应用PCR-DGGE和Q-PCR分析不同体重斑点叉尾鮰皮肤、鳃和胃肠道菌群结构

采用聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)和荧光定量-聚合酶链式反应(Q-PCR)技术分析高体重(high weight,HW)和低体重(low weight,LW)斑点叉尾鮰鮰(Ietalurus punetaus)皮肤、鳃和胃肠道菌群多样性,为斑点叉尾鮰微生态研究及筛选斑点叉尾鮰源益生菌提供理论依据。结果显示,斑点叉尾鮰菌群丰富度由低到高依次为鳃、皮肤、前肠、后肠和胃。肠杆菌科(Enterobacteriaceae)和气单胞菌属(Aeromonas)是皮肤的优势菌群;肠杆菌科(Enterobacteriaceae)、气单胞菌属(Aeromonas)和肠球菌属(Enterococcus)是水体、鳃和胃的优势菌群;肠杆菌科(Enterobacteriaceae)、拟杆菌属(Bacteroidetes)、气单胞菌属(Aeromonas)和酵母菌属(Saccharomyces)是肠道的优势菌群。HW斑点叉尾鮰鳃菌群的香农多样性指数、均匀度和丰富度及前肠菌群的丰富度显著高于LW斑点叉尾鮰(P0.05)。皮肤的黄杆菌属(Flavobacterium),胃的肠杆菌科(Enterobacteriaceae),前肠的拟杆菌属(Bacteroidetes)和双歧杆菌属(Bifidobacterium)及后肠的拟杆菌属(Bacteroidetes)、双歧杆菌属(Bifidobacterium)和酵母菌属(Saccharomyces)的拷贝数分别是101.97、107.69、106.19、103.83、106.13、103.92和104.26,均显著高于LW斑点叉尾鮰(P0.05)。结果表明,斑点叉尾鮰皮肤、鳃、胃肠道均形成独特的菌群结构,LW和HW斑点叉尾鮰菌群结构存在明显差异,HW斑点叉尾鮰菌群多样性增加。     

酵母免疫多糖对受免斑点叉尾(鮰)免疫应答的增强作用

在饲料中添加定量的酵母免疫多糖,投喂注射接种了福尔马林灭活的嗜水气单胞菌(Aeromonas hydrophila)菌苗的斑点叉尾(鮰)(Ictalurus punctatus),28 d后通过测定供试鱼的增重量、血清中凝集抗体效价、溶菌酶活性、谷丙转氨酶活性(SGPT)、血清总蛋白含量、白细胞吞噬活性以及活菌攻毒后的免疫保护率(RPS),探讨了酵母免疫多糖对受免斑点叉尾(鮰) 免疫应答的增强作用.结果表明,用添加200.0 mg/(kg·d)酵母免疫多糖的饲料投喂受免斑点叉尾(鮰),不仅可以使受免斑点叉尾(鮰) 对灭活嗜水气单胞菌的免疫应答水平提高,增强抵抗嗜水气单胞菌人工感染的能力,而且还具有一定的促进生长和改善肝功能的作用.     

斑点叉尾(鮰)传染性套肠症及其防治

套肠症是近年来发生的一种新型细菌性传染病,危害对象是斑点叉尾(鮰)(Ictalurus punctatus),已经连续几年造成了斑点叉尾(鮰)大批发病死亡,严重地威胁着斑点叉尾(鮰)养殖的健康发展.目前初步认为该病是由嗜麦芽寡养单胞菌(Stenotrophomonas maltophilia)引起的急性致死性传染病,以发生严重的肠炎、肠套叠和脱肛为特征,在短时间内即可引起大量死亡,我们称此病为"斑点叉尾(鮰)传染性套肠症(Infectious intussusception of channel catfish,IICC)"该病的致病病原和病理变化特征在水生动物疾病中是罕见的.     

