Design of Vibrio 16S rRNA Gene Specific Primers and Their Application in the Analysis of Seawater Vibrio Community

The pathogenic species of genus Vibrio cause vibriosis, one of the most prevalent diseases of maricultured animals and seafood consumers. Monitoring their kinetics in the chain of seafood production, processing and consumption is of great importance for food and mariculture safety. In order to enrich Vibrio-representing 16S ribosomal RNA gene （rDNA） fragments and identify these bacteria further real-timely and synchronously among bacterial flora in the chain, a pair of primers that selectively amplify Vibrio 16S rDNA fragments were designed with their specificities and coverage testified in the analysis of seawater Vibrio community. The specificities and coverage of two primers, VF169 and VR744, were determined theoretically among bacterial 16S rDNAs available in GenBank by using BLAST program and practically by amplifying Vibrio 16S rDNA fragments from seawater DNA. More than 88.3% of sequences in GenBank, which showed identical matches with VR744, belong to Vibrio genus. A total of 33 clones were randomly selected and sequenced. All of the sequences showed their highest similarities to and clustered around those of diverse known Vibrio species. The primers designed are capable of retrieving a wide range of Vibrio 16S rDNA fragments specifically among bacterial flora in seawater, the most important natural environment of seafood cultivation.     

Vaccination in three different ways against vibriosis of Seriola dumerili caused by Vibrio hollisae

Bacterin was prepared by formalin-inactivating the virulent strain of Vibrio hollisae isolated from diseased Seriola dumerili (amberjack) suffering from vibriosis. Healthy S. dumerili were vaccinated by respective procedures of intramuscular injection, immersion, and orally administration. Results of the three different vaccinations were compared. Blood was drawn from the vaccinated fish every 7 days, and the antibody titers and lysozyme activities of the sera were determined. The antibody titer of injected fish was 1:40 at 7 d, and reached its peak of 1:320 at 28 d, while the fish vaccinated by immersion and orally administration exhibited weak antibody responses, the antibody titres of <1:10, 1:20, 1:160 were observed at 7 d, 14 d, 35 d respectively. Compared with the control, the vaccinated fish exhibited significantly higher lysozyme activities (P<0.05). Upon challenge with virulent strain, the relative percent survival (RPS) of injected, immersed and oral administrated fish were 75%, 45%, and 40% respectively, and the injected fish showed significantly higher RPS than immersed and oral administrated fish. The results suggested that vaccination of S. dumerili by the injection would be the best strategy to prevent the vibriosis in S. dumerili farm.     

Vaccination in three different ways against vibriosis of <Emphasis Type="Italic">Seriola dumerili</Emphasis> caused by <Emphasis Type="Italic">Vibrio hollisae</Emphasis>

Bacterin was prepared by formalin-inactivating the virulent strain of Vibrio hollisae isolated from diseased Seriola dumerili （amberjack） suffering from vibriosis. Healthy S. dumeriIi were vaccinated by respective procedures of intramuscular injection, immersion, and orally administration. Results of the three different vaccinations were compared. Blood was drawn from the vaccinated fish every 7 days, and the antibody titers and lysozyme activities of the sera were determined. The antibody titer of injected fish was 1：40 at 7 d, and reached its peak of 1：320 at 28 d, while the fish vaccinated by immersion and orally administration exhibited weak antibody responses, the antibody titres of 〈1：10, 1：20, 1：160 were observed at 7 d, 14 d, 35 d respectively. Compared with the control, the vaccinated fish exhibited significantly higher lysozyme activities （P〈0.05）. Upon challenge with virulent strain, the relative percent survival （RPS） of injected, immersed and oral administrated fish were 75%, 45%, and 40% respectively, and the injected fish showed significantly higher RPS than immersed and oral administrated fish. The results suggested that vaccination of S. dumerili by the injection would be the best strategy to prevent the vibriosis in S. dumerili farm.     

