草鱼源致病性维氏气单胞菌的鉴定及其黏附特性分析

采用表型鉴定与细菌16S rRNA基因序列分析对从患病草鱼体内分离的病原菌株16-1进行分类鉴定,综合该菌株的表型特征与16S rRNA基因序列,确定其为维氏气单胞菌。随后,对该菌株的细胞黏附特性及其携带的黏附素基因进行检测与分析。结果显示,分离菌株能以聚集性方式黏附于鲤鱼上皮瘤细胞（EPC）周围,平均黏附菌数随共孵育时间延长而增加,90 min达到峰值,说明该分离株具有良好的细胞黏附性。黏附因子检测表明,该菌株同时携带ompAI、ompAII和ahl13种黏附素基因,这些黏附素基因在分离菌株与不同来源参考株之间的核苷酸序列和氨基酸序列同源性分别介于95.8%～99.5%和95.0%～100%（ompAI）,96.3%～99.1%和97.9%～100.0%（ompAII）,78.6%～99.6%和75.5%～99.4% （aha1）,说明所携带的ompAI和ompAII黏附素基因在不同来源的维氏气单胞菌之间具有较高的保守性。     

白鲢出血病病原菌的鉴定

从患出血病的白鲢体内分离出两株细菌(编号为水_1、水_2),分别感染健康白鲢,人工复制白鲢出血病获得成功。对这两株细菌进行形态、营养需求、生长特性、以及生化反应等观察,作者认为,水_1菌为产碱假单胞菌(Pseudomonas alcaligenes);水_2菌为嗜水气单胞菌(Aeromonas hydrophila)。前者隶属于假单胞菌科(Pseudomonaceae),后者隶属于孤菌科(Vibrionaceae)。     

Analysis of exotoxins produced by atypical isolates of Aeromonas salmonicida,by enzymatic and serological methods

In this study, exotoxins produced by 62 Aeromonas salmonicida strains and the bacterium Haemophilus piscium were analysed. Enzymatic assays, zymograms and serological detection were used to monitor secretion by bacterial strains of the previously described exotoxins P1, GCAT and AsaP1 and also the extracellular P2 metallo-gelatinase and a serine caseinase, which is different from the P1 protease and has not yet been characterized. Based on the results, the strains were divided into five groups. One comprised the type strains for A. salmonicida ssp. masoucida, H. piscium and 36% of the atypical isolates, and another, a type strain for A. salmonicida ssp. smithia together with 14% of the atypical isolates. A second type strain of A. salmonicida ssp. smithia was grouped with 8% of the atypical isolates. The largest group contained the type strains for A. salmonicida ssp. achromogenes and 38% of the atypical isolates. The type strains for A. salmonicida ssp. salmonicida were in the last group with all the four typical strains and 4% of the atypical isolates. The combination of zymogram and serological detection used is recommended as the most reliable method for characterizing A. salmonicida strains according to their exotoxin secretion.     

Effect of Cinnamon (Cinnamomum zeylanicum) on Growth Performance,Feed Utilization,Whole-Body Composition,and Resistance to Aeromonas hydrophila in Nile Tilapia

Four isonitrogenous (30% crude protein) and isocaloric (4.40 kcal/g) experimental diets were formulated to contain 0.0% (control), 0.5%, 1.0%, or 1.5% cinnamon. The diet containing 1% cinnamon resulted in significantly greater (P < 0.05) specific growth rate (SGR), feed conversion ratio (FCR), feed efficiency ratio (FER), protein efficiency ratio (PER), apparent protein utilization (APU), and energy utilization (EU). In regard to body composition, there was no significant difference in dry matter, protein, lipids, and ash (P > 0.05) contents due to cinnamon supplementation. Blood plasma profile showed an improvement in hemoglobin (HB), red blood cell (RBCS), hematocrite (PCV), total protein, and total lipids, while there was a decrease in creatinin, urea, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and glucose in fish fed 1% cinnamon. Cinnamon was found to have an antibacterial activity antagonistic to Aeromonas hydrophila infection in fish. Performance and FCR improvement translated into a 10% decrease in feed costs associated with the 1% cinnamon diet.     

不同来源嗜水气单胞菌耐药基因检测

采用PCR方法,对分离于唐山和秦皇岛养殖场的宽体金线蛭(Whitmania pigra)、中国林蛙(Rana temporaria chensinensis)、牙鲆(Paralichthys olivaceus)、草鱼(Ctenopharyngodon idellua)、青鱼(Mylopharyngodon piceus)以及鲤(Cyprinus carpio)肝脏的42株不同水产养殖动物的致病性嗜水气单胞菌(Aeromonas hydrophila)进行了4种耐药基因β-内酰胺类(TEM)、氨基糖苷类(ant(3″)-I)、磺胺类(Sul3)和四环素类(tet(A))的检测。结果表明:42株嗜水气单胞菌均100%携带TEM、ant(3″)-I和Sul3,不携带tet(A)。所检不同水产动物源嗜水气单胞菌均携带TEM、ant(3″)-I及Sul3耐药基因,从分子层面表明该致病菌多重耐药或潜在耐药严重。     

Identification and Pathogenicity Analysis of Aeromonas caviae M12 from Fox

We tried to identify the bacteria and explore the mechanism of the bacteria's pathogenicity via housekeeping gene gyrB and in vitro organ culture (IVOC) of ileum and intramuscular injection. Microscope, electron microscope and scanning electron microscope were also used to observe and the structure, pathogen of M12 and changes of infected tissues. The results showed that M12 was short gram-negative bacteria, and both ends of it were obtuse and the size without flagellum was (0.6 to 1.6) μm×(0.6 to 0.7)μm while length of flagellum was about 2 to 3 times of the length of bacteria.It had 100.0% similarity with Aeromonas caviae from GenBank. IVOC test observed M12' adhesion in intestinal epithelial cell, resulting in formulation of biofilm structure and damage in intestinal epithelial cell. Histopathologic examination showed that M12 could cause damage in intestine, liver, lung, kidney, muscle tissue of rabbit.The assay would offer references for researching the pathogenic mechanism of Aeromonas caviae.     

