弧菌拮抗菌的筛选及其效果

从健康的鱼虾体内以及养殖水体中分离得到 2 19株海洋细菌 ,以鳗弧菌 (Vibrioanguillarum)、副溶血弧菌 (Vibrioparahaemolyticus)、沙蚕弧菌 (Vibrioneresis)、飘浮弧菌 (Vibrionatriegen)等病原性弧菌为指示菌 ,在 2 2 16E平板上采用十字交叉划线法、点种法和平板扩散法进行体外拮抗实验 ,从中筛选到 6株有拮抗作用的海洋细菌。用平板活菌计数法研究拮抗菌J - 10及其胞外产物对鳗弧菌的抑菌作用 ;采用平板扩散法研究拮抗菌J- 10胞外产物抑菌效果的影响因子。结果显示 ,拮抗菌J- 10在混合体系中 ,2 4h之后表现出拮抗作用 ,在第 3天使鳗弧菌的数量降为最低 ,比第 1天的鳗弧菌数量下降了 2个数量级 ,之后鳗弧菌的数量开始上升 ,而对照组鳗弧菌的数量在第 2～ 5天基本维持第 1天的水平 ,表明拮抗菌J - 10在混合体系中具有较强的拮抗作用。拮抗菌J- 10的胞外产物能较好地抑制鳗弧菌的生长 ,使实验组的弧菌数量在 3h降为最低 ,之后呈缓慢上升的趋势 ,而对照组则一直呈上升的趋势 ;拮抗菌J - 10的胞外产物对热敏感 ,对pH的变化不敏感 ,对蛋白酶K不敏感 ,Fe3 的添加会减弱其抑菌活性。根据形态特征和常规生理生化反应进行鉴定 ,J - 10为藤黄微球菌 (Micrococcusleteus)。     

致病性拟态弧菌的敏感药物和拮抗菌的筛选

采用纸片扩散法,系统测试了3株致病性拟态弧菌对8类23种抗菌药物的敏感性与联合用药的敏感性。结果显示:氨基糖甙类、头孢类、喹诺酮类和磺胺类等多种抗菌药物对病原弧菌均有抑制或杀死作用。庆大霉素与四环素类、头孢类、喹诺酮类、红霉素或氯霉素联用以及多粘菌素与头孢类、喹诺酮类、红霉素或氯霉素联用均表现出一定程度的累加或协同作用;同时,采用琼脂平板扩散法从9株非致病菌中筛选对致病性拟态弧菌具有拮抗作用的益生菌并对其抑菌活性进行测定。结果显示:HX-01和HX-05两株细菌的菌液和无细胞上清液对3株致病性拟态弧菌均具有拮抗作用。在拮抗菌—病原菌混合体系中,病原菌经24 h培养,菌数由原来的105CFU/mL增至2ⅹ108CFU/mL,但从第2天开始,随着拮抗菌产生的抗性产物浓度的增加,菌数快速下降,至第6天病原菌完全被杀死。而拮抗菌培养至第6天,菌数仍保持较高水平(2ⅹ107CFU/mL)。表明这两株拮抗菌对致病性拟态弧菌具有较强的拮抗作用。经数值鉴定法确定两株拮抗菌均为产碱杆菌。     

5株鲆鲽鱼类病原菌多克隆抗体的制备与应用

本研究制备了鳗弧菌Vibrio anguillarum、杀鲑气单胞菌Aeromonas salmonicida、副溶血弧菌V. parahaemolyticus、哈维氏弧菌V. harveyi和腐败希瓦氏菌Shewanella putrefaciens 5株鲆鲽鱼类病原菌的兔抗血清,建立了5种菌的间接ELISA检测方法,并将该检测方法用于鱼类细菌分离物病原检测。人工感染结果显示,5株菌对大菱鲆的半数致死量(LD50)在102~107CFU/fish;制备的兔抗血清效价分别为1∶2 048 000、1∶16 000、1∶16 000、1∶1 024 000、1∶128 000;交叉反应结果显示,鳗弧菌、副溶血弧菌、哈维氏弧菌3株弧菌与抗血清相互之间存在交叉反应;抗血清特异检测灵敏度分别为104、108、107、105、106cell/ml;对13株海水鱼类细菌分离物进行检测,有1株腐败希瓦氏菌阳性,两株哈维氏弧菌阳性;1株副溶血弧菌和哈维氏弧菌均为阳性,该结果与16S rDNA序列的分子分析方法一致。     

