鲟源致病性嗜水气单胞菌X1的分离鉴定与药敏特性研究

从患细菌性败血症的西伯利亚鲟(Acipenser baerii)的体内分离到一株致病菌株X1,其对西伯利亚鲟的半数致死浓度(LC50)为5.62×105CFU/mL,具有较强毒力;菌株X1为革兰氏阴性杆菌,菌体大小为1.0μm～1.2μm×2.1μm～2.4 μm,周生侧鞭毛,在兔血琼脂平板上能形成明显的β-溶血圈,经ATB细菌鉴定仪鉴定和16S rDNA序列分析,菌株X1为嗜水气单胞菌(Aeromonashydrophila)(登录号:EU669667);其系统发育分析表明,菌株X1与嗜水气单胞菌ATCC35654(登录号:X74676.1)的亲缘关系最近,其同源性为99%.此外,菌株X1对先锋必、左氟沙星等2种药物高度敏感,对妥布霉素、氟哌酸、舒普深、卡那霉素、庆大霉素、复达欣、万古霉素、新霉素、多粘菌素B,洛美沙星等10种药物中度敏感.     

西伯利亚鲟源嗜水气单胞菌致病菌的分离及其全菌苗的免疫效果

从患细菌性败血症的西伯利亚鲟(Acipenser baerii)的体内分离到一株致病菌株X1,其对西伯利亚鲟的半数致死浓度(LC50)为5.62×105 cfu/ml,具有较强毒力;经ATB细菌鉴定仪生理生化鉴定和16SrDNA序列分析,菌株X1为嗜水气单胞菌(Aeromonas hydrophila);其系统发育分析表明,菌株X1与嗜水气单胞菌ATCC35654(登录号:X74676.1)的亲缘关系最近,其同源性为99%.用0.30%福尔马林灭活,将菌株X1制成灭活全菌苗,对西伯利亚鲟进行注射免疫.研究结果表明,嗜水气单胞菌X1全菌苗能够明显提高西伯利亚鲟的血清抗体水平及总蛋白、免疫球蛋白、溶菌酶含量,而且在嗜水气单胞菌X1全菌苗中加入弗氏不完全佐剂,有利于进一步增强西伯利亚鲟血清抗体水平及总蛋白、免疫球蛋白、溶菌酶含量.此外,嗜水气单胞菌X1全菌苗对西伯利亚鲟抗嗜水气单胞菌X1人工感染也具有较好的免疫保护作用,其对西伯利亚鲟的免疫保护率为50%,而且在嗜水气单胞菌X1全菌苗中加入弗氏不完全佐剂,嗜水气单胞菌X1全菌苗对西伯利亚鲟抗嗜水气单胞菌X1人工感染的免疫保护作用更好,其对西伯利亚鲟的免疫保护率为70%.因此,将嗜水气单胞菌X1全菌苗用于西伯利亚鲟细菌性败血症的防治具有广阔的发展前景.     

嗜水气单胞菌外膜蛋白基因ompTS的高效表达及其免疫原性

根据嗜水气单胞菌外膜蛋白基因ompTS的核苷酸序列设计引物,运用聚合酶链式反应（PCR）扩增出与预期大小相符的基因片段。将此基因片段克隆至质粒pRSET A的BamHI和EcoRI位点,构建重组质粒,转化大肠杆菌BL21（DE3）,经IPTG诱导获得高效表达,SDS－PAGE蛋白电泳表明在39．9kD处出现超强特异带,占总蛋白的51％。以Ni-NTA-Conjugate抗体进行Western blot分析证明该39．9kD的蛋白为所表达的融合蛋白。纯化融合蛋白注射雄性新西兰大白兔可诱导产生特异抗体。ELISA和Western blot检测结果显示,该抗体与表达的融合蛋白和从嗜水气单胞菌中提取的36．9kD外膜蛋白均呈阳性反应,表明所表达的融合蛋白仍保持原有外膜蛋白的免疫原性,为此融合蛋白作为 疫苗的候选成份提供理论基础。     