南美白对虾肠道细菌菌群分析

对健康养殖南美白对虾胃、肝胰腺、肠道、肌肉中的需氧菌群进行研究。结果表明:胃、肝胰腺、肠道、肌肉中的异氧细菌总菌数分别为5.3×105~6.5×107cfu/g、6.6×105~5.2×106cfu/g、1.4×108~4.5×109cfu/g和1.0×104~1.1×105cfu/g。对从其肠道中分离出来的111株菌株进行属(科)的鉴定。这些菌株分属于13个属(科),发光杆菌属、弧菌属、气单胞菌属、肠杆菌科、黄单胞菌属、土壤杆菌属、芽孢杆菌属、棒状杆菌属、小球菌属、黄杆菌属、产碱杆菌属、假单胞菌属和色杆菌属,还有一株未鉴定的革兰阳性菌。其中优势菌为发光杆菌属、弧菌属,气单胞菌属、肠杆菌科、黄单胞菌属,次优势菌为土壤杆菌属、芽孢杆菌属、棒状杆菌属、小球菌属。     

不同鱼类和采样时间的养殖池塘环境微生物群落研究

微生物群落是养殖池塘生态系统重要的组成部分。本研究在不同月份采集尼罗罗非鱼和斑点叉尾鮰池塘水体样品,分析水样中的硝酸盐氮、亚硝酸盐氮、氨氮、总氮和总磷等理化指标,利用Biolog-Eco微平板技术分析水体中微生物对各类碳源代谢的平均颜色变化率,利用高通量测序技术分析水体中的菌群结构,以期了解养殖鱼种类和季节对养殖池塘环境及菌群结构的影响。结果表明：冬季淡水养殖池塘水质和菌群结构不同于其他季节,养殖鱼类种类对池塘水体理化指标和水体微生物菌群结构的影响不大。不同采样时间的池塘水体理化指标差异显著,同一采样时间不同养殖鱼类池塘水体理化指标之间无显著差异。其中,1月尼罗罗非鱼养殖池塘水样中的NH4-N含量高于其他月份,显著高于4月和7月的水样(P<0.05)；1月TP含量显著高于1月、4月和7月(P<0.05)。1月斑点叉尾鮰养殖池塘水样中的TP和NO₃-N含量显著高于其他三个月份(P<0.05)。Biolog-ECO微平板技术检测到尼罗罗非鱼和斑点叉尾鮰养殖池塘水样中的微生物群落对碳水化合物和氨基酸的利用能力较强,对酚胺类化合物的利用能力较弱,4月斑点叉尾鮰养殖池塘水体微生物对碳源的利用率高于尼罗罗非鱼养殖池塘。高通量测序结果发现厚壁菌门(Firmicutes)和拟杆菌门(Bacteroidetes)是尼罗罗非鱼和斑点叉尾鮰养殖池塘水体中的绝对优势菌门。hgcI clade、CL500-29 marine group和C39等参与氮磷循环的菌属为淡水池塘养殖水体中的主要菌属。本研究结果为了解淡水养殖环境中微生物群落结构与变化规律、更有针对性的构建淡水养殖池塘尾水生态化处理方法提供科学依据。     

斑点叉尾(鮰)"腹水症"病原的分离鉴定及体外芽孢杆菌拮抗试验

为了解斑点叉尾(鮰)(Ictalurus punctatus)"腹水症"的发病原因,提高防控技术,试验在佛山市某渔场患典型"腹水症"的病(鮰)上分离出优势菌落,通过形态学观察、生化及分子生物学鉴定确定该细菌为温和气单胞菌(Aeromonas sobria).通过对健康斑点叉尾(鮰)的回归感染实验发现,与原感染鱼发病症状相似,且分离鉴定得到相同病原菌.体外药敏试验结果显示:该温和气单胞菌菌株对恩诺沙星、氟苯尼考等7种药物敏感,而对四环素、强力霉素耐药.本研究还探讨了4种芽孢杆菌对分离的温和气单胞菌的拮抗作用,结果表明地衣芽孢杆菌对该病原的拮抗作用最好.     

