月鳢败血病病原菌（气单胞菌）的研究

1997年在南宁和柳州两地发生了严重的月鳢败血病，通过病原菌的分离培养、人工感染试验和生理生化测定，确定嗜水气单胞菌和豚鼠气单胞菌为此败血病的病原菌。     

大西洋鲑杀鲑气单胞菌的分离鉴定

从皮肤溃烂的大西洋鲑(Salmon salar)肌肉、肝、肾分离到一致病性的菌株(AB080226),经人工感染实验证实其为该病的病原菌,研究了该菌的形态、生理生化特征并对其16SrDNA序列进行了在线Classifer和Blast分析。结果显示:该菌为革兰氏阴性杆菌,葡萄糖氧化发酵阳性,氧化酶检测阳性,0%NaCl生长;能利用甘露醇,不具运动性,能发酵麦芽糖,葡萄糖产酸阳性,精氨酸双水解酶阳性,苯丙氨酸脱氨酶阴性;该菌属于气单胞菌属的细菌,在系统发育树上与杀鲑气单胞菌形成一个簇群,同源性高达99.9%以上。综合形态学、生理生化特征及16SrDNA序列鉴定其为杀鲑气单胞菌(Aeromonas salmonicida)。     

月鳢败血病病原菌(气单胞菌)的研究

１９９７年在南宁和柳州两地发生了严重的月鳢败血病,通过病原菌的分离培养、人工感染试验和详细的生理生化测定,确定嗜水气单胞菌和豚鼠气单胞菌为此败血病的病原菌。     

患病草鱼肠道病原菌的分离鉴定及毒力研究

自环洞庭湖区精养鱼池患病草鱼肠道中分离获得4种病原菌,进行16S rRNA基因和gyrB基因序列测定和在线比对,并对草鱼进行回归感染。随后进行4种病原菌的药敏特征、溶血活性试验,并利用PCR方法检测了6种毒力因子的携带情况。试验结果表明,病原菌分别为温和气单胞菌、异常嗜糖气单胞菌、类志贺邻单胞菌和格氏乳球菌;4种病原菌回归感染草鱼,均能使草鱼患病致死,自死亡的草鱼体内均能分离得到相应感染的病原菌;4种病原菌对多种抗生素具有耐药性,且在脱纤维绵羊血血平板上检测到了温和气单胞菌、异常嗜糖气单胞菌AaB007的溶血活性;PCR结果表明,4种病原菌携带多种毒力因子。研究结果可为环洞庭湖区草鱼精养鱼池健康养殖及病害防治提供科学依据。     

一种新的对虾病原菌——雷氏普罗威登斯菌

本文从患败血病的中国对虾体内分离到多株细菌，其中三株经人工感染证实为病原菌，又经４０多项形态、生理、生化特性测试，鉴定为雷氏普罗威登斯菌（Ｐｒｏｖｉｄｅｎｃｉａｒｅｔｇｅｒｉ），其主要特性为杆状，革兰氏阴性，周生鞭毛，运动，无自然游动现象，兼性厌氧，苯丙氨酸脱氨酶、柠檬酸盐利用、吲哚、甲基红反应均阳性，发酵甘露醇、肌醇、甘露糖产酸。赖氨酸、鸟氨酸脱羧酶、精氨酸双水解酶、ＶＰ反应均阴性。生长最适温度、ＮａＣｌ、ｐＨ范围分别为２５℃—３０℃、０５％—１５％、７０—９０。对羧苄青霉素、先锋霉素５号、氯霉素、ＳＭＺ＋ＴＭＰ、氟哌酸、链霉素等极为敏感。雷氏普罗威登斯菌引起对虾败血病在国内外尚属首次报道。     

