Physiological responses, desaturase activity, and fatty acid composition in milkfish (Chanos chanos) under cold acclimation

Physiological responses of milkfish (Chanos chanos) under cold shock and acclimation were investigated. The experimental milkfish, a warm-water teleost, were initially acclimated at 25 °C and then transferred directly to 15 °C; stress responses of this species were monitored for 1 week. Parameters monitored included plasma glucose, lactate, and lipids, as well as stearoyl-CoA desaturase activity and fatty acid compositions of hepatic membranes. All parameters showed significant changes in the process of cold acclimation. A hyperglycemic response indicated by a notable and steady increase in plasma glucose levels from 85 mg dl⁻¹ to the highest level of 458.2 mg dl⁻¹ in 24 h was followed by a rapid decline thereafter. The elevation in plasma glucose content under cold shock resulted from gluconeogenesis and glycogenolysis, indicated by correlated changes in plasma glucose with fructose-1,6-biphosphatase and phosphorylase a activities. Plasma lactate concentrations remarkably increased from 47 mg dl⁻¹ on day 0 to 149.6 and 120.4 mg dl⁻¹ on days 1 and 2, respectively, and then rapidly declined to the same level as the control thereafter. In contrast, plasma lipids increased gradually from 44.8 mg dl⁻¹ to 191 mg dl⁻¹ over the 5-day acclimation period, followed by a declining trend from day 6 on. Furthermore, changes in monounsaturated fatty acids were highly correlated with those of stearoyl-CoA desaturase activities in hepatic microsomes of milkfish during cold acclimation. Results indicate that in milkfish subjected to cold stress, plasma hyperglycemic and hyperlactemic responses can be used as acute stress indicators, and plasma lipids can be used as a chronic stress indicator.     

Effect of probiotics, Enteroccus faecium, on tilapia (Oreochromis niloticus) growth performance and immune response

The aim of this study was to analyze the effect of a probiotic bacterium, Enterococcus faecium ZJ4 on growth performances and immune responses of tilapia (Oreochromis niloticus). The tilapias were treated with E. faecium ZJ4 at a final concentration of 1 × 10⁷ cfu ml^− 1 in aquaria water every 4 days. Six aquaria with three replicates for treated and controls were used. After 40 days, the tilapias supplemented with the probiotic showed significantly better final weight and daily weight gain (DWG) than those fed the basal diet (Control) (P < 0.05). There was no remarkable difference (P > 0.05) in the total serum protein, albumin content, globulin concentration and A/G ratio between the treated and control tilapias. The result of lysozyme activity assay was similar to these biochemical indexes. However, the complement component 3 content, myeloperoxidase (MPO) activity and the respiratory burst activity of blood phagocytes were higher (P < 0.05) in E. faecium treated tilapias (trial 1) than the controls.     

永生化尼罗罗非鱼巨噬细胞系的建立及鉴定

为获得具有单一克隆特性的能稳定传代培养的罗非鱼巨噬细胞系,本研究从尼罗罗非鱼腹腔中分离纯化巨噬细胞,采用EB病毒(Epstein-Barr virus,EBV)感染,筛选单克隆细胞的方法建立了尼罗罗非鱼巨噬细胞系,并对其进行了EBV感染鉴定、电镜观察、端粒酶活性检测、致癌性评估、核型分析以及分子生物学鉴定。研究表明,EBV已整合到尼罗罗非鱼巨噬细胞中且稳定表达,经30代稳定传代,该细胞系仍维持较好的增殖状态;该细胞系表面不平滑,有明显的钝圆形突起和细长的伪足,表现为典型的巨噬细胞形态;端粒酶活性显著高于未经感染的巨噬细胞,而与He La细胞差异不显著,且该细胞系不具有致癌性,说明永生化细胞系构建成功。核型分析结果发现,该细胞系具有44条染色体,其核型公式为2 n=2 x=44=4 sm+17 st+1 t。PCR检测发现,该细胞系存在CD33和CD205的转录本,这些都是单核巨噬细胞的标志物,经18S r RNA检测证明该细胞系来自尼罗罗非鱼巨噬细胞。永生化尼罗罗非鱼巨噬细胞系已被成功建立,该细胞系为研究罗非鱼链球菌HSP70-肽疫苗的高保护率,以及罗非鱼的免疫防御机制提供了工具。     

