弗氏柠檬酸杆菌对河触致病性的研究

首次报道了弗氏柠檬酸杆菌对河蟹的病原性、并导致其病理变化,药对物该菌的敏感试验及生产中药物治疗效果。从垂死的病蟹肝胰脏中分离得到可疑病菌,经人工感染试验证实此菌导致健康蟹发病死亡。该菌可导致河触的肝胰腺、鳃等发生病理变化,最终引起败血症而死亡。通过16种药物对该菌的敏感试验,筛选出有效药物,应用于生产中取得了较好疗效。     

鸡大肠杆菌病病原的分离鉴定和部分生物学特性研究

无菌采取某鸡场送检的疑似大肠杆菌病的病、死鸡的心血、肝、脾、肾,进行病原菌的分离、生化鉴定、动物实验和药敏试验。生化鉴定结果显示引起该鸡场鸡只发病的病原为大肠杆菌,并且该菌对小白鼠和鸡有较强的致病力。药物敏感性试验结果显示,该菌对先锋V、头孢呋肟、头孢噻肟敏感,对庆大霉素和氧氟沙星不敏感,而链霉素、新诺明、红霉素、青霉素、氨苄西林、四环素对它完全没有抑制效果。结果表明:该鸡场出现了耐药的致病性大肠杆菌,在以后的肉鸡生产中应慎用抗生素,在使用药物治疗大肠杆菌病时,应根据药敏试验结果,选择敏感药物,且应注意交替用药,按疗程投药。     

某林麝场林麝感染假结核耶尔森菌的诊治

为寻求四川宝兴某家养林麝死亡的原因及控制措施,采集一例病死林麝各组织器官进行病理切片观察和细菌分离,对分离到的病菌进行形态学观察、16S rDNA序列分析、小鼠致死试验、生化鉴定及药敏试验。结果显示,切片中各组织含有大量细菌团;从肠系膜淋巴结、肝、脾、肾和肺中只分离到1株病菌;16S rDNA测序分析发现该菌与耶尔森菌16S基因的相似性高达100%,生化及鉴别试验最终判定为假结核耶尔森菌。该菌对头孢噻肟、头孢西丁、丁胺卡那霉素等药物敏感。结果证实,此次林麝死亡的原因为假结核耶尔森菌引起的败血症,并及时应用敏感抗生素控制了病情。     

凹甲陆龟沙门氏菌的分离鉴定及毒力因子基因的PCR检测

从成都动物园因腹泻死亡的凹甲陆龟体内分离到一株病源菌,经选择性分离培养、生化试验、血清型鉴定等,确定该病源菌为D群肠炎沙门氏菌,其抗原结构式为1,9,12∶ g,m∶ -.该菌株对小鼠有较强的致病性,能引起人工感染小鼠大量死亡,且从其体内分离到相同特性的菌株;药物敏感性试验证实该分离菌对头孢曲松、氧氟沙星、卡拉霉素、多粘菌素、复方新诺明、呋喃唑酮等敏感,对链霉素、四环素、氯霉素等耐药;同时根据GenBank中已报道的沙门氏菌毒力因子ivnA和ivnE的基因序列,设计两对特异性引物对该菌进行PCR检测,结果在该菌中成功扩增并检测出ivnA和ivnE基因.本试验尝试了沙门氏菌的PCR检测方法,为该分子诊断技术的全面推广奠定了良好基础,也为今后该疾病的预防、诊断和治疗提供了科学的实验数据.     

低氧胁迫和螺原体感染对中华绒螯蟹存活和细胞凋亡的影响

为了探究低氧胁迫对螺原体(Spiroplasma eriocheiris)感染中华绒螯蟹(Eriocheir sinensis)的影响,以中华绒螯蟹为研究对象,在低氧胁迫后,取中华绒螯蟹肝胰腺和鳃组织加入组织固定液,进行HE染色。在低氧条件下使螺原体感染中华绒螯蟹,计算螺原体拷贝数,并对血淋巴细胞进行细胞凋亡、细胞坏死和线粒体膜电位检测。结果显示,与对照组相比,处于长时间低氧胁迫状态下的中华绒螯蟹肝胰腺组织疏松,出现大量小空泡,鳃轴结构弥散,组织结构被破坏。此外,低氧组的中华绒螯蟹感染螺原体后的死亡速度相对于常氧组明显加快,血细胞内的螺原体数量、线粒体膜电位、血细胞凋亡率和坏死率相较于常氧组均显著升高。以上研究说明低氧胁迫可以加速螺原体的感染,使河蟹死亡速度变快,使血淋巴细胞凋亡和坏死更显著,不利于河蟹的生理生化。     