皱纹盘鲍幼鲍溃烂病病原菌的研究

从患有溃烂病的皱纹盘鲍幼鲍体上，分离到一种致病性细菌。经人工感染试验，证实该菌为皱纹盘鲍幼鲍溃烂病的病原菌。显微镜观察该细菌为革兰氏阴性，极生１～３根鞭毛，能运动的杆菌，大小为０．７～０．８×２．３～２．８ｕｍ。经细菌分类试验鉴定，该菌生长最适温度为２０～３０℃，最适ｐＨ值５．５～８．５。该菌在培养基Ｂ上产生蓝色的荧光素。能利用葡萄糖产酸不产气，果糖、Ｄ－半乳糖、甘露糖、木糖、阿拉伯糖、海藻糖发酵反应阳性，不能发酵乳糖、麦芽糖、鼠李糖、蔗糖，可分解山梨醇、肌醇、肌苷。过氧化氢酶、脲酶、氧化酶、及明胶液化反应阳性；能利用柠檬酸，不还原亚硝酸盐，不产生吲哚和硫化氢，ＭＲ和Ｖ．Ｐ．试验阴性。初步鉴定该病原菌为荧光假单胞杆菌。还进行了病原菌药敏试验，该菌对卡那霉素、呋喃唑酮、恶喹酸敏感，这些药物可作为防治该病的首选药。     

蟹源温和气单胞菌的分离及鉴定

从中华绒螯蟹中分离到一株温和气单胞菌，定名为CAS—l菌株，经常规菌种鉴定和SWF(A)菌鉴系统鉴定，主要特性为革兰氏阴性，具动力短杆菌，氧化酶、触酶阳性，发酵葡萄糖产酸产气，鸟氨酸脱羧酶、赖氨酸脱羧酶、精氨酸双水解酶、苯丙氨酸脱氨酶阴性，蔗糖、甘露酵、β—半乳糖苷酶反应阳性，肌醇、山梨醇、昆李糖、七叶苷反应阴性，菌株培养物县溶血性毒素。     

一株致病性异育银鲫源维氏气单胞菌的分离鉴定

从急性患病异育银鲫Carassius auratus gibelio肝胰脏中分离到一株优势菌。对该菌株进行了显微和超微结构观察、生理生化表型鉴定、16S rRNA和毒力基因分析及人工感染实验。结果显示:分离菌呈短杆状、两头钝圆、端生鞭毛,为革兰氏阴性菌;发酵葡萄糖产酸,氧化酶、触酶试验阳性,高盐浓度不生长;16S rRNA序列与气单胞菌属Aeromonas的基因相似性为98%以上,系统发育分析进一步表明该菌为维氏气单胞菌A.veronii,在该菌中可检测到肠毒素基因,且回接感染能导致健康银鲫发病,表明该菌具有致病性。     

水生动物的病原菌(上)

病原微生物又可称为病原菌,是指能入侵宿主引起感染的微生物,有病毒、细菌、真菌等。以下要对水产养殖中的病原细菌进行阐述。1.斑点气单胞菌特点:产气单胞菌属,革兰氏阴性菌,发酵型产酸。极端单鞭毛,逗点状,无芽孢,染色均匀。生存环境:存在于正常鱼的肠道、皮肤或水体、水下污泥中。     

大黄鱼源气单胞菌的分离鉴定及致腐表型

为鉴别大黄鱼腐败菌气单胞菌的致腐能力,采用生理生化和分子方法(16S r RNA)鉴定冷藏大黄鱼货架期终点不产H2S的菌株。分析菌株在体外培养条件下的生长、胞外酶活性、三甲胺(TMA)和生物胺形成的腐败表型,将菌株接种在灭菌鱼汁中测定其致腐能力。结果显示,分离株AE03和AE04在0~37°C及0~60的盐度下生长良好,在4和25°C条件下AE03菌株的生长速率显著高于AE04菌株。2株细菌不产H2S,能还原氧化三甲胺(TMAO),分解尿素和液化明胶,氧化酶、赖氨酸、鸟氨酸脱羧酶、精氨酸水解酶等都呈阳性,能利用葡萄糖。结合16S r RNA鉴定发现分离株AE03为杀鲑气单胞菌,AE04为软体动物气单胞菌。在体外培养中,2种气单胞菌均能产生蛋白酶、形成TMA和生物被膜,其中AE03菌株生成各指标的能力高于AE04菌株,同时AE03菌株还具有脂酶活性,产生生物胺,其中尸胺含量较高。气单胞菌AE03在灭菌鱼汁中产生的挥发性盐基氮(TVB-N)显著高于AE04。通过紫色杆菌CV026和根癌农杆菌A136 2种报告菌检测发现,2株气单胞菌是高丝氨酸内酯(AHLs)产生菌,其中AE03菌株产生的AHLs含量显著高于AE04菌株。研究表明,杀鲑气单胞菌AE03为冷藏大黄鱼的强致腐菌,且能分泌高含量的AHLs,研究为从群体感应角度阐明气单胞菌的致腐机理奠定了良好的基础。     