嗜水气单胞菌外膜蛋白W 基因的表达及其免疫原性分析

从患暴发性败血病的草鱼病灶处分离鉴定了嗜水气单胞菌(Aeromonas hydrophila)Wp3菌株。以其基因组DNA为模板扩增外膜蛋白W基因(OmpW),该基因全长为865 bp,开放式阅读框(ORF)为615 bp,与标准株ATCC7966的OmpW基因的同源性为99.8%。根据ORF序列设计引物扩增OmpW成熟肽编码序列并将其插入到表达载体pQE30中,转化大肠杆菌,经诱导可表达分子量为24.7 kD的带His标签的融合外膜蛋白His-W。用此融合蛋白免疫草鱼,所得草鱼血清经ELISA分析显示呈现阳性反应,说明重组蛋白能诱导产生抗体。采用实时荧光定量PCR分析草鱼头肾组织IgM基因表达水平的变化,结果显示免疫组IgM的表达量均明显高于空白组,其中低浓度免疫组(2μg/g)与空白对照组的差异显著(P<0.05),说明融合蛋白可使草鱼产生良好的免疫应答并上调抗体基因表达、产生高效抗体。保护性实验显示,不同免疫剂量均可使免疫组获得较高保护率(57%?86%)。结果显示,重组嗜水气单胞菌外膜蛋白W可作为草鱼嗜水气单胞菌基因工程亚单位疫苗。     

嗜水气单胞菌S蛋白的提纯及特性分析

电镜观察表明,嗜水气单胞菌J-1株具有S层结构,在菌体外层呈晶格样规则排列。菌体经酸性甘氨酸缓冲液处理,S层从菌体上脱落,离心上清液即为粗提S蛋白。进一步经Sephadex G200凝胶层析和DEAE-纤维素离子交换层析纯化,获得的S蛋白呈单一多肽,分子量为51500。氨基酸组分分析结果表明,S蛋白含有天门冬氨酸等15种氨基酸,其中丙氨酸等疏水性氨基酸占36.8％。生物学活性显示,S蛋白对Vero细胞有轻微的细胞毒性,但没有溶血性,对鲫鱼和小鼠也无致死作用。用自制的嗜水气单胞菌J-1株S蛋白抗血清PM及PR和国外提供的嗜水气单胞菌TF7株S蛋白抗血清PF1分别作免疫转印和间接ELISA,检测来源于不同地区和不同动物种类的20株嗜水气单胞菌的S蛋白。结果表明,S蛋白的抗原性存在着菌株间的差异。另外,某些菌株不具有S层。     

嗜水气单胞菌致病性研究进展

嗜水气单胞菌(Aeromonas hydrophila,AH)是我国养殖鱼类的重要病原,对其侵袭机制和毒力因子的研究有重要的意义。本文对嗜水气单胞菌的生物学特性、致病机制、毒力因子及其防治措施进行了综述。     

嗜水气单胞菌主要外膜蛋白对欧洲鳗鲡的免疫保护试验

制备出4株分属3个不同血清型嗜水气单胞菌的主要外膜蛋白(MOMP)免疫刺激复合物(ISCOMs)亚单位疫苗,腹腔注射免疫欧洲鳗鲡,ELISA法测得免疫后第14d和第30d所采集的血清的平均抗体效价在1∶320-1∶1280之间。免疫印迹结果显示,在印迹膜上对应的位置均可检测到MOMP的主要蛋白条带。第40d的免疫保护试验表明,免疫欧鳗对TPS-30菌株约100倍半数致死剂量的攻击无免疫保护作用,其中的3组被测免疫欧鳗对TPS-30菌株约10倍半数致死剂量的攻击的免疫保护率皆在80%以上。上述试验结果提示,嗜水气单胞菌的主要外膜蛋白是重要的保护性抗原,在制备成ISCOMs亚单位疫苗的情况下,较小的剂量免疫一次即可显示出较高的相对免疫保护率,且没有明显的血清型特异性。       