患病大鲵中弗氏柠檬酸杆菌的分离与鉴定

【目的】确定导致大鲵(Andrias davidianus)细菌性感染死亡的病原。【方法】从大鲵肝脏中分离细菌,通过Biolog微生物自动鉴定系统及分子生物学方法对纯培养的细菌进行鉴定,再用大鲵和鲫鱼分别进行人工感染试验,以确定分离菌的致病性,同时对分离到的病原菌进行药物敏感试验。【结果】从患病大鲵肝脏中分离到一株致病菌JZ01,经人工感染健康大鲵,可复制与自然发病相同的症状,且从人工感染病鲵体内再次分离到相同的病原菌。该致病菌对健康鲫鱼也有致病性。经Biolog微生物自动鉴定系统的鉴定,以及进一步的16S rDNA基因序列和系统发育分析都表明,此致病菌为弗氏柠檬酸杆菌。药物敏感性试验表明,该菌株对氨曲南、头孢三嗪、先锋噻肟等9种药物高度敏感。【结论】弗氏柠檬酸杆菌是大鲵的一种致病菌。本文在国内外首次报道了该菌对大鲵具有致病性。     

鳜源致病性荧光假单菌的分离与鉴定

为确定导致鳜（Siniperca chuatsi）细菌性感染死亡的病原,从患病濒死的鳜肝中分离出一株优势菌B01,经人工回感实验确定其分离菌的致病性,并利用VITEK-2全自动微生物鉴定仪及16S rRNA序列分析对纯培养细菌进行鉴定。此外,对分离的菌株进行药敏实验。研究结果表明,菌株B01对鳜鱼具有致病性。经VITEK-2全自动微生物鉴定仪鉴定菌株B01为荧光假单胞菌（Pseudomonas fluorescens）（表1）,并在紫外灯可以产生荧光（图1）。进一步的16S rRNA基因序列和系统发育树分析表明,该菌与荧光假单胞菌同源性达到了97%以上,并和（图2）。药物敏感性实验结果显示,该分离株对恩诺沙星、诺氟沙星、链霉素和四环素药物等6种药物高度敏感（表2）。     

麂子源蜂房哈夫尼亚菌的分离鉴定及生物学特性

【背景】蜂房哈夫尼亚菌是革兰阴性杆菌,是一种机会致病菌、腐生菌,常见于人和动物肠道、污水、土壤和乳制品中,能引起人和动物败血症,而且具有潜在的致腹泻作用。【目的】为对昆明轿子雪山自然保护区内死亡麂子体内潜在的致病菌进行分离鉴定及生物学特性分析。【方法】无菌采集死亡麂子的部分肠道组织进行细菌的分离培养和鉴定,并对分离获得的菌株进行药物敏感性试验及动物回归试验。【结果】鉴定分离菌株为蜂房哈夫尼亚菌,编号KMJZXS0312。药敏试验结果表明,该菌对青霉素、头孢噻吩等7种抗生素耐药,对氟苯尼考、卡那霉素、呋喃唑酮、阿莫西林中介,对恩诺沙星、复方新诺明等13种抗生素敏感。动物回归试验表明,该菌能致小鼠死亡,引起小鼠胃和肠道胀气,肠道薄而透亮,肝脏点状出血,肺脏有针尖大小出血点,肝脏病理切片显示,肝细胞轻度水样变性,肝细胞肿胀,胞质疏松淡染。【结论】本实验从麂子肠组织分离到一株具有致病性的蜂房哈夫尼亚菌,对其致病机制进行了分析,并进行了药物敏感试验,提供了菌株新的生物学信息,具有重要的公共卫生学意义。     

锦鲤中吉氏库特菌的分离与鉴定

对从患病锦鲤(Cyprinus carpio L.)肝组织中分离的1株纯培养菌(HC050630C-1)进行了形态特征、主要理化特性、对健康鲤鱼的致病作用、药物敏感性等方面的检验;同时测定了该株菌的16S rRNA基因序列,构建了系统发育树。结果表明,被检菌为吉氏库特菌(Kurthia gibsonll),对健康鲤鱼未显示明显的致病作用;所测菌株的16S rRNA基因序列长度1459bp,在GenBank中登录号为EF611423;该菌株16SrRNA基因序列与GenBank数据库中吉氏库特菌的16S rRNA基因序列同源性在99%;药敏试验结果显示,对供试的氨苄青霉素等27种药物呈现高度敏感或敏感,对头孢吡肟等6种呈低度敏感,对苯唑青霉素等4种耐药。     