Bacterial diversity of tilapia (Oreochromis niloticus) cultured in brackish water in Saudi Arabia

The bacterial flora occurring in brackish pond water, sediment, gills and intestine of healthy tilapia cultured in Saudi Arabia were estimated both quantitatively and qualitatively, and the isolates were identified to genus or species level. Total viable count of bacteria ranged from 1.4±1.5×10³ to 8.6±2.7×10³ cfu ml⁻¹; 1.2±3.1×10⁶ to 7.3±1.1×10⁷ cfu g⁻¹; 8.7±1.9×10⁵ to 2.1±0.9×10⁶ cfu g⁻¹; and 2.8±2.4×10⁷ to 1.0±1.6×10⁸ cfu g⁻¹ in the pond water, sediment, gills and intestine of brackish water tilapia, respectively. In total, 19 bacterial species were identified. The bacteria were predominantly Gram-negative rods (87%). Pond water and sediment bacteria influenced the bacterial composition of gills and intestine of tilapia. In contrast to gill bacteria, more diversification was observed in intestinal bacteria. The predominant (prevalence >10%) bacterial species were Vibrio parahaemolyticus, Vibrio carchariae, Vibrio alginolyticus, Chryseomonas sp., Vibrio vulnificus, and Streptococcus sp. in all the populations with the exception of the sediment population where Streptococcus sp. was replaced by Shewanella putrefaciens. Vibrio spp. (58% of the total isolates) dominated the total bacterial population.     

Effects of dietary starch and energy levels on maximum feed intake, growth and metabolism of Nile tilapia, Oreochromis niloticus

The aim of this study was to gain insight into how Nile tilapia (Oreochromis niloticus) regulate feed and energy intake in response to diets low and high in starch and cellulose. It was hypothesized that high-starch diets would reduce feed intake due to the effect of high blood glucose level, and that stomach volume may limit feed intake of fish fed diets low in energy. Four experimental diets, low starch–no cellulose inclusion, high starch–no cellulose inclusion, low starch–with cellulose inclusion, and high starch–with cellulose inclusion, were formulated. The high-starch diets and diets with cellulose inclusion were 17.5% more energy-diluted than the low-starch diets and diets without cellulose inclusion, respectively. Male tilapia were fed to apparent satiation for six weeks. Feed and digestible energy intake of fish fed diets with cellulose inclusion increased and decreased by 8.3% and 5.5%, respectively, compared to fish fed diets without cellulose inclusion. This suggests the role of stomach volume in restricting feed consumption. Fish fed high-starch diets achieved only 0.5% more feed intake and 13.9% less digestible energy intake than fish fed low-starch diets. The lower increase in feed intake and higher decrease in digestible energy intake of fish fed high-starch diets than of fish fed diets with cellulose inclusion suggests that high blood glucose suppresses feed intake in Nile tilapia. An alternative explanation for the differences in feed and digestible energy intake of fish fed different diets was based on the fact that heat production was not influenced by starch nor cellulose-inclusion levels. Thus, under satiation feeding, oxygen uptake capacity may determine feed and digestible energy intake in fish rather than blood glucose or stomach volume.     

