Serum resistance as a good indicator for virulence in Aeromonas hydrophila strains isolated from diseased fish in South-East Asia

Abstract. Aeromonas hydrophila strains isolated from diseased fish in South-East Asia were studied for their virulence in naive tilapia, Oreochrommis aureus (Steindachner), and blue gourami, Trichogaster trichopterus (Pallas). Five of the most virulent strains used in this study shared a common resistance to the killing effect of naive fish serum. Other factors such as lack of autoagglutination in 0.2% acriflavine, instability after boiling, production of an S-layer, and proteases and haemolysins did not correlate well with virulence. In addition, serotyping could not group all the virulent strains. Therefore, serum resistance is considered as a good indicator for screening virulence of A. hydrophila strains isolated from diseased fish in South-East Asia.     

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.     

Aeromonas hydrophila B-haemolysin: purification and examination of its role in virulence in 0-group channel catfish, Ictalurus punctatus (Rafinesque)

Abstract. A single step procedure for purification of B-haemolysin from Aeromonas hydrophila is described which recovered 94% of the haemolytic activity with a sixfold increase in specific activity. Toxicity of haemolysin in 0-group channel catfish was 0–6 mg of purified protein per gram offish. Of six strains of A. hydrophila evaluated for differential toxin production and virulence in 0-group channel catfish, three B-haemolysin-deficient strains were virulent while three B-haemolysin-producing strains were avirulent.     

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.     

左旋咪唑对罗氏沼虾免疫功能及抗病力的影响

以0(对照)、50、100和150 mg/kg饲料的剂量将左旋咪唑(LMS)添加于基础饲料中制成颗粒饲料投喂罗氏沼虾(Macrobrachium rosenbergii)14 d,采样测定了罗氏沼虾血细胞吞噬活性、血清酚氧化酶(PO)、溶菌酶(LSZ)及超氧化物岐化酶(SOD)活性,并以6×105cells/mL浓度的致病性嗜水气单胞菌(Aerom onas hydrophila)对罗氏沼虾进行肌肉注射(20μL/尾),记录接种7 d后罗氏沼虾的累积死亡率。结果表明,3个LMS处理组的罗氏沼虾血细胞吞噬百分比和吞噬指数、血清PO、LSZ及SOD活性均显著地高于对照组(P<0.05);LMS处理组的罗氏沼虾对嗜水气单胞菌的抵抗力明显增强。因此,饲喂适量的LMS能促进罗氏沼虾的免疫力和抗病力;在本试验条件下,100 mg/kg饲料的剂量为最适添加剂量。     

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。     

Biochemical and physiological characteristics and plasmid profiles of Aeromonas hydrophila strains, isolated from freshwater fish and from fresh water

Abstract. Twenty-five Aeromonas hydrophila strains, isolated from freshwater fish and from freshwater samples were identified, using API 20NE and CH50. They were screened for the one plasmid (20 kb), and a third group (four strains) contained two or three plasmids. A 20 kb plasmid was common to all plasmid-positive strains. Although their plasmid profile differs, all groups are very similar with regard to their biochemical and physiological characterization. However, the differences observed using clustering can be explained by characteristics, some of which have been reported as virulence associated. No relationship between these factors and the presence of plasmids is found. The possibility of differentiating A. hydrophila strains on the basis of biochemical characteristics using a cluster analysis is considered.     

In vitro markers for virulence in Yersinia ruckeri

In this study, different traits that have been associated with bacterial virulence were studied in Yersinia ruckeri. Two isolates that had been shown to cause disease and mortality in experimentally infected rainbow trout were compared with five avirulent isolates. Both virulent isolates showed high adhesion to gill and intestinal mucus of rainbow trout, whereas the majority of non‐virulent strains demonstrated significantly lower adhesion. A decrease in adherence capability following bacterial treatment with sodium metaperiodate and proteolytic enzymes suggested the involvement of carbohydrates and proteins. All strains were able to adhere to and invade chinook salmon embryo cell line (CHSE‐214), fathead minnow epithelial cell line (FHM) and rainbow trout liver cell line (R1). One non‐virulent strain was highly adhesive and invasive in the three cell lines, whereas the virulent strains showed moderate adhesive and invasive capacity. The internalization of several isolates was inhibited by colchicine and cytochalasin‐D, suggesting that microtubules and microfilaments play a role. For all strains, intracellular survival assays showed a decrease of viable bacteria in the cells 6 h after inoculation, suggesting that Y. ruckeri is not able to multiply or survive inside cultured cells. Analysis of the susceptibility to the bactericidal effect of rainbow trout serum demonstrated that virulent Y. ruckeri strains were serum resistant, whereas non‐virulent strains were generally serum sensitive.     