嗜水气单胞菌外毒素和外膜蛋白双基因融合表达载体的构建和高效表达

用基因融合方式 ,以嗜水气单胞菌基因组DNA为模板 ,设计引物通过聚合酶链式反应 (PCR)把去除部分毒性活性编码区的细胞毒肠毒素基因 (act)与去除信号肽的外膜蛋白基因 (OmpTS)连接一起 ,两基因之间插入一个link er(Gly4Ser) 3 经BamHⅠ和HindⅢ双酶切 ,得到 2 .1kb的双基因融合片段 ,克隆于表达质粒pQE 30中 ,构建了双基因重组表达载体pQE30 /act GS ompTS ,转化大肠杆菌M15 (pREP4 ) ,经IPTG诱导 ,表达出预期大小 (81.0kD)的融合蛋白Act GS OmpTS ,此蛋白占菌体总蛋白的 4 2 %。Westernblot检测结果显示 ,该蛋白与抗Act兔血清和抗OmpTS兔血清都呈阳性反应 ,表明融合蛋白保留了外毒素和外膜蛋白的反应原性 ,为进一步研究此融合蛋白作为疫苗候选成分提供了理论依据     

嗜水气单胞菌外膜蛋白基因ompTS的高效表达及其免疫原性

根据嗜水气单胞菌外膜蛋白基因ompTS的核苷酸序列设计引物,运用聚合酶链式反应（PCR）扩增出与预期大小相符的基因片段。将此基因片段克隆至质粒pRSET A的BamHI和EcoRI位点,构建重组质粒,转化大肠杆菌BL21（DE3）,经IPTG诱导获得高效表达,SDS-PAGE蛋白电泳表明在39.9kD处出现超强特异带,占总蛋白的51％。以 Ni-NTA-Conjugate抗体进行Western blot分析证明该399kD的蛋白为所表达的融合蛋白。纯化融合蛋白注射雄性新西兰大白兔可诱导产生特异抗体。ELISA和Western blot检测结果显示,该抗体与表达的融合蛋白和从嗜水气单胞菌中提取的36.9 kD外膜蛋白均呈阳性反应,表明所表达的融合蛋白仍保持原有外膜蛋白的免疫原性,为此融合蛋白作为疫苗的候选成份提供理论基础。     

西伯利亚鲟嗜水气单胞菌与其它3株气单胞菌间的免疫交叉反应

采用间接免疫荧光技术分析了西伯利亚鲟细菌性败血症致病菌嗜水气单胞菌(Aeromonas hydrophlia)X1菌株、豚鼠气单胞菌(Aeromonas caviae)XL2-T菌株、致病性温和气单胞菌(Aeromonas sobria)W1菌株与无致病性嗜水气单胞菌(Aeromonas hydrophlia)M3菌株等水产养殖主要病原菌与抗血清之间的免疫交叉反应。结果显示具有致病性的同属菌株X1菌株、XL2-T菌株、W1菌株交叉反应程度较大,说明这3株菌表面存在较多相同抗原决定簇。而无致病性菌株M3与其他3株致病性菌株免疫交叉反应程度较小。     

西伯利亚鲟(Acipenser baerii)致病性维氏气单胞菌的分离鉴定

摘要：【目的】本研究旨在寻找引起养殖西伯利亚鲟鱼(Acipenser baerii)病害的致病因子。【方法】从北京地区自然患病的西伯利亚鲟鱼体内分离到致病菌株X-1-06909,采用生理生化鉴定结合16S rRNA基因序列的系统发育学分析确定该菌株的系统发育地位。同时采用琼脂扩散法对抗菌类药物的敏感性进行测定。【结果】菌株X-1-06909与Aeromonas veronii ATCC 35624T的16S rRNA基因序列相似性达99.6％;结合形态特征与生理生化测定结果,革兰氏阴性杆菌,具极生单鞭毛     

O-Serogrouping and surface components of Aeromonas hydrophila and Aeromonas jandaei pathogenic for eels

Abstract The relationship between virulence, O-serogroup, and some cell-surface features (self-pelleting [SP] and precipitation after boiling [PAB], profile of lipopolysaccharides [LPSs]) and outer membrane proteins [OMPs] was investigated in strains of the pathogenic species Aeromonas hydrophila and A. jandaei isolated from eels. Virulent strains of A. hydrophila reacted mostly with O:19 antiserum, and those of A. jandaei reacted with O:4, O:11, O:15 and O:29 antisera (Guinée and Jansen system). Regarding the PAB and LPS profiles two groups could be distinguished; (i) five PAB⁺ strains of serotype O:19 that possessed a homogeneous O polysaccharide side chain and (ii) thirteen PAB⁻ strains antigenically diverse that either exhibited a heterogenous side chain or were side chain deficient. A major 50 kDa protein was only found in the PAB⁺ strains, whereas major OMPs detected in PAB⁻ strains ranged from 33 to 45 kDa irrespective of the species. Epizootic eel isolates of A. hydrophila belong to serotype O:19 and share cell-surface features with the Aeromonas highly virulent for other hosts. In contrast, epizootic A. jandaei isolates were antigenically diverse. These findings reinforce the importance of an O-serotype as an epidemiological marker in motile Aeromonas strains pathogenic for eels.     

