorrhage, and inflammation in the skin, gill, fin, spleen, liver, kidney, intestine, eye, and brain. Moreover, the innate immune enzyme activities in serum such as acid phosphatase and alkaline phosphatase were increased significantly at 24 and 48 h post infection (hpi) and then decreased at 168 hpi. The transcriptional profile of immune associated gene in T. fulvidraco following bacterial infection was detected at each point of time, and the results revealed clear transcriptional activation of those genes, which proving their reacting and regulatory role during the response of the host against S. iniae infection. The results revealed that S. iniae was an etiological agent in the mass mortalities of T. fulvidraco and this research will be conducive for increasing our understanding on pathogenesis and host defensive system in S. iniae invasion.

Establishment of a cobia (Rachycentron canadum) gill cell line: A valuable tool for immune response studies.

Cobia (Rachycentron canadum), a commercially important marine fish, has been used to develop a novel gill cell line, designated CG, for the first time. The CG cell line was cultured in Leibovitz's-15 medium with 5% fetal bovine serum (FBS) and successfully sub-cultured more than 110 passages. It underwent verification through sequencing of the mitochondrial cytochrome C oxidase subunit I (COI) gene. Optimal growth rate was achieved when the CG cell line was cultured in a medium supplemented with 5% FBS, 1% Penicillin-Streptomycin (P/S), and 5 parts per thousand (ppt) of coral sea salt water, maintained at a temperature of 27 °C. The addition of 5 ppt of salt in the growth medium suggests that this cell line could be a viable in vitro tool for marine ecosystem toxicological studies or for culturing marine parasitic microorganisms. The CG cell line was also successfully transfected using the pTurbo-GFP plasmids, showing an 18% efficiency, with observable GFP expression. Furthermore, the cell line has been effectively cryopreserved. Gene expression analysis indicated that the CG cell line exhibits responsive regulation of immune gene expression when exposured to various stimulants, highlighting its potential as an in vitro platform for immune response studies. This makes it suitable for exploring dynamic immune signaling pathways and host-pathogen interactions, thereby offering valuable insights for therapeutic development.

Impacts of astaxanthin-enriched Paracoccus carotinifaciens on growth, immune responses, and reproduction performance of broodstock Nile tilapia during winter season.

The growth, immune response, and reproductive performance of broodstock of Nile tilapia (Oreochromis niloticus) under winter stress conditions were investigated the effects of supplementary diets with astaxanthin-enriched Paracoccus carotinifaciens. Throughout an eight-week period in the winter season, male and female tilapia were fed with diets containing different levels of P. carotinifaciens dietary supplementation: 0 g/kg (T1; control), 5 g/kg (T2), 10 g/kg (T3), and 20 g/kg (T4). Subsequently, a four-week mating system was implemented during the winter stress period. The results revealed that there were no significant differences observed in growth, hematological indices, and blood chemical profiles among all treatment groups for both male and female tilapia. However, a significant increase in cholesterol content was noted in both male and female tilapia fed with the T4 diet (p<0.05). The total carotenoid content in the muscle was evaluated, and significantly higher values were found in both male and female tilapia that fed T4 supplementation (p<0.05). Moreover, immunological parameters such as myeloperoxidase and antioxidant parameters in the liver including superoxide dismutase activity and catalase enzyme activity showed significant increases in tilapia fed with the T4 diet. The impact of P. carotinifaciens supplementation on broodstock tilapia indicated a significant increase in spermatozoa concentration in males and increased egg production in females after consumption of the T4 diet (p<0.05). Thus, this study highlighted that the presence of astaxanthin-enriched P. carotinifaciens in the diet of broodstock Nile tilapia can lead to the accumulation of carotenoids in their muscle tissue, improvement in antioxidant status, enhancement of immune function, and potential enhancement of reproductive capabilities, even under overwintering conditions.

Immune response and protective efficacy of Streptococcus agalactiae vaccine coated with chitosan oligosaccharide for different immunization strategy in nile tilapia (Oreochromis niloticus).

