一株致病性溶藻弧菌的多重耐药与毒力基因分子分析

从发病的凡纳滨对虾中分离并经鉴定得到一株溶藻弧菌(命名为:V_A-76),通过对虾回感实验和药物敏感性实验分别对V_A-76菌株的耐药表型和致病能力进行了研究,并运用PCR方法检测了该菌的毒力相关基因、整合子和耐药基因的携带情况。对虾感染实验结果表明,溶藻弧菌V_A-76能够导致凡纳滨对虾发病死亡。经PCR检测发现,该菌携带8种毒力相关基因:tdh、tlh、tox S、collagenase、fla A、omp W、asp A和fur。药敏结果显示,V_A-76对环丙沙星、氯霉素、链霉素、红霉素、磺胺甲噁唑、复方新诺明和利福平7种药物表现出较高的耐药水平,且该菌包含1类整合子、4类超级整合子SXT和1型插入序列共同区(ISCR1)。此外,该菌携带arr-2、dfr A27、str A、str B、floR、cat和qnrv C耐药相关基因。从上述研究结果可知,菌株V_A-76既具有一定的感染能力,同时对多种抗菌药物表现出较高的耐药水平,提示应对养殖源致病性弧菌的耐药性现状予以更多的关注。     

副溶血弧菌养殖对虾分离株耐药性及耐药基因分析

文章采用琼脂稀释法检测从养殖患病对虾中分离的36株副溶血弧菌(Vibrio parahaemolyticus)对16种药物的耐药性,并用PCR法检测喹诺酮类耐药基因qnrA、qnrB、qnrS和qnrVC,酰胺醇类耐药基因cat、optrA、floR和cfr,四环霉素类耐药基因tetA、tetB和tetM,磺胺类耐药基因sul1、sul2和sul3,氨基糖苷类耐药基因strA、strB、aadA和aacA,利福霉素类耐药基因arr,β-内酰胺类耐药基因bla CARB和大环内酯类耐药基因erm的携带状况,分析其耐药表型和基因型之间的相关性。结果显示,36株副溶血弧菌对β-内酰胺类药物氨苄西林耐药率最高(88.9%),其次为磺胺类药物磺胺甲噁唑(66.7%),硫酸新霉素、庆大霉素、头孢曲松和美罗培南呈现100%敏感。多重耐药副溶血弧菌比例高达61.1%(22/36),其中1株对6类抗菌药耐药。喹诺酮类耐药基因qnrVC检出率最高达72.2%(26/36);其次为氨基糖苷类耐药基因srtB,检出率58.3%(21/36);大环内酯类、利福霉素类耐药基因均未检测到。耐药基因检出率与耐药表型没有表现出一一对应的关系,提示副溶血弧菌耐药的复杂性。     

海南地区溶藻弧菌耐药性及其质粒多样性的研究

调查了海南省的30株溶藻弧菌环境菌株对24种常见抗生素的耐药性,并对菌株携带质粒及其多样性进行了研究,所有菌株对洁霉素、乙酰螺旋霉素、万古霉素、氯洁霉素、制霉菌素5种抗生素完全耐药,对24种抗生素的耐药率为20.8%～66.7%,表明该地区溶藻弧菌具有严重的多重耐药性;30株溶藻弧菌中有22株不携带质粒,8株携带1～3个质粒,质粒为1.20～12.5kb;8株携带质粒的菌株共形成了7种类型的质粒指纹图谱。试验结果表明,该地区溶藻弧菌的耐药性与其是否携带质粒无明显关系。分离菌株的耐药性可能主要是由染色体相关基因引起的。     