Intraperitoneal injection urocortin-3 reduces the food intake of Siberian sturgeon (Acipenser baerii)

Urocortin-3 (UCN3), one of the corticotropin releasing factor (CRF) family peptides, which was discovered in 2001, has a variety of biological functions. However, the researches of UCN3 in fish were scarce. In order to understand whether UCN3 play a role in regulating food intake in fish, we first cloned the ucn3 cDNAs sequence of Siberian sturgeon (Acipenser baerii Brandt), and investigated the ucn3 mRNA levels in 11 tissues. The Siberian sturgeon ucn3 cDNA sequence was 1044 bp, including an open reading frame (ORF) of 447 bp that encoded 148 amino acids with a mature peptide of 40 amino acids, a 5ʹ-terminal untranslated region (5ʹ-UTR) of 162 bp and a 3ʹ-terminal untranslated region (3ʹ-UTR) of 435 bp. The result of tissue distribution showed that ucn3 widely distributed in 11 tissues with highest expression in brain. We also assessed the effects of periprandial (pre- and post-feeding), fasting and re-feeding on ucn3 mRNAs abundance in brain. The results showed the expression of ucn3 mRNA in brain was significantly elevated after feeding, decreased after fasting 17 days and increased after re-feeding. To further investigate the food intake role of UCN3 in Siberian sturgeon, we performed intraperitoneal (i.p.) injection of Siberian sturgeon UCN3 (SsUCN3) with three doses (60, 120 or 240 ng/g) and recorded the food intake. Acute and chronic i.p. injection SsUCN3 reduced the food intake in a dose-dependent pattern. In conclusion, this study indicates that SsUCN3 acts as a satiety factor to inhibit the food intake of Siberian sturgeon.     

The evidence of apelin has the bidirectional effects on feeding regulation in Siberian sturgeon (Acipenser baerii)

Apelin is a peptide, mainly produced in the brain, which participates in several physiologic effects. However, knowledge about the mechanism of appetite regulation in teleosts, including the role of apelin is not well understood. The aim of this study is to explore the effect of feeding status on the expression of apelin mRNA in the whole brain and the effects of injection of apelin on food intake in Siberian sturgeon (Acipenser baerii). In this study, we first cloned the apelin cDNA sequence of the Siberian sturgeon. We obtained a 1046-bp cDNA fragment, including a 237-bp open reading frame (ORF) that encoded 78 amino acids. Apelin was widely distributed in 11 tissues related to feeding regulation, with the highest expression in thewhole brain, followed by the spleen and trunk kidney. In addition, we measured the effects of periprandial (preprandial and postprandial) change, fasting and re-feeding on apelin mRNA expression in whole brain. The level of apelin mRNA was significantly decreased 1 h after feeding. The results of the fasting experiment showed that the expression of apelin mRNA in the brain was significantly reduced after 1 day of fasting but consistently increased throughout the 15-day food deprivation period. When the 15-day fasted fish were re-fed, apelin mRNA expression in the brain was significantly increased as compared to that of the control. These results suggest that apelin may play a bidirectional role in the regulation of food intake in the Siberian sturgeon. In order to further examine the effect of apelin on feeding regulation in Siberian sturgeons, acute and chronic intraperitoneal (i.p.) injection experiments were performed and food intakes were recorded. Results showed that acute i.p. injection of apelin-13 reduced food intake, however, chronic i.p. injection apelin-13 increased the food intake for 7 days in Siberian sturgeons. In conclusion, our results show that apelin has a bidirectional effect on feeding regulation in Siberian sturgeons by acting as a satiety factor in short-term feeding regulation and a starvation factor in long-term feeding regulation.     

