CD40 induces an antimicrobial response against the intracellular pathogen Streptococcus agalactiae in Nile tilapia,Oreochromis niloticus

Streptococcus agalactiae is a Gram‐positive facultative intracellular bacterium that leads to severe economic loss of tilapia worldwide. Previous studies demonstrated that CD40 contributes to host protection against intracellular injection. In this study, CD40 was characterized from Nile tilapia (Oreochromis niloticus), named OnCD40. Sequence analysis showed that open reading frame of OnCD40 was 933 bp, containing a single peptide, a transmembrane domain and four cysteine‐rich domains. The qRT‐PCR revealed that OnCD40 was expressed in all examined tissues with the most abundant ones in spleen and thymus. After S. agalactiae stimulation, the expression of OnCD40 was significantly induced in most of the detected organs. Moreover, OnCD40‐overexpressing fish elicited significant protection against subsequent S. agalactiae challenge; approximately 10000‐fold fewer bacteria were detected in spleen of OnCD40‐overexpressing fish in comparison with control fish. Thus, CD40 had protecting function in Nile tilapia against intracellular pathogens.     

Development of attenuated erythromycin‐resistant Streptococcus agalactiae vaccine for tilapia (Oreochromis niloticus) culture

Streptococcus agalactiae is an important pathogen in fish, causing great losses of intensive tilapia farming. To develop a potential live attenuated vaccine, a re‐attenuated S. agalactiae (named TFJ‐ery) was developed from a natural low‐virulence S. agalactiae strain TFJ0901 through selection of resistance to erythromycin. The biological characteristics, virulence, stability and the immunization protective efficacy to tilapia of TFJ‐ery were determined. The results indicated that TFJ‐ery grew at a slower rate than TFJ0901. The capsule thickness of TFJ‐ery was significantly less (p < 0.05) than TFJ0901. When Nile tilapia were intraperitoneally (IP) injected with TFJ‐ery, the mortality of fish was decreased than that injected with TFJ0901. The RPS of fish immunized with TFJ‐ery at a dose of 5.0 × 10⁷ CFU was 95.00%, 93.02% and 100.00% at 4, 8 and 16 weeks post‐vaccination, respectively. ELISA results showed that the vaccinated fish produced significantly higher (p < 0.05) antibody titres compared to those of control at 2 or 4 weeks post‐vaccination. Taken together, our results suggest that erythromycin could be used to attenuate S. agalactiae, and TFJ‐ery is a potent attenuated vaccine candidate to protect tilapia against S. agalactiae infections.     

Dietary vitamin C requirement of genetically improved farmed Tilapia,Oreochromis Niloticus

A 12‐week growth experiment was conducted to quantify the appropriate dietary vitamin C requirement for GIFT tilapia Oreochromis niloticus. Triplicate groups of 25 experimental tilapia [initial body weight: (70.0 ± 1.6) g] were cultured in 5.6‐m³ aquaria (r = 1.5 m,h = 0.8 m) and fed with semi‐purified diets containing six levels [6.1 (un‐supplemented diet], 23.8, 41.9, 85.1, 167.4 and 339.0 mg kg^?1 diet respectively) of vitamin C (supplied as L‐ascorbyl‐2‐polyphosphate). The results showed that, increasing dietary vitamin C level up to 41.9 mg kg^?1 diet increased weight gain rate of tilapia, beyond which it remained nearly unchanged. Vitamin C contents in liver and muscle presented first increased linearly then tended to reach saturation at high vitamin C (167.4 and 339.0 mg kg^?1) treatments. Muscle collagen content significantly increased with increasing dietary vitamin C levels. Whole‐body lipid content significantly increased, whereas ash content significantly declined, but moisture and crude protein content showed no significant difference with the increasing of dietary vitamin C. The blood chemistry analysis showed that dietary vitamin C had significant effects on enzyme activities of serum alkaline phosphatase, aspartate aminotransferase and alanine aminotransferase. Significant effects were also observed on albumin and total protein content of serum, but haemoglobin content showed no significant differences among all the treatments. The vitamin C requirement for GIFT tilapia was estimated to be 45.0 mg kg^?1 diet based on maximum growth, 114.9 and 118.6 mg kg^?1 diet based on maximum liver and muscle vitamin C concentration respectively.     

