罗非鱼无乳链球菌的分子鉴定

对罗非鱼致病菌株TL60829NA及其人工感染后分离菌株TL60829NA1、TL60829NA2应用原核生物16S rRNA基因通用引物进行分子分类学鉴定.对这些菌株进行16srRNA基因的克隆、序列分析,核酸序列同源性分析表明,TL60829NA及其人工感染后分离菌株TL60829NA1、TL60829NA2为同一种细菌.其中,TL60829NA2与GenBank登陆号为DQ303183的无乳链球菌(Streptococcus agalaciate)菌株ATCC13813序列同源性最高(99.8%).同时,通过与常引起罗非鱼链球菌病的S.agalaciate、S.iniae代表菌株16srRNA基因构建的发育进化树表明,该菌株及其人工感染后分离菌株与S.agalaciate代表菌株构成一个进化分枝,而4株S.iniae代表菌株的16srRNA基因则构成另一个分枝.本研究证实了从发病罗非鱼肝脏组织中分离到的致病性链球菌为无乳链球菌.     

罗非鱼海豚链球菌16srRNA基因的序列测定和系统进化分析

为了从分子水平上对～株来源于发病罗非鱼的链球菌进行分类学鉴定，本研究利用原核生物16srRNA基因通用引物对该菌株进行了16srRNA基因的克隆及序列分析。结果扩增出长约1．5kbp目的片段，测序得到一条长度为1447bp核苷酸序列。核苷酸同源性分析表明，该序列与NCBI公布的海豚链球菌（Streptococcus iniae，s．iniae）SCCF5L菌株16srRNA基因核苷酸序列同源性最高（99．496），暂称为中国广西株（S．iniae-CGX）。同时，亲源关系较近的S．iniae、S．difficile和S，agalaciate代表菌株构建的系统发育进化树显示，该菌株与S．iniae代表菌株组成同一进化分支，与s．agalaciate代表菌株组成的另一进化分支距离较近（95．596），而与S．difficile代表菌株组成的进化分支距离较远（92．3％）。本研究证实，从发病罗非鱼脑组织分离得到的致病性链球菌为海豚链球菌。     

罗非鱼海豚链球菌16S rRNA基因的序列测定和系统进化分析

为了从分子水平上对1株致病性罗非鱼链球菌进行分类学鉴定,利用原核生物16SrRNA基因通用引物对分离纯化的罗非鱼致病性链球菌进行16S rRNA基因的克隆及序列分析。结果扩增出长约1.5 kp目的片段,测序得到1条长度为1 447 bp核苷酸序列。核苷酸相似性分析表明,序列与NCB I公布的海豚链球菌(Streptococcus iniae,S.iniae)SCCF5L菌株16SrRNA基因核苷酸序列相似性最高(99.4%),暂称为中国广西株(S.iniae-CGX)。同时,亲源关系较近的S.iniae、S.difficilis和S.agalactiae代表菌株构建的系统发育进化树显示,所得菌株与S.iniae代表菌株组成同一进化分支,与S.agalactiae代表菌株组成的另一进化分支距离较近(95.5%),而与S.difficilis代表菌株组成的进化分支距离较远(92.3%)。上述研究证实,本试验从发病罗非鱼脑组织分离到的致病性链球菌为海豚链球菌。     

