工厂化养殖暗纹东方鲀致病菌的分离和鉴定

从不同养殖场患病暗纹东方鲀（Takifagu obscurus）的鳃、肠、肝、肌肉及溃疡部位分离到9株菌株，经人工感染致病性试验，其T-22、T-11、J-2、H-3、H-5表现出较强的致病性，出现症状与自然发病鱼相同。通过对菌体形态特征、培养特性和API20E细菌生化快速鉴定试条的测定，分别鉴定为温和气单胞菌（Aeromonas sobria）、豚鼠气单胞菌（Aeromonas calyioe）、嗜水气单胞菌（Aeromonas hydrophila）、类志贺邻单胞菌（Plesiomonas shigelloides）、霍乱弧菌（Vrbrio cholerae）。证实这5株菌为致病菌。药敏试验结果表明9种药物对温和气单胞菌、豚鼠气单胞菌、霍乱弧菌均有较好的抑菌效果；嗜水气单胞菌表现出明显的耐药性。氟嗪酸、环丙沙星、复合磺胺对类志贺邻单胞菌有一定抑菌效果。     

异育银鲫气单胞菌病原菌鉴定和药敏试验

从患病异育银鲫体内分离到5株可疑菌株,经API 20NE细菌生化鉴定,YCS07-03和YZ07-11株鉴定率为99.2%和99.3%。注射感染中5×108 CFU.mL-1剂量组表现出较强的致病性,12 d死亡率100%。菌株在营养肉汤培养液中呈均匀混浊状;在营养琼脂平皿中呈灰白色半透明菌落;在血琼脂培养基中呈光滑灰白色菌落,YZ07-11株出现透明的β溶血环;在TCBS培养基呈黄色菌落;在SS和麦康凯培养基中形成无色透明的扁平菌落。经负染电镜观察,菌株两端圆,呈直杆状,无芽孢,极生单鞭毛,菌株大小(短径×长径)分别为1.53μm×2.71μm和0.80μm×2.12μm。菌株经生化实验鉴定为温和气单胞菌(Aeromonas sobria)和嗜水气单胞菌(Aeromonas hydrophila)。试验结果表明:菌株对丁胺卡那霉素、头孢噻肟、庆大霉素、左氟沙星、复合磺胺、氟嗪酸、洛美沙星、复方新诺明、链霉素和新霉素均产生高度敏感;对万古霉素为中度敏感;氨苄青霉素、羧苄青霉素、强力霉素和青霉素产生耐药。     

30种中药及其复方对温和气单胞菌的抗菌作用

为评价中药对温和气单胞菌的体外抑菌效果,取鱼腥草、五倍子、黄芩等30种中药,采用水提法制备药液,通过试管二倍稀释法测定各药液对温和气单胞菌的最小抑菌浓度(MIC)及最小杀菌浓度(MBC),筛选出抗菌作用较强的中药作为主药,与牡丹皮、龙胆草、蒲公英等18种中药组成复方,测定中药复方对温和气单胞菌的体外抑制效果。结果表明:五倍子、山茱萸、白芍对温和气单胞菌有较强抑菌活性(MIC≤12.5mg/mL),选取8种主药与清热凉血、清热燥湿、清热解毒药分别组成复方,其中五倍子、诃子、五味子、乌梅与其他多种中药组成的复方抗菌作用增强。     

具有多种胞外酶的对虾肠道黏附菌的分离和鉴定

用对虾饲料培养基从健康凡纳滨对虾肠道分离出500株黏附细菌,以产淀粉酶、脂肪酶和蛋白酶能力为指标,筛选出产该3种消化酶的细菌90株,占总菌株的18%.对其中生长较快的69株进行16S rDNA 基因测序,确定其分类地位.结果显示,69株菌分别属于不动杆菌属(Acinetobacter)、芽孢杆菌属(Bacillus)、葡萄球菌属(Staphylococcus)、假交替单胞菌属(Pseudoalteromonas)、气单胞菌属(Aeromonas)、嗜盐单胞菌属(Halomonas)、利斯顿氏菌属(Listonella)、莫拉氏菌属(Moraxella)等,其中数量最多是芽胞杆菌属,占鉴定细菌总数的53.62%,数量最少是气单胞菌属和嗜盐单胞菌属,均占鉴定细菌总数的2.90%.表明对虾肠道黏附菌群中具有较多能分泌多种消化酶的细菌,可进一步开发为促进对虾消化功能的益生菌     

