乳酸杆菌L1对致病弧菌的抑制作用

以致病弧菌T1、T2为检测菌,采用平板抑菌圈检测法,研究乳酸杆菌L1及其代谢产物的抑菌能力。结果表明:L1发酵上清液对两种弧菌的抑制效果和发酵液对弧菌的抑菌作用相似,即对弧菌T1的抑制作用强于T2;L1 发酵液对弧菌T1、T2的抑制能力强于上清液,表明乳酸杆菌及其代谢物质对弧菌具有协同抑制作用;L1的发酵液经3倍稀释仍对弧菌T1有抑制作用;乳酸杆菌L1生长不同时期的代谢产物对弧菌的抑菌活性不同,代谢产物的抑菌能力在衰退期>稳定期>生长期;L1的发酵液经沸水浴处理15 min其抑菌活性不变,表明抑菌代谢物质具有较好的耐热性。     

大蒜对印度明对虾黑鳃病原耐药菌株哈维氏弧菌抗菌活性的研究

本研究针对大蒜提取物对印度明对虾(Fenneropenaeus indicus)黑腮病病原多重耐药哈维氏弧菌(Vibrio harveyi)体外和体内的抗菌活性进行了评价。先利用市售的几种抗生素记录致病性哈维氏弧菌的抗菌谱。然后,利用琼脂扩散法来研究大蒜对哈维氏弧菌的抗菌活性。将哈维氏弧菌接种在琼脂平板上,对每种大蒜提取物(鲜榨大蒜提取物—FSGE,冻干大蒜提取物—FDGE以及用甲醇提取的大蒜提取物—MGE)设计三种不同的浓度。结果表明,鲜榨大蒜提取物对哈维氏弧菌的抑菌活性明显优于冻干大蒜提取物和用甲醇提取的大蒜提取物。本研究分别在对虾基础饲料中添加0%(对照)、0.5%和1%浓度梯度的大蒜来评价大蒜治疗感染哈维氏弧菌的对虾的效果。结果表明,综合大蒜提取物的短期和长期防治(1%含量)的效果分析,对虾累积死亡率降低了75%。本研究探讨了鲜榨大蒜提取物替代抗生素在印度明对虾饲养防病上的优点。     

Application of silver nanoparticles immobilized on TEPA-Den-SiO2 as water filter media for bacterial disinfection in culture of Penaeid shrimp larvae

In the present study, we prepared silver nanoparticles immobilized on silica beads (Ag/TEPA-Den-SiO₂) and examined their potential for removing luminous Vibrio sp. Persian1 from seawater used to culture Penaeus (Litopenaeus) vannamei post-larvae. The Ag/TEPA-Den-SiO₂ sample was characterized by TEM, FE-SEM/EDS, FT-IR and ICP-OES and its antibacterial activity assayed by a test tube test and flow test using water filters filled with Ag/TEPA-Den-SiO₂ as the filter media_. The results of the test tube test indicated that no Vibrio sp. Persian1 was detectable after 2-h contact. The filter column that contained Ag/TEPA-Den-SiO₂ + silver absorbent inactivated 100% of the bacteria after passage of seawater through the column for 12-h at a flow rate of 0.5 l/min. The survival rate and growth performance of post-larvae cultured in seawater treated with an antibacterial filter improved significantly compared with the control.     

常用中草药对4种病原弧菌体外抗菌效果的研究

采用琼脂扩散法(打孔法)测定了五倍子等18种中草药对鳗弧菌、副溶血弧菌、河口弧菌及霍乱弧菌等4种病原弧菌的抑制作用。结果表明,五倍子、苏木、地锦草、石榴皮、黄芩、虎杖和山楂等7种中草药对病原弧菌具有较好的抑菌效果,而金银花等11种中草药抑菌作用较弱;同时采用试管连续稀释法,测定了五倍子等抑菌效果较强的7种中草药对4种病原弧菌的抑菌和杀菌浓度。结果表明,苏木、五倍子、地锦草和石榴皮4种中药具有较好的抑菌和杀菌作用,此4种中药对鳗弧菌、副溶血弧菌、河口弧菌及霍乱弧菌等4种病原弧菌的最小抑菌浓度(MIC)为0.78～6.25mg/ml,最小杀菌浓度(MBC)为3.12～12.5mg/ml。     

