外排泵抑制剂对海水养殖源弧菌酰胺醇类药物耐药性的影响

通过检测从海水养殖中分离的对氟苯尼考低敏感弧菌菌株中常见酰胺醇耐药基因的携带情况,来分析外排泵抑制剂对弧菌酰胺醇耐药性的影响,旨在研究主动外排机制在我国海水养殖源弧菌耐药中的作用。本研究从前期分离的海水养殖源弧菌中挑选出41株对氟苯尼考低敏感的菌株,运用琼脂稀释法测定了这些菌株对酰胺醇类药物(氯霉素和氟苯尼考)的敏感性;利用PCR方法检测了这些菌株的酰胺醇外排泵耐药基因和整合子的携带情况,包括外排泵基因floR、cmlA、pexA、fexA、fexB和optrA,以及Int1、SXT和ISCR1整合子相关基因;同时研究4种外排泵抑制剂对弧菌氯霉素、氟苯尼考最小抑菌浓度(MIC)的影响,利用琼脂稀释法检测41株弧菌分别加入甲基吡咯烷酮(N-methyl-2-pyrrolidone,NMP)、利血平(reserpine)、羰基氰氯苯腙(carbonyl cyanide m-chlorophenyl hydrazone,CCCP)和苯丙氨酸-精氨酸-β萘酰胺(Phe-Arg-β-naphthylamide,PAβN)4种外排泵抑制剂前后对氯霉素和氟苯尼考的MIC值的变化情况。副溶血弧菌(14株)、溶藻弧菌(17株)、哈维氏弧菌(8株)、创伤弧菌(2株)共41株氟苯尼考低敏感弧菌敏感度检测结果显示,氟苯尼考耐药菌株39株、氯霉素耐药菌株11株。41株氟苯尼考低敏感弧菌菌株中,酰胺醇类外排泵基因floR检出率为100%,而cmlA的检出率仅为17.1%,其他4个外排泵基因均未检出;整合子相关基因Int1、SXT和ISCR1的检出率分别为46.3%(19/41)、46.3%(19/41)和63.4%(26/41)。41株弧菌中,添加NMP、利血平、CCCP和PAβN后,分别有0株、2株、10株和2株弧菌为氯霉素外排阳性菌;分别有1株、1株、15株和3株弧菌为氟苯尼考外排阳性菌。研究表明,CCCP被验证可以作为有效治疗酰胺醇类药物耐药弧菌的外排泵抑制剂。     

养殖刺参溃疡病病原菌RH2的鉴定及其生物学特性分析

从患溃疡病养殖刺参(Apostichopus japonicus)病灶处分离出3株优势菌(RH1、RH2、RH3),以浸浴、体腔注射、体壁肌肉注射等方式进行感染实验,证实菌株RH2为养殖刺参溃疡病的病原菌,其对刺参的LD50(半数致死量)每尾为5.68×106CFU,并证实浸浴、体腔注射的方式无法感染刺参,该菌可能通过体表创伤侵入的方式感染刺参。经形态学观察、生理生化特性分析、16SrRNA和HSP60基因测序确定菌株RH2为溶藻弧菌(Vibrio alginolyticus)。基于16S rRNA基因构建的系统发育树显示,菌株RH2与溶藻弧菌(AF513447)亲缘关系最近,置信度为50%;HSP60基因序列分析表明RH2与溶藻弧菌(AY332570、AF230931)聚为一个分支,且置信度高达99%。菌株RH2的最适生长盐度为25~35,最适生长pH为7.2~8.0,最适生长温度为14~22℃。对18种药物的敏感实验证实菌株RH2对诺氟沙星、复方新诺明、壮观霉素等较为敏感。     

3株O3血清型鳗弧菌灭活疫苗的免疫原性和免疫保护效果

鳗弧菌(Vibrio anguillarum)O3血清型菌株是感染鱼类的重要病原菌,本文研究了3株O3血清型鳗弧菌(SMP1、SMP3和SMP4)灭活疫苗的免疫原性和免疫保护。首先在牙鲆(Paralichthys olivaceus)体内对3株鳗弧菌进行复壮;检测复壮后的菌株毒力,检测的3株鳗弧菌对蓝蔓龙(Trichogaser trichopterus)的半数致死量(LD50)分别为105.1 CFU/ml(SMP1)、104.7 CFU/ml(SMP3)和105.4 CFU/ml(SMP4);制备了3株菌的甲醛灭活疫苗,注射免疫牙鲆,免疫后第7天可检测到牙鲆的血清特异性抗体产生,免疫后第28天的血清特异性抗体效价为1:1280(SMP1)、1:640(SMP3)和1:905(SMP4),提供的免疫保护率(Relative percent survival rate,RPS)为94.4%(SMP1)、100%(SMP3)和73.7%(SMP4)。研究表明,3株致病性O3血清型鳗弧菌菌株具有良好的免疫原性,其中SMP3为最适疫苗候选株。本研究为鳗弧菌O3血清型疫苗的开发应用奠定了基础。     

