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1.
Distribution and abundance of resting cysts of the toxic Alexandrium spp. (Dinophyceae) in sediments of the western Seto Inland Sea, Japan 总被引：1，自引：0，他引：1
MINEO YAMAGUCHI SHIGERU ITAKURA KEIZO NAGASAKI YUICHI KOTANI 《Fisheries Science》2002,68(5):1012-1019
Sediment samples were collected from 135 stations in the western part of the Seto Inland Sea (Iyo Nada, Suo Nada, Beppu Bay, Tokuyama Bay, Hiroshima Bay, Aki Nada, Hiuchi Nada and Bingo Nada) to determine the horizontal distribution and abundance of resting cysts of Alexandrium spp. ( A. tamarense + A. catenella ). Enumeration of the cysts was performed using the primuline-staining direct count method. Cysts of Alexandrium spp. were rarely found in Iyo Nada, Suo Nada and Beppu Bay, but were widely distributed in Tokuyama Bay, Hiroshima Bay, Aki Nada, Hiuchi Nada and Bingo Nada. Cyst concentrations ranged from not detected (ND) to 14, ND to 17, ND to 4, 93 to 8137, 8 to 4454, ND to 6, ND to 18 and 4–29 cysts/cm3 wet sediment in Iyo Nada, Suo Nada, Beppu Bay, Tokuyama Bay, Hiroshima Bay, Aki Nada, Hiuchi Nada and Bingo Nada, respectively. The majority of cysts occurred in Tokuyama Bay and Hiroshima Bay, where higher densities were observed in the inner bay and along the coastal margin. Relatively higher cyst concentrations were observed at stations with a higher mud content. The abundance of Alexandrium spp. cysts in western Seto Inland Sea is lower than in the eastern Seto Inland Sea, except for Tokuyama Bay and Hiroshima Bay. However, because sporadic blooms of Alexandrium have been observed, continuing monitoring is necessary to prevent paralytic shellfish poisoning outbreaks in this area. 相似文献
2.
ABSTRACT: Paralytic shellfish poisoning (PSP) toxins produced by Alexandrium isolates from Korea were analyzed by high-pressure liquid chromography. Species designation of the regional isolates was determined by morphological criteria and ribotyping inferred from sequences of the 28S rDNA D1-D2 region. Toxin analysis performed at the exponential growth phase, revealed that the two strains of A. fraterculus were non-toxic, while the strains of A. tamarense and A. catenella were toxic. Toxic isolates DPC7 and DPC8 of A. catenella produced GTX1, 2, 3, 4, 5, dcGTX2, 3, C1, 2, neoSTX and STX with trace or non-detectable levels of C3 and C4, while isolates UL7, KDW981, SJW97043, SJW97046, KJC97111 and KJC97112 of A. tamarense produced GTX1, 2, 3, 4, dcGTX3, C1, 2, neoSTX with trace or non-detectable levels of C3, 4, dcSTX and STX, and no GTX5 and dcGTX2. The major toxins produced by A. catenella were C1 +2, and those of A. tamarense were C1 +2 and GTX4 in most of the isolates. A. tamarense strains other than SJW97046 produced a relatively high proportion of carbamate toxins, reflecting the high toxicity scores of shellfish intoxication in sampled coastal areas. Two representative toxic isolates, A. tamarense SJW97043 and A. catenella DPC7, were cultured for 30 days in batch mode and subjected to toxin analysis at 5-day intervals. Comparison of toxin productivity in terms of total toxin content, toxin components, and their variations with culture age revealed marked differences between the two strains. 相似文献
3.
ABSTRACT: The toxic dinoflagellates Alexandrium tamarense (Lebour) Balech and A. catenella (Whedon and Kofoid) Balech produce potent neurotoxins, such as saxitoxin and gonyautoxin and have been mainly responsible for paralytic shellfish poisoning (PSP) in Japan. To prevent a negative effect on the fishery industry, it is necessary to identify these toxic species precisely and rapidly before and during the bloom. In this paper, a rapid and simple protocol of a fluorescence in situ hybridization (FISH) method using ribosomal RNA (rRNA)-targeted probes has been established for identifying the cultured strains and natural cells of A. tamarense and A. catenella . Using the FISH method established in this study, it was possible to identify these toxic species species-specifically and rapidly, within 30 min. The procedure of detection constituted three steps: (i) fixation/dehydration; (ii) hybridization; and (iii) washing; this made the identification simple. Moreover, this method did not require either special techniques or equipment, and the cost for detection was low. The specificity, rapidity, and simplicity of the developed method suggest that it might be useful for routine monitoring of these toxic microalgae. 相似文献
4.