Selection response for Streptococcus agalactiae resistance in Nile tilapia Oreochromis niloticus

The potential of selection to improve resistance to streptococcosis was evaluated in a commercial population of Nile tilapia in Thailand. The base generation (G0) consisted of offspring from 98 sires and 149 dams using a partly nested design. At 60 days post‐hatch, 30 fish from each family were injected intraperitoneally with a Streptococcosis agalactiae solution (1 × 10⁹ CFU/ml) and evaluated for 14 days. Disease resistance was recorded as the number of days from challenge until death (DD) and as a binary (BIN) trait (dead/alive) on day 14. Three models were used for genetic analyses: Cox frailty model for DD; animal model for DD; and animal model for BIN. Age at challenge was fitted as a covariate and contemporary group as fixed or random effect, depending on the model. Fish from the 18 most resistant families were selected to produce the first generation (G1). Heritability estimates for G0 were 0.22, 0.14 ± 0.02 and 0.11 ± 0.02 for the Cox, linear DD and linear BIN models, respectively. Selection response indicated that the risk of death decreased to 54%, survival time increased to 3.4 days and survival rate increased to 21%. These results suggest that genetic improvement is possible for this population.     

Maternal immunity of tilapia broodstock vaccinated with polyvalent vaccine and resistance of their offspring against Streptococcus agalactiae

This study aimed to examine the use of Streptococcus agalactiae polyvalent vaccine in tilapia broodstock and the effect of maternal immunity and resistance on their offspring against S. agalactiae strain. The broodstock was injected with polyvalent vaccine of S. agalactiae at a dose of 10⁸ CFU per fish at 2nd gonadal maturity until spawning. Challenge test was carried out on the offspring at the 5, 10, 15 and 20 days after hatching using NK₁, N₁₇O, N₁₄G, N₃M, N₄M strain respectively and combination of them. We observed immunological parameters in broodstock, eggs and larvae and relative per cent survival (RPS) of larvae after challenged with pathogenic S. agalactiae. The results showed that the leukocytes, phagocytic activity, respiratory burst, lysozyme activity and antibody levels of vaccinated broodstock had higher level compared with unvaccinated broodstock. The high level of the lysozyme activity, antibody levels and recombination activating gene 1 (RAG1) were also observed in eggs and larvae from vaccinated broodstock. Larvae produced from vaccinated broodstock when challenged with variety strain of pathogenic S. agalactiae had RPS value more than 50% until 20 days after hatching. In conclusion, polyvalent vaccine of S. agalactiae administrated in the broodstock could enhance immunity in the broodstock and protect their offspring from pathogenic S. agalactiae.     

Effects of ferulic acid on growth performance,immunity and antioxidant status in genetically improved farmed tilapia (Oreochromis niloticus) fed oxidized fish oil

A study was conducted to characterize the effects of dietary oxidized fish oil on the growth performance, immunity and antioxidant status of genetically improved farmed tilapia (Oreochromis niloticus) and to determine the role of ferulic acid on the oxidative damage induced by the oxidized fish oil. The tilapia (13.73 ± 0.31 g) were fed four experimental diets containing untreated (peroxide value, POV: 2.2 meq/kg) and highly oxidized (POV: 120.6 meq/kg) fish oil either with or without ferulic acid (0 or 400 mg/kg) supplementation for 12 weeks. From the results, the oxidized fish oil treatments increased antioxidant enzyme activities and MDA values but decreased the weight gain and the immunological parameters in tilapia. Meanwhile, the serum biochemical indices were significantly affected by the oxidized fish oil. Besides, the addition of ferulic acid partially counteracted the free radical‐induced damage and improved the health status of tilapia. In conclusion, the oxidized fish oil may induce oxidative stress, destroy liver, dysregulate lipid metabolism as well as reduce non‐specific immunity, and eventually result in growth inhibition of tilapia. The ferulic acid supplementation partially offset the negative effects of the oxidized fish oil on tilapia.     

