天津市售主要经济贝类中麻痹性贝类毒素污染现状及特征分析

使用液相色谱串联质谱法(LC-MS/MS)检测9种天津市售主要经济贝类中13种麻痹性贝类毒素(paralytic shellfish poisoning,PSP)的含量,分析PSP的污染现状和成分特征。结果显示,共计6个品种(紫贻贝、虾夷扇贝、牡蛎、毛蚶、脉红螺、菲律宾蛤仔)检出PSP。其中虾夷扇贝、紫贻贝、毛蚶3个品种的检出率较高且含量较高,检出率分别为66.7%、58.3%、58.3%。PSP中检出率较高的毒素包括N-磺酰胺甲酰基类毒素C1、C2和膝沟藻毒素GTX1、GTX2、GTX3,毒力占比主要以膝沟藻毒素GTX1-4为主,毛蚶蓄积STX毒素的能力强于其他贝类。全年的PSP含量季节变化为夏季最高,冬季最低。PSP主要存在于贝类的消化腺内,全年不同月份的PSP含量均在安全食用范围内。     

Purification and characterization of sulfotransferase specific to O-22 of 11-hydroxy saxitoxin from the toxic dinoflagellate Gymnodinium catenatum (dinophyceae)

ABSTRACT: A novel sulfotransferase (O-ST), which transferred the sulfate group of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to O-22 of 11-α,β-hydroxy saxitoxin (STX) and produced GTX2 + 3, was purified to homogeneity from the cytosolic fraction of clonal-axenic vegetative cells of the toxic dinoflagellate Gymnodinium catenatum GC21V. After four purification steps, including affinity chromatography and anion exchange chromatography, the enzyme was purified 500-fold and the yield was 4%. On affinity chromatography with a PAP-agarose column, O-ST was observed in the bound fraction, and N-ST specific to N-21 of STX and GTX2 + 3 was found in the unbound fraction. The molecular mass of the purified enzyme was determined by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) to be 65 kDa. Gel filtration chromatography showed a native molecular mass of 67 kDa, indicating that O-ST is a monomeric enzyme. The enzyme was optimally active at pH 6.0 and 35°C. O-ST did not require metal cations for its activity. O-ST required PAPS as the sole source of sulfate. O-ST transferred a sulfate group from PAPS to only O-22 of 11-α,β-hydroxy STX and not to N-21 of these toxins. These observations suggested that two ST, N-ST and O-ST, participate in the sulfation of PSP toxins.     

Development of a quantification assay for the cysts of the toxic dinoflagellate <Emphasis Type="Italic">Alexandrium tamarense</Emphasis> using real-time polymerase chain reaction

ABSTRACT:   The cysts of toxic dinoflagellate Alexandrium tamarense are the seed population for the bloom responsible for paralytic shellfish poisoning (PSP). However, it is impossible to identify the Alexandrium spp. cyst on the basis of morphological features. In this study, we prepared A. tamarense cysts by sexual conjugation in laboratory conditions and developed an efficient DNA extraction method for polymerase chain reaction (PCR) assay. Using the A. tamarense cysts, we established the identification and quantification method showing the species specificity and the high sensistivity for A. tamarense cysts using real-time PCR. This assay was also able to detect and quantify the A. tamarense cysts accurately when mixed with excess cysts of A. catenella (Whedon and Kofoid) Balech prepared by conjugation experiment.     

Development and application of fluorescence in situ hybridization (FISH) method for simple and rapid identification of the toxic dinoflagellates Alexandrium tamarense and Alexandrium catenella in cultured and natural seawater

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.     

营养环境对微小亚历山大藻C4生长和产毒的影响

为考察营养成分以及培养基础液再利用对一株微小亚历山大藻的生长和产毒的影响,采用单因素实验分别研究了氮(NaNO_3)、磷(NaH_2PO_4)、微量元素(FeCl_3、Na2EDTA、CuSO_4、Na_2MoO_4、ZnSO_4、CoCl_2、MnCl_2)、维生素(Vitamin B12、Vitamin H、Vitamin B1)、碳(NaHCO_3)的不同含量以及海水(培养基础液)利用方式(不循环与循环利用)对微小亚历山大藻C4生长、毒素含量(μmol/L)与毒素组成的影响。结果显示,氮、磷浓度对C4藻的毒素总含量(μmol/L)有显著影响,其他因素影响均不显著;氮、磷、碳及微量元素的浓度对GTX1/4(GTX1+GTX4)在总毒素(GTX1+GTX2+GTX3+GTX4)中所占比例均影响显著,而维生素浓度和海水循环利用对GTX1/4所占比例影响均不明显。氮浓度在0~883μmol/L范围内,毒素含量与氮浓度呈正相关关系,氮浓度进一步增加,毒素含量没有明显变化;磷浓度在0~145.2μmol/L范围内增加,毒素含量先增后降,最终保持稳定的趋势。最佳的产毒条件为氮883μmol/L,磷18.15μmol/L,微量元素为f/2海水培养基中微量元素的0.5倍,碳不添加。     