5.
Utilization of dissolved organic phosphorus by the two toxic dinoflagellates,
Alexandrium tamarense and Gymnodinium catenatum (Dinophyceae) 总被引：2，自引：0，他引：2
SEOK JIN OH TAMIJI YAMAMOTO YUKIHIRO KATAOKA OSAMU MATSUDA YUKIHIKO MATSUYAMA YUICHI KOTANI 《Fisheries Science》2002,68(2):416-424
ABSTRACT: The utilization of dissolved organic phosphorus (DOP) by the two toxic dinoflagellates Alexandrium tamarense (Lebour) Balech and Gymnodinium catenatum Grahamm which were isolated from Hiroshima Bay, Japan, was studied. Alexandrium tamarense grew poorly on fructose-6-phophate, glucose-1-phosphate, glycerophosphate, and ribose-5-phosphate with a phosphomonoester bond, although it grew well on the nucleotides adenosine-5-diphosphate (ADP) and adenosine triphosphate (ATP), as well as on dissolved inorganic phosphorus (DIP; as metaphosphate, pyrophosphate, tripolyposphate and orthophosphate). The results imply that A. tamarense was able to utilize DOP and DIP from ambient water using nucleotidase, pyrophosphatase and poly-phosphatase, which hydrolyze phosphodiesters. In contrast, G. catenatum was able to utilize DOP compounds of various molecular weights and structures as well as DIP. In time-course experiments, alkaline phosphatase activity (APA) was induced at orthophosphate concentrations of 0.43 mmol/L and 3.3 mmol/L for A. tamarense and G. catenatum , respectively, and APA increased with orthophosphate depletion. The experiments also demonstrated that APA was maximum at the optimum temperatures for the growth of A. tamarense and G. catenatum ; that is, 15°C and 25°C, respectively. These results suggest that the DIP-depleted conditions in Hiroshima Bay might have led to the outbreaks of noxious dinoflagellates in recent years. 相似文献
6.
采用显微镜细胞计数和标准藻毒监测方法—小鼠生物法,研究了4种磷浓度0µmol•L-1、1.8µmol•L-1、3.6µmol•L-1和5.4µmol•L-1对有毒甲藻塔玛亚历山大藻(Alexandrium tamarense)和微小亚历山大藻(Alexandtium minutum)的生长和产毒力的影响。结果表明,磷浓度对两种甲藻的生长和产毒能力都有显著性影响(p<0.05)。3.6µmol•L-1磷浓度组的塔玛亚历山大藻密度显著高于其它3个磷浓度组,0µmol•L-1磷浓度组显著低于其它3个磷浓度组。微小亚力山大藻在1.8µmol•L-1、3.6µmol•L-1和5.4µmol•L-1磷浓度下生长没显著性差异(p>0.05)。但在0µmol•L-1磷浓度下生长缓慢,藻密度显著低于其它3个有磷组。0µmol•L-1磷浓度下塔玛亚历山大藻和微小亚历山大藻的产毒能力最高, 显著高于其它磷浓度组(p<0.05),分别为3 MU•10000cells-1和5.2MU•10000cells-1。其它磷浓度组两种有毒甲藻的产毒能力没有显著性差异(p>0.05)。 相似文献
7.