迁延性黑点癣1例

目的报道病程30余年、由紫色毛癣菌感染所致成人黑点癣1例。方法对头发及鳞屑标本进行多次真菌直接镜检和培养,对病原菌进行形态学鉴定和药物敏感性试验。结果鉴定为紫色毛癣菌。体外药敏试验该菌对特比萘芬、伊曲康唑敏感。结论本病例证实为紫色毛癣菌感染导致黑点癣,患者经口服伊曲康唑和局部治疗痊愈。     

中华绒螯蟹水瘪子病不同发病阶段的病理变化特点

为了解中华绒螯蟹(Eriocheir sinensis)“水瘪子”病发生发展的过程, 揭示疾病的本质, 对病蟹进行了较全面的病理特点分析研究。根据病蟹肝胰腺颜色, 将病蟹划分为浅黄、浅粉、乳白和灰白4个发病阶段。研究结果表明, 随着病情的发展, 病蟹肝胰腺与蟹重比逐渐减少, 腔液与蟹重比逐渐增加, 肝胰腺管直径逐渐缩小, 各发病阶段病蟹的这些指标都与健康蟹有极显著差异(P<0.01), 病蟹肝胰腺与蟹重比除乳白阶段与灰白阶段之间和腔液与蟹重比除浅粉阶段与乳白阶段和乳白阶段与灰白阶段之间以及肝胰腺管直径除乳白阶段与灰白阶段之间的差异均不显著(P>0.05)和浅粉阶段与乳白阶段之间的差异显著(P<0.05)外, 其他各发病阶段之间这些指标均有极显著差异(P<0.01); 4个发病阶段病蟹6种组织器官中肝胰腺、鳃、肌肉组织和心脏组织病理变化具有相似性, 浅黄阶段主要是以组织器官水肿和萎缩为主, 是一种可逆的组织病理过程, 但在浅粉阶段肝胰腺和肌肉组织出现细胞坏死, 以后发病阶段更为严重, 鳃和心脏组织在乳白阶段出现细胞坏死, 坏死是一种不可逆的组织病理过程, 神经团和肠道组织病理变化不明显。根据不同发病阶段的病理特点, 在未来确定病因后通过消除病因等方法, 只有在病理变化处于可逆状态的浅黄阶段, 病蟹才有恢复的可能, 才有防控和挽救价值, 而浅粉阶段以后的病蟹由于主要器官组织细胞崩解坏死处于不可逆的病理过程, 即使病因消除病蟹已不可能恢复, 这样的病蟹应及时清除, 减少继续养殖造成进一步的经济损失。     

Characterization of two C-type lectin-like domain (CTLD)-containing proteins from the cDNA library of Chinese mitten crab Eriocheir sinensis

Chinese mitten crab Eriocheir sinensis is one of the most important aquaculture crustacean species in China. A cDNA library was constructed from mixed tissues of E. sinensis challenged with LPS. Eight genes involved in immune response were identified from 319 single colonies. Among them, two different C-type lectin-like domain (CTLD)-containing proteins were firstly identified in Chinese mitten crab. The full-length cDNA sequences of two C-type lectin-like domain (CTLD)-containing proteins named EsCTLDcp-1 and EsCTLDcp-2 were cloned by 5' RACE. The deduced amino acid sequences of EsCTLDcp-1 and EsCTLDcp-2 possessed several conserved features of C-type lectin subfamily. The tissue distribution of EsCTLDcp-1 and EsCTLDcp-2 was examined by Real-time PCR. In the normal Chinese mitten crab, the expression of EsCTLDcp-2 was detected in all tested tissues such as haemolymph, muscle, intestine, gill, heart, gonad and hepatopancreas, whereas in muscle, intestine, gill, heart and hepatopancreas for EsCTLDcp-1. The highest expressions of EsCTLDcp-1 and EsCTLDcp-2 were both observed in hepatopancreas. LPS significantly induced the expression of EsCTLDcp-1 and EsCTLDcp-2 in the hepatopancreas at the different time points. The induced fold change of EsCTLDcp-1 and EsCTLDcp-2 increased significantly from 2 h for EsCTLDcp-1 and 4 h for EsCTLDcp-2, and reached a maximum at 12 h, then dropped at 24 h. A differential pattern was found in Chinese mitten crab challenged with Chinese mitten crab pathogen Aeromonas hydrophila. The expression of EsCTLDcp-1 increased significantly at 2 h post-challenge crabs with A. hydrophila, then decreased at 4 h and 8 h, after that increased at 12 h and 24 h. The expression of EsCTLDcp-2 was decreased at the all time points. All these data suggest a differential role of EsCTLDcp-1 and EsCTLDcp-2 in the crab innate immune response to bacterial infection.     

