高丝氨酸内酯酶AI-96对嗜水气单胞菌NJ-1浸浴攻毒斑马鱼保护效应的研究

为评价斑马鱼口服高丝氨酸内脂酶AI-96对嗜水气单胞菌NJ-1浸浴攻毒的保护效应,实验设置基础料与实验料两组饲料,实验料是在基础料中按3 U/g饲料添加N酰基高丝氨酸内酯酶AI-96(以下简称AI-96),通过高浓度(2.5×108 cfu/mL)及低浓度(0.7×108 cfu/mL)两组剂量的嗜水气单胞菌NJ-1(以下简称NJ-1)分别浸浴攻毒斑马鱼,在12h、24 h、3d、7d和14d取鳃丝、肠道壁、肝和肾样,采用实时荧光定量PCR法检测取样器官中NJ-1量,并统计攻毒周期内的死亡率,来评价高丝氨酸内酯酶AI-96的保护力.结果显示:在攻毒周期内所取组织内均检测到NJ-1,按菌数肠＞鳃＞肝＞肾,其中高NJ-1剂量未加酶组各组织NJ-1数量均分别明显高于低剂量处理组.在高剂量攻毒条件下,加酶组各组织NJ-1数量均显著低于未加酶处理组(P＜0.05),鳃除外;在低剂量攻毒条件下,未加酶组的NJ-1数量在鳃(3 d)、肠(0.5、1、3、7及14 d)、肝(3 d)和肾(7 d)显著高于加酶组(P＜0.05),其余差异不显著(P＞0.05).此外无论在高、低剂量攻毒条件下,加酶组的死亡率均低于未加酶组,其中低剂量攻毒7d及以后其死亡率显著低于对照(P＜0.05).结果表明,口服AI-96可以有效预防NJ-1≤0.7×108 cfu/mL范围内的侵袭.     

甘露寡糖对草鱼感染嗜水气单胞菌后细胞因子表达的调节

在饲料中添加0.2%甘露寡糖(Mannan oligosaccharide,(MOS)),饲养草鱼(Ctenopharyngodon idellus)28 d后,草鱼经腹腔注射3.2×106cell/mL的嗜水气单胞菌(Aeromonas hydrophila,Ah)0.1 mL,在注射嗜水气单胞菌48 h、72 h和96 h后,分别取草鱼头肾、脾脏和肝脏,利用荧光定量PCR分析甘露寡糖对草鱼感染嗜水气单胞菌后各组织中IL-1β和IL-10的表达影响。结果显示,在草鱼头肾中,MOS/I(0.2%甘露寡糖+注射Ah)组IL-1β的表达在注射嗜水气单胞菌后48 h和72 h显著高于CON/I(基础饲料+注射Ah)组;MOS/I组中1L-10的表达在注射嗜水气单胞菌72 h显著高于CON/I组,在96h时显著低于CON/I组。在草鱼脾脏中,MOS/I组IL-1β的表达在注射后72 h和96 h显著低于CON/I组;IL-10的表达在注射嗜水气单胞菌后48 h和96 h显著低于CON/I组。在草鱼肝脏中,MOS/I组IL-1β和IL-10的表达在注射嗜水气单胞菌后48 h显著高于CON/I组,但在注射后72 h时该2种基因的表达显著低于CON/I组。表明甘露寡糖在鱼体感染嗜水气单胞菌时能快速调节头肾、脾脏和肝脏中细胞因子的表达,增强鱼体抵抗细菌性病原的免疫力。     