S E Keeling C L Brosnahan C Johnston R Wallis N Gudkovs W L McDonald 《Journal of fish diseases》2013,36(5):495-503
A real‐time PCR assay using a molecular beacon was developed and validated to detect the vapA (surface array protein) gene in the fish pathogen, Aeromonas salmonicida. The assay had 100% analytical specificity and analytical sensitivities of 5 ± 0 fg (DNA), 2.2 × 104 ± 1 × 104 CFU g?1 (without enrichment) and 40 ± 10 CFU g?1 (with enrichment) in kidney tissue. The assay was highly repeatable and proved to be robust following equivalency testing using a different real‐time PCR platform. Following analytical validation, diagnostic specificity was determined using New Zealand farmed Chinook salmon, Oncorhynchus tshawytscha (Walbaum), (n = 750) and pink shubunkin, Carassius auratus (L.) (n = 157). The real‐time PCR was run in parallel with culture and all fish tested were found to be negative by both methods for A. salmonicida, resulting in 100% diagnostic specificity (95% confidence interval). The molecular beacon real‐time PCR system is specific, sensitive and a reproducible method for the detection of A. salmonicida. It can be used for diagnostic testing, health certification and active surveillance programmes. 相似文献
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9.
ABSTRACT: Standard molecular techniques, such as sequencing and restriction fragment length polymorphism analysis after polymerase chain reaction (PCR) amplification are relatively complicated, and species identification can take a long time when using such techniques. We established a quick method, using PCR with species-specific TaqMan Minor Groove Binder (MGB) probes based on single nucleotide polymorphism (SNP) to distinguish the two eel species Anguilla japonica and Anguilla anguilla . This method can be used in processed products. Partial sequences of the mitochondrial 16S rRNA gene were compared between A. japonica and A. anguilla to design a primer pair common to both A. japonica and A. anguilla and probes specific to A. japonica and A. anguilla . Different fluorescence intensities were produced in two PCR microtubes each containing A. japonica - and A. anguilla -specific probes for one target sample. We observed the fluorescence intensity of PCR products in microtubes under ultraviolet transillumination, with similar results to those obtained by real-time PCR. Therefore, SNP-based PCR is a powerful tool for identifying materials of processed foods from either A. japonica or A. anguilla . 相似文献
10.
Vibrio anguillarum , an opportunistic fish pathogen, is the main species responsible for vibriosis, a disease that affects feral and farmed fish and shellfish, and causes considerable economic losses in marine aquaculture. In this study, we used polymerase chain reaction (PCR) to detect V. anguillarum . PCR specificity was evaluated by amplifying the rpoS gene, a general stress regulator, in six strains of V. anguillarum and 36 other bacterial species. PCR amplified a species-specific fragment (689 bp) from V. anguillarum . Furthermore, the PCR assay was sensitive enough to detect rpoS expression from 3 pg of genomic DNA , or from six colony-forming units (CFU) mL−1 of cultured V. anguillarum . However, the assay was less sensitive when genomic DNA from the infected flounder and prawn was used (limit of detection, 50 ng and 10 ng g−1 tissue, respectively). These data demonstrate that PCR amplification of the rpoS gene is a sensitive and species-specific method to detect V. anguillarum in practical situations. 相似文献
11.
条斑紫菜提取物对4种赤潮微藻生长的抑制作用 总被引：2，自引：1，他引：1
研究条斑紫菜水溶性抽提液对前沟藻、中肋骨条藻、米氏凯伦藻和塔玛亚历山大藻等4种赤潮微藻生长的影响,在此基础上,利用甲醇、丙酮、乙酸乙酯、氯仿和石油醚等有机溶剂浸泡条斑紫菜干粉,经抑藻圈方法检测条斑紫菜水溶性抽提液的抑藻活性。通过测定藻细胞密度和细胞体积,观察藻细胞形态,分析藻细胞内叶绿素、蛋白质和多糖等生理生化指标的变化,对抑藻活性最大的提取物对前沟藻、米氏凯伦藻、中肋骨条藻和塔玛亚历山大藻赤潮微藻生长的抑制作用进行分析,并依次以石油醚、乙酸乙酯和正丁醇为提取溶剂,采用液液分离法对此提取物做了进一步分离。结果表明,当条斑紫菜水溶性抽提液浓度超过16g/L时能显著抑制4种赤潮微藻的生长,尤其是对前沟藻和米氏凯伦藻具有很强的抑制作用。在5种有机溶剂提取物中,甲醇提取物的抑制作用最为明显。进一步研究发现此提取物对4种赤潮微藻的生长抑制显著且具有浓度效应,在16g/L时,此提取物对前沟藻、米氏凯伦藻、中肋骨条藻和塔玛亚历山大藻的生长抑制率分别为70.5%、79.9%、67.1%和65.1%。同时,致使米氏凯伦藻、中肋骨条藻和塔玛亚历山大藻等3种赤潮微藻体积变小,运动能力下降,藻细胞出现空洞、细胞破碎和色素减褪等现象;... 相似文献
12.
