拟态弧菌的绿色荧光蛋白标记及其在感染草鱼体内的动态分布

为了示踪研究拟态弧菌感染草鱼的动态过程,将增强型绿色荧光蛋白编码基因EGFP克隆至质粒pBAD24,并转化到拟态弧菌04-14菌株构建荧光标记重组菌.重组菌经阿拉伯糖诱导后,能高效表达EGFP蛋白;荧光显微镜观察和流式细胞仪检测均发现重组菌能够发出明显的绿色荧光信号,且传至30代后质粒稳定率仍为100％;生物学特性检测结果显示,与野生株相比,重组菌的形态、生长特性和细胞黏附性均未发生明显改变.用标记重组菌浸泡感染草鱼,定点采集鳃、肠道、肌肉、头肾、脾脏和肝脏,借助荧光信号检测4d内细菌在不同组织脏器中的动态分布.结果发现感染4h后即可在肠道和鳃中检测到绿色荧光信号,标记菌检出量分别为3.60×108和2.36×106 CFU/g,直至10 h,其含量无明显变化,12 h后含菌量逐渐下降,但持续存在直至鱼死亡.标记菌在肌肉、头肾、脾脏和肝脏中呈现相似的动力学,感染24 h后才检测到荧光信号,24～ 85 h时间段含菌量呈现先增加后下降的变化,48 h达到峰值,检出量分别为9.58×104(肌肉)、8.75×104(头肾)、1.50×104(脾脏)和4.50×104 CFU/g(肝脏),但均低于肠道中的检出量,结果表明肠道是拟态弧菌黏附定植与繁殖的主要靶器官.     

草鱼对~3H—葡萄糖标记荧光极毛杆菌灭活菌苗的吸收效果

荧光极毛杆菌能利用~3H—葡萄糖达到同位素标记效果,在灭活标记菌苗的浸泡免疫试验中,包括大于0.45μm规格的各类标记抗原均能在浸泡后30分钟从草鱼肝组织中探测到比仪器本底值高约6～12倍的cpm值。莨菪佐剂能明显促进草鱼对灭活标记菌苗的吸收,浸泡后30分钟及90分钟标记菌苗在鱼体各组织器官分布的顺序为:肝>脾>前肠>肾>血液>肌肉;肌肉>肾>肝>前肠>血液。     

鳗弧菌铁吸收系统的毒力作用研究进展

鳗弧菌(Vibrio anguillarum)是鱼类弧菌病的主要病原菌,是具有极生鞭毛[1]、弯曲杆状的革兰氏阴性细菌,可经消化道、鳃及皮肤侵入鱼体[2],导致多种海水鱼(如牙鲆、鲈鱼等)和淡水鱼(例如鳗鲡等)的死亡[3]。在长期的进化过程中,鳗弧菌发展出特有的铁吸收系统(iron uptake sys-tem     

拟态弧菌感染黄颡鱼的动态病理损伤及病原分布研究

以1.0×106CFU·m L~(-1)拟态弧菌(Vibrio mimicus)浸浴感染黄颡鱼(Pelteobagrus fulvidraco),于第0、第4、第8、第16、第24、第36、第48、第60、第72小时剖杀取样,研究病理损伤及病原菌的体内动态分布。结果显示,皮肤肌肉、鳃、肠道出现病变最早,其中皮肤肌肉损伤最严重。第16小时表皮变性、坏死,鳃上皮肿胀,肠绒毛水肿;第24~第48小时表皮坏死、脱落,真皮严重出血,肌纤维变性、坏死,鳃出血,上皮局部坏死,肠上皮变性与灶性坏死;第48~第72小时皮肤肌肉坏死更严重,形成溃疡灶,鳃灶性坏死。肾、肝、脾与心36 h后相继出现不同程度的淤血、出血,变性和灶性坏死。q PCR检测发现,第4小时即在鳃、肠、皮肤肌肉检出病菌,细菌含量分别为1.3×102CFU·mg~(-1)、2.6×102CFU·mg~(-1)、4.7×102CFU·mg~(-1);鳃与皮肤肌肉中菌量随时间延长而增加,第72小时皮肤肌肉中菌量达到4.5×107CFU·mg~(-1);第24小时后相继在肾、肝、脾、心检出病菌,菌量介于1.9×10~4.7×102CFU·mg~(-1)之间。结果表明皮肤、鳃和肠道是病菌的入侵位点,并在体内多组织、器官分布。     