瘤背石磺肌肉生长抑制素基因的克隆及组织表达分析

为探究石磺由海洋向陆地进化过程中肌肉生长和发育的分子机制,实验以石磺科贝类转录组数据为基础,采用RACE方法从瘤背石磺肌肉中首次克隆到MSTN cDNA的全长并做了相应的组织表达分析。结果显示,瘤背石磺MSTN基因cDNA全长2667 bp,包括1650 bp的开放阅读框,374 bp的5′端非翻译区,643 bp的3'端非翻译区,共编码549个氨基酸。预测该基因编码的蛋白质原子总量为8776,分子式为C2774H4331N783O862S26,分子质量约为63.27 ku,理论等电点为6.02,信号肽预测结果显示,N端具有21个氨基酸长度的信号肽。瘤背石磺MSTN具有MSTN的共同特征,包括蛋白酶水解位点RSRR和C端多肽生物活性区以及9个保守的半胱氨酸残基。通过进化树分析,瘤背石磺MSTN与加州海兔MSTN的亲缘关系最近。RT-PCR结果显示,MSTN基因在各个组织中均有表达;含肌纤维组织中的表达量低于内脏器官的表达量,在肝胰腺中的表达量最高,腹足表达量最低。MSTN基因一级结构具有很高的保守性,说明该基因在进化上的限制性和功能的重要性;同时该基因在石磺非肌肉组织中表达,表明该基因不仅有抑制肌肉生长的作用,还参与其他生命活动的调节。     

The use of alfalfa leaf protein concentrates as a protein source in diets for tilapia (Oreochromis mossambicus)

Chloroplastic and cytoplasmic alfalfa (Medicago sativa) leaf protein concentrates were tested as dietary replacements for fish meal in practical diets for tilapia fingerlings. Each concentrate was used to replace 15, 25, 35, 45 and 55% of the fish meal protein in diets containing 40% protein. Diets containing cytoplasmic leaf protein gave the best results, with growth rates higher than those obtained with a fish meal-based diet when the plant protein replaced up to 35% of the fish meal protein in the diet; higher inclusion levels of plant protein depressed growth. No mortalities were recorded during the 9-week test period with any of the experimental diets. It was concluded that the alfalfa leaf protein could be included at levels of up to 35% of the dietary protein in feeds for tilapia.     

Production of a high percentage of male offspring in growth-enhanced transgenic tilapia using Oreochromis aureus ZZ selected pseudofemales

Gene transfer has offered a new tool for the development of improved fish strains for aquaculture. Monosex fish populations could minimize the impact of genetically modified organisms in the environment. In Oreochromis aureus, the use of pseudofemale spawners (sex-reversed male with a female phenotype) is an alternative technique for producing genetically male tilapia offspring. O. aureus fry were treated with 17β-estradiol at 100 mg/kg of food for 45 days. We obtained 77.1% females and 45.9% in the control group. Females randomly taken from the treated group were crossed with normal males. Fry from pseudofemales producing more than 90% male progenies were submitted to 17 β-estradiol treatment to obtain F₂ pseudofemales. The results of the sex-reversal were low and variable ranging between 66.0 and 84.3% females. F₂ pseudofemales were crossed with transgenic males from the F70 line (O. aureus × O. urolepis hornorum) and non-transgenic (O. aureus) males. The sex ratio of progeny of F₂ pseudofemale deviated significantly (P < 0.01) in favor of males in the crosses with transgenic (90.2%) and non-transgenic (89.3%) males compared to the results observed with normal females (51.0 and 52.3%, respectively). The mean fry production with pseudofemales (per m²/day) was similar to the normal females in the crosses with transgenic and non-transgenic males. To our knowledge this is the first report on the production of a near monosex population in genetically modified fish.     

Vitamin E sparing effect by dietary vitamin C in juvenile hybrid tilapia, Oreochromis niloticus×O. aureus

In order to investigate sparing effects of vitamin C on vitamin E for juvenile hybrid tilapia, fish were fed diets with or without vitamin E each supplemented with either adequate or a high (3× adequate) level of two ascorbate sources, -ascorbic acid (AA) and its derivative, -ascorbyl-2-monophosphate-Mg (C2MP-Mg). Each diet was fed to three replicate groups of fish initially weighing a mean value of 0.54±0.01 g/fish in a closed-recirculated rearing system for 8 weeks. Weight gain and feed efficiency of fish fed the vitamin E-deficient diet supplemented with adequate levels of either AA or C2MP-Mg were significantly lower (P<0.05) than fish fed the other diets. Plasma and liver -tocopherol concentrations were lower in fish fed vitamin E-deficient diets regardless of the level or source of supplemented ascorbate. Gill -tocopherolxxxxxxxx concentration was highest in fish fed vitamin E-adequate diets, intermediate in fish fed vitamin E-deficient diets supplemented with a high level of AA or C2MP-Mg and lowest in fish fed a vitamin E-deficient diet supplemented with an adequate level of AA or C2MP-Mg. Fish fed a vitamin E-deficient diet supplemented with an adequate level of AA or C2MP-Mg had higher hepatic thiobarbituric acid-reactive substances than fish fed the other diets. These data suggest that the high supplementation level (3× adequate) of ascorbate could spare vitamin E in diets for hybrid tilapia and that both AA and C2MP-Mg exhibits the same sparing effect.     