In the past decade, the outbreak of Streptococcus agalactiae has caused significant economic losses in tilapia farming. Vaccine immunization methods and strategies have gradually evolved from single-mode to multi-mode overall prevention and control strategies. In this study, an inactivated vaccine of S. agalactiae with a chitosan oligosaccharide (COS) adjuvant was constructed using different administration methods: intraperitoneal injection (Ip), immersion combined with intraperitoneal injection (Im + Ip), immersion combined with oral administration (Im + Or), and oral administration (Or). Safety analysis revealed no adverse effects on tilapia, and the vaccine significantly promoted fish growth and development when administered through Im + Or or Or immunization. Following vaccination, innate immunity parameters including SOD, ACP and CAT activities were all significantly enhanced. Additionally, specific serum IgM antibodies reached their highest level at the 6th week post vaccination. Skin and intestinal mucus IgT antibodies reached peaked at the 6th and 7th week post vaccination, respectively. The relative peak expression values for IL-8, IL-12, MHC-I, MHC-II, IgM, IgT, CD4, CD8, TNFα, IFNγ from Im + Ip group were significantly higher than those in Ip group, Im + Or group and Or group in most cases (p < 0.05). Importantly, the relative protection survival of Im + Ip group was the highest (78.6%), followed by the Ip group (71.4%), the Or group (64.3%) and the Im + Or group (57.1%). In summary, this study encourages further research on multi-channel immunization strategies of other kinds of vaccines in other aquatic economic animals to improve their disease resistance.

Insights into the functional properties of a natural free amino acid mix: Effect on growth performance, nutrient metabolism, and immune response in a carnivorous fish, Asian seabass (Lates calcarifer).

Dietary supplements containing a functional feed additive have been shown to be beneficial to fish and shellfish aquaculture. However, the functional properties of aquafeed formulations have rarely been reported in fish. This study aimed to investigate the effects of natural free amino acid mix (FAAM) supplementation as a functional solution on the growth performance and nutrient utilization in a carnivorous fish, Asian seabass (Lates calcarifer). Five isonitrogenous and isolipidic diets were prepared with graded supplementation levels of FAAM at 0 % (control group), 0.25 %, 0.50 %, 0.75 %, and 1.0 %, denoted as FAAM0, FAAM0.25, FAAM0.5, FAAM0.75, and FAAM1.0, respectively. The experimental fish were fed different dietary FAAM supplementations to apparent satiation twice daily for eight weeks. Significant improvements were observed in the growth performance of fish among the five groups (P < 0.05). Fish fed with FAAM0.75 displayed significantly increased activities of lysozyme, myeloperoxidase, catalase, and glutathione peroxidase (P < 0.05). The activities of digestive enzymes, including amylase, protease, and lipase, were enhanced by the supplementation of FAAM in the feed (P < 0.05), especially for the groups that contained more than 0.5 % FAAM in the feed. Furthermore, the morphological profile of the intestinal tract, including the mucosal fold height, width, thickness, and goblet cell, increased in fish fed with FAAM at 1.0 % (P < 0.05). Moreover, FAAM supplementation in diets not only modulated the expression of immune-related genes (glutathione peroxidase (GPx), complement (C)3, C4, and C-reactive protein) in the liver but also positively impacted the growth-ralated genes, including growth hormone (GH), GH receptor (GHR), insulin-like growth factor I (IGF-I), and IGF-II. In addition, the amounts of monounsaturated fatty acids (mainly oleic acid (C18:1n9c)) and polyunsaturated fatty acids-especially γ-linolenic acid (C18:3 n6) and α-linolenic acid (C18:3n3)-increased in fish fed with diets containing FAAMs (P < 0.05). Interestingly, the diets supplemented with FAAMs also had a positive effect on the economic indices in terms of revenue-to-cost ratios. These findings provide a scientific basis for the application of FAAMs as a functional solution that can be used in feed formulations for Asian seabass.

Assessment of Bacillus subtilis fermented Caulerpa microphysa byproduct as feed additive on the growth performance, immune regulation and disease resistance of white shrimp (Litopenaeus vannamei).