黄鳍鲷细菌性溃疡病致病菌研究

研究了黄鳍鲷细菌性溃疡病的病原。从患病濒死的黄鳍鲷体内分离到两株细菌,经人工感染试验确定为致病菌,通过测定其形态及生理生化特性得出：致病菌为革兰氏阴性杆菌,极生单鞭毛,发酵葡萄糖产酸不产气,氧化酶阳性、过氧化氢酶阴性,发酵乳糖,甘露醇发酵阳性,明胶酶阳性,在70g／LNaCl培养基中生长良好,对O／129不敏感;由于两株菌对山梨醇、水扬苷、七叶苷及麦芽糖等氧化发酵的结果不一致,分别被鉴定为嗜水气单胞菌不产气亚种（Aeromonashydropilaanaerogenes）和温和气单胞菌（A．sobria）变异亚种。药敏试验显示,两个菌株对氯霉素高度敏感而对氟哌酸中度敏感。两株菌对黄鳍鲷的半致死剂量（按每g鱼计算）分别为2．3×106个菌体和2．6×105个菌体。     

苦地胆内酯对嗜水气单胞菌感染斑马鱼保护效果的研究

在饲料中添加不同比例的苦地胆内酯,饲喂斑马鱼成鱼14 d后,采用创伤感染法构建斑马鱼嗜水气单胞菌感染模型,研究苦地胆内酯对嗜水气单胞菌感染斑马鱼的保护效果.结果显示:饲喂含苦地胆内酯0.4%、0.8%、1.6%饲料的斑马鱼,其存活率显著高于阳性对照组(p值<0.05);0.8%和1.6%的苦地胆内酯对斑马鱼的保护率分别为(73.91±6.52)%和(73.91±11.30)%,与0.1%恩诺沙星保护率(76.08±7.53)%相比无显著差异(p值>0.05).一定剂量的苦地胆内酯对嗜水气单胞菌感染斑马鱼具有保护效果.     

广东省罗非鱼主养区无乳链球菌的分离、鉴定与致病性

从广东省5个罗非鱼主养区患暴发性的罗非鱼中分离到15株菌,挑选其中的5株(不同地域、不同组织分离的菌株)进行人工感染试验,表现出自然发病的症状,确定此5株分离菌为导致罗非鱼暴发病的主要病原。通过形态学观察和生理生化实验,并结合16SrRNA基因序列分析,5株分离菌株均为无乳链球菌(Streptococcus agalactiae)。5株分离菌在形态学和生理生化实验结果上保持一致,16SrRNA基因序列同源性达98.6%～99.8%,但在致病性和对药物的敏感性上却呈现一定的差异。另外10株分离菌株通过生理生化实验鉴定也为无乳链球菌。研究表明,广东省罗非鱼流行性暴发病的病原菌主要为无乳链球菌。     

拮抗香蕉枯萎病菌海洋细菌的筛选和鉴定

从93份中国南部海域近海海泥样品中分离出1800多株海洋细菌,以对峙培养法,测定海洋细菌对香蕉枯萎菌的拮抗作用,再以琼脂划线培养法(agar streak)测定菌株的抑菌活性,结果表明:约55.6%的菌株对香蕉枯萎菌有一定的拮抗作用,其中58株菌株抑制香蕉枯萎菌生长所形成的抑菌带宽度大于10.0mm,抑菌作用较好,复筛后其中13个菌株形成的抑菌带宽在15.0mm以上。经反复转接和对峙培养,筛选出拮抗效果好、抑菌活性稳定的菌株TC-1,初步鉴定为芽孢杆菌属的细菌,TC-1对14种病原微生物都有较好的抑菌效果。     

一株拮抗芒果炭疽菌海洋细菌的鉴定和发酵培养基优化

从湛江近海海水和海泥分离的229株细菌中,选出对芒果炭疽菌(Colletotrichum gloeosporioides)具有较强拮抗作用的海洋细菌BSW03,抑菌圈直径为14.0 mm。根据菌体形态、生理生化特性和16s rDNA序列分析,BSW03鉴定为枯草芽孢杆菌(Bacillus subtilis)。从9种培养基中筛选出BDPB作为BSW03的发酵培养基,通过单因子碳源、氮源试验和L9(34)正交试验对发酵培养基成分进行优化。结果表明:发酵培养基最佳配方为乳糖20 g/L,牛肉膏15 g/L,K2HPO3.3H2O 0.75 g/L,MgSO4 0.75 g/L,pH自然。转速170 r/min、温度28℃的恒温摇床培养3 d后的无菌发酵液抑菌效果最佳,抑菌圈直径为27.3 mm。     

四种酵母对红鱼干中镰孢菌和T-2毒素消减效应

【目的】探讨四种酵母对镰孢菌生长和T-2毒素消减的控制效应,为红鱼干防霉脱毒的应用提供理论依据。【方法】将三株鱼干源酵母分离株季也蒙毕赤酵母Ym0104、奥默柯达酵母Yk0104(Yk)和阿氏丝孢酵母Yt0104(Yt)以及一株酿酒酵母BY4741的发酵液,与三株产T-2毒素(T-2)镰孢菌(鱼干源F11、F21菌株,标准菌株DL1445-5)、不同浓度T-2在红鱼干(DRF)培养基上共培养,通过测定镰孢菌生长半径以及T-2残留量来评价四种酵母发酵液对镰孢菌生长抑制作用以及T-2的消减效果。【结果】四种酵母菌对三株镰孢菌的生长均有抑制作用,其中Ykt的抑制作用最强;对T-2均有一定的消减作用,其中Yk的消减效果最好。【结论】Yk对红鱼干中镰孢菌和T-2毒素有良好的消减效应。     