斑点叉尾鮰的细菌病及其防控方法(三)

正三、斑点叉尾鮰气单胞菌败血症该病在20世纪80年代中期开始流行于我国广大淡水鱼养殖地区,会危害包括斑点叉尾鮰在内的青鱼、草鱼、鲢、鳙、鲮等主要淡水养殖鱼类。1994年前后,被暂定名为淡水鱼细菌性败血症。该病当前仍是淡水鱼类养殖的主要疾病,由该病造成的经济损失达养殖产量的15%～30%。1.病原病原为嗜水气单胞菌(Aeromonas hydrophila)和温和气单胞菌(A.sobria)等,本病中以嗜水气单胞     

斑点叉尾■的细菌病及其防控方法(一)

正一、斑点叉尾鮰肠道败血症(爱德华氏菌病,Enteric septicaemia of catfish,ESC)该病是由鮰爱德华菌(Edwardsiella ictaluri)引起的细菌性疾病,于1976年在美国阿拉巴马州和佐治亚州的斑点叉尾中被发现。1.病原病原是鮰爱德华菌,属于肠杆菌科,是一种非条件性致病菌。从宿主范围、致病性、血清学特性和质粒构型来看,鮰爱德华菌至少可分为两种类型。但是,从患病斑点叉尾鮰分离到的鮰爱德华菌     

无公害斑点叉尾鮰成鱼池塘养殖技术

斑点叉尾鮰(Ictalurws punctatws)亦称沟鲶(Chanmel catfish)属，鲶形目(Siluriflrmes)，鮰科(Ictaluridae)鱼类。斑点叉尾鮰天然分布区域在美国中部流域、加拿大南部和大西洋沿岸部分地区，以后广泛地进入大西洋沿岸．现在基本上全美和墨西哥北部都有分布，斑点叉尾鮰具有适应性强、生长速度快、抗病力强、易养殖、易     

斑点叉尾鮰套肠病的诊断与防治

<正>近几年,斑点叉尾鮰套肠病在各地频繁爆发,而且发病突然,来势凶猛,传染快,死亡率高,给斑点叉尾鮰养殖业造成了巨大损失。为此,本文详述了斑点叉尾鮰套肠病的病因分析、诊断方法以及治疗措施。斑点叉尾鮰(Ictalurus Punctatus)又称沟鲶、钳鱼,属于鲶形目、鮰科鱼类。原产于北美洲,是一种大型淡水鱼类。因其食性杂、生长快、适应性广、抗病力强、肉质上     

Bacterial Flora in Turbot Scophthalmus maximus Cultured in Deepwell Seawater of Liaodong Peninsula

The microbial flora in turbot (Scophthalmus maximus) cultured in deepwell seawater from Liaodong Peninsula, China was studied for the first time. The total amount of aerobic meso- and psychrophilic microflora from the culturing water, gills, and skin were 3.46–3.82, 3.19–4.28, and 1.70–1.95 log cfu/g, respectively. Out of 204 strains isolated from fresh turbot meat with skin, Gram-positive bacteria accounted for 19.2% of the total aerobic meso- and psychrophilic microflora, and 72.1% were Gram-negative bacteria. Phylogenetic analysis was performed using 16S rDNA sequences of microbial flora isolated from turbot. The predominant bacteria in fresh turbot were Micrococcus, Serratia liquefaciens, and Enterobacteriaceae. Meanwhile, the bacterial flora of refrigerated turbot fish were investigated, and the results indicated that Shewanella putrefaciens was the predominant spoilage bacteria.     