饲料糖脂比对吉富罗非鱼生长、生理生化指标、肠道菌群组成及抗病力的影响

为研究不同糖脂比饲料对罗非鱼幼鱼生长、生理生化指标、肠道菌群组成和抗病力的影响。实验选取平均体重为27g的罗非鱼幼鱼为研究对象,分别用等氮等能的五种不同糖脂比饲料: 44.2/4(D1)、39.6/6(D2)、35/8(D3)、30.4/10(D4)、25.6/12(D5),饲喂10周,采用营养学、生化分析结合高通量测序技术开展了相关测定研究。结果显示,糖脂比饲喂前期对罗非鱼生长具有显著影响,高糖或高脂饲料均生长较差,但饲喂到10周后,所有组间生长无显著差异 (P > 0.05)。相比之下,10周糖脂比在脏体比、肠系膜脂肪指数、肝体比、全鱼粗蛋白、肝脏甘油三酯、血清低密度脂蛋白胆固醇（LDL）和尿素、抗病力、菌群组成等方面存在显著效应。低糖脂比D4在脏体比上显著高于高糖脂比D1 (P < 0.05),而在血清尿素和抗病力上显著低于D1 (P < 0.05),在肠系膜指数上显著高于高糖脂比D1和D2 (P < 0.05),但在全鱼粗蛋白上显著低于D1和D2 (P < 0.05)。另外,D1不仅在肝体比上高于低糖脂比D5 (P < 0.05),而且在LDL和糖耐受能力上均高于其它各组 (P < 0.05)。与之相比,糖脂比虽在肠道微生物物种丰度和均一度上没有显著性差异 (P < 0.05),但在物种组成和热图显示低糖脂比D4与其它组肠道菌群组成不同。D4在聚类关系上单独一支,其门水平上衣原体门、绿弯菌门、放线菌门高于其它各组,特别是D2和D1,但厚壁菌门低于其它各组,特别是D2; 属水平上,D4组幽门螺杆菌属明显多于其它组,而乳杆菌、双歧杆菌属明显少于除D3外的其它组。综上所述,罗非鱼对不同糖脂比饲料有很强的适应和利用能力,但高糖比饲料利用上强于低糖脂比。本实验条件下,高糖组中39.6/6(D2)饲料组虽然在生长上与其它各组无显著差异,但其鱼体粗蛋白含量在各组中最高,肠道菌群组成上也优于其它各组,是罗非鱼适宜的糖脂比水平。     

Behavioral responses of tilapia (Oreochromis niloticus) to acute fluctuations in dissolved oxygen levels as monitored by computer vision

Behavioral and ventilatory parameters have the possibility of predicting the stress state of fish in vivo and in situ. This paper presents a new image-processing algorithm for quantifying the average swimming speed of a fish school in an aquarium. This method is based on the alteration in projected area caused by the movement of individual fish during frame sequences captured at given time intervals. The image enhancement method increases the contrast between fish and background, and is thus suitable for use in turbid aquaculture water. Behavioral parameters (swimming activity and distribution parameters) and changes in ventilation frequency (VF) of tilapia (Oreochromis niloticus) responded to acute fluctuations in dissolved oxygen (DO) which were monitored continuously in the course of normoxia, falling DO level, maintenance of hypoxia (three levels of 1.5, 0.8 and 0.3 mg l⁻¹) and subsequent recovery to normoxia. These parameters responded sensitively to acute variations in DO level; they displayed significant changes (P < 0.05) during severe hypoxia (0.8 and 0.3 mg l⁻¹ level) compared with normoxic condition, but there was no significant difference under conditions of mild hypoxia (1.5 mg l⁻¹ level). There was no significant difference in VF between two levels of severe hypoxia 0.8 and 0.3 mg l⁻¹ level during the low DO condition. The activity and distribution parameters displayed distinguishable differences between the 0.8 and 0.3 mg l⁻¹ levels. The behavioral parameters are thus capable of distinguishing between different degrees of severe hypoxia, though there were relatively large fluctuations.     