不同环境条件对溶藻弧菌粘附大黄鱼肠粘液的影响

研究了不同环境条件下溶藻弧菌对大黄鱼肠粘液的粘附作用。采用细菌计数法测定溶藻弧菌的粘附作用。粘附的细菌经SYBR GreenⅠ染色后在荧光显微镜下观察,用数码相机拍照后在电脑上计数。试验结果表明,溶藻弧菌对大黄鱼肠粘液的粘附量随菌浓度的升高而升高,并在1—1．5h内趋于饱和;粘附作用在15—30℃、pH偏酸时较强;盐度在5—35范围内对前肠粘液的粘附作用影响不明显,后肠粘液的粘附作用在此范围内随盐度增大而加强,在盐度为0时,溶藻弧菌对前、后肠粘液都无粘附作用;56℃热处理5min及60℃处理1h均能大幅减弱溶藻弧菌对两种肠粘液的粘附作用,表明溶藻弧菌表面的某些热敏结构在粘附作用中起着重要作用。根据以上结果,可以认为溶藻弧菌能够很好地粘附于大黄鱼肠粘液层,其粘附作用受温度、盐度、pH值等环境因子影响很大,溶藻弧菌表面的某些热敏结构可能在粘附过程中起着重要作用。研究结果表明,溶藻弧菌对大黄鱼肠粘液的粘附作用是可控制的,这对于鱼类养殖疾病的控制有一定的参考价值。     

恩诺沙星诱导溶藻弧菌耐药和交叉耐药的研究

自收集于海南海水养殖区域内的溶藻弧菌中筛选恩诺沙星敏感株,在含亚抑菌质量浓度恩诺沙星的培养基上进行质量浓度递增法体外诱导,探究耐药性的发生状况,比较诱导耐药前后诱导菌对诱导药物和6种非诱导药物的最小抑菌质量浓度,并在不含药物的培养基上连续传代考察诱导后的耐药株遗传稳定性.试验结果表明,30株养殖源溶藻弧菌中筛选出24株...     

养殖大黄鱼低温弧菌病病原的研究

浙江沿海深水网箱养殖大黄鱼冬天发生大量死亡，从病鱼体内分离出致病力较强的细菌GYC0227。人工感染试验证实这株菌为大黄鱼的病原菌。对细菌进行了形态、生理生化特性的测定，并进行了16S rRNA分子鉴定。PCR扩增获得了GYC0227菌株大小约1．5kb的16S rDNA部分基因片段，产物经回收纯化，克隆到pMD18-T载体，转化大肠杆菌TG1，对阳性克隆子进行酶切及PCR鉴定，并测序，将测定的序列递交NCBI进行BLAET同源序列比对，与溶藻弧菌的标准菌株的16S rDNA具有96％的同源性。结合菌株的生理生化特性，GYCD227菌株属于溶藻弧菌。该菌适应于低温生长，生长温度范围为7—25℃，并测定了其对14种药物的敏感性。     

养殖环境及贝源溶藻弧菌MLST分型及其毒力基因、耐药性分析

溶藻弧菌(Vibrio alginolyticus)是近年来贝类病害中最常见的细菌性病原之一,对贝类养殖产业的健康发展构成严重威胁。本研究旨在分析不同养殖环境水体和贝类组织中,溶藻弧菌的基因变异、毒力基因、耐药性及其分布规律。对12株溶藻弧菌分离株开展多位点序列分型(multilocus sequence typing, MLST)、毒力因子以及菌株耐药性分析,结果显示,12株溶藻弧菌的序列型(sequence typing, ST)分型互不相同,7株为PubMLST数据库已经收录的ST型,5株因管家基因的等位基因位点变化而形成新的ST型,贝类养殖环境中的溶藻弧菌具有较高的遗传多样性。12株溶藻弧菌都携带tlh、fur和collagenase三种毒力基因,但均未检测到tdh、trh、toxR和tcpA毒力基因。溶藻弧菌携带毒力因子的种类和数量受地区分布等因素的影响。不同来源的溶藻弧菌均具有多重耐药特征,对青霉素和氨苄西林产生抗性。本研究表明,贝类养殖环境中的溶藻弧菌具有种群复杂、遗传多样性高的特点;不同来源菌株在毒力基因携带和耐药性方面存在较大差异。本研究通过探究不同区域内不同来源的溶藻弧菌遗传变异及耐药性差异,对贝源溶藻弧菌的有效防控提供一定理论参考。     