富锶水体处理对大黄鱼耳石和肌体中锶含量的影响

利用富锶水体处理的大黄鱼(Larimichthy scrocea)耳石元素标记是小规格鱼体规模化标记的一种技术手段,而标记完成后鱼体中的锶残留量是否符合食品食用安全是增殖放流评估中重要一环。通过开展富锶水体对大黄鱼鱼苗的浸泡标记实验,探究受试鱼苗耳石Sr/Ca值对富锶水体标记过程的响应方式,研究耳石元素指纹标记稳定性,分析标记过程对受试鱼苗肌体和主要脏器中锶含量的影响。结果表明:经为期7d、锶浓度含量3倍于天然海水的富锶水体的浸泡标记,受试鱼苗在特定耳石区段的Sr/Ca值显著增大,形成了与对照组个体在该耳石区段Sr/Ca值上的极显著差异(P<0.01);该差异不随时间的推移而减弱,可视做上述处理过程所产生的耳石元素指纹标识。浸泡标记处理后,受试鱼苗肌肉、鳃、肝脏和肾中的锶含量均显著高于对照组(P<0.05)。但随着时间的推移,锶含量逐渐下降;在天然海水中暂养59d后受试鱼苗肌肉和上述脏器中的锶含量均可衰减至对照组的水平。     

鱼类传染病“一看准”

1 肠道病感官鉴别 1．1 出血病 肌肉、口腔、各种鳍条的基部充血,尤以臀鳍为甚。剥去皮肤可见肌肉点状出血,严重病鱼肌肉全部发红。有时可见鱼体发红,不用剥开,就可判断。有时鳃盖、大颚、眼圈、肠道也有充血现象,鳃失去鲜红色或呈苍白色。     

长丰鲫c型溶菌酶基因的克隆及其在细菌感染条件下的表达分析

为了解c型溶菌酶基因与长丰鲫(Chang Feng Carassius auratus)的抗菌效应关系,本研究利用同源克隆方法获得长丰鲫c型溶菌酶基因cDNA全长序列。结果显示:c型溶菌酶基因全长698 bp,包括5'端非翻译区60 bp,3'端非翻译区200 bp,开放阅读框438 bp,编码145个氨基酸。长丰鲫c型溶菌酶基因在肾脏组织中表达量最大,在脾脏、肠道、心脏和脑中大量表达,在肝脏和鳃中表达量相对较低,在皮肤和肌肉中几乎不表达。长丰鲫在感染迟钝爱德华氏菌、嗜水气单胞菌和金黄色葡萄球菌后,在肝脏、肾脏、脾脏和鳃等组织中基因表达量均发生显著变化,出现不同程度的上调。感染迟钝爱德华氏菌后,在脾脏中的上调幅度最大,其次为鳃、肝脏和肾脏;感染嗜水气单胞菌后,在脾脏中上调程度最大,其次是鳃;感染金黄色葡萄球菌后,在脾脏中上调幅度最大。在肝脏和肾脏中,经金黄色葡萄球菌刺激后c型溶菌酶基因的上调幅度最高;在脾脏和鳃中,经嗜水气单胞菌感染后c型溶菌酶基因上调幅度最高。三种刺激后,c型溶菌酶基因升高幅度的不同,说明不同刺激使鱼体组织产生的应激反应能力不同。     

污水对鱼类的影响

根据研究,认为污水主要通过鳃部直接侵入鱼体。鱼类依靠鳃与周围的水和气体进行交换,同时鳃又与血管连接在一起。因此,毒物经鳃而侵入到血液中。毒物被吸收到鱼体之后,给予组织器官以各种影响。浓度大的毒物能严重地损伤鳃部,使粘液崩散,并使鳃明显地变色。例如,氰酸钾可使鳃变为鲜红色,氯化汞使鳃呈灰白色,浓度大的硫酸锢使鳃呈绿色,强酸可使鳃呈黑褐色。     

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。     

军曹鱼黏膜免疫组织发育的形态学研究

运用组织学及组织化学的染色方法,对出膜后第l-44天（days post-hatch,dph）的军曹鱼（Rachycentron canadum）黏膜免疫组织的发育过程进行研究。结果显示,白细胞在黏膜免疫组织出现的时间顺序由早到晚依次为鳃、皮肤、胃、肠,同大量抗原接触鱼体组织时白细胞出现的时间顺序相符。军曹鱼胃固有层中丰富的白细胞说明胃可能在黏膜免疫中发挥着很大的作用。黏液细胞最早出现在胃;其后是皮肤和肠道;鳃最晚。在胃肠、皮肤内的黏液细胞几天后就可分泌中性至酸性黏液物质,且分泌量随鱼体分发育而增多,但在鳃组织内,黏液的分泌早于黏液细胞的出现。在采样的时间段里,黏膜免疫组织未发现浆细胞,可能是由于B细胞还没有发育成熟并具备免疫功能。从白细胞在军曹鱼黏膜组织内发育及分布的状况,可见黏膜组织具有完成免疫应答的细胞基础。     

