海豚链球菌兼职蛋白FBA的克隆表达、抗原性检测及免疫效果评价

为检测斑点叉尾鮰源海豚链球菌兼职蛋白(fructose-1,6-bisphosphate aldolases,FBA)的抗原性和潜在的疫苗价值,本实验克隆得到斑点叉尾鮰源海豚链球菌DX09(基因组登陆号LXQF01)的fba基因序列(基因登录号A7N10_RS06935),对克隆序列进行生物信息学分析,并通过原核表达得到重组FBA蛋白(r FBA),制备了兔抗r FBA血清用于FBA蛋白抗原性检测,同时通过免疫保护实验评估重组蛋白的免疫保护效果。结果显示,海豚链球菌DX09 fba基因有1个882 bp的开放阅读框(ORF),编码293个氨基酸。生物信息学分析显示,其分子式为C_(1378)H_(2172)N_(368)O_(422)S_8,分子质量为30.9 ku,理论等电点为5.01,不具有信号肽和跨膜区域;具有保守的裂解酶结构域,且与其他来源的FBA蛋白同源性达100%;具有较高的抗原指数,表明其可形成多个抗原表位。SDS-PAGE检测发现,诱导表达的重组蛋白以包涵体的形式出现在沉淀中,大小约为47 ku。Western blot分析表明,兔抗r FBA血清能特异性结合菌体蛋白。同时免疫保护实验显示,重组蛋白对斑点叉尾鮰的相对保护率可达55%,免疫后鱼体抗体水平相对对照组显著升高。本研究表明,原核表达的斑点叉尾鮰源海豚链球菌DX09 rFBA具备较好的抗原性和免疫保护作用,具有研发斑点叉尾鮰海豚链球菌亚单位疫苗的潜在价值。     

Loss of genetic diversity due to hatchery culture practices in barramundi (Lates calcarifer)

Many aquaculture hatchery practices are detrimental to the long-term viability of restocking and selective improvement programs. Small effective broodstock population sizes, differential broodstock contribution, differential larval/juvenile survival during metamorphosis and size-based grading, all have the potential to drastically reduce the level of genetic variation remaining in hatchery populations. Monitoring levels of genetic variation and maintaining detailed pedigrees on progeny is the key to circumventing these problems. In this study we used microsatellites, coupled with DNA parentage analyses, to track the loss of genetic diversity in two independent commercial barramundi (Lates calcarifer) hatcheries over three mass spawning events, where up to two females and seven males had the opportunity to participate. Initial broodstock contributions were observed to be highly skewed, with significant differences observed in both the level of contribution by females to each mass spawning, as well as in the number of males participating and subsequently contributing to the genetic composition of cohorts. Effective population sizes were around half that of census sizes. We then examined whether differential family survival through metamorphosis (27 days post-hatch) and/or first size grading further influenced the retention of genetic diversity levels initially sampled during spawning. Parentage analyses indicated that some families that had been initially represented in cohorts had been lost, or that the contribution by particular broodstock had changed. In one cohort, as many as 55% of progeny were found to be sired by a single male individual. Size grading was also found to potentially impact on genetic diversity, with data suggesting that family representation in each of the grades was non-uniform and that some families were on average faster or slower growing than others. These results illustrate that hatchery management practices have the potential to significantly impact on the retention of genetic diversity in this species.     

