人工感染嗜水气单胞菌对日本鳗鲡血清酶活力的影响

为了解日本鳗鲡(Anguilla japonica)受嗜水气单胞菌(Aeromonas hydrophila)感染后血清酶活力的变化规律,将日本鳗鲡分为感染组和对照组,每组30尾,腹腔分别注射0.1 mL浓度为3×106CFU/mL的菌液和等量灭菌生理盐水。在注射后0、1、5、9、13、17、21、25、29、33 d从两组各取3尾日本鳗鲡,尾静脉取血,测定其酸性磷酸酶(ACP)、超氧化物歧化酶(SOD)、一氧化氮合酶(NOS)活力的变化规律。结果显示:注射嗜水气单胞菌一段时间后,实验组鳗鲡的ACP和SOD的活力都显著高于对照组,且都在21 d时达到最高值,随后逐渐下降。结果表明:嗜水气单胞菌感染后,日本鳗鲡能够通过提高血清中ACP和SOD活力来提高机体对嗜水气单胞菌的免疫力,而对一氧化氮合酶的活力没有显著影响。     

Improvement of intestinal barrier,immunity, and meat quality in common carp infected by Aeromonas hydrophila using probiotics

The fish pathogen Aeromonas hydrophila causes gastrointestinal tract infections and hemorrhagic septicemia and represents a widespread risk in aquaculture. This study aimed to determine whether compound probiotics could improve the intestinal barrier, immunity, and meat quality of common carp infected by A. hydrophila by feeding them compound probiotics. Carp were assigned to one of four groups: (1) control (no infection, no probiotics); (2) control?+?probiotic; (3) A. hydrophila infected; and (4) the A. hydrophila?+?probiotic group. At the beginning of the experiment, the carp were injected with either saline (0.86%) or A. hydrophila (4.87?×?10⁷ CFU/mL). After 2 weeks of the feeding regime, results suggested that in A. hydrophila infected carp, dietary probiotics regulated the intestinal microflora as evidenced by a reduced abundance of Proteobacteria and increased amounts of Firmicutes and Fusobacteria in the intestine. In addition, dietary probiotics ameliorated both villus swelling and the decrease in villus height induced by A. hydrophila (P?<?0.05). Moreover, probiotics prevented the A. hydrophila-induced decline of Occludin, Claudin-1, and ZO-1 levels in the intestine (P?<?0.05). The addition of probiotics into the feed also increased acid phosphatase (ACP), alkaline phosphatase (AKP), and lysozyme (LZM) activity in the serum (P?<?0.05), in comparison to the A. hydrophila group. Importantly, when compared to A. hydrophila-infected carp, compound probiotics alleviated the decrease in muscle nutrient quality (P?<?0.05). In summary, we show that dietary compound probiotics can prevent the impact of A. hydrophila infection on the intestinal function and disease resistance, while ensuring carp muscle quality.

     

Cell-mediated protection in carp, Cyprinus carpio L., against Aeromonas hydrophila

Abstract. The mechanism of protection against Aeromonas hydrophila infection in carp was studied. Recipient fish, into which pronephric cells from carp previously immunized by dipping in bacterial crude lipopolysaccharide (LPS) at 25°C for 2h were transferred, demonstrated almost the same level of protective ability as an immunized control group. The protective ability was transmitted by non-adherent (to nylon fibre) immune pronephric cells. These non-adherent cells were damaged by anti-carp thymocyte serum and were, thus, considered to be T-like cells. The protective ability was depressed in immunized carp treated with anti-carp thymocyte serum in vivo and was also remarkably reduced in immunized carp whose macrophage function was impaired by Dextran sulphate-500 treatment. These results indicate that the protection shown by carp immunized by dipping in crude LPS is dependent on cellular immunity regulated by a T-like cell-macrophage system.     