西伯利亚鲟海豚链球菌和鲟疱疹病毒Ⅱ型混合感染的诊断与病理损伤

2018年8月,四川彭州与邛崃的养殖西伯利亚鲟发生一种以出血为临床特征,高死亡率的传染病,为明确其病因,本研究对发病鲟的肝脏、肾脏进行病原菌分离、鉴定和鲟疱疹病毒Ⅱ型(AciHV-2)的PCR检测。从患病鲟体内分离到2株G+链状球菌,其16S rRNA序列(MN416231、MN416230)与GenBank中海豚链球菌16S rRNA序列同源性达99%,在以16S rRNA序列构建的系统发育树上,分离菌与海豚链球菌聚为一支;同时基于海豚链球菌lctO基因的特异性PCR检测为阳性,鉴定2株分离菌为海豚链球菌。提取患病鲟肝脏、肾脏组织DNA进行AciHV-2特异性PCR检测,扩增出501 bp的特异性条带,在以AciHV-2 polymerase基因序列构建的系统发育树上,检测样本与AciHV-2聚为一支。病理组织学上,患病鲟的多组织器官发生明显损伤,尤其是肝脏、肾脏、鳃、脾脏和肠的损伤较为严重,表现为明显的变性、坏死、出血以及炎症细胞浸润;电镜下,观察到肝脏、肾脏组织内大量直径200～220 nm的疱疹病毒样颗粒与0.7～0.8μm的链球菌入侵细胞,并导致细胞损伤。综上,诊断患病西伯利亚鲟的病因是海豚链球菌与AciHV-2混合感染。     

南方鲇溃疡综合症病原菌的分离与鉴定

从南方鲇(Silurus meriordinalis Chen)病鱼体表溃疡部及内脏分离出细菌12株,经人工感染证实其中6株治病,生化鉴定6株为同一种菌即为引起该鱼溃疡综合症的病原菌,命名为DKN-1。该菌为革兰氏染色阴性,兼性厌氧发酵型短杆菌,极生单鞭毛,对除葡萄糖、麦芽糖、甘露糖、海藻糖、甘露醇、水杨苷、七叶苷以外多种糖不利用,氧化酶、过氧化氢酶、DNA酶、脂酶、蛋白酶、精氨酸双水解酶阳性,MR、VP阳性。在以该菌16S rDNA序列和GenBank及RDP数据库内同源性较高的细菌16S rDNA序列构建的系统发育树中,DKN-1与豚鼠气单胞菌(Aeromonas caviae)同源性达到99.6%,综合该菌在形态、生理生化的结果,鉴定其为豚鼠气单胞菌。该病病原对美满霉素、大观霉素、妥布霉素高度敏感。     

一株引起香鱼出血症的嗜水气单胞菌的鉴定

从发生出血症的香鱼体腔液分离到一高致病性的菌株(ayu-ah0201),经人工侵染试验证实该菌能引起香鱼出血症.对该菌的形态、生理生化及16S rDNA序列分析结果表明,其为革兰氏阴性,短杆状,极生鞭毛.对蔗糖、阿拉伯糖、七叶苷、氧化酶、精氨酸双水解酶、赖氨酸脱羧酶呈阳性,鸟氨酸脱羧酶呈阴性.可利用葡萄糖产酸产气.在以该菌16S rDNA序列和GenBank数据库内同源性较高的细菌16S rDNA序列构建的系统发育树,表明分离菌ayu-ah0201与嗜水气单胞菌聚在一簇,与嗜水气单胞菌核苷酸序列同源性最高,为99.6%～99.7%,与其他相关气单胞菌属成员的核苷酸序列同源性小于98.9%.结合形态和生理生化特点将其鉴定为嗜水气单胞菌.     

广东省养殖罗非鱼、海鲈、尖吻鲈海豚链球菌感染调查

利用细菌分离培养方法结合特异PCR技术,对广东省珠三角地区养殖罗非鱼(Oreochromis spp.)、海鲈(Lateolabrax japonicus)及尖吻鲈(Lates calcarifer)的海豚链球菌(Streptococcus iniae)感染情况进行了周年调查。每月固定时间在特定养殖区域采集目标鱼的脑、肝、脾、肾和肌肉等组织,并对其进行海豚链球菌的细菌分离培养鉴定。仅从已经患病的尖吻鲈中分离到3株链球菌,经生理生化鉴定和16S rDNA测序确定为海豚链球菌。利用海豚链球菌特异PCR技术对上述养殖鱼类不同组织进行检测,发现罗非鱼、海鲈、尖吻鲈的海豚链球菌感染率分别为30.21%、23.53%、14.55%,其中罗非鱼脑和肌肉的感染率明显较其他组织高(P<0.05),分别为20.65%和23.75%;海鲈的脑部和肌肉感染率也较其他组织高(P<0.05),分别为12.1%和10%;而尖吻鲈各组织感染率没有较大差异(P>0.05)。另外,研究结果还表明采集样本的海豚链球菌感染率随着其体长的增加而呈现下降趋势。     