抗弧菌光合细菌的分离鉴定及对氨氮和亚硝态氮的降解特性

该研究采用双层平板涂布法和划线法,从不同地区的海洋环境样品中分离纯化光合细菌,以副溶血弧菌(Vibrio parahemolyticus)、创伤弧菌(V.vulnificus)、鳗弧菌(V.anguillarum)为指示菌,采用牛津杯法测定海洋光合细菌菌株的抑菌作用,采用靛酚蓝分光光度法和盐酸萘乙二胺分光光度法测定不同菌株对氨氮(NH_4~+-N)和亚硝态氮(NO_2~--N)的降解作用,筛选出具有抗弧菌并高效降解NH_4~+-N和NO_2~--N复合功能的优良菌株。结果显示,从30个海水、海泥等样品中分离得到3株光合细菌,分离自连云港车牛山岛海水样品的菌株P-3,对3种弧菌均具有较强的抑制作用,其中对鳗弧菌的作用最强,抑菌圈直径为5.3 mm。3株光合细菌均具有一定的降解NH_4~+-N和NO_2~--N作用,菌株P-3的降解作用最强,在含有50 mg·L~(-1) NH_4~+-N和NO_2~--N的培养基中培养4 d,降解率分别为89.68%和94.98%。经形态学观察、生理生化试验和16S rDNA序列分析,确定P-3为沼泽红假单胞菌(Rhodopseudomonas palustris)。     

抗弧菌海洋细菌的分离筛选及其抗菌作用测定

采集连云港海域海水、海泥以及海洋动植物等样品86个,分离到海洋细菌176株.以病原性弧菌鳗弧菌为指示菌,采用平板对峙法在2216E培养基上测定不同海洋细菌菌株的抗菌作用, 其中XDY-7-4、YC-6-3、BM-1、BN-1、BN-2和XS1-5等6个海洋细菌菌株有明显的抗菌作用,菌株BN-1的抑菌带为11.2 mm,抗菌作用最强;其次为菌株XS1-5 、XDY-7-4 和YC-6-3,抑菌带宽度为10.4、10.2、9.9 mm;菌株BN-2和BM-1对鳗弧菌的抑菌作用较弱. 采用打孔法测定不同海洋细菌菌株无菌发酵液的抗菌作用,结果表明,菌株BN-1对鳗弧菌生长的抑制作用最明显,其次为菌株YC-6-3和XDY-7-4,菌株BM-1、XS1-5和BN-2的抗菌效果较弱.     

对虾养殖池中一株弧菌拮抗菌的分离鉴定

从对虾养殖池中分离出1株编号为2013082515(简称菌株15)的菌株,以鳗弧菌(Vibrio anguillarum)、哈维氏弧菌(Vibrio harveyi)和副溶血弧菌(Vibrio parahaemolyticus)为指示菌,分析了菌株15对指示菌的抑菌效果及最低抑菌浓度,同时,分析了菌株15对其他7株弧菌的抑菌效果.结果显示,菌株15对3株指示菌均具有抑菌效果,对鳗弧菌、哈维氏弧菌及副溶血弧菌的最低抑菌浓度分别为2.50×104、2.50×105、2.50×105 CFU/ml;对其他弧菌也具有一定的抑菌效果.采用注射及浸泡感染方法分析了菌株15对对虾的生物安全性,结果显示,在高浓度时,菌株15对对虾具有潜在毒性.分别用细菌全细胞脂肪酸气相色谱法和16S rDNA序列分析比对法对该菌进行分类鉴定,表明菌株15为一株假交替单胞菌(Pseudoalteromonas sp.).     