塔玛亚历山大藻对中国明对虾肝胰腺和鳃SOD、GST和MDA的影响 总被引：2，自引：0，他引：2
本文选择在我国分离得到的一株能典型产生麻痹性贝毒(PSP,Paralytic Shellfish Poison)的有毒赤潮甲藻塔玛亚历山大藻(Alexandrium tamarense,ATHK株),研究其是否能通过引发中国明对虾的脂质过氧化作用, 使机体产生过多活性氧自由基, 导致氧化损伤而发挥其毒性作用。塔玛亚历山大藻粗提液经肌肉注射方式染毒中国明对虾,于染毒后1、3、6、12、24和48 h 测定肝胰腺和鳃SOD,GST活力和MDA含量的影响。结果显示,染毒后 1-6 h, 对虾肝胰腺和鳃组织SOD,GST酶活力均增加,12和48h鳃组织的上述指标受到抑制。对虾肝胰腺MDA含量除1h外未见明显改变,鳃中MDA含量随时间增加呈升高趋势。研究表明：塔玛亚历山大藻粗提液在中国明对虾体内代谢过程中能诱导对虾产生活性氧自由基,塔玛亚历山大藻粗提液能诱导MDA含量增加,降低SOD和GST活力,能够引发鳃的脂质过氧化,对鳃造成氧化损伤。 相似文献
13.
研究了塔玛亚历山大藻（Alexandrium tamarense）ATHK株对海湾扇贝受精卵孵化的影响,并探讨了可能的影响机制。将海湾扇贝受精卵暴露于不同密度的塔玛亚历山大藻ATHK中,观察其对胚胎孵化各阶段的影响,并利用光镜和透射电镜对实验组和对照组胚胎的外部形态和内部结构进行连续观察。结果表明：该藻对海湾扇贝受精卵孵化有显著的抑制作用,其IC50约为800 mL-1;2000 mL-1的ATHK延缓或破坏了胚胎的正常发育,胚胎内部出现大量溶酶体,原肠胚腔不能形成,且部分细胞出现胞质肿胀、线粒体自溶的现象,细胞不能进行正常的功能性分化;观察发现实验组中有些担轮幼虫的外形畸变,鞭毛发育迟缓;至24 h（D型幼虫）时,ATHK组中的D型幼虫数量远远低于对照组（P<0.05）,且死亡率明显高于对照组。观察还发现ATHK藻细胞和胚胎碰撞接触的现象,这种碰撞接触可能对有害物质的释放和转移有一定的刺激作用。 相似文献
14.
Chin-I Chang Chia-Che Wu Ta Chih Cheng Jyh-Ming Tsai & King-Jung Lin 《Aquaculture Research》2009,40(10):1182-1190
A multiplex nested-polymerase chain reaction (PCR)-based (m-nested PCR) method was developed for simultaneous detection of four important freshwater/marine fish pathogens in subtropical Asia, including Aeromonas hydrophila, Edwardsiella tarda, Photobacterium damselae and Streptococcus iniae . The specificity of the oligonucleotide primers used for PCR detection was confirmed to generate specific amplicons for the corresponding pathogens. Moreover, non-specific amplicons were observed when the primers were tested using pure DNA extracted from 31 related bacterial strains belonging to 23 species or tissue homogenates of infected tilapia. This m-nested PCR approach could detect 19 colony forming unit (CFU) for A. hydrophila , 62 CFU for E. tarda , 280 CFU for P. damselae subsp. piscicida and 179 CFU for S. iniae in infected tilapia kidney homogenates, consistent with the results derived from bacteriological methods. The assay described in this paper is a sensitive and effective method for simultaneous detection of multiple fish pathogens. 相似文献
15.