西伯利亚鲟海豚链球菌和鲟疱疹病毒Ⅱ型混合感染的诊断与病理损伤

2018年8月,四川彭州与邛崃的养殖西伯利亚鲟发生一种以出血为临床特征,高死亡率的传染病,为明确其病因,本研究对发病鲟的肝脏、肾脏进行病原菌分离、鉴定和鲟疱疹病毒Ⅱ型(AciHV-2)的PCR检测。从患病鲟体内分离到2株G+链状球菌,其16S rRNA序列(MN416231、MN416230)与GenBank中海豚链球菌16S rRNA序列同源性达99%,在以16S rRNA序列构建的系统发育树上,分离菌与海豚链球菌聚为一支;同时基于海豚链球菌lctO基因的特异性PCR检测为阳性,鉴定2株分离菌为海豚链球菌。提取患病鲟肝脏、肾脏组织DNA进行AciHV-2特异性PCR检测,扩增出501 bp的特异性条带,在以AciHV-2 polymerase基因序列构建的系统发育树上,检测样本与AciHV-2聚为一支。病理组织学上,患病鲟的多组织器官发生明显损伤,尤其是肝脏、肾脏、鳃、脾脏和肠的损伤较为严重,表现为明显的变性、坏死、出血以及炎症细胞浸润;电镜下,观察到肝脏、肾脏组织内大量直径200～220 nm的疱疹病毒样颗粒与0.7～0.8μm的链球菌入侵细胞,并导致细胞损伤。综上,诊断患病西伯利...     

广西卵形鲳鲹海豚链球菌基因分型、耐药谱型以及毒力基因检测

为了解近年来广西卵形鲳鲹海豚链球菌流行菌株的基因型信息以及菌株间的差异,对2015—2016年从广西地区7个养殖场的患病卵形鲳鲹体内分离得到的17株海豚链球菌菌株分别进行了基因型分析、耐药谱测定以及毒力基因检测。采用随机扩增多态性DNA标记技术(RAPD)和基因组重复序列PCR(rep-PCR)分析其基因型。结果显示,RAPD和rep-PCR指纹图谱结果一致,17株海豚链球菌可分为2种基因型。对海豚链球菌7种主要的毒力相关基因特异PCR检测,所有菌株均为sim A+scp I+pdi+sag A+cps D+pgm A+cfi+毒力基因型,表明这2种基因型的海豚链球菌似乎均为毒力较强的菌株。采用K-B法进行了20种抗生素敏感实验分析其耐药谱,结果表明归于基因1型的菌株耐药谱为AZT,基因2型菌株则出现3种相似的耐药谱,分别为SIZ/T/S/PEN/AZT/SPE/CAZ/PB、SIZ/T/S/PEN/AZT/SPE/CAZ/CRO和SIZ/T/S/PEN/AZT/SPE/CAZ/PB/RIF,证实基因型相同的菌株耐药谱型也相似,2种基因型的菌株耐药谱型差异显著,因此基因型和耐药谱型存在相关性。此结果为卵形鲳鲹海豚链球菌病流行病学研究、疫苗研制以及疫病监测提供理论依据。     

温度对尼罗罗非鱼无乳链球菌毒力的影响

为了解温度对鱼源无乳链球菌毒力的影响机制,本实验研究了温度对无乳链球菌毒力相关参数(生长、粘附、入侵、毒力基因表达以及对罗非鱼致死率)的影响。结果发现,不同培养温度下(25~40 ℃)无乳链球菌的生长速度不同,37 ℃为其最适生长温度,25 ℃时生长速度最慢;25~34 ℃内无乳链球菌粘附在惰性基质上的菌体对应的吸光值(OD_590nm)之间无显著差异(P>0.05),37 ℃时显著增加,40 ℃时又急剧下降;罗非鱼在人工感染无乳链球菌后各时间点(6、12、24和48 h),鱼体脑组织中菌量随注射菌体培养温度的增加呈先上升后下降的趋势,且与不同培养温度下的无乳链球菌对罗非鱼的致死率呈正相关;无乳链球菌毒力基因的表达也与培养温度相关,hly和cfb基因的表达量随菌体培养温度的升高先增加后降低,分别在34和37 ℃处达到峰值,不同培养温度下无乳链球菌sip基因表达的变化幅度较小,而scpB基因的表达则随培养温度增加而下降;无乳链球菌对罗非鱼的致死率随其培养温度的升高呈先升高后降低趋势,低温(25和28 ℃)培养条件下的菌体对罗非鱼致死率较低(<20%),37 ℃时致死率最高66.67%±6.67%。以上结果表明温度参与无乳链球菌生长、粘附、转移、入侵和部分毒力基因表达的调控,它们共同影响无乳链球菌对罗非鱼的毒力。     