人工感染溶藻弧菌对大黄鱼免疫功能的影响

为了解大黄鱼在抗溶藻弧菌感染时免疫功能的变化规律,将160尾健康大黄鱼分为感染组和对照组,通过腹腔分别注射0.2 mL浓度为2×107 CFU·mL-1的溶藻弧菌和灭菌生理盐水,在注射0,1,3,7,11,15,19 d后从两组各取6尾大黄鱼,尾静脉取血,进行外周血的血相、NBT阳性细胞数、血清抗菌活力、抗体效价等免疫学指标的测定。结果显示：在人工感染的初期,感染组大黄鱼外周血的红细胞、白细胞、淋巴细胞和NBT阳性细胞的数量及血清抗菌活力等指标均较对照组有显著提高;在注射后1 d外周血粒细胞的数量显著低于对照组;抗体效价在7 d开始增加,15 d达到峰值,且用间接ELISA和试管凝集两种方法所得结果具有非常高的同步性。结果表明：在感染溶藻弧菌后,大黄鱼能通过红细胞和白细胞增殖、释放抗菌物质、产生特异性抗体等方式提高其对溶藻弧菌的免疫力;在感染的早期阶段非特异性免疫因子起主要作用,在感染后期阶段特异性免疫因子起重要作用;NBT阳性细胞数可以作为细菌感染的指标。图2表1参24     

致病性溶藻弧菌生物膜形成特性研究

采用改良的微孔板法研究静置培养条件下致病性溶藻弧菌ND-01在酶标板上成膜情况。结果显示,溶藻弧菌的生物膜的OD590在16h达到峰值后,随培养时间延长出现下降;在102～108cfu/mL范围内,不同的初始菌密度对生物膜的OD590影响并不显著;NaCl质量分数为4.5%时,所形成生物膜的OD590最高;30℃,溶藻弧菌的生物膜OD590显著高于其他温度组(P0.05);在胰蛋白胨大豆肉汤培养液中加入质量分数1.5%的CaCl2,能够显著促进生物膜形成,而加入MgCl2对于生物膜的影响则不显著;经过大黄鱼表皮黏液包被后,96孔板中生物膜的OD590显著高于其他部位组织提取液包被组(P0.01)。致病性溶藻弧菌ND-01能够在聚苯乙烯材料的96孔酶标板表面形成稳定而明显的生物膜,且其生物膜的形成与多种环境因素有着密切关系。     

大菱鲆致病性溶藻弧菌SR1的外膜蛋白及其抗原性分析

用十二烷基肌氨酸钠(Sarkosyl)抽提结合超速离心的方法提取了一株大菱鲆致病性溶藻弧菌(Vibrio alginolyticus)SR1和其他7株弧菌的外膜蛋白。通过SDS-PAGE图谱分析比较了这8株弧菌外膜蛋白的组成,结果表明,8株弧菌的外膜蛋白电泳一般可得到6-12条条带,其分子量多集中在65-106 kD和28-48 kD,其中36 kD的蛋白带为8株弧菌所共有。用兔抗SR1全菌血清进行Western-blot印迹显示,菌株SR1的外膜蛋白条带中有6条发生了阳性反应,其分子量分别为73 kD、48 kD4、5 kD3、9 kD、36 kD和32 kD。而其他7株弧菌的外膜蛋白与兔抗SR1血清也发生程度不等的阳性反应,这些阳性反应条带的分子量集中在65-73 kD、45-48 kD和36-41 kD之间,其中36 kD的外膜蛋白在8株弧菌中均出现明显的阳性反应,说明36 kD的外膜蛋白是这8株弧菌共有的特异性抗原。     

Oral bovine serum albumin immune‐stimulating complexes improve the immune responses and resistance of large yellow croaker (Pseudosciaena crocea,Richardson, 1846) against Vibrio alginolyticus