Diversity of bacteria isolated from crustacea larvae and their rearing water

1　Introduction　Blacktigershrimp (Penaeusmonodon)isthemajorshrimpspeciesculturedinIndonesia .Itsculturehasbecomemoreintensivebecauseofitshighdemandandeconomicvalue .Infact,shrimpexporthasyieldedthehighestIndonesianincomeinfisherysector .Itwasestimatedthattheproductionofculturedshrimpwas 80 0 0 0metrictonnesin 1995 .Consequently ,theneedforthefryforstockingpondculturesteadilyin creased .Thetotalnumberoffryrequiredwasabout 6millionperyear (FAO ,1995 )and 90 %oftherequiredanimalswereobtained…     

Disruption of bacterial balance in the gut of Portunus trituberculatus induced by Vibrio alginolyticus infection

Journal of Oceanology and Limnology - Gut microbiota impacts the health of crustaceans. Vibrio alginolyticus is a main causative pathogen that induces the vibriosis in farmed swimming crabs,...     

不同养殖模式虾池弧菌对抗菌药物的耐药性与虾池水质评价

研究了湛江东海岛3种不同养殖模式虾池弧菌对抗菌药物的耐药性,并对虾池水域环境的水温、盐度、DO、pH、叶绿素a、DOC、无机氮、无机磷、硅酸盐、细菌总数和弧菌数量变化进行了监测和评价。结果表明,调查的三个虾池的弧菌对万古霉素产生耐药性,高位新池和传统精养池的弧菌对利福平产生耐药性,对其他抗菌药尚未形成耐性,3个虾池的弧菌对恩诺沙星和氯霉素高度敏感,传统精养池和天然虾池的弧菌对复方新诺明、庆大霉素、TMP和环丙沙星高度敏感;在水平差异上,高位新池的弧菌比传统精养池和天然虾池对12种抗菌药更具耐药性,同时多重耐药菌株在高位新池中出现,可能与虾苗来源和配合饲料中添加了抗菌药物有关。水质监测结果表明,养殖过程中,虾池水域环境逐渐富营养化,无机氮和Chla超过富营养化阈值,细菌总数增加;两个精养池的水体富营养化程度高于天然虾池;调查期间的弧菌数量在对虾发病的感染剂量阈值范围之内,尚无弧菌病害发生的潜在危险。     

哈维氏弧菌GST核酸疫苗制备及其对斜带石斑鱼的免疫保护作用

通过同源引物从致病性哈维氏弧菌(Vibrio harveyi)ZJ0603基因组中克隆GST的开放阅读框(ORF),构建真核表达质粒pcDNA-GST。大量抽提重组质粒后,于背鳍基部肌肉注射重组质粒免疫斜带石斑鱼(Epinephelus coioides),分析重组质粒的免疫效果。通过核酸水平检测重组质粒在鱼体肝、肌肉、头肾和脾脏组织的分布;用ELISA法检测鱼体血清的抗体水平,用Western-blot检测目的蛋白的表达情况。结果表明:该序列全长615 bp;免疫7 d后,鱼体中均有质粒分布;斜带石斑鱼血清中产生抗GST的高效抗体(1∶4 096);相应的目的蛋白也在鱼体中成功表达。攻毒后,疫苗免疫保护率达80%,表明GST可作为防治哈维氏弧菌病有效候选抗原。     

Historical Evolution of Mariculture in China During Past 40 Years and Its Impacts on Eco-environment

The rapid development of mariculture in China in recent decades has satisfied people's demand of seafood, and has made a great contribution to economic development. However, mariculture has also caused some negative impacts on the eco-environment. By statistically analyzing national data(as well as data for four main provinces) regarding the mariculture area and cultivation species in China since reform and opening up, the historical evolution of mariculture during the past 40 years was analyzed, the driving factors related to policy, market and technology innovation were discussed, and the potential impacts of mariculture on the coastal eco-environment were also illustrated. The statistical results indicated that the maricultural area increased radically during the past 40 years, from 1979(116.47 × 10~3 ha) to 2012(2205.65 × 10~3 ha), with an increase of nearly 20-fold. Shandong Province represented the fastest-growing region, with an increase of 31-fold. Moreover, the cultivated species had gradually become diversified. Initially, shellfish and algae were major species, and then it developed to various species including fish, and currently shellfish and crustaceans were the major species. The development of mariculture was driven by government policies, market economy and technology innovation. Rapid development of mariculture also caused significantly adverse impacts on the eco-environment of the coastal regions. For example, the sea reclamation for mariculture decreased the area and function of coastal wetlands and the contaminants originating from mariculture destroyed the coastal aquatic environment. Fortunately, the implementation of ‘13 th Five-Year Fisheries Planning' is expected to help both improve the seafood quality and reduce the contamination in coastal aquatic environment. This current study will provide reference for management and structure adjustment of mariculture in the future.     