Differential pathogenicity of five Streptococcus agalactiae isolates of diverse geographic origin in Nile tilapia (Oreochromis niloticus L.)

Streptococcus agalactiae is an emerging pathogen of fish and has caused significant morbidity and mortality worldwide. The main objective of this study is to assess whether pathogenic differences exist among isolates from different geographic locations. Nile tilapia (Oreochromis niloticus L.) were administered an intraperitoneal injection of suspension containing USA, Brazil, Honduras, Israel, or Kuwait S. agalactiae isolates at concentrations ranging from 10² to 10⁷ cfu mL^?1. The LD₅₀ values 7 days after challenge were as follows: USA (1.0 × 10² cfu mL^?1), Brazil (1.5 × 10³ cfu mL^?1), Honduras (6.8 × 10³ cfu mL^?1), Israel (1.0 × 10⁴ cfu mL^?1) and Kuwait (7.2 × 10⁵ cfu mL^?1). Fish from all groups exhibited lethargy, anorexia, exophthalmia, corneal opacity, erratic swimming, petechiae and mortality. Opercular clearing and ascites were only found after infection with certain geographic isolates. The findings in this study indicate that S. agalactiae isolates of different geographic origin can cause significant mortalities after experimental challenge and can have different pathogenic capacities. Isolates from the Americas (USA, Brazil and Honduras) were more pathogenic to Nile tilapia than isolates from the Middle East/Asia (Israel and Kuwait).     

Experimental early pathogenesis of Streptococcus agalactiae infection in red tilapia Oreochromis spp.

Streptococcus agalactiae causes a severe systemic disease in fish, and the routes of entry are still ill‐defined. To address this issue, two groups of 33 red tilapia Oreochromis spp. each of 10 g were orally infected with S. agalactiae (n = 30), and by immersion (n = 30), six individuals were control‐uninfected fish. Three tilapias were killed at each time point from 30 min to 96 h post‐inoculation (pi); controls were killed at 96 h. Samples from most tissues were examined by haematoxylin–eosin (H&E), indirect immunoperoxidase (IPI) and periodic acid‐Schiff; only intestine from fish infected by gavage was evaluated by transmission electron microscopy. The results of both experiments suggest that the main entry site of S. agalactiae in tilapia is the gastrointestinal epithelium; mucus seems to play an important defensive role, and environmental conditions may be an important predisposing factor for the infection.     

Characterization of bacteriophage HN48 and its protective effects in Nile tilapia Oreochromis niloticus against Streptococcus agalactiae infections

Streptococcus agalactiae is a causative agent responsible for massive mortalities of tilapia that has led to catastrophic losses to tilapia culture globally. Bacteriophages represent a new class of antimicrobials against bacteria. In this study, we characterized the bacteriophage HN48, which formed small and round‐transparent plaques on a double‐layer plate. With a hexagonal head and a long tail, this phage may belong to the Caudovirales according to the International Committee on Taxonomy of Viruses. HN48 was found to have a relatively wide and highly specific host range, to be sensitive to high temperature (60–80°C) and low pH (3–5), and to be relatively stable at alkaline pH (8–10). Intraperitoneal injection with HN48 had no adverse effects on tilapia and effectively inactivated the bacteria in the kidney. Fish that received phage therapy had 60% ± 3.3% survival rates and a delayed mean death time of about 3 days when compared to the control group. To the best of knowledge, this is the first study of tilapia streptococcal phage. Overall, the results indicated that phage HN48 could prevent tilapia from experimental S. agalactiae infection, suggesting it has the potential to control this disease.     