Difference in the level of paralytic shellfish poisoning toxin accumulation between the crabs <Emphasis Type="Italic">Telmessus acutidens</Emphasis> and <Emphasis Type="Italic">Charybdis japonica</Emphasis> collected in Onahama,Fukushima Prefecture

ABSTRACT:     The difference in paralytic shellfish poisoning (PSP) toxin accumulation in the crabs Telmessus acutidens and Charybdis japonica was investigated at Onahama, Fukushima Prefecture, from 2002 to 2005. The level of toxin accumulation in the hepatopancreas of T. acutidens corresponded to that of mussels when examined on a yearly basis. In 2003, some crabs had a high toxicity of approximately 1000 MU, which compares to one-third of the human minimum lethal dose. Therefore T. acutidens should be monitored as a vector species of PSP toxin. The toxin profile of T. acutidens was also investigated. Because an increase in highly toxic species of PSP toxins with a decrease in low toxic species, such as N -sulfocarbamoyl-11-hydroxysulfate toxins, was not clearly observed between consecutive samples, toxin transformation in T. acutidens was considered to have a minimal impact on toxicity. PSP toxins were also detected in several specimens of C. japonica , but the highest toxicity was only 7.4 MU/g in the hepatopancreas. Because C. japonica is widely distributed in the coastal waters of Japan, investigation of toxin accumulation in the crab should be continued, including its investigation in different areas of water from Onahama, where the causative dinoflagellates of PSP occur in different conditions.     

广东东部沿海麻痹性贝类毒素成分特征分析

为了弄清广东东部沿海贝类中麻痹性贝类毒素(Paralytic shellfish poisoning,PSP)的主要成分及其藻类来源,运用液相色谱—柱后衍生荧光检测技术,对2005-2006年间在粤东近岸重点养殖区采集的主要经济贝类进行了麻痹性贝类毒素成分分析。分析结果表明,在染毒的贝类样品中共检出PSP的12种成分,各成分的检出率时空分布特征有所不同,其中N-磺酸氨甲酰基类毒素C1和氨基甲酸酯类毒素GTX1的检出率最高,均达78%以上;其次为GTX4,达71%。在毒素含量方面,C1普遍较低,春季含量较其他季节高,而在春秋两季贝类中,GTX1和GTX4含量普遍较高。总体上,麻痹性贝毒在粤东海域的分布具有明显的地域性、季节性和贝类种间差异。地域上以柘林湾污染较为严重,毒素成分最多,且含量较高;季节上春季为染毒高峰期,秋季次之;主要染毒贝类为牡蛎(Crassostrea spp.)、结蚶(Tegillarca nodifera)、泥蚶(Tegillarca granosa)和蚶(Scapharca spp.),柘林湾2005年9月14日的结蚶毒性最高,其毒性当量值达到370μgSTX/100g。与其他贝类相比,蚶类对高毒力的GTX毒素有更强的积累能力。     

Accumulation of paralytic shellfish poisoning toxin by the swimming crab <Emphasis Type="Italic">Charybdis japonica</Emphasis> in Kure Bay,Hiroshima Prefecture

The accumulation of paralytic shellfish poisoning (PSP) toxin in the crab Charybdis japonica was investigated in Kure Bay, when a bivalve Mytilus galloprovincialis, in the same water, showed toxicity caused by PSP toxin. In 2005, 17 specimens of the crab had PSP toxin over 4 MU/g in the hepatopancreas, and the highest toxicity was at 37.4 MU/g. Since the regulation limit of PSP for crab hepatopancreas was set in 2004, this is the first observation of regulatory level of PSP toxin in C. japonica. Unlike in the hepatopancreas of the crab, the hazardous level of the toxin was not detected in the muscle of the cephalothorax and the appendage. The toxin accumulation in the crab was also investigated by feeding toxic mussels to the crab. The crab retained the toxin mainly in the hepatopancreas, and the ratio of retention in the crab was from 12.9 to 24.6%. The toxin profiles, shown in the feeding experiments, suggest that the transformation of PSP toxin occurs in the crab because dcGTX2 and dcGTX3 was detected in all crab specimens despite the lack of these analogs in the mussels used as feed.     