为了筛选链状亚历山大藻特异表达的发光相关基因,应用抑制消减杂交技术,构建了链状亚历山大藻特异表达的cDNA消减文库,共获得500个克隆,通过基因PCR初步筛选表明插入片段的大小主要集中在250～1000 bp,随机挑选10个阳性克隆进行双向测序和序列比对分析,有2个克隆基因片段与已知基因序列高度同源,分别为荧光素结合蛋白和羧化酶/加氧酶,另有8个克隆基因片段在GenBank中未查找到相应的同源基因,可能为未知新基因序列.这些基因的获得为进一步研究链状亚历山大藻特异表达基因,阐明其发光机理及建立特异甲藻的新型检测方法提供重要依据. 相似文献
16.
Hiroshi Yokoyama Tetsushi Kageyama Kenichi Ohara Tetsuya Yanagida 《Fisheries Science》2007,73(3):633-639
ABSTRACT: A new myxosporean parasite was found in the body cavity and caudal peduncle of the freshwater goby Rhinogobius sp. Orange type (OR) collected from the Nagara River, Gifu Prefecture, Japan. Infected fish exhibited substantial swelling of the abdomen caused by large parasitic cysts approximately 10 mm in size, formed in the visceral cavity. The cyst was a compacted aggregate of several smaller cysts, similar to a bunch of grapes in appearance. Histological examination showed that plasmodia developed within the renal capsule, and finally occupied the visceral cavity. Spores were ovoid with an attenuated anterior end. Sutural ridges were conspicuous with several folds on the edge. Average spore size was 11.9 (10.5–13.5) μm long, 9.0 (8.0–10.0) μm wide, and 6.5 (6.0–7.0) μm thick. Two equal polar capsules were 5.5 (4.5–6 0) μm long and 3.0 (2.5–4.0) μm wide. Partial small subunit ribosomal DNA sequences of the myxosporean were distinct from those of other myxozoan species in GENBANK. A new species name, Myxobolus nagaraensis , is proposed for this parasite. 相似文献
17.
S Bartkova B Kokotovic H F Skall N Lorenzen I Dalsgaard 《Journal of fish diseases》2017,40(2):231-242
Furunculosis, a septicaemic infection caused by the bacterium Aeromonas salmonicida subsp. salmonicida, currently causes problems in Danish seawater rainbow trout production. Detection has mainly been achieved by bacterial culture, but more rapid and sensitive methods are needed. A previously developed real‐time PCR assay targeting the plasmid encoded aopP gene of A. salmonicida was, in parallel with culturing, used for the examination of five organs of 40 fish from Danish freshwater and seawater farms. Real‐time PCR showed overall a higher frequency of positives than culturing (65% of positive fish by real‐time PCR compared to 30% by a culture approach). Also, no real‐time PCR‐negative samples were found positive by culturing. A. salmonicida was detected by real‐time PCR, though not by culturing, in freshwater fish showing no signs of furunculosis, indicating possible presence of carrier fish. In seawater fish examined after an outbreak and antibiotics treatment, real‐time PCR showed the presence of the bacterium in all examined organs (1–482 genomic units mg?1). With a limit of detection of 40 target copies (1–2 genomic units) per reaction, a high reproducibility and an excellent efficiency, the present real‐time PCR assay provides a sensitive tool for the detection of A. salmonicida. 相似文献
18.