罗非鱼海豚链球菌病的病理学观察

采用光学显微镜和透射电子显微镜技术分别对患海豚链球菌病的罗非鱼的病理学变化进行了研究。分离的病原菌革兰氏染色呈阳性,透射电镜负染观察菌体球形或卵圆形,直径0.6~1.0μm,多数呈链状排列。组织学病变主要表现为全身多组织、器官水肿,出血、变性、坏死以及炎症反应,特别是肝、脾、肾和脑分别表现为肝炎,脾炎,间质性肾炎和脑膜炎。超微结构观察发现病鱼肝、脾、肾、脑、心肌和骨骼肌等器官的细胞超微结构都有较为严重的破坏,细胞核畸形,染色质浓缩或边集,粗面内质网囊泡化及脱粒,线粒体肿胀,嵴断裂或溶解消失。研究表明,海豚链球菌能造成罗非鱼全身性组织器官病变,致使器官功能障碍,正常生理代谢调节紊乱,最后导致死亡。     

罗非鱼源无乳链球菌cpsE基因的克隆和分子特性分析

利用特异性引物,采用PCR扩增出分离自患病罗非鱼无乳链球菌强毒株的cpsE基因,将其克隆到pMD19-T载体上,通过菌落PCR鉴定和利用限制性内切酶BamHⅠ和HindⅢ对重组质粒进行双酶切鉴定之后送测序公司测序,并利用生物信息学软件Clustal X 2.0、MEGA 4.1、Bioedit 7.0、TMHMM、NetPhos 2.0、NetNGlyc 1.0、SignalP 3.0 Server、PSIpred、SAM_T08以及CUSP等分析cpsE基因的分子特性。结果显示,罗非鱼源无乳链球菌cpsE编码氨基酸序列具有高度保守性,与人源、动物源无乳链球菌亲缘性达100%,具有1个参与催化糖基元转运的Glycosyltransferases超级家族结构域,具有与蛋白翻译后修饰功能相关的磷酸化位点3个,编码多肽链中亲水区大于疏水区,且存在跨膜区。密码子偏爱性分析表明,罗非鱼源无乳链球菌cpsE基因密码子使用频率差异较大,密码子偏爱性更接近真核生物。     

BALB/c小鼠用于评价尼罗罗非鱼无乳链球菌毒力的研究

实验采用BALB/c小鼠作为实验动物,旨在建立尼罗罗非鱼无乳链球菌毒力测定的BALB/c小鼠模型。BALB/c小鼠经腹腔注射尼罗罗非鱼源无乳链球菌建立感染模型,比较了尼罗罗非鱼源无乳链球菌分别感染尼罗罗非鱼和小鼠的LD_(50)差异,分别测定了不同毒力尼罗罗非鱼无乳链球菌对尼罗罗非鱼和小鼠的毒力。结果显示,小鼠经腹腔注射无乳链球菌,在24 h内出现死亡现象,且对小鼠脑、肝脏、脾脏、肾脏等组织造成损伤。3次测定尼罗罗非鱼无乳链球菌TFJ0901对尼罗罗非鱼和小鼠LD_(50)分别为7.7×10~7、2.2×10~8、3.5×10~9 CFU/mL和405、361、419 CFU/只。将无乳链球菌TFJ0901和THN0901感染尼罗罗非鱼(1.0×10~7 CFU/mL)和小鼠(100 CFU/只),尼罗罗非鱼和小鼠存活率分别为100%、6.7%±5.8%和100%、0,其存活率都具有显著性差异。将无乳链球菌TFJ0901和TFJ-F感染尼罗罗非鱼(3.0×10~8 CFU/mL)和小鼠(2 500 CFU/只),尼罗罗非鱼的存活率分别为73.3%±11.5%和80.0%±10.0%,存活率差异不显著,小鼠存活率分别为13.3%±11.5%和100.0%,存活率具有显著性差异。研究表明,本实验成功建立了BALB/c小鼠作为尼罗罗非鱼源无乳链球菌毒力测定的稳定模型,测定不同毒力的尼罗罗非鱼源无乳链球菌对小鼠毒力与对尼罗罗非鱼毒力一致,且该模型能够区分尼罗罗非鱼模型难以区分的毒力相近的无乳链球菌。     