咪唑类物质KK-42对日本沼虾α2-巨球蛋白基因表达的影响

为探讨KK-42提高感染嗜水气单孢菌的日本沼虾幼虾成活率的机制,实验首次克隆了α2M部分序列,研究了α2M基因的时空表达以及KK-42对其表达和活力的影响。将体长3.5~5.0 cm的日本沼虾幼虾随机分为2组,分别用1.95×10-4mol/L的KK-42溶液和不含KK-42的溶液浸泡处理1 min,12 h后,每组再分为2个亚组,分别向虾腹部注射嗜水气单胞菌菌悬液(菌攻毒实验组和菌攻毒对照组)或生理盐水(实验组-1和对照组-1),于不同时间点测定幼虾的成活率、α2M基因表达水平和活力的变化。结果显示,KK-42预处理可显著提高感染嗜水气单胞菌的日本沼虾幼虾成活率。经BLAST比对,克隆的α2M部分mRNA序列与罗氏沼虾α2M具有90%以上同源性。实时荧光定量PCR实验显示,α2M基因表达水平在血细胞中最高,且以蜕皮前期最为显著。KK-42处理的幼虾血细胞α2M基因的表达无显著性差异;嗜水气单胞菌刺激使血细胞α2M mRNA水平在3 h比0 h提高了580%,其活力在24 h增幅为47.5%;而菌攻毒实验组mRNA水平在6~48 h明显高于菌攻毒对照组,尤其在12 h,增幅为511%,其活力水平也呈同样的变化趋势。研究表明,KK-42预处理能上调感染嗜水气单胞菌的幼虾血细胞α2M基因的转录水平,诱导α2M活力增强,这可能增强了幼虾的免疫水平,从而提高成活率。     

嗜水气单胞菌在浸泡感染团头鲂的组织动态分布

利用具有绿色荧光蛋白基因标记的嗜水气单胞菌(WJ-8G FP)对团头鲂(Megalobrama amblycephala)进行浸泡攻毒试验,探究温度对浸泡感染后嗜水气单胞菌在团头鲂各组织分布的影响。实验设立A组(水温25℃),B组(水温32℃),C组(水温25℃),其中C组为对照组。用菌株WJ-8G FP对实验组A、B进行浸泡攻毒,试验组C不进行攻毒处理,攻毒后分别于2 h、4 h、8 h、12 h、24 h采集各组鱼血液、脾、肾、鳃、肠道、肌肉,培养法统计分析各组织器官上的荧光细菌数量。实验结果显示,在各取样时间段实验组A(25℃)和实验组B(32℃)团头鲂各组织在均能检测到荧光嗜水气单胞菌,对照组未检测到嗜水气单胞菌;最高菌量出现在鳃,且鳃上嗜水气单胞菌数量显著大于其他组织(P0.05),其次是脾、肾;组织内的菌量随时间大体呈现先上升后下降的趋势;B实验组中各组织菌量显著大于A实验组(P0.05)。研究结果表明,鳃是嗜水气单胞菌浸泡感染团头鲂的主要组织器官,与25℃相比较,在水温32℃时团头鲂被嗜水气单胞菌感染的风险更高。     

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。     

团头鲂Toll样受体Ⅱ基因的克隆与表达分析

为了研究团头鲂(Megalobrama amblycephala)TLR2(ma TLR2)在抗嗜水气单胞菌(Aeromonas hydrophila)感染中的作用,本实验克隆了ma TLR2基因的c DNA全长。结果显示:ma TLR2基因c DNA的全长包括2923 bp,编码792个氨基酸。预测得到的ma TLR2的结构域包括一个信号肽、氨基端的亮氨酸重复基序(LRRs)、跨膜结构域(TM)和一个胞内的Toll/白介素(IL)-1受体区(TIR)。在嗜水气单胞菌感染后,团头鲂头肾中,TLR2的表达量在6 h时显著升高。TLR2下游相关的炎症细胞因子TNF-α的表达量也显著上调。结果表明ma TLR2在嗜水气单胞菌感染团头鲂后的免疫应答中起到了重要作用。     