In vitro and in vivo passage of bacteria across restricted areas and isolated tissues of trout, Salmo trutta L. and S. gairdneri Richardson

Abstract. Isolated areas of the skin and gills of rainbow and brown trout were subjected to hyperosmotic infiltration with E. coli both in vitro and in vivo and examined for the passage of bacteria. Bacteria were recovered from the isolated gill arches only after treatment with balanced salt solution at x 10 concentration, followed by the bacterial suspension. All the skin flap experiments were negative. Bacteria were recovered from the blood offish when that part of the animal anterior to the pectoral fins was exposed to the infiltrating bacteria. They were only recovered when the animal was exposed to the full hyperosmotic infiltration schedule. The evidence strongly supports the view that the bacteria enter the animal through the gills.     

Specific immune response in mucosal and systemic compartments of Cirrhinus mrigala vaccinated against Edwardsiella tarda: In vivo kinetics using different antigen delivery routes

Mucosal immune barriers confer protection against invading fish pathogens. Here, we conducted an experiment for 60 days to assess the mucosal and systemic immune response in Mrigal (Cirrhinus mrigala), an Indian major carp. Fish were immunized with inactivated Edwardsiella tarda by four different routes, namely, oral, immersion, injection, and anal intubation. An indirect enzyme‐linked immunosorbent assay (ELISA) was used to measure the specific immune response (antibody) in serum and mucus (collected from skin, gill, and gut) of the fish on 0, 15, 30, 45, and 60 days postimmunization. For specific immune response in the serum, significantly higher (p < 0.05) optical density (OD) values were obtained in the anal group (0.52 ± 0.03) and in the oral group (0.48 ± 0.03). In the skin mucus, significantly higher OD values were obtained in the oral group (0.48 ± 0.04) and immersion group (0.32 ± 0.03). In the gill mucus, significantly higher OD values were obtained in the oral group (0.82 ± 0.08) and the immersion group (0.73 ± 0.03). In the gut mucus, significantly higher OD values were obtained in the immersion group (0.080 ± 0.007) compared to the rest of the treatments. Fish from all the groups were challenged with LD₅₀ dose of E. tarda at the end of the experiment. We conclude that oral and immersion immunization routes offer better protection of C. mrigala compared to other antigen delivery routes.     

哈维氏弧菌浸浴后凡纳滨对虾肠道组织病理变化及转录水平的免疫应答

肠道是弧菌进入对虾机体的有效手段,因此弧菌攻毒下的对虾肠道组织的形态及免疫反应对对虾疾病防控至关重要。本文研究了凡纳滨对虾肠道在不同浓度哈氏弧菌的浸浴攻毒作用下的组织形态及免疫相关基因的表达水平。通过组织切片探究对虾肠道组织形态在攻毒过程中的动态变化,采用实时荧光定量PCR测定抗脂多糖因子基因(ALF)、对虾素基因(Pen-4c)和Crustin基因(Cru)以及溶菌酶基因(LZM)、脂肪酸结合蛋白基因(Fabp)的表达水平变化,并对肠道及水体中的弧菌数目进行监测。结果表明,随着弧菌浓度的提高和攻毒时间的延长,中肠组织损伤情况逐渐加重,出现上皮细胞形态改变直至溃散,黏膜褶皱形态发生皱缩及脱落,肌层发生损伤。攻毒后40 h内,对虾肠道组织形态持续恶化,未见组织修复。肠道受到哈氏弧菌侵染后8~16 h,ALF、Pen、Cru及 LZM表达量能够迅速上调,Fabp表达量未显著增加。攻毒后,三种攻毒剂量下弧菌数目均处下降趋势,弧菌在0-8h经口大量进入对虾肠道,至32h菌数回落。     

The ontogenetic development of the reticulo-endothelial system in the rainbow trout, Salmo gairdneri Richardson

Abstract The phagocytosis of colloidal carbon in the adults and fry of Salmo gairdneri Richardson was investigated. Adult fish accumulated trapped carbon in the spleen, kidney and heart. Fry, as early as 4 days post-hatch, had an efficient phagocytic system, the carbon being engulfed by free macrophages which accumulated in the connective tissue, under the skin, in the gut and in the gills. By 18 days post-hatch, the situation was similar to that found in the adult. The trapping of carbon in the gills of young fry is discussed, and it is suggested that it acts as a special mechanism to protect the thymus from undesirable, and possible tolerigizing antigen exposure before immunocompetence is attained.     