中国南方地区罗非鱼无乳链球菌的分子流行病学研究

从广东省以及海南省等地区养殖的患病罗非鱼体内分离、收集到多株致病菌,经生化分析和分子生物学鉴定,均为无乳链球菌。对这些菌株分别进行了耐药谱测定、分子分型试验以及分子血清型分析。药敏试验结果表明,2007—2010年分离到的无乳链球菌耐药谱基本相似;多位点可变数目串联重复序列分析(MLVA)试验中,选择5个高变异指数的可变数目重复位点(VNTR)进行分子分型,结果表明,所有鱼源无乳链球菌菌株为同一MLVA型,而作为对照的牛源无乳链球菌则明显不同;为了对这些菌株进一步分型,分别进行了分子血清型和表面蛋白抗原基因的检测,结果表明,鱼源无乳链球菌的分子血清型均为Ⅰa型,表面蛋白抗原均为alpha-C蛋白。这进一步说明了不同年份和不同地区的鱼源无乳链球菌在基因水平上为同一分子类型,具有相同的起源或传染源。同时也说明,我国南方地区罗非鱼无乳链球菌在这几年中未发生明显的遗传变异。这些结果为罗非鱼无乳链球菌病疫苗研制,疫病监测及药物防治的研究提供理论依据。     

中国七种水生动物源无乳链球菌的分子特征及其对斑马鱼的致病性

为了解中国水生动物源无乳链球菌的分子流行特征,揭示其传播和流行规律,本实验对分离得到并鉴定的10株7种水生动物源无乳链球菌通过分子血清型、多位点序列分型(MLST)分型、毒力基因型和前噬菌体分型等方法进行分子分型;其次,通过斑马鱼评价7种水生动物源无乳链球菌的致病性。分子血清型分析结果表明,10株无乳链球菌可分为3种血清型,即Ⅰa、Ⅰb和Ⅲ型;MLST分型结果表明,Ⅰa型无乳链球菌均为ST7型,Ⅰb无乳链球菌均是ST261型,只有Ⅲ型无乳链球菌是ST739型。进一步分型结果表明,10株无乳链球菌可分为3种毒力基因型和4种前噬菌体基因型。根据4种分型结果可知,10株水生动物源无乳链球菌可分为4种类型,其中虎纹蛙源无乳链球菌具有独立的分子血清型、MLST型、毒力基因型和前噬菌体基因型,即Ⅲ-ST739-V1-P3;罗非鱼源无乳链球菌的基因型有3种,即Ⅰa-ST7-V2-P1、Ⅰa-ST7-V2-P2和Ⅰa-ST7-V3-P4;红尾皇冠鱼、鳙和罗非鱼源无乳链球菌的基因型相同:Ⅰa-ST7-V2-P2;卵形鲳鲹、宝石鲈和罗非鱼源无乳链球菌具有相同的基因型:Ⅰa-ST7-V2-P1;鲮和罗非鱼源无乳链球菌的基因型相同,即Ⅰb-ST261-V3-P4。致病性研究表明,7种水生动物源无乳链球菌对斑马鱼均有强致病性。研究表明,两栖类虎纹蛙源无乳链球菌和鱼源无乳链球菌的基因型明显不同,它们之间遗传变异较大,因此,无乳链球菌在两栖类和鱼类之间相互传播的可能性较小。鱼源无乳链球菌有3种基因型,且这3种基因型均在罗非鱼中流行,这表明无乳链球菌在鱼类中相互传播的可能性较大,尤其是在罗非鱼与其他鱼类之间。     