鲤细胞因子多克隆抗体的制备及检测

为从蛋白质水平研究细胞因子在鲤体内免疫应答过程中的合成变化,本研究采用PCR技术克隆TNF-α、IL-1β、IL-6、IL-12、IL-10和TGF-β基因含有部分抗原决定簇的片段,引入双酶切位点Bam HⅠ和HindⅢ后连接至p ET-32a/21a,构建相应的表达载体,制备多克隆抗体。采用ELISA检测抗体效价,并以此作为实验工具,检测经嗜水气单胞菌感染后鲤血清中炎性细胞因子的合成变化。结果显示,基因TNF-α、IL-1β、IL-6、IL-12、IL-10和TGF-β融合蛋白分子量分别约为31.8、31.7、35.3、32.5、18.0和33.6 ku;抗体效价达到2.4×106;在病原菌感染后的不同阶段,促炎细胞因子TNF-α、IL-1β、IL-6、IL-12和抗炎细胞因子IL-10、TGF-β呈现出不同的合成变化。研究表明,制备的抗体具有较高的效价、亲和力和特异性,可用于鲤细胞因子的定量研究,该抗体的获得为鲤免疫应答与细胞因子合成的系统研究奠定了基础。同时,获取的鲤细胞因子TNF-α、IL-1β、IL-6、IL-12、IL-10和TGF-β的抗体亦可用于其他鱼类细胞因子蛋白质水平的定量研究。     

干酪乳杆菌发酵人参茎叶提取物对锦鲫免疫及抗氧化功能的影响

为了研究干酪乳杆菌发酵人参茎叶提取物对锦鲫的免疫及抗氧化功能的影响，先用3%、4%、5%、6%（记为L3、L4、L5、L6）的干酪乳杆菌菌液发酵人参茎叶提取物，再分别添加到基础饲料（记为L0）中，对初始平均体重为（25.00±?0.05）g 的锦鲫进行为期五周的饲喂试验。周期性取样，采集锦鲫血清、肝胰脏、中肾、脾脏、全肠，以检测相关免疫指标的变化。在饲喂试验结束后进行嗜水气单胞菌攻毒保护性试验。结果显示，与L0组相比，L5组碱性磷酸酶（AKP）、免疫球蛋白M（IgM）、溶菌酶（LZM）、超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、谷胱甘肽过氧化物酶（GSH-PX）含量显著升高，L5组丙二醛（MDA）含量显著低于L0组。各组织中IL-10、TNF-α、TGF-β、IFN-γ、IL-1β基因表达水平均有不同程度的升高，表现出时空差异，其中IL-10的基因水平表达升高更为敏感。在攻毒保护性试验中，L5组的存活率达到30%，相较于其他组而言存活率最高。本实验条件下，当干酪乳杆菌的接种量为5%时，人参茎叶提取物发酵对锦鲫的应用效果最好，能够提高锦鲫抗氧化能力及免疫相关基因的表达，并对降低嗜水气单胞菌的感染有较好的预防作用。     

A3α肽聚糖增强灭活嗜水气单胞菌疫苗免疫效果的研究

为了探讨 A_(3α)肽聚糖对灭活嗜水气单胞菌疫苗免疫效果的影响,用添加肽聚糖的饲料投喂注射接 种经甲醛灭活的嗜水气单胞菌菌苗的彭泽鲫,测定鲫鱼白细胞吞噬活性、血清和体表粘液溶菌酶活性、抗体 效价以及活菌攻毒后的免疫保护率。结果表明,肽聚糖与灭活菌苗联合使用,鱼体表粘液和血清溶菌酶活性、 白细胞吞噬活性、抗体效价以及活菌攻毒后的免疫保护率显著提高,表明 A_(3α)肽聚糖可增强灭活菌苗的免疫效 果。     