Effects of dietary supplementation with freeze‐dried powder of Ampithoe sp. on the growth performance,energy metabolism,and ammonia‐nitrogen tolerance of the Pacific white shrimp,Litopenaeus vannamei

This study evaluated the influence of dietary supplementation with freeze‐dried powder of Ampithoe sp. (FDPA) on the growth, energy metabolism, and resistance to ammonia‐nitrogen stress in Litopenaeus vannamei. There were four treatment groups: a 0% group (no FDPA addition), a 33% group, a 66% group (33% and 66% of the shrimp diet, respectively, replaced with FDPA), and a 100% group (only FDPA). The results of this study suggested a positive effect of FDPA supplementation on shrimp survival: the supplemented groups had significantly higher survival than the 0% group (p < 0.05). The body length, body weight, and specific growth rate (SGR) of the 33% group were higher than those of the other groups and were significantly higher than that of the 100% group (p < 0.05). FDPA feeding had a negative effect on carbohydrate metabolism pathways and energy consumption due to decreases in pyruvate kinase (PK), phosphofructokinase (PFK), succinate dehydrogenase (SDH), lactic dehydrogenase (LDH), and respiratory electron transport system (ETS) activity in shrimp fed FDPA during the culture period. The shrimp in the 33% group exhibited good resistance to ammonia‐nitrogen stress. Additionally, the glycolysis pathway and energy consumption of shrimp in the 33% group were enhanced during the ammonia‐nitrogen stress period. Consequently, it was inferred that FDPA supplementation could improve the resistance of shrimp to ammonia‐nitrogen stress (in the 33% group), which might be related to the effects of the supplement on energy metabolism pathways, particularly in terms of enhancing glycolysis to provide sufficient energy for the stress response.     

Lutein enrichment of the rotifer Brachionus sp. using freeze‐dried Muriellopsis sp. cells

Rotifers, used in aquaculture as feed for early larval stages, are poor in carotenoids compared with copepods, the natural most common prey item of marine fish larvae. A minimal sufficient level of carotenoids in fish larvae may be essential for a correct development. The freshwater microalga Muriellopsis sp. has been identified as a potential source of the carotenoid lutein with possible application in aquaculture. Lutein is a potent antioxidant, with potentially beneficial effects as a carotenoid for fish larvae. This study describes the lutein enrichment of the rotifer Brachionus sp. when incubated with homogeneously dispersed suspensions of freeze‐dried, lutein‐rich Muriellopsis sp. in seawater. A short enrichment time (2 or 2.5 h) resulted in a higher lutein content of the rotifers than a long enrichment time (13.5 or 24 h). Although the concentration of lutein in enriched rotifers, 0.06–0.16 mg (g dry weight)^?1 was low compared with its content in Muriellopsis sp. [about 3 mg (g dry weight)^?1], it was in the range reported for natural copepod populations. Therefore, we conclude that freeze‐dried biomass of Muriellopsis sp. can be used to enrich rotifers with lutein, providing an adequate level of this carotenoid for marine fish larvae.     

Vibrogen‐2 vaccine trial in lumpfish (Cyclopterus lumpus) against Vibrio anguillarum

Lumpfish (Cyclopterus lumpus), a native fish of the North Atlantic Ocean, is utilized as cleaner fish to biocontrol sea lice infestations in Atlantic salmon aquaculture. However, bacterial infections are affecting cleaner fish performance. Vibrio anguillarum, the aetiological agent of vibriosis, is one of the most frequent bacterial infections in lumpfish, and effective vaccine programmes against this pathogen have been identified as a high priority for lumpfish. Vibrogen‐2 is a commercial polyvalent bath vaccine that contains formalin‐inactivated cultures of V. anguillarum serotypes O1 and O2, and Vibrio ordalii. In this study, we evaluated Vibrogen‐2 efficacy in lumpfish against a local isolated V. anguillarum strain. Two groups of 125 lumpfish were bath‐immunized, bath‐boost‐immunized at four weeks post‐primary immunization, and intraperitoneally (i.p.) boost‐immunized at eight weeks post‐primary immunization. The control groups were i.p. mock‐immunized with PBS. Twenty‐seven weeks post‐primary immunization, the fish were i.p. challenged with 10 or 100 times the V. anguillarum J360 LD₅₀ dose. After the challenge, survival was monitored daily, and samples of tissues were collected at ten days post‐challenge. Commercial vaccine Vibrogen‐2 reduced V. anguillarum tissue colonization and delayed mortality but did not confer immune protection to C. lumpus against the V. anguillarum i.p. challenge.     