嗜水气单胞菌对鲫三项血液指标的影响

用嗜水气单胞菌(浓度9×108CFU/mL)感染健康鲫(剂量为0.3mL/尾),4d后观察鲫的发病情况,并对鲫三项血液生化指标进行测定。结果表明:感染鲫表现出典型的嗜水气单胞菌引起的败血症症状,与对照组比较甘油三酯含量极显著下降,总蛋白含量显著下降,而谷丙转氨酶活性极显著升高。     

嗜水气单胞菌感染翘嘴鳜头肾转录组分析

嗜水气单胞菌(Aeromonas hydrophila)是严重危害翘嘴鳜(Siniperca chuatsi)养殖生产的主要病原之一,为揭示嗜水气单胞菌感染翘嘴鳜后宿主基因表达水平的变化,筛选免疫相关基因,解析翘嘴鳜应答病原细菌感染的分子机制,本研究以病原嗜水气单胞菌感染翘嘴鳜,于感染24 h后,采集感染组与对照组翘嘴鳜头肾组织,采用Illumina Hiseq 2000进行了RNA-Seq分析,原始数据拼接后组装共获得53 040个单基因(unigene)。基因差异表达分析结果显示,感染组和未感染组翘嘴鳜存在526个差异表达基因,包括254个上调基因和272个下调基因,其中,免疫相关的显著上调的差异基因主要有炎症和免疫原性细胞因子白介素、补体系统、MHCⅠ型抗原提呈、溶菌酶、丝氨酸蛋白酶抑制因子、泛素蛋白连接酶等。GO富集分析发现,差异基因主要涉及免疫应答反应和炎症反应等,经KEGG富集分析显示,89个通路富集显著,免疫相关的代谢通路主要有内吞作用和吞噬体等。此外,实时荧光定量PCR验证结果表明,所选取7个差异表达免疫相关基因与RNA-seq结果具有相似的表达趋势。本研究为揭示翘嘴鳜对病原微生物感染的防御分子机制奠定了理论基础。     

载铜蒙脱石对嗜水气单胞菌粘附尼罗罗非鱼上皮细胞的影响

采用尼罗罗非鱼上皮细胞培养模型,观察嗜水气单胞菌对鳃上皮、皮肤上皮、肠上皮细胞的粘附率,研究载铜蒙脱石对细菌粘附的阻断作用及其对细菌粘附引起鱼上皮细胞膜生物特性变化的影响。结果显示：嗜水气单胞菌与鱼上皮细胞均有不同程度的粘附作用,粘附率大小顺序为鳃上皮〉皮肤上皮〉肠上皮细胞。载铜蒙脱石均显著降低了嗜水气单胞菌对鳃、皮肤和肠上皮细胞的粘附率（P〈0．05）,而且对不同上皮细胞的粘附阻断率无显著差异。嗜水气单胞菌粘附肠上皮细胞后细胞膜生物特性发生了显著变化,与正常对照组相比,嗜水气单胞菌粘附鱼上皮细胞后,胞浆游离钙和细胞膜磷脂酶A2活性显著上升。载铜蒙脱石可显著降低由于嗜水气单胞菌粘附鱼上皮细胞引起的胞浆游离钙和细胞膜磷脂酶A2活性的升高。结果表明,载铜蒙脱石可有效阻断病原菌粘附,从而防治细菌感染和细菌移位。     