Cloning, expressing, and hemolysis of tdh, trh and tlh genes of Vibrio parahaemolyticus

Vibrio parahaemolyticus (VP) is one of the pathogenic vibrios endangering net-cage cultured Pseudosciaena crocea,Fennerpenaeus chinensis, and shellfish in coastal areas of China. Several types of hemolysins produced by Vp have been characterized as major virulence factors.They are thermostable direct hemolysin (TDH),TDH-related hemolysin (TRH) and thermolabile hemolysin (TLH). In this study, we cloned tdh, trh, and tlh genes from the genome DNA of VP by polymerase chain reaction (PCR).We ligated the three genes into prokaryotic expression vector pET-28a (+),and transformed the recombinant plasmids into Es-cherichia coli BL21 (DE3). The expression of recombinant proteins was induced by isopropyl-β-D-thiogalacto-pyranoside (IPTG). The recombinant proteins were expressed in a form of inclusion bodies and thus purified with Ni-NTA affinity chromatography. Western blotting results showed that recombinant proteins,TDH, TRH and TLH, could be recognized by rabbit anti-VP serum. The three purified proteins were renatured by gradient dialysis.The renatured proteins exhibited hemolytic activity except for TLH in the presence of phosphatidylcholine. These results not only are helpful for better understanding these genes’ functions under a single factor level, but also provide evidence for VP vaccine engineering.     

Construction and characterization of a fosmid library for pathogenic bacterium <Emphasis Type="Italic">Vibrio anguillarum</Emphasis>

Vibrio anguillarum is a common bacterial pathogen in fish.However,little is known about its pathogenic mechanism,in part,because the entire genome has not been completely sequenced.We constructed a fosmid library for V.anguillarum containing 960 clones with an average insert size of 37.7 kb and 8.6-fold genome coverage.We characterized the library by end-sequencing 50 randomly selected clones.This generated 93 sequences with a total length of 57 485 bp covering 1.4% of the whole genome.Of these sequences,58...     

海洋弧菌鉴定与分类相关功能基因研究进展

【目的】探究功能基因在海洋弧菌物种鉴定中的作用,为海洋弧菌多样性研究以及致病性弧菌的监测和防控提供参考。【方法】根据已有的弧菌分类学研究成果,阐述16S rRNA基因、管家基因和毒力基因等相关功能基因在海洋弧菌鉴定与分类研究中的应用进展。【结果】16S rRNA基因和功能基因可用于海洋弧菌的分类学及其快速检测鉴定的研究,对致病性弧菌的诊治具有重要的意义。【结论】功能基因和16S rRNA基因在海洋弧菌菌种鉴定中的广泛应用,提高了海洋弧菌种间分类的准确性,对海洋弧菌的多样性研究以及水产养殖和人类健康的风险评估有重要价值。探索新型功能基因在海洋弧菌分类学研究和快速鉴定中的应用有现实意义。     

红笛鲷弧菌病血清流行病学研究方法的建立

以红笛鲷Lutjanus sanguineus弧菌病的主要病原菌——溶藻弧菌Vibrio alginolyticus为抗原制备兔抗血清,以辣根过氧化酶标记的羊抗兔血清为酶标二抗,建立了酶联免疫吸附试验检测技术。通过棋盘滴定法测定,得出溶藻弧菌菌悬液的最佳工作浓度为5×108mL-1,兔抗溶藻弧菌血清的最佳稀释度为1∶16000,酶标羊抗兔抗体的最佳稀释度为1∶2000。应用本实验建立的间接双抗体夹心法对红笛鲷进行现场检测,患病红笛鲷特异性抗体的检出率为70%,外观健康红笛鲷的检出率为20%。结果表明,本实验所建立的酶联免疫吸附试验(ELISA)检测法可以用于红笛鲷弧菌病的血清流行病学研究,而且可以检测出隐性感染状态下的红笛鲷。     

Characterization of a Mutant of Alteromonas aurantia A18 and Its Application in Mariculture

Mutant J61321 with enhanced siderophore production of Alteromonas aurantia AI8 was obtained after a series of chemical-physical mutageneses. It was found that the antibacterial activity against Vibrio anguillarum W-1 and siderophore production of the mutant were higher than those by the original strain A 18 which had been used in mariculture. The results of the specific assay（Csaky and Arnow methods） of siderophore showed that the sidrophore with hydroxamate group was produced by mutant J61321 and the original strain A 18, respectively, while the siderophore with catechol group was yielded by strain W-1 （Aibrio anguillarum）. Meanwhile, the siderophore yield, antibacterial activity and anti-chelator activity of strain J61321 were higher than those of its parent strain A 18.     