2株副溶血弧菌不同盐度下致病性和毒力基因差异分析

副溶血弧菌(Vibrio parahaemolyticus)是常见的食源性致病菌和海水养殖动物致病菌,目前已知的毒力基因有tlh、tdh、trh、T3SS、pirA、pirB、toxR/S、orf8等。盐度是影响细菌基因表达的关键生态因子之一,为进一步探求副溶血弧菌的致病机理,对2株分别分离自海水和淡水养殖发病凡纳滨对虾(Litopenaeus vannamei)中的副溶血弧菌在不同盐度下的生长速率、致病性进行检测,并用q-PCR方法对菌株毒力基因携带和表达情况进行定量研究。实验结果表明:海水菌株383生长速率快于淡水菌株V9,但菌株383在生长稳定期的细菌浓度明显低于菌株V9;菌株383对凡纳滨对虾的致病性明显高于菌株V9,前者的48 h半致死浓度(LD_(50))低于后者2个数量级;2株副溶血弧菌皆携带毒力基因tlh、T3SS1和pirA/B,但未检测到tdh、trh、T3SS2、toxR/S和orf8。部分毒力基因表达量检测结果显示,菌株383和菌株V9均为vcrD1表达量最高,其次是pirA,vopD1表达量最低;4个毒力基因不仅在2菌株间的表达量差别较大,而且盐度对同一菌株不同毒力基因表达的影响也是不同的。2株副溶血弧菌的毒力与pirA和vcrD1的表达量呈正相关性。研究结果为探究环境因素与副溶血弧菌致病力的关系提供了科学依据,同时也提示,淡水养殖凡纳滨对虾要防范副溶血弧菌输入的风险。     

牙鲆秦皇岛弧菌HQ010712-1外膜蛋白及其抗原性分析

采用十二烷基肌氨酸钠(Sarkosyl)和苯甲基磺酰氟(PMSF)2种方法提取秦皇岛弧菌HQ010712-1(Vibrio qinhuangdaora sp.nov.)外膜蛋白,结果显示 Sarkosyl法提取效果较好,且所提取的主要外膜蛋白分子量为102kD、45 kD、39 kD、36 kD、30 kD、28 kD、24 kD、22 kD;为比较该菌株与弧菌属其他细菌外膜蛋白组分及抗原性异同,以鳗弧菌(Vibrio anguillarum)、副溶血弧菌(Vibrio parahaemolyticus)、溶藻胶弧菌(Vibrio alginolyticus)为对照,电泳图谱显示4种弧菌外膜蛋白的分子量主要集中在22～48 kD之间;利用抗秦皇岛弧菌HQ010712-1血清的免疫印迹表明菌株HQ010712-1外膜蛋白中分子量为45 kD、36 kD的蛋白条带呈现阳性反应,其他3种弧菌外膜蛋白中均有与该抗血清反应的条带,且分子量为36 kD的反应带为菌株HQ010712-1、副溶血弧菌、溶藻胶弧菌共有.本研究旨在为进一步筛选和研究致病性弧菌的共同保护性抗原提供参考.     

对虾源副溶血弧菌强毒株HNBX-1的分离鉴定与毒性分析

副溶血弧菌(Vibrio parahaemolyticus)是海水养殖中常见的条件致病菌,具有发生率高和流行面广等特点。本文从患红体病的凡纳滨对虾(Litopenaeus vannamei)体内分离得到一株细菌HNBX-1,经生化实验结合热休克蛋白60(Heat Shock Protein 60,HSP60)基因鉴定为副溶血弧菌。毒性实验表明,弧菌HNBX-1对凡纳滨对虾的毒性极强,感染24 h时的LD50为3.5×104cfu·mL-1;对其重要毒力基因———耐热溶血毒素基因(TDH)、耐热溶血毒素相关基因(TRH)和Ⅲ型分泌系统(T3SS1和T3SS2)的分析结果显示,HNBX-1中的TDH、TRH以及T3SS2基因检测结果均为阴性,而T3SS1的4个毒力基因中的VP1689(vscK)和VP1694(vscF)基因扩增为阳性,表明该副溶血弧菌菌株的强毒特性与其携带的T3SS1密切相关。     

蟹源拟态弧菌外毒素生物学特性研究

对蟹源拟态弧菌HX-4菌株外毒素的酶活性、溶血活性、细胞毒性和致死性等生物学特性进行了系统研究。采用固体硫酸铵盐析法和层析技术提取拟态弧菌HX-4菌株外毒素,所得外毒素具有酪蛋白酶、明胶蛋白酶、溶血素等多种毒性酶活性、细胞毒性(HEp-2)、肠毒性和致死性等生物学特性,TC ID50为0.025μg,对中华绒螯蟹的半数致死量(LD50)为1.40μg。     

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.