健康养殖南美白对虾肠道细菌的抗菌活性

用双层琼脂扩散法检测从健康养殖南美白对虾肠道内分离出的111株菌对8株病原菌的抗菌活性。结果表明:111株菌对白假丝酵母菌,非01群霍乱弧菌,嗜水气单胞菌指示菌无抗菌活性,48.6%的菌株对余下的5株指示菌有抗菌活性,其中气单胞菌属和发光杆菌属对荧光假单胞菌与金黄色葡萄球菌的抑制性最强。各菌属中以气单胞菌属,发光杆菌属的抗菌谱最广,弧菌属、肠杆菌科、黄单胞菌属、芽孢杆菌属、小球菌属次之。土壤杆菌属、棒状杆菌属、黄杆菌属、产碱杆菌属、假单胞菌属和色杆菌属则没有抗菌活性。     

投喂阿维拉霉素对鲤免疫应答的影响

将阿维拉霉素按照一定的浓度添加到饲料中,投喂经注射接种福尔马林灭活的嗜水气单胞菌(Aerom onashydrophila)菌苗的鲤,于免疫后的第7 d、14 d、21 d、28 d和35 d检测供试鲤血液中的白细胞吞噬活性、血清中溶菌酶活性、抗体效价、补体C3含量及免疫保护率(RPS),探讨了阿维拉霉素对受免鲤免疫应答的影响。结果表明,饲料中添加阿维拉霉素(4 mg/kg和8 mg/kg),投喂较长时间(≥28 d)会降低受免鲤的血液白细胞吞噬活性、血清中的溶菌酶活性和抗体效价,减少受免鲤血清中补体C3的含量,减弱鲤抵抗嗜水气单胞菌人工感染的能力,每公斤饲料中添加8 mg的阿维拉霉素其免疫抑制作用更强。     

豫北地区主要淡水鱼类感染嗜水气单胞菌的流行病学调查

对豫北地区嗜水气单胞菌引起的细菌性败血病进行流行病学调查,分离鉴定致病性嗜水气单胞菌菌株,并对致病性嗜水气单胞菌菌株进行毒力验证和药敏试验。采集4个养殖场病鱼标本及水样,每月进行流行病学调查,利用生理生化及分子生物学方法检测嗜水气单胞菌的分布状况。结果显示,筛选出52株为嗜水气单胞菌,其中32株为致病性嗜水气单胞菌,20株为非致病性嗜水气单胞菌,且致病性嗜水气单胞菌主要分布在7—9月份,毒力验证试验表明,32株致病性菌株的毒力大小差异明显,筛选出强毒株XDMG(4),为后期试验的疫苗株做准备;药敏试验表明,不同菌株对同一种药物的药敏结果不同,但大部分药物的药敏结果基本一致,70%以上的致病性菌株对头孢哌酮、氟本尼考、菌必治、阿米卡星等抗菌药物表现为高度敏感,对青霉素类药物表现为高度耐药性,耐药率达100%,对氨基糖苷类(不包括阿米卡星)、磺胺类、四环素等药物呈现不同程度的耐药性,具有多重耐药性。本试验旨在丰富本地区的鱼类细菌性败血病的病原资料,并为该菌引起的人类疾病的防控提供科学依据。     

鱼源气单胞菌的毒力基因检测、分型及致病力

为调查鱼源气单胞菌毒力基因与其致病力的相关性,以2009--2018年从不同患病鱼分离的173株气单胞菌为研究对象,通过检测毒力相关基因、测定溶血活性、腹腔注射感染异育银鲫等方法开展评价.通过管家基因gyrB分子鉴定结果显示,173株气单胞菌中维氏气单胞菌(119/173,68.9％)和嗜水气单胞菌(50/173,28...     