脆肉尼罗罗非鱼与普通尼罗罗非鱼肌肉理化与营养特性的比较

脆肉罗非鱼是近年快速发展起来的特色淡水养殖产品,具有肉质爽脆、富有弹性、久煮不烂等优点,受到消费者的广泛欢迎。本研究主要是通过对脆肉尼罗罗非鱼与普通尼罗罗非鱼肌肉在质构特性、感官评价、基本化学成分、脂肪酸分布和游离氨基酸组成等进行比较研究,从而揭示脆肉尼罗罗非鱼与普通尼罗罗非鱼的肌肉理化与营养特性的差异。研究结果显示：脆肉尼罗罗非鱼肉的硬度、弹性和咀嚼性与普通尼罗罗非鱼差异显著（p<0.05）;脆肉尼罗罗非鱼肌肉中的粗蛋白和灰分与普通尼罗罗非鱼无明显差异,但水分含量显著降低,总脂含量显著升高（p<0.05）;脂肪酸组成分析显示脆肉罗非鱼肌肉中所含的多不饱和脂肪酸显著高于普通尼罗罗非鱼（p<0.05）,其中亚油酸、二十碳五烯酸和二十二碳六烯酸分别是对比组的2.12、3.36和3.71倍,脆肉尼罗罗非鱼肌肉的游离必需氨基酸总量,显著高于普通尼罗罗非鱼（p<0.05）,但鲜味氨基酸甘氨酸略低于普通尼罗罗非。研究结果表明,脆肉尼罗罗非鱼和普通尼罗罗非鱼肌肉在质构特性、含水量、脂肪酸含量和游离氨基酸分布上都存在有明显的差异。     

奥利亚罗非鱼AMH基因结构及其表达

抗缪勒氏管激素(antiMullerian hormone, AMH),也称缪勒氏管抑制物质(mullerian inhibiting substance, MIS),为肽类生长因子,属于TGFβ生长和分化因子家族。应用RTPCR和RACE法,从奥利亚罗非鱼精巢组织中分离出AMH的cDNA。分离到3′UTR长为621 bp和168 bp的两条cDNA,除了长度不一,两者之间只存在两个碱基的差别。5′UTR为 27 bp,阅读框为1 545 bp,翻译成514个氨基酸,其中,AMH-N域237个氨基酸,TGFβ域93个氨基酸。同源性分析显示,AMH保守性偏低,奥利亚罗非鱼与其它鱼类的相似性为32%~60%,与哺乳类动物只有19%~21%,而TGF-β域的保守性相对较高,与其它鱼类的相似性为54%~72%。基因结构分析显示,奥利亚罗非鱼AMH存在6个内含子,和斑马鱼AMH结构相似,和人、鼠AMH有较大差别,它们缺少奥利亚罗非鱼和斑马鱼AMH基因共有的内含子Ⅰ和Ⅵ。然而,启动子转录因子分析结果表明,奥利亚罗非鱼AMH启动子中也存在SRY, SF-1, GATA-4, Sox5, Sox9等结合位点。使用Taqman探针分析奥利亚罗非鱼AMH在成鱼不同组织中的表达以及鱼苗、鱼种、成鱼性腺中的表达量,结果显示AMH在成鱼的卵巢、精巢中均有表达,而在脑、肝、肌肉以及心脏中不表达。AMH在鱼苗性腺中不表达,在鱼种、成鱼中雄鱼AMH表达量显著高于雌鱼,而鱼种雄鱼AMH表达量是成鱼雄鱼的10倍。     