吉富罗非鱼对饲料中锌的需要量

为考察吉富罗非鱼(GIFT,Oreochromis niloticus)对饲料中锌的需要量,以蛋氨酸锌为锌源,配制锌添加水平分别为0,10,20,40,80,160 mg/kg的6组等氮等脂饲料(实测值分别为9.98,21.47,33.75,56.03,88.16,172.54 mg/kg),饲喂初始体质量为(84.44±3.04)g的吉富罗非鱼,试验鱼分为6组,每组3个重复,每个重复20尾鱼,养殖周期为10周。结果显示,随着饲料中锌含量的增加,吉富罗非鱼的增重率、特定生长率先显著升高,在锌含量达到33.75 mg/kg后趋于稳定,添加锌显著降低了饲料系数,提高了成活率。饲料锌含量对全鱼水分、粗蛋白及灰分含量无显著影响,显著降低了全鱼粗脂肪含量。饲料中添加锌对血清总胆固醇含量无影响,显著提高了血清碱性磷酸酶的活性,碱性磷酸酶活性在锌含量达到33.75 mg/kg后趋于稳定,血清甘油三酯含量,乳酸脱氢酶、谷草转氨酶和谷丙转氨酶活性则呈下降趋势。饲料锌含量显著影响脊椎骨、全鱼和肝脏锌的含量,脊椎骨和全鱼锌含量先线性上升,在锌含量达到56.03 mg/kg后趋于稳定,各组的肌肉锌含量无显著差异。以增重率、骨骼锌和全鱼锌含量为评价指标,根据折线回归分析得出,以蛋氨酸锌为添加锌源,吉富罗非鱼对饲料中锌的需要量分别为32.37、55.67和56.13 mg/kg。     

Moderate salinities enhance growth performance of Nile tilapia (Oreochromis niloticus) fingerlings in the biofloc system

The biofloc technology (BFT) has recently gained attention as an economic and environmentally sustainable system for aquaculture. The use of BFT with salinized water could be used to minimize the negative effects of nitrogenous waste and improve the growth performance of Nile tilapia. This work evaluated the growth performance, survival, gill lesions, and fillet composition of tilapia fingerlings (Oreochromis niloticus) reared during 70 days with different salinities (0, 4, 8, 12, and 16 g/L) in a biofloc previously developed. The results indicated that the use of mature bioflocs alone was sufficient to avoid fingerling mortality due to nitrite peaks. Moreover, the salinized water, especially between 4 and 8 g/L (maximum points around 6 g/L), can be recommended in BFT to improve the growth performance of tilapia in the initial culture phase. The salinity level that was evaluated did not affect fillet composition nor the occurrence of gill lesions. The total ammonia nitrogen concentration was lower in freshwater than in salinized water (p < 0.05). Nitrite peaks in salinities of 12 and 16 g/L appeared later and were higher in comparison to other treatments. Considering minimal water exchange in BFT, the use of salinized water in this system may be environmentally viable.     

Capsular polysaccharide of Streptococcus agalactiae is an essential virulence factor for infection in Nile tilapia (Oreochromis niloticus Linn.)

Streptococcus agalactiae (Group B Streptococcus, GBS) is associated with diverse diseases in aquatic animals. The capsule polysaccharide (CPS) encoded by the cps gene cluster is the major virulence factor of S. agalactiae; however, limited information is available regarding the pathogenic role of the CPS of serotype Ia piscine GBS strains in fish. Here, a non‐encapsulated mutant (Δcps) was constructed by insertional mutagenesis of the cps gene cluster. Mutant pathogenicity was evaluated in vitro based on the killing of whole blood from tilapia, in vivo infections, measuring mutant survival in tilapia spleen tissues and pathological analysis. Compared to wild‐type (WT) GBS strain, the Δcps mutant had lower resistance to fresh tilapia whole blood in vitro (p < 0.01), and more easily cleared in tilapia spleen tissue, and was highly attenuated in tilapia and zebrafish. Additionally, compared to the Δcps mutant, numerous GBS strains and severe tissue necrosis were observed in the tilapia spleen tissue infected with WT strains. These results indicated that the CPS is essential for GBS pathogenicity and may serve as a target for attenuation in vaccine development. Gaining a better understanding of the role, the GBS pathogenicity in fish will provide insight into related pathogenesis and host–pathogen interactions.     