This study aimed to investigate effects of bovine serum albumin immune‐stimulating complexes (BSA ISCOMs) on immune‐related genes expression, serum nonspecific immunity and disease resistance of large yellow croaker (Pseudosciaena crocea). Fish were fed diets containing 3.5 ml of BSA ISCOMs per kg feed (experimental group) or 3.5 ml of phosphate‐buffered saline per kg feed (control group) for 1 week. The liver, spleen, head‐kidney tissues were sampled for determining gene expression of myxovirus‐resistant protein (Mx), major histocompatibility complex class II alpha chain (MHC II α), tumour necrosis factor‐alpha (TNF‐α) and interleukin‐10 (IL‐10) 30 and 90 days after feeding. Also, blood samples were collected for determining activities of serum superoxide dismutase (SOD), interferon alpha (IFN‐α), TNF‐α and alkaline phosphatase (ALP). TNF‐α and MHCⅡα gene expression in the liver, spleen, head‐kidney, as well as IFN‐α, TNF‐α and ALP activities in the serum, of experimental fish were significantly higher 30 days after feeding; while only TNF‐α and MHC II gene expression in the head‐kidney remained upregulated 90 days after feeding. The cumulative mortality of the experimental fish was significantly lower than control. This study indicated that BSA ISCOMs improved the immune response and induced protective immunity in large yellow croaker.     

大黄鱼微卫星标记的富集与筛选

根据生物素与链霉亲和素的亲和原理，采用生物素-磁珠富集微卫星，与传统放射性同位素杂交法相结合，构建筛选大黄鱼的微卫星文库。用生物素-微卫星捕捉单链限制性酶切片段（含有接头和大黄鱼微卫星序列），经PCR扩增单链目的片段形成双链，然后连接至T载体上，转化感受态细胞。将移至硝酸纤维素膜的重组菌用^32P标记的放射性同位素探针50[γ-^32P]ATP（CA）15筛选出阳性克隆菌。测序结果发现，阳性克隆率为71.9％，105个微卫星位点。其中选取设计合成30对并筛选出22对可用引物。说明所建大黄鱼微卫星文库是一个高质量的文库，可为大黄鱼基因组结构分析、大黄鱼精密微卫星连锁图谱构建、分子进化和系统发育研究、分子标记辅助育种以及经济性状的QTL定位提供大量的微卫星标记。     

A selective and differential medium for Vibrio alginolyticus

In this paper, we present a selective and differential medium, termed Vibrio alginolyticus (VAL) agar, developed for the isolation and identification of V. alginolyticus. The presence of bile salts, high salinity and high incubation temperature allows the selective growth of moderately halophilic Vibrio species. Differentiation of bacteria is achieved by identifying species capable of sucrose fermentation, made visible by the pH indicator bromocresol purple. In this study, all of the 26 strains of V. alginolyticus and only three of the 99 strains representing 30 species (including 19 Vibrio species) other than V. alginolyticus were able to grow in the VAL medium. The remaining three strains could be further differentiated from V. alginolyticus according to colour or the diameter of colonies produced on VAL agar plates. Colonies isolated from shellfish rearing water and infected shrimp through the use of VAL agar plates were all positively identified as V. alginolyticus by conventional tests and 16S rDNA sequencing. The testing of specificity and differentiation capability of VAL shows the potential of the agar as a medium for the primary isolation of V. alginolyticus from pathological and environmental samples.     