Growth efficiency of juvenile barramundi, Lates calcarifer, at high temperatures

Temperature is recognized to be the most important environmental factor affecting growth in fish. Barramundi are cultured over a wide range of temperatures some of which approach the upper thermal tolerance for this species. A growth trial was conducted on juvenile barramundi to examine the effects of high temperatures ranging from the minimum optimal temperature (27 °C) for growth efficiency to the extreme upper thermal limits (39 °C) for feed intake, growth and growth efficiency. Juveniles (4.87 ± 0.32 g) were held at four different temperatures 27, 33, 36 and 39 °C and fed twice daily to satiation (503.5 g kg^− 1 crude protein, 182.5 g kg^− 1 lipid, 150.1 g kg^− 1 ash, 20.52 GE MJ kg^− 1). Feed intake (g·day^− 1) and SGR (%·day^− 1) increased with increasing temperature up to 36 °C. At 39 °C feed intake, growth, feed efficiency ratio, protein efficiency ratio and productive energy value were significantly lower than at the other temperatures. This demonstrates that growth was optimized at temperatures from 27 to 36 °C and that barramundi have a much wider range for maximum growth efficiency than previously thought.     

尼罗罗非鱼无乳链球菌Sip蛋白乳酸菌活载体口服疫苗的研制及其免疫效果

链球菌病是威胁我国罗非鱼养殖产业健康发展的重要病害之一。为研制出免疫效果好、操作简便的罗非鱼链球菌病疫苗,本研究构建重组表达无乳链球菌Sip蛋白的穿梭质粒pNZ8124-Sip,通过酶切和测序验证后电转化乳酸乳球菌NZ9000,获得能够诱导重组表达无乳链球菌Sip蛋白的乳酸菌活菌载体疫苗。采用SPS-PAGE电泳摸索最佳诱导浓度和诱导时间以获得最大表达量,通过镍柱纯化目的蛋白并进行Western blot检测;利用不同浓度的重组乳酸菌活载体疫苗灌胃口服免疫尼罗罗非鱼,采用间接ELISA法测定免疫后血清抗体水平变化,通过人工腹腔注射感染无乳链球菌获得相对免疫保护率。研究结果显示,构建的重组乳酸乳球菌可通过nisin诱导表达大小为48 ku特异性蛋白,与目的蛋白大小一致;PAGE电泳显示,重组蛋白主要以可溶蛋白和包涵体2种形式存在,其中胞内可溶性蛋白浓度达7.65 mg/mL;诱导表达的最佳条件为100 ng/mL nisin诱导6 h;Western blot检测结果显示,诱导蛋白可与鼠抗His标签抗体特异性结合。口服免疫结果显示,中浓度组(2.24×10~(10) CFU/mL)和低浓度组(2.24×10~9 CFU/mL)免疫2次能够显著提高尼罗罗非鱼的血清抗体水平和抗无乳链球菌感染能力,中浓度免疫组的相对免疫保护率最高为41.0%。本研究可为罗非鱼链球菌病口服疫苗的研究奠定基础,具有广阔的应用前景。     

西伯利亚鲟海豚链球菌的分离鉴定及毒力基因检测

2013年8-9月,四川雅安汉源湖养殖的西伯利亚鲟发生一种以体表溃疡、内脏出血和心外膜囊肿为特征的传染病.为明确其病因,本研究从自然发病鱼的肝、脾和肾进行了病原菌的分离、人工感染、分离菌的表型特征和分子生物学特征的检测.结果从患病鱼体内分离到一株G+链状球菌(Ab130920),人工感染实验证实了其病原性,生理生化特性与海豚链球菌(ATCC29178)基本一致;16S rDNA序列(GenBank登录号:KJ162337)与GenBank中S.iniae 16S rDNA序列同源性最高,在以16S rDNA序列及其GenBank中同源性较高的相关序列构建的系统发育树上,分离菌与海豚链球菌聚为一支;同时,在基于S.iniae lctO基因的特异性PCR检测中,从分离株基因组DNA扩增出预期大小的870 bp条带,进而鉴定分离菌Ab130920为S.iniae.在对cpsD、simA、sagA、pdi和scpI等5种S.iniae毒力基因的多重PCR检测中,分离菌均扩增出相应大小的特异性片段,表明其为一毒力较强的菌株,与人工感染实验的高致病性结果相佐证;药物敏感性检测发现其对阿莫西林、强力霉素、氟苯尼考等抗菌药物敏感,但对新生霉素、利福平、氧氟沙星耐药.     