Inhibition of Vibrio spp. by 2‐Hydroxyethyl‐11‐hydroxyhexadec‐9‐enoate of Marine Cyanobacterium Leptolyngbya sp. LT19

Seven marine cyanobacteria were isolated from two regions of the Gulf of Thailand and evaluated by the agar diffusion method for antibacterial activity. Inhibitory compound was purified from the crude methanol extract and its structure was elucidated based on extensive spectroscopic analysis, including IR, 1D and 2D NMR spectra as well as mass spectrometry. A novel antimicrobial compound produced by the marine cyanobacterium Leptolyngbya sp. LT19 was identified to be a 2‐hydroxyethyl‐11‐hydroxyhexadec‐9‐enoate which has so far never been reported in microorganisms. Biological assays revealed that this novel compound exhibited antibacterial activities against the Gram‐negative, persistent shrimp pathogens, Vibrio harveyi and V. parahaemolyticus with minimal inhibitory concentration of 250–1000 and 350–1000 μg mL^?1 respectively.     

Effects of dietary β‐glucan and glycyrrhizin on non‐specific immunity and disease resistance of white shrimp,Litopenaeus vannamei (Boone) challenged with Vibrio alginolyticus

The white shrimp Litopenaeus vannamei, fed immunostimulant‐free, 0.2%β‐glucan and 0.06% glycyrrhizin diets for 18 days, respectively, were challenged with Vibrio alginolyticus at 6.4 × 10⁴ CFU shrimp^?1. The total haemocyte count (THC), phenoloxidase (PO) activity, respiratory burst (RB) and superoxide dismutase (SOD) activity changes for a 120‐h period were investigated, and shrimp mortality was also recorded. The results showed that PO activity, RB and SOD activity were significantly higher in shrimp fed the two immunostimulant diets after 18 days than those in shrimp fed immunostimulant‐free diets. The THC and SOD activity decreased significantly from 0 to 24 h post challenge, and then reverted to normal levels at 96 and 72 h respectively. The values for PO activity and RB increased from 0 to 48 h post challenge. Compared with those fed the control diets, shrimp fed immunostimulants had significantly higher PO activity and RB values at 120 h post challenge. Mortalities after challenge with V. alginolyticus were significantly lower in shrimp fed with β‐glucan or glycyrrhizin than in those fed with a diet without immunostimulants. It was concluded that dietary β‐glucan and glycyrrhizin increased the shrimp immunity. Furthermore, β‐glucan caused an increase in some immune parameters 12 h earlier than glycyrrhizin after V. alginolyticus challenge.     

Growth,body chemical composition and trypsin activity of South American catfish,surubim (Pseudoplatystoma sp.) juveniles fed different dietary protein and lipid levels

To evaluate protein and lipid requirement of South American catfish surubim (Pseudoplatystoma sp.) juveniles, nine semi‐purified diets containing three levels of protein (40%, 45% and 50%) and three levels of lipid (12%, 16% and 20%) were tested. After 8‐week feeding trial, body weight increase averaged 2124.3 ± 295.7%. Growth performance was significantly affected by dietary level of protein (P < 0.05). At the 40% protein level, increasing level of dietary lipid had a positive effect on final individual mean weight (protein sparing effect). Whole body protein and moisture contents were affected by the dietary level of lipid (P < 0.05). Whole body lipid content positively correlated with the level of dietary lipid (P < 0.05). Cannibalism related mortality was observed despite rearing fish in 24 h dark. Fatty acid composition of fish was affected by the dietary lipid level (P < 0.05). Polyunsaturated fatty acids increased with the increasing level of dietary lipid while saturated fatty acids and monounsaturated fatty acids decreased. Trypsin activity in the digestive tract of surubim was influenced by dietary levels of protein and lipid (P < 0.05). Our preliminary results suggest that the optimum protein/lipid ratio might be close to 45/16% for surubim juveniles.     