In this study, the immunomodulatory and antioxidant activity of fermented Caulerpa microphysa byproduct (FCMB) by Bacillus subtilis was evaluated, and its potential as a feed additive for white shrimp (Litopenaeus vannamei) was explored. In vitro experiments showed that the FCMB supernatant contained polysaccharides, polyphenols and flavonoids, and exhibited antioxidant properties as assessed by various antioxidant assays. Additionally, the FCMB supernatant was found to increase the production rate of reactive oxygen species and the activity of phenoloxidase in hemocytes in vitro. Furthermore, the results of the in vivo feeding trial showed that dietary 5 g kg-1 FCMB significantly improved the weight gain and specific growth rate of white shrimp after 56 days of feeding. Although there were no significant differences in total hemocyte count, phagocytosis, superoxide anion production rate, and phenoloxidase activity among the experimental groups, upregulation of immune-related genes was observed, particularly in the hepatopancreas and hemocytes of shrimps fed with 5 g or 50 g FCMB per kg feed, respectively. In the pathogen challenge assay, white shrimp fed with 5 % FCMB exhibited a higher survival rate compared to the control group following Vibrio parahaemolyticus challenge. Therefore, it is concluded that the fermented byproduct of C. microphysa, FCMB, holds potential as a feed additive for enhancing the growth performance and disease resistance against V. parahaemolyticus in white shrimp.

Dietary Administration Effects of Exopolysaccharide Produced by Bacillus tequilensis PS21 Using Riceberry Broken Rice, and Soybean Meal on Growth Performance, Immunity, and Resistance to Streptococcus agalactiae of Nile tilapia (Oreochromis niloticus).

Overuse of antibiotics in aquaculture has generated bacterial resistance and altered the ecology. Aquacultural disease control requires an environmentally sustainable approach. Bacterial exopolysaccharides (EPSs) as bioimmunostimulants have not been extensively explored in aquaculture. This study investigated EPS produced from 5% w/v riceberry broken rice as a carbon source and 1% w/v soybean meal as a nitrogen source by Bacillus tequilensis PS21 from milk kefir grain for its immunomodulatory, antioxidant activities and resistance to pathogenic Streptococcus agalactiae in Nile tilapia (Oreochromis niloticus). The FTIR spectrum of EPS confirmed the characteristic bonds of polysaccharides, while the HPLC chromatogram of EPS displayed only the glucose monomer subunit, indicating its homopolysaccharide feature. This EPS (20 mg/mL) exhibited DPPH scavenging activity of 65.50 ± 0.31%, an FRAP value of 2.07 ± 0.04 mg FeSO4/g DW, and antimicrobial activity (14.17 ± 0.76 mm inhibition zone diameter) against S. agalactiae EW1 using the agar disc diffusion method. Five groups of Nile tilapia were fed diets (T1 (Control) = 0.0, T2 = 0.1, T3 = 0.2, T4 = 1.0, and T5 = 2.0 g EPS/kg diet) for 90 days. Results showed that EPS did not affect growth performances or body composition, but EPS (T4 + T5) significantly stimulated neutrophil levels and serum lysozyme activity. EPS (T5) significantly induced myeloperoxidase activity, catalase activity, and liver superoxide dismutase activity. EPS (T5) also significantly increased the survival of fish at 80.00 ± 5.77% at 14 days post-challenge with S. agalactiae EW1 compared to the control (T1) at 53.33 ± 10.00%. This study presents an efficient method for utilizing agro-industrial biowaste as a prospective source of value-added EPS via a microbial factory to produce a bio-circular green economy model that preserves a healthy environment while also promoting sustainable aquaculture.

Effect of dietary gamma aminobutyric acid (GABA) modulated the growth performance, immune and antioxidant capacity, digestive enzymes, intestinal histology and gene expression of Nile tilapia (Oreochromisniloticus).

Dietary GABA plays an important role in a variety of physiological functions in animals, but this has been rarely reported in fish. This study investigated the effects of dietary supplementation with GABA on growth, serum antioxidant indicators, digestive enzyme activities, intestinal morphology, and the gene expression of Nile tilapia. Diets containing three different GABA concentrations, 0 (control), 200 mg/kg (GABA200), and 500 mg/kg (GABA500), were fed to Nile tilapia (6.97 ± 0.34 g) for 56 days. The present study found that increasing dietary GABA content could increase the fish growth performance including final body weight, weight gain, specific growth rate, average daily gain, protein efficiency ratio, and feed efficiency compared to the control diet. Interestingly, the feed conversion ratio was improved by dietary GABA supplementation. The antioxidant enzyme activities against ammonia stress of fish fed the GABA diets were significantly higher than the corresponding control group throughout the 96-h ammonia exposure. Moreover, significant increases in digestive enzyme activities including protease, amylase and lipase were found in fish fed the GABA diets. Intestinal morphology analysis revealed increased heights and widths of intestinal villi as well as thickness of the intestinal muscularis in fish fed the GABA diets compared to the control diet. The supplementation of diets with GABA significantly increased the expression level of immune- and growth-related genes. The above results indicate that dietary GABA can modulate the growth, improve their immune response and antioxidant status, gut health and morphology and gene expression of Nile tilapia. Therefore, GABA is a promising feed additive for Nile tilapia aquaculture.