罗非鱼源无乳链球菌兼职蛋白EF-Tu的克隆、表达及其抗原性检测

为检测罗非鱼源无乳链球菌兼职蛋白EF-Tu(延伸因子Tu,Elongation Factor Tu)的抗原性,本实验克隆了罗非鱼源无乳链球菌HN0303的EF-Tu基因序列,并进行了蛋白相关性质的预测和系统发育树的构建。通过原核表达得到EF-Tu重组蛋白,同时利用纯化的蛋白免疫家兔获得多克隆兔抗EF-Tu重组蛋白血清以用于EF-Tu蛋白抗原性检测。结果显示,罗非鱼源无乳链球菌HN0303 EF-Tu基因有1个由1197个碱基组成的ORF,编码398个氨基酸。生物信息学分析显示其分子式为C_(1933)H_(3096)N_(532)O_(615)S_(11),分子质量为43.981 ku,理论等电点为4.749;具有多个磷酸化位点,不具有信号肽和跨膜区域;具有保守的EFTu结构域、EF-Tu-II结构域和EF-Tu-Ⅲ结构域,且与其他来源无乳链球菌的EF-Tu蛋白具有很高的同源性;具有较高的抗原指数,表明其可形成多个抗原表位。SDS-PAGE检测发现,诱导表达的重组蛋白以包涵体的形式出现在沉淀中,大小约为66.4 ku。Western Blot分析表明,兔抗EF-Tu重组蛋白血清能分别特异性结合菌体蛋白和EF-Tu重组蛋白。同时使用兔抗EF-Tu重组蛋白血清封闭罗非鱼源无乳链球菌HN0303表面的EF-Tu蛋白后,无乳链球菌HN0303粘附EPC(Epithelioma papulosum cyprini,鲤鱼上皮细胞)的能力下降了79.99%±2.43%。本研究表明,原核表达的罗非鱼源无乳链球菌EF-Tu重组蛋白具备较好的抗原性,用其制备的兔抗血清能够较好地抑制罗非鱼源无乳链球菌的粘附,推测其可能为罗非鱼源无乳链球菌亚单位疫苗的候选蛋白。     

罗非鱼湖病毒核蛋白的克隆表达、抗体制备及其组织分布

近年来罗非鱼湖病毒(TiLV)在多个国家流行,对世界罗非鱼养殖业造成严重威胁。中国是罗非鱼第一养殖大国,尽管我国大陆还没有TiLV的正式报道,鉴于吉富罗非鱼是我国重要的罗非鱼养殖品种,其对TiLV的感染特性研究具有重要意义。本实验采用TiLV对吉富罗非鱼进行人工感染,随后在肝脏组织中克隆和测定了TiLV第6片段基因。罗非鱼湖病毒第6片段基因cDNA全长1 044 bp,开放读码框(ORF)为954 bp,编码317个氨基酸,预测分子量为36.38 ku;5′非编码区(NCR)为19 bp,3′非编码区(NCR)为972 bp。系统进化树分析表明该蛋白属于TiLV核蛋白(NP)。随后在大肠杆菌中表达和提纯了GST融合NP蛋白,在新西兰大白兔上制备了多克隆抗体。通过酶联免疫吸附实验(ELISA)检测抗体效价为1∶51 200,且抗体可特异性识别感染组织中的病毒NP蛋白。对吉富罗非鱼不同组织进行苏木精—伊红(H.E)染色观察,发现肝脏组织坏死并形成合胞体,脾脏部分细胞出现空泡、坏死,含铁血黄素增多,头肾细胞坏死,鳃丝上皮细胞明显解离脱落,鳃小片黏连,脑组织细胞肿大。通过蛋白印迹法(WB)和免...     