Effects of continuous and alternate administration of β‐glucan and mannan‐oligosaccharide on the growth,immunity and resistance against Vibrio splendidus of sea cucumber Apostichopus japonicus (Selenka)

A 4‐week growth trial was conducted to compare the effects of different feeding strategies of dietary immunostimulants on the growth, immunity and resistance against Vibrio splendidus of sea cucumbers Apostichopus japonicus (Selenka). Six feeding strategies were set, including feeding immunostimulants‐free diet continuously (control), feeding dietary β‐glucan (1.25 g kg^?1 diet) continuously, feeding dietary mannan‐oligosaccharides (MOS; 2.00 g kg^?1 diet) continuously, feeding β‐glucan 2 days followed by MOS 5 days alternately, feeding β‐glucan 5 days followed by MOS 2 days alternately and feeding β‐glucan 7 days followed by MOS 7 days alternately. The sea cucumbers fed immunostimulants showed higher specific growth rate (SGR) and lower cumulative mortality than control (P < 0.05). When sea cucumbers were fed with β‐glucan continuously, total coelomocytes counts and superoxide anion were significantly higher than control on the 4th day (P < 0.05). However, these two immune parameters were not significantly higher than those in control after the 18th day (P > 0.05). While sea cucumbers continuously fed MOS, these two immune parameters were not significantly higher than control until the 15th day. All immune parameters of the sea cucumbers fed with β‐glucan and MOS alternately were significantly higher than those in control during the experiment (P < 0.05). The sea cucumbers fed with β‐glucan 7 days followed MOS 7 days alternately showed the highest SGR and second lowest cumulative mortality. It was suggested that this feeding strategy is most suitable for sea cucumbers.     

Antiparasitic activity of the antimicrobial peptide HbβP‐1, a member of the β‐haemoglobin peptide family

A family of antimicrobial peptides (AMPs) derived from the β‐subunit of haemoglobin was recently isolated from channel catfish, Ictalurus punctatus, infected with Ichthyophthirius multifiliis (ich), an important freshwater fish parasite that causes ichthyophthiriosis. We previously discovered that one of these AMPs, HbβP‐1, had strong cidal activity against ich as well as another ectoparasite, Tetrahymena pyriformis. HbβP‐1 toxicity was specific, primarily affecting the trophozoite (trophont) stage of ich. Here, we show that HbβP‐1 acts more rapidly to kill smaller (presumably less mature) trophonts of ich, taking almost twice as long to kill larger trophonts (P < 0.0001). It acts more rapidly than an unrelated AMP, piscidin 1, which is haemolytic and also lethal to ich trophonts. HbβP‐1 is potently and selectively lethal to the trophont stage of the dinoflagellate ectoparasite, Amyloodinium ocellatum, one of the most important pathogens of warmwater marine fish. HbβP‐1 has no effect on the fish gill cell line feeder layer (G1B cells) used to propagate Amyloodinium, further suggesting a highly selective action. These findings suggest that HbβP‐1 or related AMPs might function in protecting marine as well as freshwater fish and that HbβP‐1 has highly selective activity against specific life stages of important fish ectoparasites.     

Effects of dietary Nannochloropsis sp. powder and lipids on the growth performance and fatty acid composition of larval and postlarval kuruma shrimp,Marsupenaeus japonicus

The current study evaluated the effects of fish oil replacement with eicosapentaenoic acid‐enriched single‐cell microalgae in the diets of larval and postlarval kuruma shrimp. Experimental diets containing different level of Nannochloropsis sp. powder (10, 40, 70 g/kg) and lipids (2, 8, 10, 14, 35, 58 g/kg) were evaluated. The substitution of fish oil with algal powder significantly improved shrimp growth compared to that in the control group, with the highest final body weight recorded at 70 g/kg in larvae and 40 g/kg in postlarvae. Larvae fed algal lipids with a dry weight percentage in the diet of greater than 10 g/kg showed significantly lower performance than those in the control group. The neutral lipids in the tissue of the postlarvae that had been fed algal lipids had an improved fatty acid profile, as the content of highly unsaturated fatty acids increased compared to that in the other groups. The content of docosahexaenoic acid increased in the tissue, indicating the possible occurrence of the metabolism and accumulation of Nannochloropsis sp. essential fatty acids. Algal powder could replace FO up to 140 g/kg in shrimp diets without compromising growth and FA profiles.     