Evaluation of dietary chitosan effects on growth performance,immunity, body composition and histopathology of Nile tilapia (Oreochromis niloticus) as well as the resistance to Streptococcus agalactiae infection

The present investigation aimed to evaluate the effect of dietary chitosan supplementation on growth performance, body composition, immune response and histopathology of Nile tilapia, and also the in vitro antibacterial activity of chitosan against Streptococcus agalactiae (S. agalactiae). About 180 fish (average body weight 39.3 ± 0.3 g) were randomly divided into three groups according to chitosan supplementation: control group (basal diet without chitosan), Ch3 group (3 g chitosan/kg diet) and Ch5 group (5 g chitosan/kg diet). Growth performance parameters and body proximate composition were measured before infection but biochemical parameters and lysozyme and antibacterial activities before and after experimental infection. Results of the present investigation showed dietary chitosan (5 g chitosan/kg diet) significantly (p < .05) improved growth performance parameters, body composition (dry matter, crude protein, ether extract, ash, and carbohydrate) and serum biochemistry (total protein, albumin, globulin, with no effect on AST, ALT, urea and creatinine) before infection in Ch5 group than the control. After infection, liver enzymes (serum AST and ALT) were maintained lower in fish fed Ch3 or Ch5 than the control. Serum lysozyme and bactericidal activities significantly increased (p < .05) in chitosan groups before and after the challenge. The mortality rate was markedly reduced in the Ch3 group and prohibited in the Ch5 group after the experimental infection. In conclusion, feeding 3 or 5 g chitosan/kg diet increased the growth rate and improved FCR of Nile tilapia. In addition, it reduced mortality by its antibacterial and immunostimulant effects.     

Influence of traditional Chinese medicine and Bacillus species (TCMBS) on growth,immune response and disease resistance in Nile tilapia,Oreochromis niloticus

Multispecies herbs and probiotic bacteria offered to fish might enhance their immune response and increase disease resistance, but the dose effects of herbal‐probiotic application remain unclear. Therefore, the effect of herbal‐probiotic mixtures of traditional Chinese medicine (TCM) of composition Astragalus membranaceus, Angelica sinensis, Crataegus hupehensis and probiotic Bacillus species (BS) of composition Bacillus subtilis and Bacillus lincheniformis as natural immunostimulants in tilapia Oreochromis niloticus have been investigated. Fish were randomly divided into four groups: control diet (CT), TCMBS1 [TCM at 3 g/kg and BS at 7 g/kg], TCMBS2 [TCM at 5 g/kg and BS at 5 g/kg], TCMBS3 [TCM at 7 g/kg and BS at 3 g/kg]. Tilapia in the TCMBS3 group showed significant improvement in weight gain, specific growth rate, and lowered feed conversion ratio compared with other treated groups and the control. Concerning immune indexes, all treated groups significantly enhanced lysozyme, superoxide dismutase, catalase, protease and antiprotease activities, with highest values in catalase and antiprotease activities in TCMBS3 compared with control. TCMBS3 demonstrated higher expression of βeta‐defensin, lysozyme, heat shock protein 70, catalase and transforming growth factor‐βeta compared with other treated groups or the control group in both mid‐intestines and head‐kidney. After challenge with Streptococcus agalactiae, the best survival was found in TCMBS3 (97%), followed by TCMBS2 (73%), and TCMBS1 (69%) compared with the CT (35%). Collectively the present results suggest that TCMBS3 dose might potentiate a higher immune response and disease resistance in fish.     