Siderophore pattern of fish-pathogenic Vibrio anguiliarum, Aeromonas spp. and Pseudomonas spp. from the German Baltic coast

Abstract A Total of 355 strains of fish-pathogenic bacteria, including Vibrio anguillarum, Aeromonas hydrophila, A. Caviae, Pseudomonas fluorescens and P. putida , were examined for the presence of hydroxamate-or phenolate-type siderophores. These strains were isolated from diseased rainbow trout, Oncorhynchus mykiss (Walbaum), from fish farms in the Baltic Sea off the coast of the former German Democratic Republic. The production of siderophores was demonstrated in bioassays and siderophore-pattern analysed using several indicator strains. All strains tested grew under conditions of iron limitation. The strains of Vibrionacease produced at least a hydorxamate-type siderophore detected by the bioassay with A. flavercens JG-9. Only a few V. anguillarum , but 50% of A. hydrophila produced a phenolate-type siderophore. Seventy percent of V. anguillarum , 33% if A. hydrophila , 40% of A. caviae , 53% of P. fluorescens and 50% of P. putida promoted the growth of the indication strain S. typhimurium SR 1001, which can use iron by 2,3-dihydroxybenxoic acid. The pseudomonads tested produced either hydroxamate- or phenolate-type siderophores, or both.     

Dominant pathogenic species of mesophilic aeromonads isolated from diseased and healthy fish cultured in Poland

Aeromonas isolates were collected from cultured fish, characterized phenotypically and identified to species using 16S rDNA. The pathogenicity of all isolates was assayed on the basis of haemolytic and proteolytic activity and challenge tests were performed for isolates from healthy fish. A total of 131 Aeromonas isolates were obtained and identified as follows: A. hydrophila (13), A. bestiarum (23), A. salmonicida (motile biogroup) (19), A. caviae (2), A. sobria (18), A. veronii bt. sobria (42), A. jandaei (1), A. encheleia (11) and A. allosaccharophila (2). All isolates of A. hydrophila and A. bestiarum and most isolates of A. salmonicida and A. veronii were classified as pathogenic. Aeromonas hydrophila was isolated only from diseased trout except for one isolate obtained from carp fry. The other potentially pathogenic Aeromonas species were present in diseased as well as healthy fish. The pathogenicity of isolates from healthy fish was correlated with their enzymatic activity and was also tested by challenge experiments. The dominant pathogenic species were A. veronii bt. sobria, A. bestiarum and A. salmonicida in common carp and A. hydrophila in rainbow trout.     

四株嗜水气单胞菌的比较鉴定

从鲫鱼肌肉和脏器先后分离到四株嗜水气单胞菌Ｄｙ、ｘＡ、ｘＡ５和ｘＡ６。与南京农业大学惠赠的嗜水气单胞菌Ｊ—１株进行了系统的比较鉴定。结果表明：１．五株细菌在培养特性、生化特性和药敏特性方面，除在分解水杨素和蔗糖能力以及新生霉素和新霉素敏感性等指标外，其他均基本一致；２．Ｄｙ、ｘＡ５和ｘＡ６同Ｊ—１一样能致死小白鼠和鲢鱼，而ｘＡ却不能；３．在与兔抗Ｊ—１抗血清的凝集反应中，Ｄｙ、ｘＡ５和ｘＡ６呈强阳性反应，而ｘＡ呈弱反应。这些结果表明，从不同地区分离的四株气单胞菌在致病性和抗原结构方面均存在差异。     