鲫源嗜水气单胞菌毒力基因多重PCR检测及ERIC-PCR分子分型

为快速了解鲫源嗜水气单胞菌株毒力基因携带情况及与菌株基因型的相关性,建立多重PCR法和ERIC-PCR分子分型,为临床快速检测、菌株分型和菌株致病性分析提供依据。通过单重PCR法检测出标准菌株ATCC7966内5个毒力基因气溶素(aerolysin,aer)、溶血素(hemolysin,hly)、细胞毒性肠毒素(cytotoxic enterotoxin,alt)、胞外蛋白酶(extracellular protease,ahp)和细胞肠兴奋性肠毒素(intestinal cells of excitatory enterotoxin,act),其扩增产物长度依次为300 bp、592 bp、442 bp、856 bp和500 bp。在此基础上,优化并建立特异性高,敏感度达7.2×102cfu·m L-1多重PCR法,用于检测从江苏射阳地区患病水产动物体内分离的17株嗜水气单胞菌5个毒力基因携带率。结果显示,毒力基因act的携带率为100%,而80%的菌株5个毒力基因均有检出。采用ERIC-PCR分子分型技术,以标准菌株ATCC7966为对照,对17株鲫源致病性嗜水气单胞菌进行基因分型,获得两种基因型,分别描述为A型和B型,其中B型菌株14株,带型与ATCC7966一致,认为是当地的主要流行株。探究菌株基因型与毒力基因分布相关性,携带5个毒力基因的均为B型菌株,而所有A型菌株存在一或多个毒力基因缺失,有可能是此类菌株更易发生毒力基因漂变,但还需进一步研究。     

嗜水气单胞菌感染后牛蛙血清中抗菌物质的初步研究

用1×107 mL-1的嗜水气单胞菌腹腔注射感染牛蛙,注射后7 d内试验组牛蛙血清抗菌活性都高于对照组,其中在3 d、7 d时存在显著性差异,峰值出现在感染后第3天。收集第2批人工感染后3 d时牛蛙的血清。该血清经Sephadex G25凝胶柱分离后,并在波长280 nm处测定紫外吸收值,得到两个吸收峰。以嗜水气单胞菌检测分离物的抗菌活性,结果表明经诱导的血清抗菌物质主要集中在第1个吸收峰。通过对金黄色葡萄球菌等10株指示菌抗菌谱的测定,其对大肠杆菌、枯草芽孢杆菌、铜绿假单胞菌等具有明显的抗菌作用;SDSPAGE分析显示抗菌蛋白分子量较大。结果说明：牛蛙被嗜水气单胞菌感染后能很快产生多种抗菌物质,主要的抗菌物质是分子量比较大的物质,这些抗菌物质对部分革兰氏阴性菌和革兰氏阳性菌有抗菌作用。图3表1参24 关键词：牛蛙;嗜水气单胞菌;抗菌蛋白;抗菌活性     

Immune protection in carp, Cyprinus carpio L., after immunization with Aeromonas hydrophila crude lipopolysaccharide

Abstract. Vaccination with crude lipopolysaccharide (LPS) induced better protection against infection with Aeromonas hydrophila in carp than vaccination with formalin killed vaccine. Dipping fish in vaccine for 2 h at 25°C was more effective than intraperitoneal injection of the vaccine in procedural simplicity, lower stress loading and the degree of protection acquired. In carp immunized with crude LPS by the dip method, antibodies were not detected by bacteriai agglutination, passive haemagglutination and the agar diffusion tests. The results indicate that the protection against A. hydrophila infection in carp is not dependent on humoral immunity.     

鲤嗜水气单胞菌感染症及其病原生物学特性

对一起养殖鲤(Cyprinus carpio)发生的病害进行了发病情况、临床特征、病理变化等方面的检验.以3尾病(死)鲤进行病变组织中的细菌检查及细菌分离,对分离菌进行了形态特征、理化特性等较系统的表观分类学指征鉴定;同时选择代表菌株进行了16S rRNA基因的分子鉴定,测定了16S rRNA基因序列、分析了相关细菌相应序列的同源性、构建了系统发生树.结果表明,分离菌为气单胞菌属(Aeromonas Kluyver and Van Niel 1936)的嗜水气单胞菌[A.hydrophila(Chester 1901)stanier 1943],代表菌株(HC060718-1株)16S rRNA基因序列长度(不包括引物结合区)为1 454 bp(GenBank登录号:EF669478).择代表菌株做对健康鲤的人工感染试验,表明了分离鉴定的嗜水气单胞菌在被检鲤病例的相应原发病原学意义及较强的致病作用.药敏试验结果显示,对供试37种抗菌药物中的头孢噻肟等23种药物高度敏感,对氨苄青霉素等5种药物敏感,对青霉素G等9种药物耐药.     