麻痹性贝类毒素在栉孔扇贝体内的代谢轮廓

将栉孔扇贝(Chlamys farreri)直接暴露于产毒藻,通过比较各组织器官中毒素的蓄积及代谢转化特征,研究麻痹性贝类毒素(Paralytic Shellfish Toxins,PSTs)在栉孔扇贝体内危害形成的过程。结果显示,分布于中国的一株塔玛亚历山大藻(Alexandrium tamarense,AT5-3株),其生长及产毒性状稳定,产毒能力强,主要成分为Gonyautoxins-1-4(GTX14),单细胞产毒能力为7.95 fmol/cell;栉孔扇贝对该产毒藻具有较强摄食能力及PSTs蓄积能力,总体蓄积效率达到84.4%,最高蓄积浓度为1903μg STX eq./kg,不同组织蓄积能力由高到低依次为内脏团、性腺、外套膜、闭壳肌;内脏团对PSTs的代谢能力也最强,是该毒素在栉孔扇贝体内蓄积代谢的靶器官;此外,栉孔扇贝对PSTs表现出较强的生物转化能力,主要转化途径为:N-磺酰胺甲酰基类毒素(N-Sulfocarbamoylgonyautoxin-2,C1)→膝沟藻毒素(Gonyautxins-2,GTX2)/脱氨甲酰基类毒素(Decarbamoylgonyautoxins-2,dc GTX2),N-磺酰胺甲酰基类毒素(N-Sulfocarbamoylgonyautoxin-3,C2)→膝沟藻毒素(Gonyautxins-3,GTX3)。本研究中,栉孔扇贝对PSTs总体呈现出迅速蓄积和缓慢代谢的特点,同时,GTX14和NEO等高毒成分所占比例较高,造成扇贝中PSTs毒性和风险性均显著升高。本研究有助于科学评估PSTs危害的形成机制,为系统监控我国PSTs风险性提供科学支撑。     

磷浓度对２种有毒亚历山大藻生长和产毒力的影响

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)。     

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

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。     

Kinetics of diarrhetic shellfish poisoning toxins,okadaic acid,dinophysistoxin-1, pectenotoxin-6 and yessotoxin in scallops <Emphasis Type="Italic">Patinopecten yessoensis</Emphasis>

ABSTRACT:   Four toxins, okadaic acid (OA), dinophysistoxin-1 (DTX1), pectenotoxin-6 (PTX6), and yessotoxin (YTX), all associated with diarrhetic shellfish poisoning (DSP), were administered via syringe to Scallops Patinopecten yessoensis and their distribution in the hepatopancreas, adductor muscle, and combined other tissues (mantle, gill, gonad) was analyzed by liquid chromatography-mass spectrometry. Toxins exclusively remained in the hepatopancreas irrespective of the injection site, adductor muscle or hepatopancreas. When injected into hepatopancreas, OA, DTX1, and YTX were metabolized to 7- O -palmitoylOA, 7- O -palmitoylDTX1 and 45-hydroxyyessotoxin (45OH-YTX), respectively. Such metabolic changes were insignificant when toxins were injected into the adductor muscle. The residual ratio for each toxin in the hepatopancreas was less than 20%. Mortalities of scallops treated with PTX6 were lower than those treated with other toxins.     

产气荚膜梭菌α、β毒素研究进展

产气荚膜梭菌(Clostridium perfringens)是一种革兰氏阳性、产生芽胞、严格厌氧及形成特殊荚膜的梭状杆菌。该菌广泛存在于自然界的水源、土壤及人和动物肠道之中。研究发现,该菌至少可以产生15种以上的毒素,但目前仅有5-6种毒素,即α、β、ε、ι毒素及肠毒素(CPE)、β2毒素等被认为是该菌的主要致病性毒素。根据产气荚膜梭菌产生四种致死性毒素(α、β、ε、ι毒素)的能力,将其分为五种类型,即A、B、C、D、E型。产气荚膜梭菌是一种重要的人畜共患病的病原体,可引起人的食物中毒和多种动物坏死性肠炎、肠毒血症同时也是人畜创伤性气性坏疽以及人类食物中毒的主要病原菌之一。近年来,对产气荚膜梭菌α、β毒素研究取得了重大进展。现就产气荚膜梭菌α、β毒素的组成、结构、分子生物学、检测等方面的研究进展做一综述。     

Accumulation of paralytic shellfish poisoning toxins in bivalves and an ascidian fed on Alexandrium tamarense cells

SUMMARY: Cultured cells of the dinoflagellate Alexandrium tamarense were fed to four species of bivalves and an ascidian to examine the interspecific differences in the ability to accumulate paralytic shellfish toxins. The specimens of each species were reared in the same tank. All the species ingested almost all the cells fed and became toxic. Marked individual differences in toxin accumulation was observed in all the species. However, the average amounts of accumulated toxins increased during feeding. When the animals were fed on the same amount of cells, they accumulated almost the same amounts of toxins, indicating that interspecific differences in toxin accumulation observed in nature is due to the difference in ingestion behavior of the animals under natural conditions.     