罗非鱼湖病毒对吉富罗非鱼和E-11细胞的感染

为研究罗非鱼湖病毒在吉富罗非鱼体内和敏感细胞E-11中的感染特性,实验首先从人工感染罗非鱼湖病毒的吉富罗非鱼脾脏中获得罗非鱼湖病毒第4片段基因组,其cDNA全长1 250 bp,开放读码框长度为1 065 bp,编码354个氨基酸。通过进化树分析,该蛋白是罗非鱼湖病毒血凝素—酯酶融合蛋白(HEF)。随后通过在大肠杆菌大量表达和提纯GST融合HEF蛋白,免疫新西兰大白兔,制备了兔抗TiLV-HEF多克隆抗体。ELISA结果显示获得的抗血清效价高于1∶51 200,并且获得的抗体可以特异性识别病毒的TiLV-HEF蛋白。人工感染实验结果显示,TiLV的感染造成鱼体表面溃疡、全身性出血以及眼晶状体混浊等症状。H.E染色结果显示,肝脏形成合胞体,脾脏中含铁血黄素增加和部分细胞空泡变性。头肾出现淋巴细胞坏死,体肾蛋白质沉淀和肾小球坏死等病理症状。Western blot和免疫组织化学结果显示该病毒在所有组织中均有分布,其中脾脏、头肾和鳃中的病毒丰度高于肝脏、体肾和脑组织。通过细胞间接免疫荧光实验,发现TiLV感染E-11细胞后,HEF蛋白在细胞质中。TiLV可以通过感染吉富罗非鱼幼鱼的肝脏、脾脏...     

广东地区吉富罗非鱼无乳链球菌病的流行情况与耐药性

为了调查广东省惠州、肇庆、珠海、湛江4个吉富罗非鱼主养区链球菌病的流行情况和耐药性,并进一步分析β-内酰胺酶基因与青霉素类药物的耐药性关系。本实验通过传统的方法对菌株进行分离纯化,扩增特异性基因cfb以及16s r DNA对各菌株进行鉴定;采用k-b法测定分离菌株的药物敏感性;通过PCR检测β-内酰胺酶类基因在分离菌株中的分布情况,并用Statistic6.0统计分析各β-内酰胺酶基因与青霉素类药物的耐药关系。实验结果表明,吉富罗非鱼无乳链球菌的阳性率从高到低的顺序为惠州(46.46%)湛江(43.24%)肇庆(17.30%)珠海(4.17%);药敏结果显示各地区无乳链球菌分离株对青霉素(耐药率为94.29%)和磺胺二甲基嘧啶(耐药率为86.40%)普遍耐药,对恩诺沙星最为敏感(耐药率为3.99%);β-内酰胺酶基因分布与细菌耐药性统计结果显示,无乳链球菌基因组中的9个β-内酰胺酶基因在各分离菌株中的分布呈高度多态性,其中SAG0658基因与氨苄青霉素抗性显著相关,提示SAG0658基因在无乳链球菌耐氨苄青霉素过程中发挥主要作用;此外,9个β-内酰胺酶基因与青霉素抗性无相关性,说明其在菌株对青霉素耐药过程中并未发挥明显作用,提示分离菌株对青霉素的耐药性可能依赖其他途径。     

吉富罗非鱼感染无乳链球菌后肝脏组织在不同时期蛋白质组的表达差异

为研究罗非鱼感染无乳链球菌前后肝脏组织蛋白质的表达变化。本研究以吉富罗非鱼为材料,采用双向电泳技术分析其在无乳链球菌感染胁迫下24 h、48-144 h、12 d与对照组（未感染）肝脏组织蛋白质组的变化,对差异表达蛋白进行质谱分析鉴定。结果显示：吉富罗非鱼在无乳链球菌胁迫下,3个实验组的蛋白质图谱与对照组相比存在显著差异,共有30个蛋白点发生显著改变,其中13个表达上调,17个下调,2个下调蛋白点在感染12 d组消失。通过MALDI-TOF-MS MS/MS质谱分析和数据库检索对这些蛋白质进行了功能分类,发现它们涉及到能量代谢、细胞防御与应激、消化免疫、抗氧化与排毒等许多方面。推测这些蛋白可能在吉富罗非鱼对无乳链球菌胁迫的抗性反应中发挥了重要作用。研究结果为无乳链球菌疫苗的研制及吉富罗非鱼抗病品种选育奠定了基础。     