西伯利亚鲟停乳链球菌的分离、鉴定与致病性

从患暴发性流行病的两伯利亚鲟(Acipenser baerii)肝脏和心脏中各分离到1株细菌,分离纯化后获得2个分离株,编号分别为AeBF070904、AbHT070912,对分离菌进行了菌株鉴定、致病性分析及药敏实验.分别应用常规生理生化鉴定、全自动细菌测定卡API 20 STREP和ID 32STREP进行检测,结果表明,2个分离株均为停乳链球菌(Streptococcus dysgalactiae).对2个分离株的16S rRNA基因进行PCR扩增和测序,并与GenBank中收录的链球菌16S rRNA基因进行序列分析并构建系统进化树,结果显示,2个分离株的16S rRNA基因序列相同,与停乳链球菌同源性最高达97.3%,在系统进化树上与停乳链球菌聚为一簇,进一步确认2个分离株均为停乳链球菌.从人工感染后发病鱼的内脏组织再分离的细菌特性与原感染菌相同,确认停乳链球菌是西伯利亚鲟的致病菌.2个分离株对两伯利亚鲟、杂交鲟及剑尾鱼均有致死毒性,37℃培养的细菌毒力比28℃培养的细菌毒力强.2个分离株均对青霉素、诺氟沙星等7种药物敏感;对头孢唑啉、庆大霉素等2种药物耐受;对红霉素巾等敏感;对卡那霉素等8种药物菌株之间出现差异.     

患刺激隐核虫病的真鲷感染迟钝爱德华氏菌的分离与鉴定

2012年8月,福清市东瀚海区网箱养殖真鲷发生大量死亡事件.病鱼除刺激隐核虫病症状外,还出现脾、肾肿大,肝脏充血等.采用生理生化指标鉴定及16S rDNA系列同源性分析和系统发育树构建等方法,对从病鱼肝脏分离获得的优势菌株进行了鉴定,其生理生化指标符合迟钝爱德华氏菌特点,GenBank中同源序列比对结果显示,该菌与迟钝爱德华氏菌的16S rDNA序列同源性高达99％,系统进化树中与迟钝爱德华氏菌自然聚为一支.药敏实验结果表明,该菌对氟苯尼考等10种药物敏感,对链霉素等4种药物中等敏感.     

养殖澳洲宝石鱼迟缓爱德华氏菌的分离鉴定及致病基因的检测

从患病澳洲宝石鱼体内分离到一株致病菌(编号Et4),对该菌的生化特性及致病基因进行检测,并进行了药敏和人工感染实验。结果显示:菌株Et4的生化鉴定结果与迟缓爱德华氏菌(Edwardsiella tarda)标准菌株(AY77513)一致;其16S rRNA序列,经同源性比对与迟缓爱德华氏菌核苷酸相似度最高,达99%;根据迟缓爱德华氏菌的致病基因Ⅲ型分泌系统装置蛋白esaV基因序列设计引物,进行PCR扩增,得到708 bp序列,该序列与迟缓爱德华氏菌的esaV基因序列相似性达99.3%,说明菌株Et4具有esaV基因。菌株Et4对环丙沙星等较敏感,对其它药物中度或不敏感,具有较强的致病力(LD50=3.74×104CFU/mL),从病鱼中可以重新分离出此菌。综合形态学、生化特性、16S rDNA序列及致病基因序列鉴定其为迟缓爱德华氏菌。     