Production of the lethal acetylcholinesterase toxin by different Aeromonas hydrophila strains

Abstract. Seven fish pathogenic isolates of Aeromonas hydrophila , one A. sobria and one A. caviae were investigated for production of the fish lethal acetylcholinesterase toxin (AcChE-toxin). Western blotting was used for screening the ECP of these strains with a rabbit antiserum prepared against the purified toxin of strain B₃₂ and all the isolates (except A. sobria ) gave positive results with different patterns of bands. The AcChE-toxin appears to be secreted as a protein of high molecular weight which is stable at −20°C, and in 90% of the strains tested, it appears to be split into lower molecular weight fragments by the action of other components present in the ECP. The smallest, stable and highly active fragment has a MW of 15kDa.     

养殖鳗鲡致病性气单胞菌对抗生素药物的敏感性

以30种抗生素药物（包括12大类）研究32株鳗鲡致病性气单胞菌（包括嗜水、维氏、豚鼠和简达气单胞菌4种）对药物的敏感性.结果表明,多数致病性气单胞菌对庆大霉素、阿米卡星、头孢他啶、头孢噻肟、卡那霉素、洛美沙星、呋喃妥因、哌拉西林、左氧氟沙星、壮观霉素、链霉素、阿奇霉素、氟哌酸、头孢曲松、强力霉素和头孢呋辛等16种药物敏感;而多数菌株对新生霉素、头孢克罗、头孢拉定、头孢唑林、头孢噻吩、利福平、复方新诺明、氨苄西林、乙酰螺旋霉素、羧苄青霉素和青霉素等11种药物耐药.32株菌对β-内酰胺类、氨基糖苷类、氟喹诺酮类、喹诺酮类、四环素类和硝基呋喃等6类药物敏感,而对安沙霉素类、磺胺类和香豆素等3类药物耐药;除哌拉西林外,所有菌株对另外3种青霉素类药物耐药;致病菌株普遍对其中的1种大环内酯类（阿奇霉素）和4种头孢类（头孢呋辛、头孢曲松、头孢噻肟、头孢他啶）药物敏感,而对其它大环内酯类和头孢类产生了耐药性.研究发现仅维氏气单胞菌对头孢克罗、头孢拉定、头孢噻吩和头孢唑林敏感,而其余3种菌均耐药.     

致病性嗜水气单胞菌多重PCR检测方法的建立

致病性嗜水气单胞菌(Aeromonas hydrophila)是近年中国各地大规模流行的淡水养殖鱼类暴发性疾病的主要病原,本研究针对GenBank中登录的致病性嗜水气单胞菌的气溶素基因(hlyA)、溶血素基因(aerA)以及为气单胞菌属所特有的内参照基因16S rRNA保守区设计了3对特异性引物,通过进行多重PCR反应体系优化,多重PCR产物的测序鉴定与特异性和敏感性实验,试图建立一种检测致病性嗜水气单胞菌的多重PCR检测方法。对8株嗜水气单胞菌、16株相关菌株进行多重PCR检测,结果显示,非致病性分离株均未扩增出毒力基因hlyA和aerA,而致病性分离株则至少含有hlyA基因;对40份送检的水产动物样品进行多重PCR检测,结果与常规微生物学检测符合率为97.5%。多重PCR检测方法具有较高的敏感性与特异性,最低可检测模板量为10 ng的样品。该方法的建立对水产动物嗜水气单胞菌病的快速诊断和分子流行病学的调查有重要意义。     

Interaction of the fish pathogen Aeromonas hydrophila with tilapia, Oreochromis aureus (Steindachner), phagocytes

Abstract. Interaction of Aeromonas hydrophila and tilapia, Oreochromis aureus (Steindachner), phagocytes was studied in vitro. All virulent and avirulent strains of A. hydrophila tested could multiply in non-activated and Freund's complete adjuvant activated phagocytes. Activated phagocytes increased the uptake of bacteria into cells, and the rates of intracellular replication for these bacteria were faster than in non-activated phagocytes. Among the A. hydrophila strains examined, virulent strain PPD134/91 replicated at the fastest rate inside phagocytic cells and produced cytopathic effect in the phagocytes in the shortest incubation time. Opsonized avirulent A. hydrophila were sensitive to phagocyte-mediated killing or unable to grow in phagocytes. Serum components and phagocytes may together prevent the growth of avirulent A. hydrophila in fish. The release of extracellular oxygen radicals during phagocytosis was examined using chemiluminescence assay (CL). Virulent strains induced CL responses but avirulent strains did not. This suggests that the virulent strains interacted with the phagocytes somewhat differently from the avirulent strains.     