罗非鱼无乳链球菌微胶囊口服疫苗的研制及其免疫效果

为了建立罗非鱼无乳链球菌病免疫防控方法,以原核表达SIP(surface immunogenic protein)蛋白为芯材,天然多糖为壁材,制备罗非鱼无乳链球菌聚丙烯酸树脂Ⅱ微胶囊口服疫苗,SIP口服疫苗微胶囊颗粒平均直径约826.5μm,包封率为72.02%,载药量最高达6.11%。微胶囊包裹的SIP蛋白在5种缓冲液中释放效果:p H 6.80p H 7.20p H 9.18p H4.68p H 2.00。体外实验证实微胶囊颗粒在模拟胃酸环境中蛋白释放量极小,数值为395.5μg;而在模拟肠液中,释放量最高达5426.0μg,其中240 min释放量为4911.1μg,释放度达90.51%,说明微胶囊能避免胃酸的破坏且肠溶性好。SIP微胶囊口服疫苗以投喂的方式免疫"新吉富"罗非鱼,分别以每克罗非鱼体质量免疫5和10μg SIP蛋白的剂量分组测试,4次免疫过程中,5μg免疫剂量组特异性血清效价为100–1~400–1,10μg免疫剂量组特异性血清效价为200–1~1600–1。4次免疫后,攻毒实验表明,5和10μg的免疫剂量组的免疫保护率分别为44.45%和66.67%。研究表明,微胶囊口服疫苗免疫为罗非鱼无乳链球菌病的免疫防治提供方便、安全、有效的途径。     

L-半胱氨酸提高嗜水气单胞菌对氟苯尼考的敏感性

抗菌药物的应用能对细菌感染性疾病进行有效地控制和治疗,但水产动物养殖中滥用和误用抗菌药物导致耐药细菌的产生、养殖水环境污染和养殖动物药物残留等一系列问题,应对和解决抗菌药物在水产动物养殖中的滥用问题已经刻不容缓。本研究通过抗菌药物杀菌、组织细菌清除、细菌攻毒、鱼体存活率统计和药物敏感性检测等方法,探究外源L-半胱氨酸添加对氟苯尼考杀菌作用的影响。体外杀菌结果显示,4.00 mmol/LL-半胱氨酸可使20.00～200.00μg/mL氟苯尼考对嗜水气单胞菌的杀菌效率提高1.9～11.1倍,其联合用药的杀菌作用具有剂量和时间依赖效应。组织细菌清除结果显示,相比单独使用氟苯尼考,L-半胱氨酸与氟苯尼考合剂对鱼体肝脏、脾脏和肾脏组织中嗜水气单胞菌的清除能力提高4～16倍。细菌攻毒后,采用注射和口服L-半胱氨酸或/和氟苯尼考进行治疗,结果发现L-半胱氨酸与氟苯尼考合剂可大幅提高黄河鲤对嗜水气单胞菌的抗感染能力。同时,单独添加L-半胱氨酸也能促进组织细菌的清除和鱼体的抗感染能力。细菌MIC测定结果显示,添加1.00～4.00 mmol/L L-半胱氨酸使嗜水气单胞菌对氟苯尼考的敏感性提高2～4倍。由此推测,L-半胱氨酸可能通过增强嗜水气单胞菌对氟苯尼考的敏感性,从而提高抗菌药物对鱼体细菌感染的防治效果。研究表明,L-半胱氨酸作为氟苯尼考的增效剂不仅可提高抗菌药物的防治效果,而且可大幅降低抗菌药物的使用量,对嗜水气单胞菌的防控具有一定的实际应用价值。     