Use of collagenase from the hepatopancreas of the Kamchatka crab for isolating and culturing endothelial cells of the large vessels in man

The application of hepatopancreas collagenase from crab Paralithodes camtschatica for the isolation and cultivation of the endothelial cells was studied in human umbilical vein endothelium. The comparison of the enzyme from crab hepatopancreas with collagenase from Clostridium histoliticum has shown that the number of viable cells isolated from human umbilical vein by the crab enzyme was lower than in the case of microbial collagenase. However, this difference was not significant for subsequent cultivation of cells. Harvesting of the endothelial cells from the substrate during cultivation was more effective in the case when collagenase from crab hepatopancreas was used. It was shown that crab collagenase, in contrast with microbiological collagenase, was not a metal-dependent enzyme.     

Monoamine Oxidase Activity in the Hepatopancreas of the Kamchatka Crab <Emphasis Type="Italic">Paralithodes camtschaticus</Emphasis>: a Substrate–Inhibitor Specificity

A study of substrate–inhibitor specificity of mitochondrial monoamine oxidase (MAO) in the hepatopancreas of the adult Kamchatka crab Paralithodes camtschaticus revealed specific catalytic properties of the enzyme. On the one hand, crab hepatopancreas MAO, like its classical hepatic counterpart, can deaminate tyramine, tryptamine, dopamine, serotonin, noradrenalin, benzylamine, β-phenylethylamine and N-methylhistamine but shows no sensitivity to 10 mM semicarbazide. On the other hand, MAO deaminates histamine but not putrescine, two classical diamine oxidase (DAO) substrates. It was established that MAO activity was several times higher toward benzylamine, β-phenylethylamine and N-methylhistamine than toward serotonin and noradrenalin. MAO was also found to be almost 500 times more sensitive to its selective inhibitor deprenyl than to chlorogilyn. A substrate–inhibitory analysis with the use of deprenyl and chloroginyl provides an indirect evidence for the existence of a sole MAO molecular form in the Kamchatka crab hepatopancreas.     

中华绒螯蟹肝胰腺R和F细胞及其脂类储存的电镜研究

电镜下研究了中华绒螯蟹不同生长发育时期（９月份卵巢处于快速发育阶段，１２月成熟阶段，胚胎发育４－１４天和卵孵出以后）肝胰腺Ｒ和Ｆ细胞的结构及其脂类储存。肝胰腺的脂类主苛以脂肪滴（Ｌ）的形式存在于Ｒ细胞中。Ｒ细胞在不同阶段Ｌ的存在形式和脂类的储存量不同，细胞数量也有所不同，显示Ｒ细胞对不同生长时期的敏感性。Ｆ细胞的结构特征是胞质中的发达的粗面内质网系统。细胞中几乎没有脂肪滴的储存。     

Ultrastructural study on a novel microsporidian, Endoreticulatus eriocheir sp. nov. (Microsporidia, Encephalitozoonidae), parasite of Chinese mitten crab, Eriocheir sinensis (Crustacea, Decapoda)

A microsporidian pathogen, infecting the epithelial cells of the hepatopancreas of Chinese mitten crab, Eriocheir sinensis, was studied by electron microscopy. The detailed ultrastructure of life cycle of the pathogen including proliferative and sporogonic developmental stages are provided. All stages of the parasite are haplokaryotic and develop in a vacuole bounded by a single membrane in contact with host cell cytoplasm. Sporogenesis is synchronous with the same developmental stage in one vacuole. Sporogony shows a characteristic of multinucleate sporogonial plasmodia divided by rosette-like division, producing 4 or 8 sporoblasts. The mature spore is ellipsoidal, length (mean) 1.7 microm, width 1.0 microm, with a uninucleate in the center of the sporoplasm, 7 turns of the polar filament, a bell-like polaroplast of compact membranes and obliquely positioned posterior vacuole. The morphological characteristics of this novel microsporidian pathogen have led us to assign the parasite to a new species of Endoreticulatus, E. eriocheir sp. nov., that has not been reported previously from crab.     

Chronic effect of atrazine on hydromineral balance in the crab

Effect in vivo of atrazine on total body weight, hydration level, haemolymph volume, inorganic electrolytes was studied in the gill, hepatopancreas and haemolymph of the crab. Significant changes were seen in the tissues indicating disturbances in the hydromineral balance of the crab as a consequence of atrazine.