Identification and expression profiling of receptor-interacting serine/threonine-protein kinase 2 in Siberian sturgeon (Acipenser baerii)

Receptor-interacting serine/threonine-protein kinase 2 (RIPK2) is an adaptor protein of the pattern recognition receptors NOD1 and NOD2 involved in regulating inflammatory response and resisting pathogenic microbial infection. In this study, Acipenser baerii RIPK2 (AbRIPK2) was identified. The open reading frame of AbRIPK2 was 1815 bp encoding 604 amino acids. AbRIPK2 possessed the typical N-terminal kinase domain (KD) and C-terminal caspase recruitment domain (CARD). The phylogenetic tree analysis revealed that AbRIPK2 shared a relatively high identity with bony fish. Real-time fluorescence quantitative PCR (qRT-PCR) results indicated that AbRIPK2 was highly expressed in the gill, followed by muscle, liver and heart. AbRIPK2 was significantly induced in the spleen and valvular intestine after Streptococcus iniae and Aeromonas hydrophila infection. AbRIPK2 was significantly upregulated after peptidoglycan (PGN) treatment in the splenic leukocytes. This study indicated that AbRIPK2 played a potential role in resisting the pathogenic infection of Siberian sturgeon by responding to bacteria.     

Morphology and ultrastructure of Siberian sturgeon (Acipenser baerii) spermatozoa using scanning and transmission electron microscopy

BACKGROUND INFORMATION: Available data concerning the sperm morphology of teleost fishes demonstrate wide variation. In the present study, the spermatozoa of Siberian sturgeon (Acipenser baerii Brandt, 1869), a chondrostean fish, was investigated. In contrast with teleost fish, chondrostean spermatozoa have a head with a distinct acrosome, whereas other structures, such as a midpiece and a single flagellum, are present in spermatozoa of most species. RESULTS: The average length of the head including the acrosome and the midpiece was 7.01+/-0.83 microm. Ten posterolateral projections derived from the acrosome were present on a subacrosomal region, with mean lengths of 0.94+/-0.15 microm and widths of 0.93+/-0.11 microm. The nucleus consisted of electrodense homogeneous nuclear chromatin. Three intertwining endonuclear canals, bound by membranes, traversed the nucleus longitudinally from the acrosomal end to the basal nuclear fossa region. There were between three and six mitochondria, two types of centrioles (proximal and distal) in the midpiece and two vacuoles composed of lipid droplets. The flagellum (44.75+/-4.93 microm in length), originating from the centriolar apparatus, had a typical 9+2 eukaryotic flagellar organization. In addition, there was an extracellular cytoplasm canal between the cytoplasmic sheath and the flagellum. CONCLUSIONS: A principal components analysis explained the individual morphological variation fairly well. Of the total accumulated variance, 41.45% was accounted for by parameters related to the head and midpiece of the sperm and the length of the flagellum. Comparing the present study with previous studies of morphology of sturgeon spermatozoa, there were large inter- or intra-specific differences that could be valuable taxonomically.     

嗜水气单胞菌HBNUAh01 外膜蛋白A 基因在烟草叶片细胞中的瞬时表达

【目的】从嗜水气单胞菌(Aeromonas hydrophila)HBNUAh01中克隆外膜蛋白A(outer membrane proteinA,ompA)基因并在烟草(Nicotiana tabacum)叶片细胞中瞬时表达该蛋白。【方法】以嗜水气单胞菌HBNUAh01为模板进行嗜水气单胞菌外膜蛋白A(AhompA)基因片段的PCR扩增,并将其克隆到pEASY-Blunt Simple载体中以进行测序。测序正确的AhompA基因序列与含有黄色荧光蛋白(yellow fluorescentprotein,YFP)基因的表达载体pCAMBIA1300构建重组表达载体。将该重组表达载体转化到农杆菌(Agrobacterium tumefaciens)GV3101感受态细胞中,随后用阳性转化子转染烟草叶片细胞。使用激光扫描共聚焦成像系统(Confocal Laser Scanning Microscope)检测观察融合表达AhompA基因的黄色荧光蛋白并采用RT-PCR检测AhompA基因在烟草叶片中的转录情况。【结果】从嗜水气单胞菌HBNUAh01中克隆出大小为1032 bp的AhompA基因序列,并在烟草叶片中成功表达AhompA和YFP的融合蛋白。【结论】AhompA基因在烟草叶片细胞中的成功表达为进一步研究利用植物疫苗防治嗜水气单胞菌引起的水产动物疾病奠定了基础。