Impacts of mariculture practices on the temporal distribution of macrobenthos in Sandu Bay,South China

Sandu Bay is located in the East China Sea and is characterized by high-density fish farming and kelp culture. Despite this, little is known about the impacts of these different mariculture practices on the local environment. We investigated the temporal variation in macrobenthos and environmental conditions at three sites in the bay (fish farming site, kelp culture site, and a control site). We collected water and sediment samples during nine cruises between May 2009 and February 2010. The density of macrobenthos peaked at the fish farming site in July (655 ind./m2) whereas density did not fluctuate as widely at the other two sites. Biomass varied significantly at both the control and kelp culture sites, but had only a single peak at the fish farming site in June (21.90 g/m2). The dominant species varied throughout the study period at the control and kelp culture sites, whereas a single terebellid species (Lysilla pacifica) dominated the macrobenthos at the fish farming site. The diversity index H’ increased at the control site beginning in February then decreased after May, whereas H’ was low at the other sites in December. The mean dissolved oxygen level was highest at the control site (6.59 mg/L) and lowest at the fish farming site (5.54 mg/L). DO levels were lowest at all sites in summer (July and August). The sediment acid volatile sulfide content was higher at the fish farming site (1.46 mg/g dry weight) than those at the kelp culture and control sites (1.22 and 0.14 mg/g, respectively). Our results suggest that mariculture practices have a clear impact on the benthic environment/ community in Sandu Bay.     

Significant effects of fishing gear selectivity on fish life history

Over the past few decades, extreme changes have occurred in the characters of exploited fish populations. The majority of these changes have affected the growth traits of fish life history, which include a smaller size-at-age, an earlier age-at-maturation and among others. Currently, the causes of these life history traits changes still require systematic analyses and empirical studies. The explanations that have been cited are merely expressed in terms of fish phenotypic adaptation. It has been claimed that the original traits of fish can be recovered once the intensity of exploitation of the fish is controlled. Sustained environmental and fishing pressure will change the life history traits of most fish species, so the fish individual’s traits are still in small size-at-age and at earlier age-at-maturation in exploited fish populations. In this paper, we expressed our view of points that fishing gear has imposed selectiv-ity on fish populations and individuals as various other environmental factors have done and such changes are unrecoverable. Ac-cording to the existing tend of exploited fish individual’s life history traits, we suggested further researches in this field and provided better methods of fishery management and thereby fishery resources protection than those available early.     

A VIBRIO ANGUILIARUM STRAIN ASSOCIATED WITH SKIN ULCER ON CULTURED FLOUNDER, PARALICHTHYS OLIVACEUS

The characteristics of a bacterium strain M3, isolated from cultured flounderParalichthys olivaceus with remarkable external sign of skin ulcer during an epizootic outbreak, indicated that the bacterium belonged to the speciesVibrio anguillarum. Challenge by I.M. (intramuscular injection), bath, and oral administration with M3 showed that it was highly pathogenic forParalichthys olivacues. The LD₅₀ dose was 5.144×10³ CFU/ per fish infection by I.M. injection. Recovered inoculated bacteria from the surviving fish revealed that the asymptomatic carriers could be a latent contagious source. Study of the effect of bacterial culture CFS (cell-free-supernatant) showed that the exotoxins produced by M3 play an important role in its pathogenicity for flounder. The resistance of M3 to 36 out of 41 antibiotics indicated that the bacterial disease outbreak was mainly attributable to the frequent and excessive use of antimicrobial agents; and that vaccination would be an effective precaution against bacterial disease. Contribution No. 4107 from Institute of Oceanology, Chinese Academy of Science. Contribution No. 270 from Experimental marine Biology Laboratory, CAS. Funded by projects under the Major State Basic Research Development Program, Grant G1999012003.     