鲤嗜水气单胞菌感染症及其病原生物学特性

对一起养殖鲤(Cypr inus carp io )发生的病害进行了发病情况、临床特征、病理变化等方面的检验。以3尾病(死)鲤进行病变组织中的细菌检查及细菌分离, 对分离菌进行了形态特征、理化特性等较系统的表观分类学指征鉴定; 同时选择代表菌株进行了16S rRNA 基因的分子鉴定, 测定了16S rRNA 基因序列、分析了相关细菌相应序列的同源性、构建了系统发生树。结果表明, 分离菌为气单胞菌属(Aeromonas K luyv er and Van N iel 1936) 的嗜水气单[A.hydrophila (Chester 1901) Stan ier 1943] , 代表菌株( HC060718- 1株) 16S rRNA基因序列长度( 不包括引物结合区)为1 454 bp( GenBank登录号: EF669478)。择代表菌株做对健康鲤的人工感染试验, 表明了分离鉴定的嗜水气单胞菌在被检鲤病例的相应原发病原学意义及较强的致病作用。药敏试验结果显示, 对供试37种抗菌药物中的头孢噻肟等23种药物高度敏感, 对氨苄青霉素等5种药物敏感, 对青霉素G等9种药物耐药。     

一种佐剂新材料在嗜水气单胞菌灭活疫苗浸泡免疫鲫中的应用效果

为研究蓖麻油聚乙二醇单酯葡萄糖苷(CPMG)在细菌灭活疫苗浸泡免疫中的佐剂作用,将CPMG和嗜水气单胞菌灭活疫苗联合施用,二次浸泡免疫异育银鲫后饲养在室内玻璃缸中,用实时荧光定量PCR(RT-PCR)检测加强免疫后2、4、7、11、14和21 d脾脏中Ig M、IL-1β、C3、C-凝集素和溶菌酶的m RNA表达量,首次免疫30 d后用同源菌株活菌攻击实验鱼;同时,用含CPMG、山莨菪碱复合佐剂的灭活疫苗浸泡免疫异育银鲫一次后饲养在池塘网箱中,定期抽取血液检测血清抗体效价并用同源菌株人工感染实验鱼。结果显示,CPMG佐剂组和无佐剂组的5个免疫相关基因的相对表达量显著高于对照组,CPMG组Ig M基因较无佐剂组更早表达,补体C3、C-凝集素以及溶菌酶的基因表达量更高或表达持续时间更长。血清抗体效价在免疫后2周达到160,6周达到峰值640,12周降低为40。室内玻璃缸中二次免疫30 d后,CPMG组的平均相对保护率达到70.6%,复合佐剂组达到88.2%,均显著高于无佐剂组的56.0%。池塘网箱中一次浸泡免疫30、60和120 d后,平均相对保护率分别为54.6%、44.0%和12.5%。研究表明,CPMG与嗜水气单胞菌灭活疫苗共同浸泡免疫异育银鲫,增强了脾脏中Ig M、IL-1β、C3、C-凝集素以及溶菌酶基因表达,提高了异育银鲫抗嗜水气单胞菌感染的能力。     

嗜水气单胞菌研究进展

嗜水气单胞菌(Aeromonashydrophila)属于弧菌科、气单胞菌属,是嗜温、有动力的气单胞菌群,普遍存在于淡水、污水、淤泥、土壤和人类粪便中,对水产动物、畜禽和人类均有致病性,是一种典型人—兽—鱼共患病病原[1],可引起多种水产动物的败血症和人类腹泻,往往给淡水养殖业造成惨重的经济损失,已引起国内外水产界、兽医学界和医学界学者的高度重视。目前国内外学者对嗜水气单胞菌的研究已有了很大进展。本文将国内外有关嗜水气单胞菌研究现状从其毒力因子以及诊断与检测技术方面综述如下。1　毒力因子嗜水气单胞菌可产生各种毒力因子,包括…     