The in‐vitro antibacterial effects of organic salts,chemical disinfectants and antibiotics against pathogens of black disease in fairy shrimp of Thailand

The antibacterial effects of organic salts, chemical disinfectants and antibiotics were evaluated on cultures of Aeromonas hydrophila C03, Aeromonas sobria C26, A. sobria C29, Aeromonas caviae C24 and Acinetobacter sp. SH‐94B, the pathogens that cause black disease found in fairy shrimps (Streptocephalus sirindhornae Sanoamuang et al. (2000) and Branchinella thailandensis Sanoamuang, Saengphan & Murugan) of Thailand. The minimal inhibitory concentrations (MICs) of organic salts (sodium chloride and potassium chloride) and antibiotics (oxytetracycline dihydrate, streptomycin sulphate, kanamycin monosulphate, chloramphenicol and ampicillin) were determined using the agar‐dilution method. The effect of chemical disinfectants (sodium hypochlorite and chlorine dioxide) was evaluated by exposing bacteria to different concentrations of these chemicals for different periods of time. Interestingly, all strains were intrinsically resistant to 0.25–3% sodium chloride and potassium chloride. The effect of sodium hypochlorite was greater than that of chlorine dioxide, and 5–20 μg mL^?1 of sodium hypochlorite was sufficient to inhibit the growth of these bacteria, but the exposure time varied, depending on the bacterial species. Of the antibiotics tested, chloramphenicol and oxytetracycline dihydrate completely inhibited the selected strains. Chloramphenicol showed the highest antibacterial effect against all pathogenic species – the MIC and minimal bactericidal concentration (MBC) ranged from 0.8 to 3.1 μg mL^?1 from 3.1 to 6.25 μg mL^?1, respectively. To achieve control of black disease during cultivation of fairy shrimp, data derived from this study can be used as a basis for further toxicity tests in vivo.     

Experimental infection of brown‐marbled grouper,Epinephelus fuscoguttatus (Forskal), with Vibrio parahaemolyticus identifies parvalbumin beta‐2 subunit I,alpha‐2‐macroglobulin,nattectin and immunoglobulin light chain,differentially expressed in resistant grouper

The mechanisms through which brown‐marbled grouper accomplishes resistance to infection, particularly against Vibrios, are not yet fully understood. In this study, brown‐marbled grouper fingerlings were experimentally infected with Vibrio parahaemolyticus, to identify disease resistance grouper, and the serum proteome profiles were compared between resistant and susceptible candidates, via two‐dimensional gel electrophoresis (2‐DE). The results showed that putative parvalbumin beta‐2 subunit I, alpha‐2‐macroglobulin, nattectin and immunoglobulin light chain proteins were among proteins that significantly overexpressed in the resistant fish as compared to the susceptible group of fish, whereas apolipoprotein E and immunoglobulin light chain proteins were observed to be differentially overexpressed in the susceptible fish. Further analysis by peptide sequencing revealed that the immunoglobulin light chain proteins identified in the resistant and susceptible groups differed in amino acid composition. Taken together, the results demonstrated for the first time that putative parvalbumin beta‐2 subunit I, alpha‐2‐macroglobulin, nattectin and immunoglobulin light chain are among important proteins participating to effect disease resistance mechanism in fish and were overexpressed to function collectively to resist V. parahaemolyticus infection. Most of these molecules are mediators of immune response.     

The effects of a dietary protease‐complex on performance,digestive and immune enzyme activity,and disease resistance of Litopenaeus vannamei fed high plant protein diets

The effects of a dietary protease‐complex on growth performance, body composition, digestive and immune enzyme activity of Litopenaeus vannamei and its resistance to a pathogenic Vibrio parahaemolyticus were assessed in a 9‐week trial. A high fish meal diet (HF) containing 200 g kg^?1 fish meal and a low fish meal diet (LF) containing 100 g kg^?1 fish meal were designed as a positive and negative control respectively. Three other diets (LF+125P, LF+150P, and LF+175P) were manufactured by supplementing graded level of a protease‐complex (125, 150 and 175 mg kg^?1, respectively) to the LF diet. All diets were formulated to be iso‐proteic and iso‐energetic. Most performance indices of shrimp fed the LF+175P diets were similar to the HF diet. Among the digestive enzymes, trypsin, lipase and amylase activity in hepatopancreas of shrimp fed LF+175P diets (4576 U mg^?1 protein and 16, 32 U g^?1 protein, respectively) were higher than those fed the LF diets but lower than the HF diets (P < 0.05). Total superoxide dismutase and polyphenol oxidase contents in both serum and hepatopancreas were higher and serum malondialdehyde content and the cumulative mortality during disease challenge tests were lower for the diets containing the protease‐complex than those fed the LF diets (P < 0.05) with no difference with those fed the HF diets.