超高效液相色谱荧光法检测贝类中三种高毒性麻痹性贝毒

建立灵敏可靠的麻痹性贝类毒素(PSP)检测技术是保障我国贝类水产品质量安全的有力手段。本研究选取3-(2-呋喃甲酰基)-喹啉-2-羰醛(FQ)为荧光衍生试剂,利用超高效液相色谱(UPLC)和柱前衍生荧光检测技术,建立了贝类中3种高毒性PSP毒素成分(STX、GTX1及NEO)的检测方法,对影响荧光衍生效率和色谱分离效果的各主要实验因素分别进行了优化。结果表明,在优化后的最佳实验条件下,3种PSP毒素成分线性方程的相关系数(r)均大于0.998,保留时间(tm)及峰面积(PA)的日内及日间精密度RSD值分别小于3.1%和5.6%,当信噪比(S/N)等于3时,检测限范围为7～14μg.kg-1,样品加标回收率为82%～92%,RSD值小于5.2%。该方法灵敏、稳定且可靠,可用于贝类中三种高毒性PSP毒素成分的日常分析检测。     

贝类毒素固相吸附示踪监测技术研究进展

固相吸附毒素示踪监测技术(Solid Phase Adsorption Toxin Tracking,SPATT)是一项新的贝类毒素监测技术。该技术模拟滤食性贝类摄食过程,利用内含吸附剂的采样器富集水体中的贝类毒素,结合高效液相色谱-质谱检测技术或酶联免疫检测技术,可以对产毒藻类的暴发以及贝类毒素污染进行示踪研究。SPATT 监测技术与贝类监测法、浮游藻类监测法相比,具有取样操作简单、检测成本低等优点,已成为贝类毒素污染监测技术研究的热点。本综述针对 SPATT 技术的研究进展及应用进行阐述,对技术适用监测的毒素种类、吸附材料的优劣等进行分析。     

Confirmation of yessotoxin production by the dinoflagellate <Emphasis Type="Italic">Protoceratium reticulatum</Emphasis> in Mutsu Bay

ABSTRACT:   Yessotoxin (YTX) is a shellfish toxin and its contamination in bivalves has seriously damaged shellfish industries. The biogenetic origin of YTX was identified as the dinoflagellate Protoceratium reticulatum (Claparède et Lachmann) Bütschli collected in New Zealand and Yamada Bay, Iwate in Japan. Scallops cultured in Mutsu Bay, Japan, were frequently contaminated with YTX, however, occurrence of P. reticulatum in this bay and YTX production by the local strains have not been investigated. Eight strains of P. reticulatum , isolated from the bay, were cultured in the laboratory, and analyzed by fluorometric high-pressure liquid chromatography and liquid chromatography/mass spectrometry methods for YTX production and composition. All strains tested were confirmed to produce YTX, and none of them produced known YTX analogs. Toxin amount and composition differed from strain to strain. This result is also confirmation of one of the biogenetic origins of YTX in Mutsu Bay.     

环境因子对链状亚历山大藻生长的影响

链状亚历山大藻是一种能产生麻痹性贝毒的有害赤潮藻类,研究环境因子对链状亚历山大藻生长特性的综合影响,找到链状亚历山大藻生长的适宜条件,对有效预报赤潮形成具有十分重要的意义.本文设计了四因素三水平的正交实验,研究了温度、盐度、营养盐、光照强度等环境因子对链状亚历山大藻生长特性的影响.结果表明,在实验范围内,链状亚历山大藻...     

Distribution and abundance of resting cysts of the toxic Alexandrium spp. (Dinophyceae) in sediments of the western Seto Inland Sea, Japan

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.     

Lactic acid bacteria effective for regulating the growth of contaminant bacteria during the fermentation of <Emphasis Type="Italic">Undaria pinnatifida</Emphasis> (Phaeophyta)

ABSTRACT:   Lactic acid fermentation of seaweed is a recent topic and quite limited information is available on culture conditions. To know the suitable strains for use as a starter culture for seaweed fermentation, 14 lactic acid bacteria (LAB) strains, including 11 species, were tested in culture conditions prepared with or without salt. A commercial product of Undaria pinnatifida powder was used as a substrate for fermentation without sterilizing. Starter-suitability of the LAB strains was assessed from their predominance after culture. Among the tested strains, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus casei and Lactobacillus rhamnosus showed high (>90%) predominance in their cultures, while control cultures prepared without inoculation of LAB did not show any detectable growth of acid producing bacteria and spoiled. A total of 102 strains not showing acid producing activity were isolated and characterized from spoiled cultures, and all the isolates were observed as Bacillus strains, including 64 strains (62.7%) and 16 strains (15.7%) of a Bacillus cereu s-related and B. fusiformis -related species, respectively. The Undaria substrate before fermentation contained culturable microorganisms at 1.4–3.1 × 10² CFU/g, but the Bacillus cereu s-related strain was not a major composition, suggesting a concern of selective growth of the Bacillus cereu s-related strain during the spoiled fermentation.