齐口裂腹鱼HMGB1基因克隆及其对嗜水气单胞菌胁迫的响应

为了解齐口裂腹鱼高迁移率蛋白B1（Schizothorax prenanti high mobility group box 1,SpHMGB1）的序列特征及其与嗜水气单胞菌胁迫的相关性,实验根据齐口裂腹鱼脾脏TSA库中获得的序列设计引物,采用克隆技术克隆SpHMGB1的核心序列并进行生物信息学分析,采用实时荧光定量PCR（qPCR）技术检测嗜水气单胞菌在体和离体胁迫后组织和巨噬细胞中SpHMGB1的响应情况。序列分析结果显示,SpHMGB1开放阅读框长为615 bp,编码204个氨基酸,理论分子量为23.5 ku,等电点为6.79,包含2个基本的HMG盒：A盒和B盒,以及一个酸性尾巴。SpHMGB1二级结构主要由47.06%的α-螺旋和50%的无规则卷曲构成。系统进化树分析显示,SpHMGB1与鱼类HMGB1聚为一支,与斑马鱼和鲫的亲缘关系最近,氨基酸一致性分别为90.7%和90.4%。qPCR检测结果显示,嗜水气单胞菌感染后SpHMGB1具有不同的时空表达趋势,其中血液在72 h表达量最高,肾脏在6 h表达量最高,脾脏在120 h表达量最高;热灭活嗜水气单胞菌刺激巨噬细胞后,SpHMGB1的表达量在0.5~24 h下调,24 h表达量最低,细胞因子IL-1β和TNF-α表达量均有上调趋势。qPCR检测结果提示,SpHMGB1可能参与齐口裂腹鱼细菌感染后的免疫响应。本实验可为进一步研究SpHMGB1在齐口裂腹鱼细菌性疾病中的免疫调节机制提供参考。     

Rare serotype occurrence and PFGE genotypic diversity of Streptococcus agalactiae isolated from tilapia in China

Previously, we reported 10 PEGE types of 85 tilapia Streptococcus agalactiae(GBS), which shifted from Streptococcus iniae in China, by using PEGE method. Presently, larger and more representative tilapia GBS were isolated, for the ?rst time in China, to characterize their serotypes and genetic diversities more precisely than had done before. 168 GBS strains were distributed in ?ve provinces of China, in which Guangdong, Guangxi and Hainan were the major ones, holding36.9%(62/168), 37.5%(63/168) and 19.6%(33/168), respectively. Serotypes, Ia, Ib and III, were observed in these strains and the most predominant one was Ia(95.2%), which mainly distributed in Guangdong, Guangxi and Hainan. Ia initially occurred in 2009, it shoot up to 32.1% in 2010,but decreased to 16.1% in 2011 before went up to 45.2% in 2012. Ib sporadically occurred during2007–2011, III onlyoccurred in 2012. 14 different PFGE types, including 4 new types(N, O,P and Q), were observed, in which B, D, F and G were the predominant types, holding 83.9%(141/168) of the total GBS strains. Ia corresponded to 11 PFGE types(A–H, N–P), in which type D predominated(51%). Ib represented 3 genotypes(I, J and Q) and III harbored only 2genotypes(N and F). Type N and Fsynchronously presented in Ia and III. In summary, the genetic diversity of tilapia GBS varied by serotypes and changed with geographical locations and years.Although Iastillpredominated, new rareserotypeIII alreadyoccurred in China.     

Growth hormone (GH) and reproduction: a review

Interaction between growth and reproduction occurs in many vertebrates and is particularly obvious at certain stages of the life cycle in fish. Endocrine interactions between the gonadotropic axis and the somatotropic axis are described, the potential role of GH being emphasised. A comparative analysis of these phenomena in mammals, amphibians and fish, suggests a specific role of GH in the physiology of puberty, gametogenesis and fertility. It also shows the original contribution made by studies on the fish model in this field of investigations.     

Purification and partial characterization of GtHs (cfLH and cfFSH) from Indian walking catfish (Clarias batrachus) (L.) and development of a homologous ELISA for cfLH