嗜水气单胞菌毒力因子研究进展

嗜水气单胞菌是一种广泛分布于自然界中的革兰氏阴性菌,是一种人一兽一鱼共患病的条件致病菌。其危害的产生与其外毒素、胞外蛋白酶及表面分子等毒力因子的分泌、表达相关。研究其毒力因子有利于深入了解该菌的致病机理,探索有效的防治方法。本文综述了嗜水气单胞菌毒力因子的相关研究进展。     

In vitro inhibition of epithelial cell invasion by Aeromonashydrophila Vibrio species by fish Aeromonas hydrophila major adhesin

A virulent strain of Aeromonas hydrophila (PPD 134/91) was obtained from the Primary Production Department, Singapore. Its major adhesin was isolated and purified by potassium thiocyanate extraction and Bio-Gel P-100 gel filtration. The ability of the protein in peak 1, termed major adhesin, to inhibit bacteria from adhering to and invading host cells was studied in vitro using epithelioma papillosum cells of carp (EPC). Results showed that a concentration of 10 μg ml^–1 of this major adhesin could competitively inhibit 28% of A. hydrophila PPD 134/91 from invading EPC cells in vitro. When the concentration was increased to 40 μg ml^–1, the major adhesin significantly cross-inhibited nine other virulent or weakly virulent strains of A. hydrophila. In addition, the major adhesin significantly inhibited not only another bacterial strain from the same family, Aeromonas sobria, but also strains of Vibrio spp. tested. Therefore, we suggest that the major adhesin of this virulent A. hydrophila strain has the potential to be used as a vaccine against the heterogeneous Aeromonas and Vibrio species.     

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。     

一株分离自团头鲂的嗜水气单胞菌病原学及其与ST251型菌株的全基因组比较

为确定湖北仙桃一团头鲂(Megalobrama amblycephala)专养池塘的发病病原体及病原特征,并从全基因组层面初步分析该病原菌的毒力和耐药特征,本研究从患病团头鲂的肝脏分离得到一株病原菌LHW39,经理化性状及16S rRNA鉴定,表明LHW39为嗜水气单胞菌(Aeromonas hydrophila).多...     

嗜水气单胞菌疫苗免疫效果研究

选取对鲤鱼毒力强的嗜水气单胞菌菌株制成灭活疫苗,研究疫苗的免疫保护效应.采用甲醛灭活制备疫苗,分别对鲤鱼进行注射和浸泡试验,测定白细胞吞噬活性、淋巴细胞转化能力、血清、皮肤黏液的抗体效价及活菌攻毒后的免疫保护率.结果表明,受免鱼各项指标均明显高于对照组,注射组免疫保护率达90%,其中浸泡组各项略低于注射组.嗜水气单胞菌灭活疫苗对鲤鱼有显著免疫保护效应,可作为预防细菌性败血症感染的疫苗.     

Proteases of the Aeromonas hydrophila complex: identification, characterization and relation to virulence in channel catfish, Ictalurus punctatus (Rafinesque)

Abstract. Traditional biochemieal techniques and a stain to detect proteases in polyacrylamide gels were used to identify and partially characterize three proteases, P1, P2 and P3, produced by Aeromonas hydrophila strain Ah 22. P1 was found to be a heat-labile serine protease with an optimum pH of 7·5, while P2 is a heat-stable metalloprotease with an optimum pH of 8·0, and P3 is a moderately heat-stable metalloprotease with peak activity beween pH 7 and 11. A comparison of 17 other strains of the A. hydrophila complex indicated that four produced P1, P2 and P3. Two strains produced just P1 and P3; one produced only P3; six produced two different serine proteases, P2a and P2b; and two produced a number of uncharacterized proteases. Virulence studies in age-0 + channel catfish indicated no correlation between either quantitative or qualitative protease production and virulence.     

芽孢杆菌R1共培养对嗜水气单胞菌NJ-1毒力基因表达的影响

为探讨芽孢杆菌潜在的淬灭靶点,本研究通过芽孢杆菌R1与嗜水气单胞菌NJ-1共培养,并对NJ-1生物量、信号分子产生量、毒力因子基因和群体感应(QS)关键调控基因表达量进行检测。结果显示:1NJ-1单独培养时,信号分子BHL和HHL的产量与NJ-1生长趋势一致,在27 h时达到峰值并启动Lux R表达,进而调控毒力因子金属蛋白酶、丝氨酸蛋白酶、肠毒素、气溶素和血溶素等表达,其在27~30 h时呈现峰值表达,通路Qse B在27~30 h启动表达,48 h时达到峰值,而种间调控Lux S基因表达一直处于低水平状态;2与芽孢杆菌R1共培养时,仅痕量AHLs信号分子被检出,毒力因子相关基因表达量均显著下调;Lux R表达受抑制,Lux S在36 h时表达量显著上调,未影响Qse B正常表达;3AHLs与R1共培养可被显著降解。研究表明,芽孢杆菌R1抑制NJ-1毒力因子相关基因表达可能与其对NJ-1多QS系统进行调控相关联。