Actin rearrangements accompanying Aeromonas hydrophila entry into cultured fish cells

Aeromonas hydrophila can enter fish cells and exist as intracellular parasites. Phase-contrast and confocal microscopy were used to examine morphological changes and various cytoskeletal components of infected fish cells. Four fish cell lines were included in this study: (1) AS, (2) BF2, (3) CHSE-214, and (4) EPC cells. Virulent but not avirulent strains of A. hydrophila PPD 134/91 invaded fish cells, causing morphological changes, and inducing microfilament (F-actin) rearrangement. Morphological changes were observed in all infected fish cell lines and could be classified into three different stages. In stage I, the cells became detached from each other and pointed ends were observed. In stage II, tubular cytoplasmic extensions formed at contact points connecting neighbouring cells. The monolayers formed a satellite-like organization and became less confluent. Finally (stage III), cells were heavily infected with bacteria, and bacteria containing vacuoles occupied most of the cells. They eventually detached and lysed. Rearrangement of F-actin was observed as local polymerization (actin clouds) in stage I and massive reorganization in stage III of infection. Actin clouds could have been induced by A. hydrophila for ‘assisted' uptake into the cells. The massive reorganization of actin in stage III may be due to products released by the bacteria and the growth of vacuoles. Pretreatment of fish cells with the microfilament inhibitors such as cytochalasins induced a similar effect. There were little if any rearrangements in intermediate and microtubule filaments during bacterial entry (stages I and II). These results suggest that A. hydrophila may bind to the surface and trigger a signal to the microfilament which then generates the force necessary for bacterial uptake.     

In vivo transfer of plasmid pRAS1 between Aeromonas salmonicida and Aeromonas hydrophila in artificially infected Cyprinus carpio L.

This study investigated the possible in vivo transfer of plasmid pRAS1 between Aeromonas salmonicida and A. hydrophila inhabiting two different organs of Cyprinus carpio L. To distinguish transconjugants from naturally occurring antibiotic resistant bacteria, twelve luminescent transposon‐tagged A. hydrophila strains using miniTn5luxCDABEKm2 transposon were generated. In conjugal transfer experiments, fish were conditioned with the donor bacteria and subsequently immersed in water containing the recipient strain. Bacteria were recovered from gills and intestines and isolated by growth on selective plates. Transconjugants were identified by their resistance to the pRAS1 encoded antimicrobials and by light emission. In vivo transfer frequencies ranged between 10^?3 and 10^?6 and were somewhat lower in intestines, compared to gills. Transfer frequencies were also smaller relative to those obtained in vitro. The minimal amount of donor and recipient bacteria needed to yield detectable transconjugants in vivo was 1 × 10⁴ CFU mL^?1. Implications of this plasmid transfer in natural settings and its possible consequences to human health are discussed.     

两种鲤鱼对侵袭性病原体及嗜水气单胞菌抗病力的比较

调查同塘饲养的高寒鲤,散鳞镜鲤当年鱼对侵袭性病原体及人工感染嗜水气单胞菌耐受力进行比较,侵袭性病原体检出8种,其中绦虫只有散鳞镜鲤感染,感染率达到29％,其余种类二种鲤鱼体表,鳃都有寄生,但寄生的程度有所不同,高寒鲤的抵抗力要比散鳞镜鲤强,对人工感染的嗜水气单胞菌的耐受力高寒鲤比散镜鲤高,这可能与品种对病原菌耐性不同有关。     

Isolation and characterization of fish Aeromonas hydrophila adhesins important for in vitro epithelial cell invasion

The molecular mechanisms involved in the invasion of Aeromonas hydrophila (strain PPD 134/91) into host cells were studied in vitro using a carp epithelial cell line. Bacterial fractions were extracted with potassium thiocyanate (KSCN) to investigate the adhesins involved. Two groups of adhesins were found. The major group was high molecular weight proteins with the largest component being a 43-kDa protein. Amino terminal sequence analysis indicated that this may have been an outer membrane porin. This supports previous suggestions that a 43-kDa outer membrane protein may be important in adhesion of a human isolate of A. hydrophila . The minor group of adhesins were low molecular weight proteins likely to be less effective in mediating bacterial adhesion and invasion into carp epithelial cells.     