Data on exopolysaccharides produced by Bacillus spp. from cassava pulp with antioxidant and antimicrobial properties.

This data evaluated the capacity of Bacillus spp. isolated from Thai milk kefir to produce exopolysaccharide (EPS) on cassava pulp and tested its antioxidant and antibacterial properties. Thailand's starch industry generates million tons of cassava pulp, which is underutilized or bio-transformed into higher-value bioproducts. Antioxidant and antibacterial bacterial exopolysaccharides are beneficial in the food, feed, pharmaceutical, and cosmetic industries. Moisture, ash, fat, protein, fiber, starch, sugar, neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL) were analyzed from cassava pulp as an EPS substrate. After 3 days of bacterial fermentation, EPS generation, culture pH, reducing sugar amount, and bacterial count were recorded. Antioxidant activities and bioactive content including hydroxyl radical scavenging activity, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, ferric reducing antioxidant power (FRAP), total phenolic and flavonoid content (TPC and TFC), and antimicrobial activity against two Nile tilapia pathogens (Streptococcus agalactiae and Staphylococcus aureus) from different Bacillus species were evaluated. Proximate analysis, dinitrosalicylic acid assay, pH value record, bacterial count using spread plate method, antioxidant activity and bioactive content assays via spectrophotometry, and agar disk diffusion were the main approaches. This study used microbial cell factories to convert agro-biowaste, such as cassava pulp, into EPS bioproducts which accords with a bio-circular green economy model.

Nutritional profile of the land snail Cyclophorus saturnus, a rich-in-nutrients food item from Thailand.

Cyclophorus saturnus is an edible land snail traditionally harvested for human food, yet little is known about its nutritional value, especially in Thailand. This study aimed to investigate its nutritional potential as an alternative food resource. In the present study, proximate composition, essential mineral content, amino acid, and lipid profiles of the meat were evaluated. Proximate analysis showed that C. saturnus contained 80.04% moisture, 11.88% protein, 6.04% carbohydrate, and 0.93% fat, with 80.01 kcal/100 g fresh matter. For minerals, calcium was the most abundant element in the meat. Its protein contained glutamic and aspartic as the major amino acids, while it was not a good source of tryptophan and methionine but was considered a very rich source of other essential amino acids (amino acid scores greater than 100). Its lipid fraction showed a higher proportion of mono and polyunsaturated fatty acids (MUFA and PUFA, 67.69%) and a lower proportion of saturated fatty acids (SFA) (32.31%). The PUFA/SFA ratio (1.56), hypocholesterolemic/hypercholesterolemic ratio (HH; 5.58), atherogenicity index (AI; 0.48), and thrombogenicity index (TI; 0.20) are considered nutritionally healthy for humans. Overall, this study demonstrates the nutritional potential of C. saturnus to serve as a nutritious part of the human diet and as an alternative ingredient in food systems; therefore, its production and consumption should be more extensively promoted.

Modulation of immune genes in the mucosal-associated lymphoid tissues of cobia by Sarcodia suae extract.

Rachycentron canadum (cobia) is a marine fish species of high economic value in aquaculture due to its fast growth rate and good feed conversion efficacy. Regrettably, the industry has been affected by significant setbacks from high mortality due to diseases. Consequently, an improved perception of innate immunity correlated to each mucosal-associated lymphoid tissue (MALT) in teleost fish is necessary to understand hosts' response towards infections better. The utilization of polysaccharides in seaweed to stimulate the immune system has gathered unprecedented attention. The present study examined the immunostimulatory effects of Sarcodia suae water extracts (SSWE) on in vivo gill-, gut- and skin-associated lymphoid tissues (GIALT, GALT, and SALT) via immersion and oral ingestions. The GIALT genes (TNF-α, Cox2, IL-1ß, IL-6, IL-8, IL-17 A/F1-3, IL-11, IL-12, IL-15, IL-18, MHCIa, IgM, and IgT) except IL-10 recorded positive upregulations in a dose-dependent manner post 24 h immersion in SSWE, indicating the algae extract contained bioactive compounds that could stimulate the immune genes. The upregulation of IL-12, IL-15, and IL-18 in the gills and hindgut post-SSWE immersion indicated that the extract could promote Th1-related responses in the MALTs. The modulation of immune gene expressions in the feeding trial was less potent than in the SSWE immersion. These findings indicated that the SSWE stimulated robust immune responses in both the GIALT and GALT of cobia. This suggests that the SSWE could be further explored as an effective immersive stimulant for fish, enhancing their immune system against pathogens.