cel-EIIB蛋白调控罗非鱼无乳链球菌强毒、弱毒株毒力相关基因的表达模式

罗非鱼无乳链球菌纤维二糖-磷酸转移酶系统（cel-PTS）的EIIB蛋白对强毒株毒力影响有限，但对弱毒株毒力似乎存在潜在的影响，但具体机制仍不清晰，有必要弄清该蛋白调控强、弱毒株的毒力相关基因表达模式。在前期研究中，通过同源重组技术，构建了无乳链球菌强毒株cel-EIIB基因缺失株，本研究通过类似的方法，获得弱毒株该基因的缺失株。用无乳链球菌强毒、弱毒株及它们的cel-EIIB缺失株分别感染斑马鱼，结果显示，cel-EIIB缺失后，导致弱毒株毒力明显返强，而强毒株毒力则轻微减弱。qPCR检测发现，cel-EIIB缺失可致cel-PTS系统的cel-EIIA、双组分信号转导系统（TCS）的DltR和CiaH以及毒力基因sodA、cpsD和cpsG在强、弱毒株中呈现相反的表达模式；此外，TCS系统的RgfC、DltS和CsrR以及毒力基因cspA和pavA在强毒突变株中表达未受影响，但在弱毒突变株中的表达却显著上调。研究揭示，EIIB蛋白可能通过调控上述毒力相关基因表达而负调控弱毒菌株的毒力。     

罗非鱼无乳链球菌的分离、鉴定及致病性研究

从患暴发性流行病罗非鱼的肝脏中分离获得一株细菌TL60829NA,将TL60829NA对罗非鱼进行人工感染致病性试验,试验感染罗非鱼表现出自然发病症状,确认分离菌株为罗非鱼暴发性流行病的致病菌.分离株菌经形态学观察、Bio Merieux Vitek全自动微生物分析系统GPI测定卡分析和16S rRNA特异性基因序列与NCBI中收录的其它引起罗非鱼病害的链球菌的16S rDNA序列构建的系统发育进化树显示,分离细菌与其它无乳链球菌的16S rDNA序列构成一个进化分支,而海豚链球菌则构成另一分支,确定分离菌株为无乳链球菌(Streptococcus agalaciate).分离菌株对氨苄西林、青霉素G、卡那霉素等28种试验药物敏感,对妥布霉素、复方新诺明、环丙沙星等15种试验药物不敏感.由无乳链球菌引起的罗非鱼暴发性流行病的主要病理变化为鳃小片结构崩解,鳃上皮增生、融合;心肌纤维变性、肌间白细胞浸润;肝脏颗粒变性和脂肪变性;肠道粘膜上皮变性、坏死、脱落、固有膜炎性白细胞浸润;肾脏组织大量嗜中性白细胞浸润,肾小管上皮细胞核固缩,小动脉血管壁玻璃样变性;眼睛的脉络膜和眶骨膜组织炎性坏死,晶状体纤维断裂和脱离.     

Suitability of the copepod, Acartia clausi as a live feed for Seabass larvae (Lates calcarifer Bloch): Compared to traditional live-food organisms with special emphasis on the nutritional value

Though artificial propagation of Asian seabass Lates calcarifer (Bloch) in captivity through induced breeding techniques is standardized under Indian conditions, larval and nursery rearing techniques including suitable nursery feeds have to be standardized to obtain better survival and growth. Feeding experiments in triplicate were conducted to evaluate the suitability of the marine copepod Acartia clausi as live prey for fourteen day-old seabass larvae (6.53 ± 0.06 mm; 8.58 ± 0.33 mg) and compared with the traditional live prey, rotifers and Artemia nauplii. While A. clausi and rotifers were mass produced using algae Isochrysis galbana, Chaetoceros affinis and Chlorella marina, Artemia nauplii were produced using cysts. Nutritional quality of cultured copepods was evaluated based on the proximate composition, amino acid and fatty acid composition, and compared with that of rotifers and Artemia nauplii. Proximate composition varied significantly (P < 0.05) among the different live feeds. A. clausi showed higher protein (63.12%) and lipid (16.65%) content than Artemia nauplii and rotifers. Total essential amino acids content was 2% lower in A. clausi compared to that in Artemia nauplii. Fatty acid profiles of the live feed organisms showed that A. clausi is a rich source of n − 3 fatty acids. The total n − 3 fatty acid content of A. clausi was 33.94%. Length, weight overall weight gain and survivorship were significantly (P < 0.05) different among the dietary treatments, and weight gain was comparatively higher in A. clausi fed larvae. Survival of seabass larvae fed A. clausi was obtained highest as 58.13% against the lower values of 39.93% and 41.62% in larvae fed rotifer and Artemia nauplii respectively. Final carcass composition of the larvae of L. calcarifer fed different live-food organisms showed significant differences (P < 0.05) among the dietary treatments. The fatty acid composition of the dietary treatments was reflected to a certain extent in the fatty acid composition of the seabass larvae. The present investigation revealed the nutritional value of calanoid copepod and thus underlining its usefulness as a suitable live-food organism for rearing larvae of the commercially valuable Asian seabass.     