Evaluation of Enteromorpha prolifera as a feed component in large yellow croaker (Pseudosciaena crocea,Richardson, 1846) diets

A 9‐week feeding trial was conducted to investigate the effects of dietary Enteromorpha prolifera on the growth performance and body composition of juvenile large yellow croaker (Pseudosciaena crocea) (Richardson, 1846) (11.41 ± 1.59 g) in floating sea cages (1.5 × 1.5 × 2.0 m). Four isonitrogenous and isoenergetic diets were formulated to contain graded levels (0%, 5%, 10% and 15%) of E. prolifera. Survival ranged from 98.7% to 99.7%, and was independent of dietary treatment (P>0.05). There were no significant differences in the feeding rate among dietary treatments (P>0.05). The specific growth rate (SGR) increased with increasing levels of E. prolifera. When the supplementation of E. prolifera was >5%, SGR was significantly higher compared with the control group (0%). The feed efficiency ratio (FER) in fish fed the diet with 5%E. prolifera (diet 2) was higher than that of the other groups, while in fish fed the diet with 10%E. prolifera (diet 3), it was the lowest (P<0.05). The protein retention (PR) decreased as the level of E. prolifera increased in diets (5%, 10% and 15%). The protein body content displayed a trend similar to that of PR. No significant difference was observed in body moisture and ash among the dietary treatments. An increase in minerals of potassium, magnesium and sodium in body was observed with an increase in dietary seaweed concentrations. On basis of the SGR and FER, supplementation levels of E. prolifera can reach at least 15% without affecting the growth and still maintain a high survival rate for juvenile large yellow croaker.     

Effects of dietary lutein/canthaxanthin ratio on the growth and pigmentation of large yellow croaker Larimichthys croceus

This study was conducted to investigate the effects of dietary lutein/canthaxanthin ratio on the growth and skin coloration of large yellow croaker. Five carotenoids supplemented diets were formulated to contain 75/0, 50/25, 37.5/37.5, 25/50 and 0/75 mg kg^?1 of lutein/canthaxanthin. The diet without carotenoids supplementation was used as the control. Fish of the similar size (13.83 ± 0.04 g) were fed with these experimental diets for 8 weeks in sea cages. Results showed that there were no significant differences in survival rate, specific growth rate and feed conversion ratio among the all treatments (P > 0.05). The ventral skin lightness was not affected by dietary treatments (P > 0.05). However, the dorsal skin lightness in the treatment of control was significantly lower than those in the treatments with supplemented dietary carotenoids (P < 0.05). The lowest values of yellowness, redness and carotenoid content both in ventral and dorsal skin were found in the control. Yellowness and carotenoid content both in ventral skin and in dorsal skin decreased with the decreasing of the proportion of dietary lutein. Meanwhile, the redness increased with the increasing of the proportion of dietary canthaxanthin. Fish fed with the control diet had higher melanin content in the dorsal skin, although no significant differences were found. Coloration parameters were linearly related to the carotenoid content in skin. Meanwhile, yellowness, redness and carotenoid content were linearly related to the proportion of dietary lutein. In conclusion, under present conditions, both lutein and canthaxanthin are needed in the diet for large yellow croaker. Compared to the lutein, higher dietary canthaxanthin contents are better for the skin redness.     

Isolation and characterization of pathogenic Vibrio alginolyticus from diseased postlarval abalone, Haliotis diversicolor supertexta (Lischke)

Outbreaks of serious mortality among cultured abalone postlarvae have occurred across Southern China since July 2002. Five motile bacterial strains were isolated from diseased abalone postlarvae on tryptic soy agar supplemented with 1% NaCl (TSA1) and/or thiosulphate citrate bile salt (TCBS) sucrose agar plates during an outbreak in August 2003 in Shanwei, Guangdong province. All isolates were characterized and identified as Vibrio alginolyticus on the basis of biochemical characteristics and comparisons with those of the reference strain V. alginolyticus ATCC 17749. Strain 19 (a representative of five similar isolates) was virulent to abalone postlarvae with an LD₅₀ value of1.00 × 10⁴ colony‐forming units mL^?1. All abalone postlarvae exhibited the same signs as in natural outbreaks. The same bacterium could be re‐isolated from abalone postlarvae after bacterial challenge using TSA1 and TCBS plates. The results reveal that V. alginolyticus is an infectious agent of abalone postlarvae.