Outbreaks of Streptococcosis associated with Streptococcus iniae in Siberian sturgeon (Acipenser baerii) in China

Streptococcus iniae has emerged as an important fish pathogen over the past few decades causing high losses in aquaculture farms all over the world. At least 27 species of fish have been documented to be infected by S. iniae, including cultured and wild populations. In August and October 2013, a serious infectious disease characterized by body ulcer, internal organs haemorrhages and nodules showing on epicardium occurred on the Acipenser baerii farms in Ya'an country, China. Histological examination revealed a multisystemic, necrotising inflammatory response that was particularly marked in liver, kidney, heart and brain. Mass mortality (>40%) was observed in infected fish and two Gram‐positive cocci (Ab130920 and Ab131025) were obtained from kidneys and livers of diseased fish. Experimental infections with these two isolates resulted in marked symptoms in the sturgeons similar to those observed in natural outbreaks, and the LD₅₀ values of the two isolates were 5.1 × 10⁵ and 6.4 × 10⁵ cfu per fish respectively. The two microorganisms were identified as S. iniae through physiological and biochemical tests, 16S rRNA and lctO gene sequence analysis. Both two isolates showed a similar antibiotic susceptibility, which were sensitive to ampicillin, amoxicillin, cefazolin, amikacin, deoxycycline, florfenicol, azithromycin, ciprofloxacin, vancomycin and resistant to streptomycin, gentamicin, kanamycin, norfloxacin and sinomin (SMZ/TMP). Multiplex PCR assay for virulence genes showed both isolates possessed six main virulence genes: simA, scpI, pdi, pgm, cpsD and sagA genes. These results indicated that S. iniae could act as a pathogen of farmed A. baerii. This is the first report of S. iniae infection associated with mass mortality in A. baerii.     

Identification and phylogenetic analysis of Vibrio vulnificus isolated from diseased Trachinotus ovatus in cage mariculture

In October 2005, an infectious disease occurred as outbreaks of high mortality within one week, which was responsible for important economic losses in intensive culture of Trachinotus ovatus around the Gulf Coast of Yangjiang city (Guangdong Province, China). The present study documented the causative agent of the diseased fish suffering from external haemorrhages and ulcers, haemorrhagic gills, livers and intestine. The strain was isolated from the diseased fish by ZoBell 2216 E agar and confirmed the pathogenicity by challenge experiments. Biochemically, the strain showed properties and biochemical characteristics similar to Vibrio vulnificus, with the exception of several atypical biochemical characteristics, especially the sucrose-positive. Meanwhile, sequencing of the 16S rRNA gene and the hemolysin gene revealed that the isolated strain was highly homogeneous with V. vulnificus. To sum up, the isolated strain was confirmed as V. vulnificus on the basis of phenotypic characteristics, phylogenetic analysis of the 16S rRNA sequences and sequence analysis of the hemolysin gene. To our knowledge, this is the first report on the V. vulnificus infection in T. ovatus.     

吉富罗非鱼与奥利亚罗非鱼自繁与杂交 F1遗传特性与抗病力分析

以吉富罗非鱼与奥利亚罗非鱼为繁育亲本,采用完全双列杂交繁育4组F1,将初始规格基本一致的4组罗非鱼饲养100 d后,运用“加性-显性”遗传分析模型,分析了4组F1罗非鱼8个生长相关性状杂种优势、遗传效应以及与性状间的相关性.结果表明:(1)F1群体平均优势为0.129 4～0.368 4.除尾柄长超亲优势较大外,其他性状的群体超亲优势较小或表现出负向超亲优势.(2)8个性状的广义遗传率(HB)为0.714 2～0.995 3,表明加性效应和显性效应对性状的遗传变异影响极显著(P＜0.01).除尾柄长外,其他性状的狭义遗传率(HN)介于0.469 4～0.737 9,表明加性遗传方差在表型方差中所占比率较高.(3)体质量、全长、体长、体高、体宽、头长、尾柄长、尾柄高性状之间表型相关在0.776 6～0.999 7范围内,而遗传相关在0.994 1～1.000 0之间,表明这些性状间都存在极显著的正相关.取样结束后,采用3.95×106 CFU/mL的海豚链球菌菌液进行腹腔感染,吉富罗非鱼自繁组F1代12 h后出现死亡,而奥利亚罗非鱼自繁组F1192 h后才出现死亡.384 h后,吉富罗非鱼自繁组F1死亡率为40％,正反交组F1分别为20％和23.3％,奥利亚罗非鱼自繁组F1死亡率最低,为6.67％.研究结果表明,除尾柄长外,杂交F1的其他性状不具备超亲优势,然而杂交可以提高选育后代的抗病力.     