Immunodiagnosis of virulent strains of Aeromonas hydrophila associated with epizootic ulcerative syndrome (EUS) using a monoclonal antibody

Abstract. A virulent strain of Aeromonas hydrophila associated with epizootic ulcerative syndrome (EUS) was used to produce monoclonal antibodies that identified virulent strains of A. hydrophila. Antibodies from a clone, designated as F26P5C8, were found to identify the A. hydrophila serotype I isolates associated with EUS fish, and which were found to be virulent after subsequent inoculation studies. Immunodiagnosis of a large number of A. hydrophila from Australia and Japan showed some additional isolates to be identified by F26P5C8, but the status of their virulence is presently unknown.     

三种鲫鱼对暴发性鱼病的抗病力

本文报道彭泽鲫、异育银鲫及白鲫对嗜水气单胞菌引起的暴发性鱼病抗病力的研究结果。在菌液浓度为6.0×10~6个/ml,注射剂量为0.3ml/尾时,半数致死量(LD_(50))分别为彭泽鲫 10~(-1.?),异育银鲫 10~(-1.61),白鲫 10~(-2.21),差异极显著(P<0.01)。从三个方面进一步研究了造成以上差异的机理:(1)补体总量——彭泽鲫 6.40 单位/ml,异育银鲫 5.97 单位/ml,白鲫 2.20 单位/ml,差异极显著(P<0.01);(2)补体替代途径(C_d 旁路)杀菌能力——彭泽鲫和异育银鲫极显著优于白鲫(均值多重分析 D>D_(0.01)),彭泽鲫好于异育银鲫,但差异不显著;(3)白细胞吞噬功能——三种鲫鱼间存在极显著差异(P<.01),吞噬百分率是彭泽鲫极显著优于白鲫和异育银鲫(D> D_(0.01)),吞噬指数是彭泽鲫和异育银鲫显著优于白鲫(D>D_(0.05))。彭泽鲫是对暴发性鱼病抗病力较强的品种,白鲫是对暴发性鱼病抗病力最差的品种。     

6株鱼源嗜水气单胞菌的分子鉴定与毒力基因分析检测

为了分子鉴定6株鱼源嗜水气单胞菌,并从分子层面验证通过检测毒力基因以推测嗜水气单胞菌潜在致病性的可行性。实验采用PCR扩增16 S rDNA和gyrB基因并结合系统发育树的构建和分析进行菌种的分子鉴定,检测气溶素（ aerolysin, aer）、溶血素（ haemoly-sin, hly）、丝氨酸蛋白酶（ serine protease, ahp）、热稳定细胞肠毒素（ heat-stable cytotonic enterotoxin, ast）和热敏感细胞肠毒素（ heat-labile cytotonic enterotoxin, alt）5种毒力基因,且使用Mega 5．2对核苷酸和氨基酸序列进行分析。结果显示6株菌均为嗜水气单胞菌Aero-monas hydrophila,检测出5种毒力基因中的至少4种,其中均检测出溶血素和2种肠毒素,序列分析表明气溶素、溶血素和丝氨酸蛋白酶的氨基酸序列高度保守。本研究基于16 S rD-NA和gyrB基因可以准确地对嗜水气单胞菌进行分子鉴定,6株菌的毒力基因丰富预示着一定的致病性, aer、 hly和ahp基因相对保守,编码的毒力因子高度同源,在临床分子诊断中建议使用aer、 ahp和hly基因对嗜水气单胞菌的潜在致病性进行检测。