Immune response and protective efficacy of mannosylated polyethylenimine (PEI) as an antigen delivery vector, administered with a Streptococcus agalactiae DNA vaccine in Nile tilapia (Oreochromis niloticus).

This study examined the effectiveness of a DNA vaccine for S. agalactiae that was delivered by mannose-based polyethyleneimine (Man-PEI). The results showed that Man-PEI/pcDNA-Sip stimulated a higher serum antibody titer compared to control or other vaccine groups (p < 0.05). Additionally, it induced higher expression of immune-related genes, and increased activities of superoxide dismutase (SOD), acid phosphatase (ACP) and alkaline phosphatase (AKP). Furthermore, the Man-PEI/pcDNA-Sip group showed an improved relative percent survival (RPS) of 85.71%. These results demonstrate the potential value of Man-PEI as a vaccine delivery vehicle, and suggest that it can be effective in boosting the immune protective rate induced by pcDNA-Sip vaccines.

Comparative study of the effects of Montanide™ ISA 763A VG and ISA 763B VG adjuvants on the immune response against Streptococcus agalactiae in Nile tilapia (Oreochromis niloticus).

Streptococcus agalactiae is regarded as a major bacterial pathogen of farmed fish, with outbreaks in Nile tilapia causing significant losses. Vaccination is considered the most suitable method for disease control in aquaculture, with the potential to prevent such outbreaks if highly efficacious vaccines are available for use. Several vaccines have been produced to protect against S. agalactiae infection in tilapia, including inactivated vaccines, live attenuated vaccines, and subunit vaccines, with variable levels of protection seen. Two commercial adjuvants, Montanide™ ISA 763A VG and ISA 763B VG, have been developed recently and designed to improve the safety and efficacy of oil-based emulsions delivered by intraperitoneal injection. In particular, their mode of action may help identify and stimulate particular immunological pathways linked to the intended protective response, which is an important tool for future vaccine development. Therefore, this study aimed to characterize the potential of two adjuvanted-bacterial vaccines against S. agalactiae (SAIV) comparatively, to determine their usefulness for improving protection and to analyse the immune mechanisms involved. Nile tilapia were divided into four groups: 1) fish injected with PBS as a control, 2) fish injected with the SAIV alone, 3) fish injected with the SAIV + Montanide™ ISA 763A VG, and 4) fish injected with the SAIV + Montanide™ ISA 763B VG. Following immunization selected innate immune parameters were analysed, including serum lysozyme, myeloperoxidase, and bactericidal activity, with significantly increased levels seen after immunization. Cytokines associated with innate and adaptive immunity were also studied, with expression levels of several genes showing significant up-regulation, indicating good induction of cell-mediated immune responses. Additionally, the specific IgM antibody response against S. agalactiae was determined and found to be significantly induced post-vaccination, with higher levels seen in the presence of the adjuvants. In comparison to the protection seen with the unadjuvanted vaccine (61.29% RPS), both Montanide™ ISA 763A VG and Montanide™ ISA 763B VG improved the RPS, to 77.42% and 74.19% respectively. In conclusion, Montanide™ ISA 763A VG and Montanide™ ISA 763B VG have shown potential for use as adjuvants for fish vaccines against streptococcosis, as evidenced by the enhanced immunoprotection seen when given in combination with the SAIV vaccine employed in this study.

Effects of Bacillus spp. Mixture on Growth, Immune Responses, Expression of Immune-Related Genes, and Resistance of Nile Tilapia Against Streptococcus agalactiae Infection.