Dietary supplementation of Tribulus terrestris extract improves growth and reproductive performances of the male Nile tilapia (Oreochromis niloticus)

Effects of dietary supplementation of Tribulus terrestris (TT) and 17α‐methyl testosterone (MT) on growth performance and reproductive efficiency of male Nile tilapia, Oreochromis niloticus (N = 75, initial weight 60 ± 3.251 g), were evaluated. Five experimental treatments in triplicate were fed on basal diet (30% crude protein) (control group, CG), basal diet containing 60 mg MT (MT group) and basal diet containing 250, 500 and 750 mg/kg diet TT (TT250, TT500 and TT750 groups), respectively, for consecutive 45 days. Results revealed that the fish received TT in diet showed better growth performance (final body weight, weight gain, feed conversion ratio, specific growth rate, testes weight and gonadosomatic index) compared with MT and CG (p < .05), as well as for haemoglobin content and red blood cell count (p < .05). The highest level of testosterone, 11‐keto testosterone, sperm concentration and vitality was recorded in TT750 followed by TT500, TT250 and MT groups, while the lowest value was recorded in the CG (p < .05). The highest seminiferous tubule diameter was recorded in TT750 followed by TT500, TT250 and CG, while the lowest diameter was recorded in the MT group (p < .05). Fish received TT showed normal dilated seminiferous tubule filled with large amount of free sperms within their lumen in a dose related effect. In conclusion, male Nile tilapia dietary supplementation with different inclusions of Tribulus terrestris extract specially 500 and 750 mg/kg diet improves growth performance, health condition, semen quality and reproductive efficiency without any harmful effect on water quality.     

Combined effects of dietary malic acid and Bacillus subtilis on growth,gut microbiota and blood parameters of Nile tilapia (Oreochromis niloticus)

The study investigated effect of dietary supplementation with malic acid, Bacillus subtilis and a mixture of the two (3 × 2 factorial trial) on Nile tilapia (Oreochromis niloticus) health. Treatment groups (T1–T6) were fed diets containing three levels of malic acid (0.0, 5.0 and 10.0 g/kg), each of which was supplemented with 0 and 1.1 × 10⁵ cfu/g B. subtilis, respectively. Each treatment group was assigned randomly to triplicate groups of 35 fish (5.26 ± 0.06 g) in 18 tanks for 84 days. The results indicated that survival was higher in all dietary treatments as compared to the control fed fish. The highest values of FBW, WG and SGR (%), PER, PPV and ER were recorded in groups T4 and T6, which were fed diets supplemented with 5 g malic acid/kg and 1.1 × 10⁵ cfu/g B. subtilis and 10 g malic acid/kg and 1.1 × 10⁵ cfu/g B. subtilis, respectively. The best value of FCR was obtained in groups T4 and T6. The lowest total bacterial count in the gut and faeces was detected in fish from group T6. The values of haematocrit, haemoglobin, red blood cells, white blood cells, total protein, albumin and globulin were significantly higher (p < .05) in fish from groups T4 and T6 (diets supplemented with malic acid and B. subtilis). As compared to the control fed fish, te mixture of these substances are promising as immune enhancher in aquacultured fish.     

Efficacy of feed‐based adjuvant vaccine against Streptococcus agalactiae in Oreochromis spp. in Malaysia

This study was conducted to determine the systemic, mucosal immunity and protective capacity of the feed‐based adjuvant vaccine (FAV) of Streptococcus agalactiae following oral vaccination against streptococcosis in tilapias. Two hundred and sixteen red tilapia fish were divided into three major groups. Each major group consisted eight tilapia kept in nine 2000 L glass aquaria. At day 0, all fish from the FAV group were fed with feed that had been incorporated with an adjuvant, while fish in the feed‐based vaccine (FNV) group were fed with vaccine incorporated into the pellet without adjuvant. Fish in the control‐unvaccinated group, FC, were fed with normal commercial pellet. Booster dose was performed on day 14 post immunization. Fish from each group were sacrificed on a weekly basis for the entire 7 weeks. Serum, body mucus and gut lavage fluid were evaluated for antibody responses by indirect ELISA, while histological examination was carried out on the gut following intraperitoneal challenge. The FAV group had a significantly higher protection (P < 0.05) following challenge with 3.4 × 10⁹ CFU mL^?1 of live S. agalactiae than FNV group. This level of protection may be due to high antibody responses, increase in size of gut‐associated lymphoid tissue and high number of lymphocytes in the FAV group.