Two gonadotropins (GtH; Qa and Qb) were purified by gel filtration and ion exchange chromatography from the pituitaries of Indian walking catfish (Clarias batrachus). The presence of GtH during purification was assessed by in vitro oocyte maturation and in vivo steroidogenic activity, and their identities were determined by elution profiles, molecular weight, biological activities and yield. The molecular weights of Qa and Qb were 37 and 42 kDa, respectively, and composed of distinct subunits (Qa: 20 and 14 kDa and Qb: 26 and 18 kDa). Polyclonal antibodies raised against Qa immunostained Qa, Qb and pituitary GtH cells. A competitive Qa‐ELISA was developed whose sensitivity was 6.25 ng mL^?1 (1.25 ng well^?1) with intra‐ (3.5%) and inter‐ (12.4%) assay coefficients of variation. Displacement curves parallel to the standard were obtained with plasma and pituitary extracts of catfish, Qb and carp GtHII. The assay was validated by measuring the plasma Qa levels after LHRH treatment and in relation to ovarian growth in the female catfish during different reproductive phases. Based on the results, Qa and Qb corresponded to fish LH and FSH respectively. The findings will increase the knowledge of the mechanisms controlling fish reproduction and identification of sensitive phases in fish in captivity for hormonal manipulation.     

Controlled infection of Poecilia reticulata Peters (guppy) with Tetrahymena by immersion and intraperitoneal injection

Tetrahymena is a protozoan parasite, which infects guppy, Poecilia reticulata Peters, and causes substantial economical losses in commercial farms worldwide. Studies of guppy infected by Tetrahymena require standardized infection protocols. The LD50 for Tetrahymena infection of guppies by intraperitoneal (IP) injection was calibrated, and the level obtained was 946 parasites per fish. Guppy infection with Tetrahymena by immersion, imitating the natural route of infection via the integument, was studied under normal or stress conditions. Exposure to cold and netting (CNI) and to cold only (CI) followed by immersion exposure to 10 000 Tetrahymena per mL resulted in 22.5% and 19.2% mortality, respectively, as compared to 14.2% and 10% in groups that were netted only (NI) or non‐stressed (I). Histopathology revealed that immersion infection resulted in a systemic infection. Lysozyme levels, measured 3 weeks after infection, were significantly higher in the CNI group (288 μg per mg protein) compared with CI‐, NI‐ and I‐treated groups (94.5, 64 and 62.3 μg mg^?1, respectively). There was no evident parasite immobilization activity in body homogenates, suggesting no development of acquired immunity. Re‐infection by IP injection revealed no increase in protection in any of the treatment groups, mortality range of 56.3–75%, higher than in the non‐exposed control (40.6% mortality).     

Imported ornamental fish are colonized with antibiotic‐resistant bacteria

There has been growing concern about the overuse of antibiotics in the ornamental fish industry and its possible effect on the increasing drug resistance in both commensal and pathogenic organisms in these fish. The aim of this study was to carry out an assessment of the diversity of bacteria, including pathogens, in ornamental fish species imported into North America and to assess their antibiotic resistance. Kidney samples were collected from 32 freshwater ornamental fish of various species, which arrived to an importing facility in Portland, Oregon from Colombia, Singapore and Florida. Sixty‐four unique bacterial colonies were isolated and identified by PCR using bacterial 16S primers and DNA sequencing. Multiple isolates were identified as bacteria with potential to cause disease in both fish and humans. The antibiotic resistance profile of each isolate was performed for nine different antibiotics. Among them, cefotaxime (16% resistance among isolates) was the antibiotic associated with more activity, while the least active was tetracycline (77% resistant). Knowing information about the diversity of bacteria in imported ornamental fish, as well as the resistance profiles for the bacteria will be useful in more effectively treating clinical infected fish, and also potential zoonoses in the future.     

Effect of iodophor disinfection of non‐hardened Salmo trutta eggs on their bacterial and fungus load

This study investigated the efficiency of iodophor disinfection (135 ppm active iodine for 15–30 min) of non‐hardened Salmo trutta eggs against different groups of bacteria and against fungus. Egg samples were taken from non‐disinfected and from disinfected eggs, microorganisms were cultured on specific nutrient media and their mass was measured by turbidimetric methods. Bacteria and fungus mass of non‐hardened eggs could be reduced but not eliminated by iodophor disinfection with 135 ppm active iodine for 15 min. The extent of reduction was 47–65% (Experiment 1). The efficiency of disinfection increased with disinfection time as the reduction in bacteria and fungus mass was 40–55% after 15 min and 58–74% after 30 min (Experiment 2). Disinfection efficiency of iodophor solution diluted in water (reduction 49–57%) and of iodophor solution diluted in sodium chloride solution iso‐osmolar to the oocytes (reduction 52–61%) was similar (Experiment 3). The reduction in bacteria and fungus mass was persistent as it was 39–72% lower in embryos deriving from disinfected eggs than in embryos deriving from non‐disinfected ones (Experiment 4). In conclusion, the tested disinfection method is inadequate to eliminate pathogens completely but it could positively influence immune defence of eggs and embryos.     