The ontogenetic development of the reticulo-endothelial system in the rainbow trout, Salmo gairdneri Richardson

Abstract. The phagocytosis of colloidal carbon in the adults and fry of Salmo gairdneri Richardson was investigated. Adult fish accumulated trapped carbon in the spleen, kidney and heart. Fry, as early as 4 days post-hatch, had an efficient phagocytic system, the carbon being engulfed by free macrophages which accumulated in the connective tissue, under the skin, in the gut and in the gills. By 18 days post-hatch the situation was similar to that found in the adult. The trapping of carbon in the gills of young fry is discussed, and it is suggested that it acts as a special mechanism to protect the thymus from undesirable, and possible tolerigizing antigen exposure, before immunocompetence is attained.     

In vivo fish models for visualizing Aeromonas hydrophila invasion pathway using GFP as a biomarker

The invasion pathway of Aeromonas hydrophila in vivo was studied in Crucian carp (Carassius auratus gibelio) with a virulent strain A. hydrophila J-1 transformed with a plasmid encoding green fluorescent protein (pGFPuv) (A. h J-1^GFP), which had similar virulence characteristics (haemolysin, extracellular proteases production, toxicity for EPC cells, survival in fish serum and LD₅₀ value) as the parent strain. Fish were divided into four experimental groups: (1) normal fish; (2) bacteria bath challenged, unwounded fish; (3) skin artificially wounded by scalpel, bacterial bath challenged fish; and (4) skin mucus layer partially removed by paper towel, bacterial bath challenged fish. The number of bacteria from blood, gills, kidney, muscle, liver and intestine were detected at 2, 4, 8, 12, and 24 h post-challenge. High bacterial numbers were observed in the muscle of the artificial wound group, in the kidney of the mucus removed group and in the gills of all groups. In conclusion, the gills and damaged skin are likely to be the main routes of entry for A. hydrophila, and GFP can be used as a real-time biomarker to study intimate host–pathogen interaction in fish.     

Use of probiotic bacteria in the rearing of Senegalese sole (Solea senegalensis) larvae

Three candidate probiotics, which had shown antimicrobial activity in vitro against two fish pathogens, were used in the rearing of Senegalese sole larvae and postlarvae. These probiotics improved the survival of starved sole yolk‐sac larvae. A feeding experiment of sole larvae and postlarvae comprised three treatments: A, B and C. Cultures of a Vibrio sp. (2J18) were added to treatment A, whereas a gram‐positive (J84) and a Shewanella sp. strain (2J27), were added to treatment B, while in a control treatment C no bacteria were added. Addition of bacteria in treatment B increased survival of larvae in the first phase of the experiment [0–20 days after hatching (DAH) (P<0.05)] and decreased the numbers of colony‐forming units (CFU) in larval gut 5 DAH compared with the control treatment (P<0.05). No differences were observed in survival (25–47%) during the second phase of the experiment (20–60 DAH). Nevertheless, the total numbers of CFU in fish gut 40 DAH were significantly lower in treatment B (P<0.05). The colonization rates of the added bacteria were the highest 5 and 11 DAH, and the highest average values reached were 65%, 82% and 17% of the total CFU count for the strains 2J18, 2J27 and J84 respectively.     

Examination of the early infection stages of koi herpesvirus (KHV) in experimentally infected carp,Cyprinus carpio L. using in situ hybridization

Koi herpesvirus (KHV) causes a highly infectious disease afflicting common carp and koi, Cyprinus carpio L. Various molecular and antibody‐based detection methods have been used to elucidate the rapid attachment and dissemination of the virus throughout carp tissues, facilitating ongoing development of effective diagnostic approaches. In situ hybridization (ISH) was used here to determine the target tissues of KHV during very early infection, after infecting carp with a highly virulent KHV isolate. Analysis of paraffin‐embedded tissues (i.e. gills, skin, spleen, kidney, gut, liver and brain) during the first 8 h and following 10 days post‐infection (hpi; dpi) revealed positive signals in skin mucus, gills and gut sections after only 1 hpi. Respiratory epithelial cells were positive as early as 2 hpi. Viral DNA was also detected within blood vessels of various tissues early in the infection. Notable increases in signal abundance were observed in the gills and kidney between 5 and 10 dpi, and viral DNA was detected in all tissues except brain. This study suggests that the gills and gut play an important role in the early pathogenesis of this Alloherpesvirus, in addition to skin, and demonstrates ISH as a useful diagnostic tool for confirmation of acutely infected carp.