Streptococcus iniae infection and tissue distribution in hybrid striped bass (Morone chrysops×Morone saxatilis) following inoculation of the gills

This study was designed to test the possibility that Streptococcus iniae enters through the gills and causes infection in hybrid striped bass. To determine the dose response, four groups of fish were inoculated with S. iniae via the gills with a dose of 5.0×10⁵, 2.6×10⁶, 5.0×10⁶, or 1.0×10⁸ CFU/fish. One group of fish was inoculated with tryptic soy broth (TSB) via the gills to serve as controls. The cumulative percent mortality was 13%, 27%, 100% and 100% for 5.0×10⁵, 2.6×10⁶, 5.0×10⁶ and 1.0×10⁸ CFU/fish, respectively. We also examined the tissue dissemination of S. iniae at 0.5, 4, 8, 12, 24, 48 and 72 h after experimental gill inoculation. Fish were inoculated with 2.6×10⁶ or 5.0×10⁶ CFU/fish, which caused low and high mortality, respectively. Within 48 h, fish inoculated with the 2.6×10⁶ dose were culture positive on the gill surface, blood of the first and second gill arches, blood of the third and fourth gill arches and the nares. However, for the dose of 5.0×10⁶ CFU/fish, S. iniae was also isolated from the olfactory, optic and cerebellum regions of the brain, eye, head kidney, trunk kidney, spleen and liver at 48 h. For the 2.6×10⁶ dose, S. iniae was not isolated until 48 h post-inoculation, but was isolated at 12 h for the 5.0×10⁶ dose. The results of this study indicate that S. iniae can enter hybrid striped bass through the gills. However, mortality at similar S. iniae doses was lower than we previously observed by inoculation of the nares.     

日本沼虾cytMnSOD和mtMnSOD基因全长cDNA克隆及表达

利用RACE技术从日本沼虾肝胰腺中克隆了cytMnSOD和mtMnSOD基因cDNA全长序列。cytMnSOD基因cDNA全长1 233 bp,开放阅读框为858 bp,编码286个氨基酸,N端含有60个氨基酸残基组成的延伸区;mtMnSOD基因cDNA全长1 113 bp,开放阅读框为654 bp,编码218个氨基酸,N端含有20个氨基酸残基组成的信号肽;cytMnSOD和mtMnSOD预测蛋白分子量及等电点分别为31.33、24.05 ku和5.62、7.12。日本沼虾cytMnSOD推导的氨基酸序列与其mtMnSOD的相似性为40%,二者均含有MnSOD的特征肽段(DVWEHAYY)、4个Mn2+结合位点和2个N-糖基化位点。Real-time PCR结果表明,cytMnSOD和mtMnSOD在日本沼虾肝胰腺、肌肉、血细胞、大颚器官、卵巢和鳃等组织均有表达,其中肝胰腺表达量最高;肝胰腺cytMnSOD和mtMnSOD基因的表达量在蜕皮间期最高,蜕皮后期和蜕皮前期较低。嗜水气单胞菌刺激后3 h,肝胰腺cytMnSOD和mtMnSOD的表达量显著增加,推测MnSOD是参与机体免疫防御反应的一种重要分子。