斜带石斑鱼消化道乳酸菌在模拟胃肠道环境中的存活

从斜带石斑鱼(Epinephelus coioides)幼鱼消化道分离到3株乳酸菌,通过抑菌试验筛选出抑菌效果较好的2株菌(MM1和MM4),经生理生化鉴定结合16SrRNA基因测序分析,MM1菌株鉴定为乳酸乳球菌(Lactococcus lactis),MM4菌株鉴定为屎肠球菌(Enterococcus faecium)。进一步研究了MM1和MM4的体外生长特性,及其对不同pH值的人工胃液、含有不同浓度胆盐的人工肠液及高温的耐受能力。结果表明,MM1和MM4均在培养6h左右进入对数生长期。对人工胃液的耐受能力相似,pH4.0和3.0的人工胃液对2株菌存活的影响较小,而pH2.0的人工胃液对2株菌的影响较大,经2h处理后两者的存活数即下降到105CFU/mL,经4h处理后的存活数低于5.0×103CFU/mL。MM1和MM4对含不同浓度胆盐的人工肠液均具有较好的耐受能力,含中低浓度胆盐(0.1%～0.3%)的人工肠液对2株菌的存活影响很小,处理3h后2株菌的存活数均维持在107CFU/mL以上;高浓度胆盐(0.5%～0.7%)处理3h后2株菌的存活数明显下降,但活菌数仍维持在105CFU/mL以上。MM1和MM4具有一定的耐高温能力,60℃处理30min二者存活数在107CFU/mL以上;80℃处理30min二者存活数在106CFU/mL以上。结论认为,2株乳酸菌具有生长速度快、抑菌能力强、对胃肠道环境和高温耐受能力较强的特点,具有作为益生菌的应用潜力。     

凡隆气单胞菌(Aeromonas veronii)拮抗菌4-1-3的筛选鉴定及其活性物质初探

凡隆气单胞菌是泥鳅养殖中容易发生而且致死率较高的病原菌之一.为筛选凡隆气单胞菌拮抗菌、研发防治此病害的生物制剂,采集泥鳅养殖池底泥样品,通过滤纸抑菌圈法筛选凡隆气单胞菌拮抗细菌,结合形态观察、生理生化实验和16S rRNA基因序列分析进行菌株鉴定.采用硫酸铵沉淀和离子交换层析方法分离纯化抑菌物质,电泳检测其分子量.结果显示,从样品中分离到17株细菌,其中筛选出的拮抗菌4-1-3,能够高效抑制凡隆气单胞菌,抑菌圈直径达到15.2 mm.经菌株鉴定后命名为Pseudomonas fluorescens 4-1-3.该菌产生的抑菌物质为胞外蛋白,分子量约为100 kDa,推测该物质是荧光假单胞菌产生拮抗物质的一种新类型,有望进一步研制成防治水产病害的生物制剂.     

凡隆气单胞菌(Aeromonas veronii)拮抗菌4-1-3的筛选鉴定及其活性物质初探

凡隆气单胞菌是泥鳅养殖中容易发生而且致死率较高的病原菌之一。为筛选凡隆气单胞菌拮抗菌、研发防治此病害的生物制剂,采集泥鳅养殖池底泥样品,通过滤纸抑菌圈法筛选凡隆气单胞菌拮抗细菌,结合形态观察、生理生化实验和16S rRNA基因序列分析进行菌株鉴定。采用硫酸铵沉淀和离子交换层析方法分离纯化抑菌物质,电泳检测其分子量。结果显示,从样品中分离到17株细菌,其中筛选出的拮抗菌4-1-3,能够高效抑制凡隆气单胞菌,抑菌圈直径达到15.2 mm。经菌株鉴定后命名为Pseudomonas fluorescens 4-1-3。该菌产生的抑菌物质为胞外蛋白,分子量约为100 kDa,推测该物质是荧光假单胞菌产生拮抗物质的一种新类型,有望进一步研制成防治水产病害的生物制剂。     