利用美国分析化学家协会(Association of Official Analytical Chemists, AOAC)所推荐的小鼠生物法(mouse bioassay, MBA), 测定了塔玛亚历山大藻(Alexandrium tamarense)(ATHK藻株)毒素粗提液的麻痹性贝毒(paralytic shellfish poisoning, PSP)毒性;采用浸浴方式, 研究了该藻株对中国对虾的急性毒性; 采用HE染色方法, 分别对有毒藻浸浴处理和毒素粗提液注射处理后96 h中国对虾(Fenneropenaeus chinensis)的鳃和肝胰腺石蜡切片进行观察。结果表明, 该藻株麻痹性贝毒毒性为3.9×10–5 MU/cell (相当于7.3 pg STX Equal/cell), 在同种藻株中属低毒藻株; 在96 h急性毒性实验中, 塔玛亚历山大藻对中国对虾的半致死浓度(LC50)为1.0×104 cells/mL, 安全浓度(SC)为1.0×103 cells/mL; 石蜡切片观察发现, 塔玛亚历山大藻与毒素粗提液分别引起了鳃和肝胰腺组织出现细胞肿胀、空泡化等病理变化。以上研究结果提示, 塔玛亚历山大藻对中国对虾有急性致死作用。为了保证中国对虾的健康养殖, 养殖水体中的塔玛亚历山大藻浓度至少应该控制在1.0×103 cells/mL以下; 鳃的病变直接导致对虾窒息死亡可能是塔玛亚历山大藻暴露后对虾急性致死作用的最重要原因;塔玛亚历山大藻所产的PSP毒素可引起虾体代谢和解毒的主要器官—肝胰腺的病变。 相似文献
19.
Induction and characterization of a lysogenic bacteriophage of Lactococcus garvieae isolated from marine fish species 
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下载免费PDF全文 This study investigated the presence of prophages in Lactococcus garvieae isolated from several marine fish species in Japan. Representative strains of 16 bacterial genotypes (S1–S16) selected from more than 400 L. garvieae isolates were used to induce lysogenic bacteriophages. These strains were treated with 500 ng mL?1 freshly prepared mitomycin C. A cross‐spotting assay was performed to validate the lysogenic and indicator strains. The lysogenic strains were selected for isolation and concentration of the phages. Phage DNA was digested with EcoRI for biased sinusoidal field gel electrophoresis analysis. Polymerase chain reaction (PCR) was used to detect integrated prophage DNA. Of the 16 representative bacterial genotypes, 12 strains integrated prophages as indicated by the PCR assay, and 10 phages were detected and isolated using two indicator bacterial strains. Analysis of genomic DNA showed that these phages were homologous and named as PLgT‐1. Transmission electron microscopy revealed that the morphology of PLgT‐1 was consistent with the virus family Siphoviridae. PCR analysis of the prophage DNA revealed that all of the S1 genotype strains were lysogenic (30/30), but none of the S16 genotype strains were lysogenic (0/30). This is the first study to investigate lysogenic bacteriophages from L. garvieae. 相似文献
20.
Rapid and sensitive detection of Vibrio harveyi by loop‐mediated isothermal amplification combined with lateral flow dipstick targeted to vhhP2 gene 下载免费PDF全文
下载免费PDF全文 Kanittada Thongkao Siwaporn Longyant Kun Silprasit Paisarn Sithigorngul Parin Chaivisuthangkura 《Aquaculture Research》2015,46(5):1122-1131
Vibrio harveyi is a causative agent of the Vibriosis or luminescent bacterial disease in worldwide aquaculture industry. A reliable assay for identification of V. harveyi infection is important to prevent the bacterial spread. In this study, biotinylated loop‐mediated isothermal amplification (LAMP) amplicons were produced by a set of four designed primers that recognized specifically the V. harveyi vhhP2 gene, encoding a putative outer membrane protein with unknown function, followed by hybridization with an fluorescein isothiocyanate (FITC)‐labelled probe and lateral flow dipstick (LFD) detection. A novel set of PCR primer was also designed specifically to vhhP2 gene and appear to be a species‐specific tool for V. harveyi detection. The optimized time and temperature conditions for the LAMP assay were 90 min at 65°C. The LAMP‐LFD and PCR methods accurately identified 22 isolates of V. harveyi but did not detect 16 non‐harveyi Vibrio isolates, and 34 non‐Vibrio bacterial isolates. The sensitivity of LAMP‐LFD for V. harveyi detection in pure culture was 1.1 × 102 CFU mL?1 or equivalent to 0.6 CFU per reaction, while that of PCR was 6 CFU per reaction. For spiked shrimp sample, the sensitivity of LAMP was 1.8 × 103 CFU g?1 or equivalent to 5 CFU per reaction, while that of PCR was 50 CFU per reaction. In conclusion, the established LAMP‐LFD methods provided a valuable tool for rapid identification of V. harveyi and can be used to distinguish V. harveyi from V. campbellii. 相似文献