Isolation and characterization of AHL-degrading bacteria from fish and pond sediment

Quorum sensing(QS)disruption is considered as a potential alternative strategy to combat bacterial diseases in aquaculture.In this study,we isolated and identified bacteria degrading QS molecules from pond sediment and fish intestine.A total of 132 strains were obtained in the enrichment culture,of which two strains were identified as Enterobacter sp.f003 and Staphylococcus sp.sw120,being isolated from the fish intestine and pond sediment,respectively.We found that strains f003 and sw120 could degrade acyl-homoserine lactones(AHLs)and cause no hemolysis of sheep red blood cells.The AHL lactonase(aiiA)homologous gene in the two strains was detected in PCR amplification and the high-degrading activity to N-hexanoyl-L-homoserine lactone(C6-HSL)and AHLs secreted from pathogenic Aeromonas hydrophila was assessed.Meanwhile,the artificial infection of cyprinid Carassius auratus gibelio with intraperitoneal injection showed that the two strains were avirulent.Therefore,the obtained indigenous bacteria are candidate probiotics against pathogenic A.hydrophila in aquaculture.     

大亚湾网箱养殖水体弧菌种类组成及变化

研究了海水网箱养殖海域弧菌数量变化、种类组成及其相关因素,探讨弧菌数量的变化与鱼病的关系。结果表明:网箱内水体弧菌数量为390~230 mL-1,平均为20.6×102mL-1,网箱外水体为130~1450 mL-1,平均为530 mL-1,非养殖对照海区为10~390 mL-1,平均为190 mL-1。养殖海区弧菌数量呈现明显的季节性变化,尤其是网箱水体,对照海区只在9月数量较高,其他时间变化不大。平均数量网箱内大于网箱外,网箱外大于对照海区。水体中出现的弧菌种类有河流弧菌Vibrio fluvialis,创伤弧菌Vibrio vulnifgicus,霍乱弧菌Vibrio cholerae,副溶血弧菌Vibrio parahaemolyticus,溶藻弧菌Vibrio alginolyticus,美人鱼弧菌Vibrio damsela,拟态弧菌Vibriomimicus和好利斯弧菌Vibrio hollisae,美人鱼弧菌、溶藻弧菌和霍乱弧菌占有较大优势。弧种数量与海水温度、盐度及营养盐有相关性,但其相关性因不同种和不同点而不同。检测期间,网箱养殖鱼类无明显流行病发生,但弧菌的高峰期明显与报道的鱼病高峰期相吻合。     

3种免疫途径对嗜水气单胞菌灭活疫苗保护作用的影响

应用间接ELISA方法,研究经嗜水气单胞菌疫苗浸泡、口服、注射免疫后鳜皮肤、肠道黏液和血清中抗体的变化关系,揭示鳜对3种免疫途径的免疫应答效果。结果显示:黏液抗体产生快(7 d达到峰值),但持续时间短,抗体水平低(213);血清抗体产生慢(28 d方达峰值),但持续时间长,抗体水平高(225)。不同方式免疫鳜后均产生远高于对照组的抗体滴度(P<0.05)。注射组产生的抗体水平高(225),免疫保护率最理想(70.6%);口服组相对另两个实验组,其抗体峰值(215)和免疫保护率(41.2%)均较低;浸泡组在皮肤黏液产生水平和溶菌酶含量方面,产生较好的免疫效应,分别为213和178μg/mL,但相对保护率仅为47.1%,低于注射组的70.6%。浸泡和口服组可诱发局部黏膜免疫,在抗体动态变化方面表现出相似的规律且峰值时间早于注射组。初步推断浸泡和口服可以直接刺激鱼体黏膜组织产生局部的特异性抗体。     

鲢、鳙溶血性腹水病研究

报道了鲢、鳙鱼溶血性腹水病的病原和防治方法。研究结果表明，该病无病毒性病原存在，病原为嗜水气单胞菌嗜水亚种（Aeromonashydrophilasubsp.hydrophila）。采用作者研制的鱼康乐（Y·K·L）进行治疗。治愈率达86．5‰。     

Comparative analysis on microbial community associated with different gastrointestinal regions of wild northern snakehead Channa argus Cantor, 1842