Microflora of Arctic charr, Salvelinus alpinus (L.): gastrointestinal microflora of free-living fish and effect of diet and salinity on intestinal microflora

Abstract. The adherent aerobic bacterial flora present in the gastrointestinal tract and faeces of free-living Arctic charr, Salvelinus alpinus (L.), from Lake Takvatn, Northern Norway, were identified both qualitatively and quantitatively. Approximately 10⁵ bacteria g⁻¹ were found in both the small and large intestines. The predominant bacterial species were identified as Aeromonas , Enterobacteriaceae, Micrococcus and Lactobacillus. Other microorganisms isolated included Acinetobacter, Cytophaga, Flavobacterium, Moraxella, Pseudomonas, Vibrio , Coryneforms and Streptococcus. The intestinal microflora of free-living fish was dominated by Aeromonas and Lactobacillus , but the intestinal bacterial flora of wild fish transferred to hatchery was affected by feeding them either a capelin roe diet or a commercial feed in fresh and sea water. Approximately 55% of the bacterial flora in intestinal contents from fish fed the capelin roe diet was Enterobacteriaceae when the fish were held in fresh and sea water. However, when the wild-caught charr were fed a commercial diet in fresh water, Aeromonas and Pseudomonas dominated in faeces, while Vibrio and Pseudomonas were predominant in the diet group held in sea water.     

温度和pH值对斜带髭鲷蛋白酶、淀粉酶活性的影响

研究了温度和pH值对斜带髭鲷Hapalogenys nitens蛋白酶、淀粉酶活力的影响。结果表明，斜带髭鲷胃内pH值范围为4.9～5.4；肝胰脏pH值为5.8～6.2；肠道pH值为6.5～6.9。蛋白酶活性随温度的上升而增加，在40℃达最大，40℃以上酶活性随温度的上升而下降。在不同消化器官中，蛋白酶活性大小顺序为：胃〉前肠〉幽门盲囊〉后肠〉肝胰脏。在15～50℃范围内，斜带髭鲷消化道不同部位淀粉酶活性的最适温度均为35℃，淀粉酶活性由高到低顺序为：肝胰脏〉幽门盲囊〉前肠〉后肠〉胃。在pH值为2.2～7.6范围内，胃蛋白酶活性的最适pH值为2.8；在pH值为4.8～8.0范围内，其他消化器官蛋白酶的最适pH值均为7.2；在最适pH值下，各消化器官中的蛋白酶活性由高到低顺序为：前肠〉幽门盲囊〉后肠〉肝胰脏。在pH值为4.8～8.0范围内，胃淀粉酶活性的最适pH值为6.0，肠、肝胰脏与幽门盲囊淀粉酶的最适pH值均为6.8，活性由高到低顺序为：肝胰脏〉幽门盲囊〉前肠〉后肠〉胃。在最适温度和pH值下，蛋白酶活性由高到低顺序为：胃〉前肠〉幽门盲囊〉后肠〉肝胰脏；淀粉酶活性由高到低顺序为：肝胰脏〉幽门盲囊〉前肠〉后肠〉胃。     

野生与养殖银鲳消化道菌群结构中产酶菌的对比分析

实验对野生和养殖银鲳胃、幽门盲囊、前肠、中肠、后肠菌群结构进行了定性对比分析,并对产蛋白酶、淀粉酶、脂肪酶、纤维素酶的菌株进行了鉴定。结果发现,野生和养殖种群的消化道菌群结构存在较大差异,且同一种群消化道各部分之间菌群结构也存在较大差异。尽管养殖和野生银鲳均在幽门盲囊中具有最多的可培养细菌菌株,但野生银鲳消化道内主要菌群为嗜冷菌属(Psychrobacter)和Pseudochrobactrum,养殖银鲳消化道主要菌群为不动杆菌属(Acinetobacter)和假单孢菌属(Pseudomonas),两种群共有细菌仅一株,即Psychrobacter piscatorii strain VSD503,但其分别存在于野生与养殖种群银鲳消化道的不同部位。在产酶菌株筛选中发现,野生银鲳消化道内分离到16株产酶菌,其中44%可培养菌能产蛋白酶,56%能产淀粉酶,11%能产脂肪酶,56%能产纤维素酶,部分菌株可产2株以上的消化酶,其中产3种以上酶的菌株有5株,且产酶量丰富。相对于野生银鲳,养殖银鲳消化道内分离到22株产酶菌,主要以产蛋白酶和淀粉酶为主,70%可培养菌可产蛋白酶,21%可产淀粉酶,仅Bacillus thuringiensis strain VITGS可产纤维素酶,无一株菌产脂肪酶,其中只有Bacillus thuringiensis strain VITGS产3种酶但产酶量相对较少。研究可为银鲳人工养殖中潜在益生菌的筛选提供理论依据。     