The purpose of this study was to evaluate the effect of Bacillus spp. mixture (Bacillus subtilis TISTR001, Bacillus megaterium TISTR067, and Bacillus licheniformis DF001) (1 × 106 CFU/g) on growth, immune parameters, immune-related gene expression, and resistance of Nile tilapia against Streptococcus agalactiae AAHM04. Fish were fed different concentrations of Bacillus spp. 0 (control; T1), 1 (T2), 3 (T3), and 5 (T4) g/kg diets for 120 days. The results showed that weight gain, average daily gain, specific growth rate, feed conversion ratio in T3 diet were significantly higher than the control group and other tested diets (p < 0.05). Immune parameters, such as myeloperoxidase and lysozyme, were significantly higher in the T3 and T4 diets compared to the control group (p < 0.05). Similarly, IL-1ß and TNF-α gene expressions in the spleen of fish fed T2, T3, and T4 diets were significantly higher than the control group (p < 0.05). However, no significant differences in survival rate, hematology, blood chemical indices, malondialdehyde (MDA) levels, body chemical composition, and organosomatic indices (p > 0.05) were noticed in all treatments. No significant differences in survival rate after the challenge test with S. agalactiae AAHM04 were found in fish fed Bacillus spp. mixture diets, except for the T3 diet. These results suggest that Bacillus spp. mixture diet at 3 g/kg diet (T3) could improve growth, immune response, and disease resistance of Nile tilapia.

Effect of supplementation and withdrawal of selenium-enriched kale sprouts on productivity and egg selenium concentration of laying hens.

OBJECTIVE: The aim of this trial was to investigate the effect of supplementation and withdrawal of selenium-enriched kale sprouts (SeKS) on productivity and egg Se concentration of laying hens. Selenium from commercial Se-enriched yeast (SeY) was used as a comparative Se source. METHODS: One-hundred and eighty 61-week-old laying hens were randomly divided into 5 treatment groups with 4 replicates (9 hens each) in a 2×2+1 Augmented Factorial Experiment in a completely randomized design. The experimental diets were basal diet, basal diet supplemented with 0.2 and 0.4 mg Se/kg from SeKS and SeY, respectively. The 8-week feeding trial was divided into 2 periods, namely the Se supplemental period (week 1 to 4) and the Se withdrawal period (week 5 to 8). RESULTS: Productive performance, egg quality and egg Se concentration of laying hens were not affected by sources of Se (SeKS and SeY) during both, the Se supplemental and withdrawal periods. Egg production and egg Se concentration increased (p<0.05) with increasing levels of Se supplementation. The egg Se concentration increased and reached a peak 1 week after Se supplementation. However, concentration of Se in eggs of hens fed Se from both sources decreased rapidly from the second week of the Se withdrawal period to reach the same egg Se concentration of hens fed the basal diet by the fourth week of the Se withdrawal period. CONCLUSION: The efficacy of Se from SeKS on productivity and egg Se concentration in laying hens was comparable to commercial SeY. Thus, SeKS can provide an alternate organic Se source for production of Se-enriched eggs.

Effects of Red Yeast (Sporidiobolus pararoseus) on Growth, Innate Immunity, Expression of Immune-related Genes and Disease Resistance of Nile Tilapia (Oreochromis niloticus).

The purpose of this study was to evaluate the effects of red yeast (Sporidiobolus pararoseus) produced from crude glycerol, as a by-product of the biodiesel production process, on the growth, innate immunity, expression of immune-related gene, and resistance of Nile tilapia against challenge with Streptococcus agalactiae. Fish were fed diets supplied with different concentrations of S. pararoseus dried cells at 0.0 (control; T1), 5.0 (T2), 10.0 (T3), and 20.0 (T4) g kg-1 diets for 90 days. The results showed that final body weight, weight gain, and average daily gain were significantly higher in fish fed T3 and T4 compared to the control group (p < 0.05). Likewise, significant (p < 0.05) increases in total carotenoid content, liver superoxide dismutase activity (SOD), and serum lysozyme and albumin were observed in Nile tilapia fed S. pararoseus, with the highest (p < 0.05) values displayed in fish fed the T4 diet. Moreover, up-regulation of IL-1ß transcription in Nile tilapia spleen and liver was observed in fish feeding group T4. In a challenge test against S. agalactiae, the fish survival rate was significantly higher in fish fed red yeast compared to the control group (p < 0.05). The highest bactericidal activity found in the T4 group (p < 0.05). However, no significant differences were found in hematology, blood chemical, malondialdehyde (MDA), body chemical composition, organosomatic indices, and myeloperoxidase (p > 0.05) in all treatments. The present results suggested that red yeast S. pararoseus (20.0 g kg-1) can be used as a potential supplementation on growth, immune response, and disease resistance of Nile tilapia.