尼罗罗非鱼铁调素调节蛋白在宿主抵御病原菌侵染和调节铁稳态中的功能

为了研究铁调素调节蛋白(hemojuvelin,HJV)在硬骨鱼中抵御病原菌感染和维持自身铁稳态过程中的作用,实验扩增了尼罗罗非鱼铁调素调节蛋白基因(Onhjv)的开放阅读框(ORF),分析其在健康尼罗罗非鱼各组织中的分布模式及在抵御病原菌感染和调节铁稳态中的相关作用。结果显示,Onhjv的ORF全长由1 248个碱基组成,编码415个氨基酸,在不同物种之间具有一定的保守性。实时荧光定量PCR(qRT-PCR)结果显示,Onhjv在尼罗罗非鱼各组织中广泛分布,并在肝脏中的表达量最高。在无乳链球菌或嗜水气单胞菌感染后,Onhjv在肝脏、脾脏、肠和鳃中的表达量均显著上调。体外头肾单核/巨噬细胞和肝细胞中Onhjv表达量在受到这2种病原菌应激下也显著上调。此外,在1和10μmol/L FeCl_3溶液刺激后,Onhjv表达量在肝脏、脾脏、肠和鳃等组织,以及头肾单核/巨噬细胞和肝细胞中的表达量也呈显著上调。受重组罗非鱼IL-6蛋白[(r)OnIL-6]刺激后,头肾单核/巨噬细胞中Onhjv表达量显著上调,表明炎症因子可以促进Onhjv表达。研究表明,尼罗罗非鱼铁调素调节蛋白在宿主抵御病原菌感染和维持铁稳态的过程中发挥作用。本实验为探究HJV在硬骨鱼中的生物学功能提供了参考,同时为进一步研究铁代谢在宿主防御病原菌感染过程中的重要作用提供理论指导。     

尼罗罗非鱼Foxp1a/b的cDNA克隆及表达模式研究

为探讨低等脊椎动物Foxp1与免疫细胞活化及机体雌激素水平的相关性,本研究首次分离克隆了尼罗罗非鱼 (Oreochromis niloticus) Foxp1的开放阅读框序列,并通过real-time PCR对其mRNA表达水平进行检测。结果显示,尼罗罗非鱼具有两种不同基因编码的Foxp1分子,分别命名为OnFoxp1a与OnFoxp1b;OnFoxp1a为1710 bp,由15个外显子编码569个氨基酸,OnFoxp1b为2040 bp,由16个外显子编码679个氨基酸;OnFoxp1a/b均含有Foxp亚家族的特征性结构,即锌指结构、亮氨酸拉链样结构和叉状螺旋结构;与OnFoxp1a相比,OnFoxp1b与高等脊椎动物Foxp1具有更近的亲缘关系。Real-time PCR检测结果显示,OnFoxp1a/b几乎在所有组织中均有表达,OnFoxp1a在精巢中有高水平表达, OnFoxp1b在心脏中有高水平表达,同时OnFoxp1b在免疫相关组织如鳃、脾脏、肾脏、肠等均有中等水平表达;淋巴细胞多克隆刺激剂PHA、PMA、LPS刺激尼罗罗非鱼外周血单个核细胞（PBMC）,结果显示,50 μg/mL PHA和50 ng/mL PMA分别刺激6、12、24 h均显著增强OnFoxp1b的表达 (p＜0.05),20 μg/mL LPS刺激后,OnFoxp1b的表达出现先降低后升高的趋势 (p＜0.05);而OnFoxp1a的表达除50 μg/mL PHA 刺激24 h后有所升高,其余均无显著变化;雌激素处理6月龄雄性尼罗罗非鱼48 h,OnFoxp1a/b在肠、肾脏中的表达显著上调 (p＜0.05),而脾脏中无显著变化 (p>0.05)。综上所述,低等脊椎动物硬骨鱼类两种不同基因编码的Foxp1a/b均为哺乳动物Foxp1的同源分子,但其序列、结构特征、表达模式迥异,提示其功能发生歧化;同时两者的表达与淋巴细胞活化及机体雌激素水平密切相关。