红鳍笛鲷弧菌病病原的研究

从广东湛江特呈岛鱼排取患病红鳍笛鲷进行病原分离、纯化,得到一株病原菌Ls 1。该菌 为革兰阴性短杆菌,有运动性,菌落半透明,最适温度28～30℃,需盐生长,氧化酶反应阳性,发酵 葡萄糖不产气,不发酵肌醇,对弧菌抑制剂O/129敏感。经API ID32E细菌鉴定系统及弧菌科细 菌生化鉴定管鉴定,该菌为一株溶藻弧菌(Vibrioaginolyticus);其对红鳍笛鲷的半数致死量为6.32 ×105mL-1。药敏试验结果表明,该菌对阿莫西林、先锋V不敏感,对新生霉素、卡那霉素、多粘菌 素B、四环素中度敏感,而对庆大霉素、氟哌酸、氯霉素、红霉素、链霉素和复方新诺明等有较高的敏 感性。     

Assessing mariculture water quality with the structural and functional characteristics of a ciliate community

Ciliated protozoa play important roles in micro-ecosystems, especially in marine biotopes. However, few studies have been carried out on the periphytic, or aufwuch, forms in mariculture waters so far. In this study, we sampled periphytic ciliate communities in two closed mariculture ponds (ponds CP1 and CP2) and a natural seawater reservoir (pond RP) using a glass slide method to evaluate their colonizing processes and general ecological features, as well as their application as water quality indicators. We analyzed species compositions, structural parameters (species number, richness, diversity, evenness, abundance and dBP) and functional parameters (G, Seq and T90%). Pond RP was characterized by higher levels of structural parameters (except for abundance and dBP) and more equal proportion of the major taxonomic groups. The values of Seq were significantly higher in pond RP and similar in both pond CP1 and CP2. It was also demonstrated that environmental factors, including NO2-H, NO3-H, NH3-H, soluble reactive phosphate, temperature and pH, were the first principal factors affecting the communities. Among them, temperature and chemical factors were all significantly and negatively correlated with species number (P<0.01), richness (P<0.01), diversity (P<0.01), and positive correlated with abundance (P<0.01). Opposite correlations between pH and structural parameters were observed. This study showed that there were significant differences in species composition, structural parameters and functional parameters of the periphytic ciliate communities among the ponds, which were in agreement with the water quality. Results of this study confirmed the periphytic ciliate communities to be useful bioindicators of water quality in intensive mariculture waters.     

大亚湾网箱养殖水体弧菌种类组成及变化

研究了海水网箱养殖海域弧菌数量变化、种类组成及其相关因素,探讨弧菌数量的变化与鱼病的关系。结果表明:网箱内水体弧菌数量为390~230 mL-1,平均为20.6×102mL-1,网箱外水体为130~1450 mL-1,平均为530 mL-1,非养殖对照海区为10~390 mL-1,平均为190 mL-1。养殖海区弧菌数量呈现明显的季节性变化,尤其是网箱水体,对照海区只在9月数量较高,其他时间变化不大。平均数量网箱内大于网箱外,网箱外大于对照海区。水体中出现的弧菌种类有河流弧菌Vibrio fluvialis,创伤弧菌Vibrio vulnifgicus,霍乱弧菌Vibrio cholerae,副溶血弧菌Vibrio parahaemolyticus,溶藻弧菌Vibrio alginolyticus,美人鱼弧菌Vibrio damsela,拟态弧菌Vibriomimicus和好利斯弧菌Vibrio hollisae,美人鱼弧菌、溶藻弧菌和霍乱弧菌占有较大优势。弧种数量与海水温度、盐度及营养盐有相关性,但其相关性因不同种和不同点而不同。检测期间,网箱养殖鱼类无明显流行病发生,但弧菌的高峰期明显与报道的鱼病高峰期相吻合。     

胶东半岛海产养殖地下咸水分布特征及开发利用现状

胶东半岛位于山东省胶莱河以东,区内海岸线全长约1 700km,沿海滩涂面积约500km2。由于该区特殊的自然地理和地质环境条件,赋存有适宜海产养殖的地下咸水。该文依据大量的基础资料,阐述了胶东半岛区内海产养殖地下咸水的分布特征、富水地段资源量及开发利用现状,并针对开发利用中引发的地质环境问题提出了相应的保护建议,对于指导该区海产养殖地下咸水资源科学合理开发利用具有重要的意义。