Molecular characterization of the evolutionary conserved signaling intermediate in Toll pathways (ECSIT) of soiny mullet (Liza haematocheila).

Mammalian evolutionary conserved signaling intermediate in Toll pathways (ECSIT) is an important intracellular protein that involves in innate immunity, embryogenesis, and assembly or stability of the mitochondrial complex I. In the present study, the ECSIT was characterized in soiny mullet (Liza haematocheila). The full-length cDNA of mullet ECSIT was 1860 bp, encoding 449 amino acids. Mullet ECSIT shared 60.4%∼78.2% sequence identities with its teleost counterparts. Two conserved protein domains, ECSIT domain and C-terminal domain, were found in mullet ECSIT. Realtime qPCR analysis revealed that mullet ECSIT was distributed in all examined tissues with high expressions in spleen, head kidney (HK) and gill. Further analysis showed that mullet ECSIT in spleen was up-regulated from 6 h to 48 h after Streptococcus dysgalactiae infection. In addition, the co-immunoprecipitation (co-IP) assay confirmed that mullet ECSIT could interact with tumor necrosis factor receptor-associated factor 6 (TRAF6). Molecular docking revealed that the polar interaction and hydrophobic interaction play crucial roles in the forming of ECSIT-TRAF6 complex. The resides of mullet ECSIT that involved in the interaction between ECSIT and TRAF6 were Arg107, Glu113, Phe114, Glu124, Lys120 and Lys121, which mainly located in the ECSIT domain. Our results demonstrated that mullet ECSIT involved in the immune defense against bacterial and regulation of TLRs signaling pathway by interaction with TRAF6. To the best of our knowledge, this is the first report on ECSIT of soiny mullet, which deepen the understanding of ECSIT and its functions in the immune response of teleosts.

Combining segments 9 and 10 in DNA and recombinant protein vaccines conferred superior protection against tilapia lake virus in hybrid red tilapia (oreochromis sp.) compared to single segment vaccines.

Tilapia lake virus (TiLV) now affects Nile tilapia culture worldwide, with no available commercial vaccine for disease prevention. DNA and recombinant protein-based vaccines were developed and tested following viral isolation and characterization. The viral strain isolated from diseased hybrid red tilapia (Oreochromis sp.) shared high levels of morphological and genomic similarity (95.49-99.52%) with other TiLV isolates in the GenBank database. TiLV segment 9 (Tis9) and segment 10 (Tis10) DNA vaccines (pcDNA-Tis9 and pcDNA-Tis10) and recombinant protein vaccines (Tis9 and Tis10) were prepared and tested for their efficacy in juvenile hybrid red tilapia. Fish were immunized with either single vaccines (pcDNA-Tis9, pcDNA-Tis10, Tis9 and Tis10) or combined vaccines (pcDNA-Tis9 + pcDNA-Tis10 and Tis9 + Tis10) by intramuscular injection and intraperitoneal injection for DNA and protein vaccines, respectively. Negative controls were injected with PBS or a naked pcDNA3.1 vector in the same manner. An experimental challenge with TiLV was carried out at 4 weeks post-vaccination (wpv) by intraperitoneal injection with a dose of 1 × 105 TCID50 per fish. Relative percent survival (RPS) ranged from 16.67 ± 00.00 to 61.11 ± 9.62%. The Tis10 and pcDNA-Tis10 vaccines conferred better protection compared to Tis9 and pcDNA-Tis9. Highest levels of protection were observed in pcDNA-Tis9 + pcDNA-Tis10 (61.11 ± 9.62%) and Tis9 + Tis10 (55.56 ± 9.62%) groups. Specific antibody was detected in all vaccinated groups at 1-4 wpv by Dot Blot method, with the highest integrated density at 2 and 3 wpv. In silico analysis of Tis9 and Tis10 revealed a number of B-cell epitopes in their coil structure, possibly reflecting their immunogenicity. Findings suggested that the combination of Tis9 and Tis10 in DNA and recombinant protein vaccine showed high efficacy for the prevention of TiLV disease in hybrid red tilapia.