穿心莲对草鱼肠内细菌的影响

给不同组草鱼分别投喂含0％、0.5％、1％和2％穿心莲的人工配合饲料，在不同时期取样检测各组草鱼前、中、后肠需氧和兼性厌氧菌的种类和数量。结果显示，草鱼肠内细菌的种类，特别是优势菌群的种类没有受到穿心莲的影响。各组草鱼肠内各部位的优势菌群均为气单胞菌Aeromonas和肠杆菌Enterobacteriaceae。草鱼肠内细菌数量为105～108CFU·g-1，分析后发现，各组草鱼肠内细菌数量之间没有显著差异(P＞0.05)。但1％及2％的穿心莲能降低气单胞菌的组成。上述结果表明，一定浓度的穿心莲可以通过降低优势菌群气单胞菌的组成而对草鱼肠内微生态系产生影响。     

军曹鱼消化系统的形态及组织学研究

利用活体解剖和光镜技术对军曹鱼Rachycentron canadum消化系统进行形态及组织学研究。结果显示，军曹鱼消化道由口咽腔、食道、胃、幽门盲囊、前肠、中肠和后肠几部分组成。口咽腔大，粘膜上皮为复层鳞状上皮，内含少量杯状细胞。食道很短，但上皮包括复层鳞状上皮区和单层柱状上皮区。胃膨大，呈Y形，在贵门和幽门部可观测到杯状细胞，胃体粘膜层下有大量的胃腺细胞。幽门盲囊发达，肠短但分3部分，前肠、中肠和后肠，肠道系数为0．43，肠道由前向后杯状细胞和粘膜皱褶不断减少。消化腺包括肝脏和胰腺，肝小叶分界不明显，肝细胞内脂肪含量丰富，胰腺属于散在性的，其外分泌部有许多腺泡组成，胰岛分散于外分泌部间。     

Culture-independent characterization of the autochthonous gut microbiota of grouper Epinephelus coioides following the administration of probiotic Enterococcus faecium

Probiotics have been applied extensively in aquaculture and demonstrated a range of benefits, but the mechanisms mediating such responses remain largely unknown. A clear illustration of the effect of probiotics on the autochthonous gut microbiota is the first step in understanding the underlying mechanisms. In this study, the autochthonous microbiota in the foregut, midgut and hindgut of juvenile grouper Epinephelus coioides following the dietary administration of probiotic Enterococcus faecium MM4 for 60?days were assessed using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) with subsequent sequencing analysis. Generally similar DGGE patterns, with 15 common bands, were observed in the foregut, midgut and hindgut of fish fed the probiotic diet or the control diet, while more visible bands were present in fish fed the probiotic diet. The similarity dendrogram showed that the foregut samples formed an independent cluster distinctly different from the midgut and hindgut samples, but a high similarity (81.4%) was observed among the three gut sections. Significantly increased species richness and Shannon index were observed in the midgut and hindgut samples in the probiotic group compared with the control group, which suggested that probiotic E. faecium could elevate the autochthonous microbial diversity in the midgut and hindgut. Sequences analysis showed that probiotic E. faecium stimulated the growth of some unidentified bacteria and potentially beneficial bacteria, while exerted no apparent effect on potentially harmful Staphylococcus saprophyticus.     