Resistance of genetically different common carp, Cyprinus carpio L., families against experimental bacterial challenge with Aeromonas hydrophila

The objective of this study was to determine the differences in disease resistance against artificial infection with Aeromonas hydrophila between genetically different common carp families. Four strains differing in their origin and breeding history were selected from the live gene bank of common carp maintained at the Research Institute for Fisheries, Aquaculture and Irrigation (HAKI, Szarvas, Hungary) to establish families with wide genetic background: Szarvas 15 (15), an inbred mirror line; Tata (T) scaly noble carp; Duna (D), a Hungarian wild carp and Amur (A), an East Asian wild carp. A diallele mating structure was used to allow the assessment of genetic variation within and between the tested 96 families for a variety of traits. The existing technologies of fertilization and incubation of carp eggs, as well as larval and fingerling rearing had been modified because of the large number of baseline populations. Two challenge trials of the 96 families of carp with Aeromonas hydrophila were done. The 10 most resistant and 10 most susceptible families to A. hydrophila were identified from these two challenges. The crosses that produced the most resistant families were mainly those having parents from Tata and Szarvas 15 domesticated strains, while the most susceptible families were from the wild strains Duna and Amur.     

鱼类细菌群落中的致病嗜水气单胞菌

鱼类细菌群落中的致病嗜水气单胞菌汤伏生，曾勇，张兴忠（中国水产科学院长江水产研究所．沙市４３４０００）朱晓燕（湖北农学院水产系，荆州４３４１０３）关键词鱼类，细菌群落，嗜水气单胞菌，致病性ＰＡＴＨＯＧＥＮＩＣＡＥＲＯＭＯＮＡＳＨＹＤＲＯＰＨＩＬＡＩＮ...     

壳聚糖硫酸酯提高异育银鲫抗嗜水气单胞菌感染的研究

对异育银鲫连续7 d分别投喂添加壳聚糖硫酸酯0.1%、0.2%和0.4%的配合饲料,然后用嗜水气单胞菌进行攻毒,以检测壳聚糖硫酸酯对病原菌的抗感染能力。实验结果显示,与对照组相比,试验组呈现出不同程度的免疫保护作用,其中以壳聚糖硫酸酯添加量为0.2%试验组的免疫保护效果最明显,异育银鲫的成活率达到46.67%;添加量为0.1%和0.4%试验组的成活率分别为23.33%和30.0%,而攻毒对照组100%死亡。然后将添加0.2%壳聚糖硫酸酯的饲料用同样的方式投喂异育银鲫,检测其血清和肝组织中的酸性磷酸酶(ACP)、碱性磷酸酶(AKP)、溶菌酶(LSZ)和超氧化物歧化酶(SOD)活性,并分析肝组织中各酶的基因表达量。结果显示,异育银鲫血清和肝组织中的ACP、AKP、LSZ和SOD活性均得到显著提高,且四种酶的活性均呈现随时间延长而先升高后降低的趋势,并于第三天达到峰值,血清和肝组织中ACP活性比对照组分别提高了54.62%和26.58%、AKP提高了37.61%和25.88%、LSZ提高了13.42%和35.43%、SOD提高了18.59%和54.62%;试验组异育银鲫肝组织中ACP、AKP和LSZ的mRNA表达量均得到显著提高,并随时间推移呈现出先升高后降低的趋势,于72 h到达峰值,而SOD的mRNA表达量与对照组相比没有显著变化。综合以上结果,壳聚糖硫酸酯能够显著提高异育银鲫非特异性免疫水平,增强抗嗜水气单胞菌的能力,表明壳聚糖硫酸酯可作为免疫增强剂用于鱼类疾病的预防与治疗。