哈维氏弧菌胶体金免疫层析试纸条的制备

用18~20nm胶体金标记抗哈维氏弧菌Vibrio harveyi抗体并制备金标垫,分别将羊抗兔Ig G和纯化后的抗哈维氏弧菌抗体包被于NC膜上作为质控线和检测线,组装制作了哈维氏弧菌胶体金免疫层析试纸条,优化了该试纸条的制作条件,测试了其性能。结果表明:所制备的哈维氏弧菌免疫层析试纸条具有较好的特异性,与费尼斯弧菌Vibrio furnissii、维氏气单胞菌Aeromonas veronii、美人鱼发光杆菌Photobacterium damselae、类志贺邻单胞菌Plesiomonas Shigelloides、鱼肠道弧菌Vibrio ichthyoenteri、鳗利斯顿氏菌Listonella anguillarum、迟缓爱德华氏菌Edwardsiella tarda、海豚链球菌Streptococcus iniae、嗜水气单胞菌Aeromonas Hydrophila等9种常见水产病原菌无明显交叉反应,检测灵敏度为3×105CFU/m L,5~10min即可得出检测结果,具有快速、简便、特异性强和适应基层推广应用等优点,可用于养殖鱼类病原哈维氏弧菌的现场检测。     

网箱养殖沉积环境中硫氧化菌的分离鉴定及生长特性

从网箱养殖区沉积物中分离硫氧化细菌,并研究其生长特性。从以硫代硫酸钠为唯一能源底物的培养基中分离得到一株硫氧化菌B1-1,经过平板菌落形态观察及其形态学特征研究,并结合16S rRNA基因序列分析及分子系统发育树的构建结果,确定菌株的种类。结果表明,菌株B1-1呈革兰氏阴性,短杆状,鉴定为Halothiobacillus hydrothermalis(GenBank登录号为KU362926)。在28℃、初始pH=8、150 r/min培养条件下,细菌在12 h进入对数生长期,36 h开始氧化硫代硫酸盐(S_2O_3~(2-)),至60 h培养液p H下降到4.8,S_2O_3~(2-)浓度从最初的2.28 g/L降到0.36 g/L,氧化率达84.2%。     

光合细菌菌剂和沼泽红假单胞菌对实验水体氮磷营养盐和微生物群落的影响

为比较光合细菌菌剂与沼泽红假单胞菌(Rhodopseudomonas palustris)的生理生态特性,分析了不同初始菌量的菌剂PG和菌株PSB-1对实验水体氨氮(NH_4~+-N)、亚硝氮(NO_2~--N)、硝氮(NO_3~--N)和活性磷(PO_4~(3-)-P)的降解效果,通过高通量测序分析了菌剂PG的优势菌组成及实验结束时水体细菌数量和微生物群落组成。结果显示,菌剂PG组对实验水体的PO_4~(3-)-P、NO_3~--N和NO_2~--N有一定的降解作用,其最大降解率分别为40.98%、28.28%和20.12%。菌株PSB-1组仅对实验水体的NO_2~--N和PO_4~(3-)-P有一定的降解效果,其最大降解率分别为14.19%和9.88%。菌剂PG的主要优势菌为红假单胞菌属(Rhodopseudomonas sp.)。实验7 d后实验组水体细菌数量和微生物群落结构发生变化,水体细菌数量增长,形成以异养细菌为优势菌的菌群结构。结果表明光合细菌菌剂PG对水质因子的降解效果优于沼泽红假单胞菌PSB-1,但与报道的高效光合细菌菌株的降解能力存在一定差距。     

刺参养殖池环境细菌群落对底质改良剂的响应

运用平板培养计数法、最大或然数法和16S rRNA 基因的 PCR-DGGE 指纹图谱技术,分析发病刺参池塘环境中不同生理类群细菌数量及群落结构对底质改良剂的响应。结果显示,加入底质改良剂后,发病刺参池塘沉积环境中的总异养菌、硝化细菌、硫酸盐还原细菌等的数量在最初的2–4 d 有所上升,但升高幅度小于对照组,且在之后的2–4 d 内下降至加改良剂之前菌量;在加入底质改良剂后第2天,弧菌和硫化细菌数量便迅速下降,明显低于对照组。PCR-DGGE 图谱及测序结果显示,刺参养殖环境细菌优势菌分别属于绿弯菌门、变形菌门的莫拉菌科、柄杆菌科和气单胞菌科以及厚壁菌门的芽孢杆菌科和乳杆菌科的某种细菌,多样性指数在2.5–3.5之间。实验组加入底质改良剂后,气单胞菌属两种细菌数量逐渐下降。研究结果表明,底质改良剂可改变沉积环境中不同细菌类群的数量,降低致病菌的数量,从而改善底质环境并对“刺参腐皮综合征”起到防治作用。     