Nitrogenous waste excretion and accumulation of urea and ammonia inChalcalburnus tarichi (Cyprinidae), endemic to the extremely alkaline Lake Van (Eastern Turkey)

The endemic, anadromous cyprinidChalcalburnus tarichi is the only fish species known to occur in alkaline Lake Van (Eastern Anatolia, Turkey). EightC. tarichi were maintained individually in Lake Van water (17 – 19°C; pH 9.8; 153 mEq·I^–1 total alkalinity; 22 total salinity) and tank water samples analyzed for 24 h in 2 to 4 h intervals. At zero time, < 1µM ammonia was present and urea was undetectable in the tank water; at 24 h, total ammonia and urea made up 114±32 and 35±25µM, respectively. Over the experimental period, ammonia-N and urea-N excretion averaged 1041±494 and 607±169moles·kg^–1 fish·h^–1, respectively. The extent of urea excretion was highly variable between specimens. Uric acid excretion was not detectable.Urea was present at high concentrations in all tissues and plasma (25 – 35moles·g^–1·ml^–1) of freshly caughtC. tarichi; total ammonia content of the tissues was by a factor of 1.9 (liver) to 3.0 (brain) lower. High arginase activity (2.4±0.2 U·min^–1·g^–1) was detected in the liver ofC. tarichi but ornithine carbamoylphosphate transferase, a key enzyme of the ornithine-urea-cycle, was absent. Ureagenesis is likely through degradation of arginine and/or uricolysis. High glutamine synthetase activity (11±0.6 U·min^–1·g^–1) and low ammonia content in brain suggest that, like other teleosts,C. tarichi has an efficient ammonia detoxification in the brain, but in no other tissue.Nitrogenous waste excretion at alkaline pH is discussed. The ability ofC. tarichi to excrete high levels of ammonia at extremely alkaline pH is unique among teleosts studied so far. The mechanism of ammonia excretion under Lake Van conditions remains to be elucidated.     

Influence of the antibacterial herbs, Solanum trilobatum, Andrographis paniculata and Psoralea corylifolia on the survival, growth and bacterial load of Penaeus monodon post larvae

The indiscriminate use of antibiotics and chemicals in shrimp hatcheries has led to biomagnification and that in turn could lead to rejection of a whole consignment. The application of the bioencapsulation technique as a tool for curative treatment in shrimp larvae was investigated. Herbs having antibacterial properties such as Solanum trilobatum, Andrographis paniculata and Psoralea corylifolia (methanolic extracts) were bioencapsulated in Artemia and fed to Penaeus monodon post larvae PL 1–25. The post larvae were reared in a medium inoculated with pathogenic bacteria such as Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhi and Vibrio sp. Post larvae reared in the non-inoculated water and fed with non-enriched Artemia exhibited 90% survival, highest specific growth rate (12.43%) and reduced bacterial load. P. monodon reared in the bacterial inoculated water and fed with the non-enriched Artemia exhibited the lowest survival (10–30%), specific growth rate (8.42–9.1%) and increased bacterial load (2.86 × 10³ to 3.76 × 10⁵ cfu/g). The methanolic extracts of the herbs helped to increase survival and specific growth rate and reduced bacterial load in the P. monodon culture system. Among the three herbal extracts, P. corylifolia enriched Artemia fed post larvae showed the tendency to higher survival (>50%), growth rate (11.5 averaged) and low bacterial load (1.12 × 10⁵ cfu/g). This revised version was published online in August 2006 with corrections to the Cover Date.