Microbial communities in different gastrointestinal regions(stomach, foregut, midgut, and hindgut) of the northern snakehead C hanna argus(Cantor, 1842) were compared by polymerase chain reaction and partial 16 S r DNA sequencing. A total of 194, 140, 212, and 122 OTUs were detected in the stomach, foregut, midgut, and hindgut, respectively. Significant dif ferences were found in the Sobs, ACE, Shannon, and Simpson indices among samples(P 0.05). The gastrointestinal microbial community of C. argus consisted predominantly of Proteobacteria with either H alomonas, Shewanella, Plesiomonas, or Sphingomonas. Fusobacteria, Firmicutes, and Bacteroidetes also existed in the gastrointestinal tracts. However, significant diff erences were found in the compositions of microbial community among the four regions( P 0.05). Cyanobacteria and Spirochetes were significantly higher in the midgut and hindgut( P 0.05). Fusobacteria and Firmicutes were dominant in the hindgut and foregut, respectively(P 0.05). Proteobacteria was the lowest in the hindgut( P 0.05). At genus level, C etobacterium and Plesiomonas were significantly higher in the hindgut than in the other three samples( P 0.05). Clostridium and Prevotella were the highest in the midgut(P 0.05). Halomonas, Shewanella, and S phingomonas were the highest in the foregut( P 0.05). Paracoccus and Vibrio were the highest in the stomach. Several genera were only detected in certain regions, as follows: stomach, P aracoccus and Vibrio; foregut, Halomonas, Shewanella, and Sphingomonas; midgut, Clostridium and Prevotella; and hindgut, C etobacterium and Plesiomonas( P 0.05). At the species level, A cinetobacter rhizosphaerae was only detected in the stomach. P revotella copri and Clostridium perfring were not detected in the foregut and midgut, respectively, whereas Prevotella copri and Faecalibacterium pra were not detected in the hindgut. These findings provide valuable information on the microbial community in each gastrointestinal region of C. argus. Moreover, this study indicated that microbial community was not only related to rearing environment but also to the physico-chemical characteristics of each gastrointestinal region.     

利用有益微生物改善养殖生态环境的研究

研究了噬菌蛭弧菌在实验室条件下对水产养殖中常见病菌的裂解能力及噬菌蛭弧菌和光合细菌对池塘生态环境的改善作用。结果表明 ,噬菌蛭菌对嗜水气单胞菌具有裂解作用 ,噬菌蛭弧菌和光合细菌结合使用对养殖环境具有明显的改善作用。 2 5d后实验组比对照组池塘中的细菌总数少 2个数量级 ,70d后实验组比对照组池塘中的细菌总数少 3个数量级 ;2 5d后对照组COD为 6 35mg L ,使用噬菌蛭弧菌浓度为 3、5、1 0mL m3 及光合细菌皆为 5mL m3 实验组的COD分别为 4 6 7、4 1 6、4 1 8mg L ;2 5d后对照组NH3 N为 0 4 9mg L ,使用噬菌蛭弧菌浓度为 3、5、1 0mL m3 及光合细菌皆为 5mL m3 实验组的NH3 N分别为 0 36、0 33、0 32mg L ,90d后对照组NH3 N为 0 4 1mg L ,使用噬菌蛭弧菌浓度为 3、5、1 0mL m3 及光合细菌皆为 5mL m3 实验组的NH3 N分别为 0 2 7、0 2 2、0 2 1mg L ;5 5d后对照组硫化物为0 0 1 1mg L ,使用噬菌蛭弧菌浓度为 3、5、1 0mL m3 及光合细菌皆为 5mL m3 实验组的硫化物分别为0 0 1 0、0 0 0 8、0 0 0 8mg L。     

Non-specific immune response of bullfrog Rana catesbeiana to intraperitoneal injection of bacterium Aeromonas hydrophila

Non-specific immune response of bullfrog Rana catesbeiana to pathogenic Aeromonas hydrophila was studied to 60 individuals in two groups.Each bullfrog in bacterium-injected group was injected intraperitoneally(i.p.) with 0.2 ml bacterial suspension at a density of 5.2 × 106 CFU/ml,while each one in control group injected i.p.with 0.2 ml sterile saline solution(0.85%,w/v).Three bullfrogs in both groups were sampled at 0,1,3,7,11,15 and 20 days post-injection(dpi) for the evaluation of non-specific immune parameters.It was observed that intraperitoneal injection of A.hydrophila significantly increased the number of leucocytes and that of NBT-positive cells in peripheral blood.Significant increases in serum bactericidal activity and serum acid phosphatase activity were also observed in the bacterium-injected frogs when compared with those in the control group.However,a significant reduction was detected in vitro in phagocytosis activity of peripheral blood phagocytes.No significant difference in changes in the number of peripheral erythrocytes,serum superoxide dismutase(SOD) activity,and lysozyme activity was detected between the two groups.It is suggested that bullfrogs may produce a series of non-specific immune reactions in response to the A.hydrophila infection.