豫北地区主要淡水鱼类感染嗜水气单胞菌的流行病学调查

对豫北地区嗜水气单胞菌引起的细菌性败血病进行流行病学调查,分离鉴定致病性嗜水气单胞菌菌株,并对致病性嗜水气单胞菌菌株进行毒力验证和药敏试验。采集4个养殖场病鱼标本及水样,每月进行流行病学调查,利用生理生化及分子生物学方法检测嗜水气单胞菌的分布状况。结果显示,筛选出52株为嗜水气单胞菌,其中32株为致病性嗜水气单胞菌,20株为非致病性嗜水气单胞菌,且致病性嗜水气单胞菌主要分布在7—9月份,毒力验证试验表明,32株致病性菌株的毒力大小差异明显,筛选出强毒株XDMG(4),为后期试验的疫苗株做准备;药敏试验表明,不同菌株对同一种药物的药敏结果不同,但大部分药物的药敏结果基本一致,70%以上的致病性菌株对头孢哌酮、氟本尼考、菌必治、阿米卡星等抗菌药物表现为高度敏感,对青霉素类药物表现为高度耐药性,耐药率达100%,对氨基糖苷类(不包括阿米卡星)、磺胺类、四环素等药物呈现不同程度的耐药性,具有多重耐药性。本试验旨在丰富本地区的鱼类细菌性败血病的病原资料,并为该菌引起的人类疾病的防控提供科学依据。     

南方鲇源豚鼠气单胞菌胞外产物活性与致病性研究

采用琼脂扩散法测定了南方鲇源豚鼠气单胞菌胞外产物酶活性和溶血活性,同时对胞外产物的细胞毒性和其致病性进行了研究。结果显示:南方鲇源豚鼠气单胞菌胞外产物具有蛋白酶、脂酶、明胶酶和脲酶活性,但不具有淀粉酶和卵磷脂酶活性,具有很强的溶血活性和细胞毒性。肌肉注射感染发现,其对南方鲇有强致病性,其LD50为每千克鱼体重0.802 mg;注射后的南方鲇肌肉、心、肝、肾、脾、肠和胃等组织发生了严重组织病理变化,骨骼肌和心肌坏死断裂,炎症细胞浸润;肝脏严重空泡变性;肾小管上皮细胞变性、坏死、间质内大量炎症细胞浸润;脾充血、出血,淋巴细胞减少,胃肠黏膜上皮细胞变性、坏死、脱落。     

中华鳖致病性嗜水气单胞菌分离鉴定与药敏试验

为了探求中华鳖致病性菌株的药物体外抗菌效果,在江西进贤某中华鳖养殖场对病死中华鳖进行细菌分离,并对细菌进行形态特征、培养特性、生化鉴定、毒力因子检测试验、小白鼠毒力试验和人工感染等试验;采用K-B法药敏试验和96孔微型板二倍稀释法测定9种抗菌药物对该菌的最低抑菌浓度(MIC)来分析抗菌效果.结果表明:中华鳖为致病性嗜水...     

褶纹冠蚌防御素基因特征与表达

褶纹冠蚌(Cristaria plicata)样本壳长(9.50±1.05) cm、壳高(6.45±0.81) cm, 暂养1周。40只随机分成试验组和对照组, 每组20只。用Trizol 法提取经诱导刺激后0、6、12、24、36、48 h的血液、外套膜、鳃、肝胰腺组织RNA, 构建褶纹冠蚌cDNA文库并从中获得防御素基因cDNA全长序列。结果表明, 褶纹冠蚌防御素序列全长为514 bp, 其中5’ UTR 有57 bp; 3’ UTR 有242 bp, 含有一个poly(A)尾; 开放阅读框长度为192 bp, 编码63个氨基酸组成的蛋白质, 该蛋白N端为1个由23个氨基酸构成的信号肽, 成熟肽由40个氨基酸组成, 理论等电点为8.72, 分子量为7.13 kD。三级结构预测显示其由一个 α 螺旋和两个 β 折叠片组成, 形成典型的CSαβ结构。通过注射10⁹ cell/mL 的嗜水气单胞菌(Aeromonas hydrophila)刺激褶纹冠蚌, 半定量分析PCR对褶纹冠蚌血液、外套膜、鳃和肝胰腺组织的防御素基因表达, 结果表明, 诱导前防御素基因在外套膜组织中表达量最高, 约为血液和鳃的1.25倍, 肝胰腺次之。在诱导0、6、12、24、36、48 h后, 对照组的防御素基因表达量在血液和鳃中无明显变化, 而在外套膜和肝胰腺中则呈现先略有下降而后上升; 试验组防御素基因在血液中 12 h 时表达量最高, 随后略有下降, 而在外套膜、鳃和肝胰腺组织中表达量均在 0 h 时出现峰值, 而后呈现下降趋势; 外套膜和鳃的表达量分别在 24 h 和 36 h 略有上升。本研究通过分析褶纹冠蚌防御素基因全序列, 并利用嗜水气单胞菌对褶纹冠蚌进行注射感染, 探讨防御素基因的表达规律, 以期为防御素对贝类非特异性免疫的影响提供基础依据。