水族箱气单胞菌的鉴定及致病特性分析

为了确定南京市某渔场发病鱼感染的病原,本研究采集患鱼脏器,采用平板培养、生化实验和特异性gyrB基因扩增测序等方法进行细菌的分离鉴定;利用PCR技术检测分离株毒力基因的分布,分析其生物学特性,并进一步通过动物实验确定菌株致病力.结果分离到1株水族箱气单胞菌,命名为LK-25.该菌株携带5种主要毒力基因:气溶素(aer)、细胞毒性肠毒素(act)、细胞兴奋性肠毒素(alt)、温敏胞外蛋白酶(epr)和丝氨酸蛋白酶(ahp),其溶血性和溶蛋白能力较强,对斑马鱼的半数致死量为1.02×103 CFU/尾,确定为强毒株.进化树分析表明,水族箱气单胞菌与嗜水气单胞菌达卡亚种亲缘关系较近.本研究在国内首次报道发现水族箱气单胞菌,为进一步预防该菌所引起的相关疾病的发生和传播提供理论基础.     

Comparison of diagnosis techniques for the protozoan parasite Bonamia ostreae in flat oyster Ostrea edulis

In recent years, the cultured production of the flat oyster Ostrea edulis has suffered a dramatic decrease in Europe partially attributed to the protozoan parasite Bonamia ostreae, the causative agent of the Bonamiosis. In this paper the results of a PCR assay for the diagnosis of B. ostreae were compared with those obtained using two classical methods of diagnosis recommended by the OIE (Office International des Epizooties) and the European Union, histology and cytology. The same samples were analyzed by two different laboratories, showing that the results obtained with the PCR method have high sensitivity and good correlation between laboratories. This method is cheaper and faster than histopathology and cytology, with no need of specifically trained personnel to perform the diagnoses. It is appropriate for fast screening of stocks of great numbers of oysters.     

Do strain differences in microalgae alter their relative quality as a food for the rotifer Brachionus plicatilis?

Brachionus plicatilis is used in aquaculture to feed larval fish and crustaceans. It is well established that different prey species alter rotifer productivity. Isochrysis galbana is one microalgal prey that is commonly fed to rotifers, and there are several strains of this flagellate available to aquaculturists. As microalgae strains may differ in their composition and growth attributes, we rigorously examined if growth and biochemical differences in I. galbana strains elicit differences in the growth and biochemical attributes of B. plicatilis. Four I. galbana strains and one strain of the flagellate Nanochloropsis were grown under standard conditions. Growth rate, cell volume, production, and composition (dry weight, carbohydrate, protein, lipid) were measured. Significant differences occurred between strains in all of these attributes (at times 2 to 3 fold), but no clear pattern emerged that one strain was superior. Of note was that for some measurements, strain differences were significantly greater than differences between species. The strains were then fed to rotifers, and a number of parameters were measured: growth rate, reproductive rate, fecundity attributes, a number of developmental rates, and composition (dry weight, carbohydrate, protein, lipid). There were significant effects of prey strain on some of these attributes, but none was dramatic (rarely more than 10% and occasionally up to 30%), suggesting that aquaculturists need not be too concerned regarding which I. galbana strain they use. However, we do indicate subtle differences, induced by different prey strains and suggest that for maximum productivity these differences should be considered.     