Association between the interannual variation in the oceanic environment and catch rates of bigeye tuna (Thunnus obesus) in the Atlantic Ocean

The environmental processes associated with variability in the catch rates of bigeye tuna in the Atlantic Ocean are largely unexplored. This study used generalized additive models (GAMs) fitted to Taiwanese longline fishery data from 1990 to 2009 and investigated the association between environmental variables and catch rates to identify the processes influencing bigeye tuna distribution in the Atlantic Ocean. The present findings reveal that the year (temporal factor), latitude and longitude (spatial factors), and major regular longline target species of albacore catches are significant for the standardization of bigeye tuna catch rates in the Atlantic Ocean. The standardized catch rates and distribution of bigeye tuna were found to be related to environmental and climatic variation. The model selection processes showed that the selected GAMs explained 70% of the cumulative deviance in the entire Atlantic Ocean. Regarding environmental factors, the depth of the 20 degree isotherm (D20) substantially contributed to the explained deviance; other important factors were sea surface temperature (SST) and sea surface height deviation (SSHD). The potential fishing grounds were observed with SSTs of 22–28°C, a D20 shallower than 150 m and negative SSHDs in the Atlantic Ocean. The higher predicted catch rates were increased in the positive northern tropical Atlantic and negative North Atlantic Oscillation events with a higher SST and shallow D20, suggesting that climatic oscillations affect the population abundance and distribution of bigeye tuna.     

Growth performance,digestive enzyme activity and immune response of Japanese sea bass,Lateolabrax japonicus fed with fructooligosaccharide

In this experiment, a feeding trial was performed to determine the effects of fructooligosaccharide (FOS) on growth performance, digestive enzyme activity and immune response of Japanese sea bass, Lateolabrax japonicus juveniles (initial weight 38.3 ± 0.5 g), and the fish were examined following feeding with six levels of FOS (0, 0.5, 1, 2, 4 and 6 g/kg) for 28 days. Significant enhancement of weight gain (WG) and specific growth rate (SGR) was found in fish fed 1 g/kg FOS incorporated diets (p < .05), while the feed conversion ratio (FCR) in the 1, 2 g/kg FOS groups reduced significantly compared with the control (p < .05). Besides, the crude lipid in the 4, 6 g/kg FOS groups increased significantly compared with the control (p < .05). On the other hand, the erepsin and lipase activities significantly elevated in intestine of fish fed 2 g/kg FOS (p < .05) and the lysozyme activity in serum of fish fed 2 g/kg FOS were significantly higher than that in the control (p < .05). Moreover, the alkaline phosphatase activities in serum of fish fed 0.5, 1, 2 g/kg FOS were significantly higher than in control (p < .05). Regression analysis showed that the relationships between dietary FOS levels and either SGR, FCR, erepsin or lysozyme activities were best expressed by regression equations, and the optimal inclusion levels are 1.37, 1.80, 3.06, 3.11, 1.93 and 1.80 g/kg for SGR, FCR, erepsin, lipase, lysozyme and total superoxide dismutase activities, respectively. Overall, this study revealed that FOS incorporated diets could beneficial for L. japonicus culture in terms of increasing the growth, digestion and immune activities. Under the present experimental condition, the optimal supplementary level of FOS in the diet of L. japonicus is 1–3 g/kg.     

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.     

Evaluation of a Fast Method Based on the Presence of Two Restriction Sites in the Mitochondrial ND5 (mt ND5) Gene for the Identification of Scomber Species

The purpose of this work was to evaluate the suitability of a method based on the presence of two restriction sites (for Hae III and Hindf I) in the mitochondrial NADH dehydrogenase subunit 5 (mt ND5) gene to identify Scomber species. The evaluation was performed on 144 reference and market samples by sequencing of the entire 505-bp fragment of the mt ND5 gene and of a 464-bp fragment of the Kocher fragment of the cytochrome b gene (mt Cytb). Sequence analysis of any of the two fragments allows the identification of each of the four Scomber species, but S. japonicus and S. colias had the same restriction sites at the ND5 amplicon and would not have been differentiated by this analysis. Similarly, loss of the Hae III site in some S. scombrus individuals would have misidentified them as not being Scomber. All the market products were correctly labeled except one acquired in Spain labeled as originating in the Atlantic and containing S. japonicus.     

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).