卵形鲳鲶消化酶活性的研究I．成鱼和幼鱼消化酶活性在不同消化器官中的分布及其比较

比较研究了卯形鲳够（Trachinotus ovatus）成鱼和幼鱼阶段消化酶（蛋白酶、淀粉酶、脂肪酶）在不同消化器官中的活性。结果表明：1）成鱼蛋白酶在不同消化器官中的活性大小依次为胃〉前肠〉中肠〉幽门盲囊〉后肠〉肝；淀粉酶活性为前肠〉后肠〉幽门盲囊〉中肠〉肝〉胃；脂肪酶活性为前肠〉中肠〉后肠〉幽门盲囊〉肝〉胃。2）幼鱼蛋白酶在不同消化器官中的活性大小依次为胃〉肠〉幽门盲囊〉肝；淀粉酶活性为肠〉幽门盲囊〉肝〉胃；脂肪酶活性为肠〉幽门盲囊〉肝〉胃。3）成鱼不同消化器官中蛋白酶和淀粉酶的活性均小于幼鱼，成鱼胃和幽门盲囊的淀粉酶活性与幼鱼的差异显著；幼鱼胃脂肪酶活性大于成鱼，但其他器官的活性均小于成鱼。卵形鲳鳕幼鱼不同消化器官中的3种消化酶活性大小顺序与成鱼基本相似。     

Lipid digestion in turbot (Scophthalmus maximus). I: Lipid class and fatty acid composition of digesta from different segments of the digestive tract

The lipid content and lipid composition of digesta from the stomach, foregut, hindgut and rectum of juvenile turbot fed a commercial diet were determined in order to examine the process of lipid digestion in this species. The moisture content of the digesta increased along the digestive tract from 71.5% in the stomach to 89.6% in the rectum. The lipid content of the digesta increased initially from 15.7% of the dry weight in the stomach to 36.1% in the foregut but thereafter decreased through 23.2% in the hindgut to 9.1% in the rectum. The proportion of triacylglycerols (TAG) in the total lipid of the digesta decreased from 63% in the stomach to 17.4% in the rectum whereas that of free fatty acids (FFA) increased from 10016 to 48.9%. The highest proportions of monoacylglycerols (MAG), diacylglycerols (DAG) and most phospholipids were observed in the lipid of the hindgut digesta. In addition, a fall in levels of neutral and phospholipid classes as digesta moved from hindgut to rectum signified absorption. Analysis of the fatty acid composition of the lipid classes TAG, DAG and MAG suggest a polyunsaturated fatty acid specificity for hydrolysis may exist. Saturated, monounsaturated and polyunsaturated fatty acids (PUFA) accounted for 17.9%, 45.4% and 37.0%, respectively of the FFA present in the foregut whereas the corresponding values for the rectum were 32.6%, 51.9% and 16.3%. Overall, the results suggest a PUFA specifity for hydrolysis may exist alongside the positional non-specific lipolytic activity associated with the hindgut regions of the digestive tract of turbot and that PUFA, released by lipolysis are more effectively absorbed from the digesta than monounsaturated and saturated fatty acids.     

Tissue localization of Aeromonas salmonicida in Atlantic salmon, Salmo salar L., following experimental challenge

Three groups of Atlantic salmon, Salmo salar L., were exposed to live, colony-forming, radiolabelled Aeromonas salmonicida bacteria in a bath challenge: (1) fish with artificial wounds; (2) fish with a reduced epidermal mucus layer caused by removal of the mucus layer on two occasions by a swabbing procedure; and (3) a control group of untreated fish. Fish were killed 2, 6 and 24 h after challenge, and radioactivity (cpm g^–1) was measured in the blood, mucus, skin, wound area, gills, anterior kidney, posterior kidney, spleen, midgut and hindgut. The highest levels of radioactivity were measured in the wound areas and in the gills. There was a significant positive correlation between the levels of radioactivity in the gills and blood, and between the mucus and skin at 2 h post-challenge. Two hours after the bath challenge, live A. salmonicida bacteria were found in the blood of fish in the 'swabbed' and 'artificial wound' groups, and not in the control group. Twenty-four hours after the bath challenge, the kidney of fish from all groups contained viable bacteria, whereas the blood was negative.