拟态弧菌OmpU蛋白的黏附功能及所介导的致病作用

为了探明拟态弧菌OmpU蛋白的黏附功能,利用同源重组技术敲除基因组中OmpU基因,并构建其互补株,再经组合PCR方法和序列测定,证实了OmpU基因的缺失和互补。对野生株、缺失株和互补株进行了遗传稳定性、生长特性、生化特性、细胞黏附性、致病性等方面比较研究。结果显示,缺失株具有遗传稳定性;在相同的培养条件下,与野生株相比,突变株的培养特性和生化特性没有明显变化,生长速率略减慢,对实验草鱼的毒力降低了4倍,对鲤上皮瘤细胞(EPC)的黏附能力显著降低,下降了66.6%,而互补株的黏附能力和毒力又得到恢复,与野生株无明显差异。研究首次确证了拟态弧菌OmpU蛋白具有黏附功能,OmpU蛋白通过黏附参与致病作用。     

Susceptibility by amberjack (Seriola dumerili), yellowtail (S. quinqueradiata) and Japanese flounder (Paralichthys olivaceus) to Neobenedenia girellae (Monogenea) infection and their acquired protection

This study investigated: 1) susceptibility differences to infection by Neobenedenia girellae (Capsalidae) between amberjack Seriola dumerili (Carangidae), yellowtail S. quinqueradiata and Japanese flounder Paralichthys olivaceus (Paralichthyidae); 2) growth and egg production of N. girellae on each fish species; 3) acquired protection of each fish species against this parasite. The number of N. girellae on S. dumerili was significantly higher than on S. quinqueradiata and P. olivaceus when these fishes were exposed to oncomiracidia in the same aquarium. Neobenedenia girellae growth on S. dumerili was fastest and, thus the number of eggs laid by parasites on S. dumerili was greater than on the other two species. Seriola dumerili and P. olivaceus, which were previously infected with N. girellae and treated by freshwater bath, acquired partial protection against re-infection by N. girellae. The relative re-infection of three S. dumerili individuals out of eleven individuals was markedly low compared with the initial infection, and the relative initial infection and re-infection on two P. olivaceus out of eleven individuals was markedly low. The results of this study could be useful to control N. girellae infections when cultivating S. dumerili, S. quinqueradiata and P. olivaceus.     

中华绒螯蟹蜕皮激素受体基因（Ers-EcR）的克隆和组织表达分析

蜕皮激素受体(ecdysteroid receptor,EcR)介导调控甲壳动物蜕皮生长、附肢再生等重要生命活动。为了解EcR在人工控制甲壳动物的繁殖和生长中的作用,采用RACE方法结合同源克隆技术,首次从中华绒螯蟹Y-器官中克隆得到蜕皮激素受体基因全长cDNA序列(Ers-EcR,登录号:KF736985),并进行了结构解析和组织表达分析。结果发现,Ers-EcR编码基因全长2 176 bp,开放阅读框为1 638 bp,编码545个氨基酸,具有DNA结合域(DBD)和配体结合域(LBD)等典型的核受体超家族结构域,但不具有信号肽结构。其中,DBD含有8个保守的Cys残基,可以形成2个锌指结构(C156-C159-C173-C176、C192-C198-C208-C211),是典型的DBD特征。多重序列比对分析表明,Ers-EcR氨基酸序列与拳手招潮蟹同源性最高,达到91%。荧光定量PCR结果显示成体中华绒螯蟹ErsEcR基因在Y-器官和肌肉组织中表达量最高,在血液、肠道、卵巢、眼柄、心脏和肝胰腺中有一定表达,在鳃、胸神经节和精巢表达量较低。这表明Ers-EcR基因在中华绒螯蟹各组织器官中的表达不具有典型的特异性,提示Ers-EcR基因可能参与体内多种生命活动的调控。