贝毒新成员--Azaspiracid shellfish poison

近年来,欧洲沿海国家发生了一系列因食用紫贻贝(Mytilus edulis)而引起人员中毒的事件。采用适宜的化学提取方法从养殖贝类的组织中提取出了azaspiracid(AZA1)及其类似物,并从养殖海区采集的厚甲原多甲藻(Protoperidinium cras-sipes)细胞中提取出了AZA1、AZA2和AZA3三种成分。鉴于这类毒素(AZAs)具有独特的化学结构和特性,把由它们引起的人员中毒事件称为AZP(azaspiracid shellfish poisoning)。根据近年来国外对AZP研究的最新进展,本文对AZAs的化学结构、来源、地理分布、毒性效应、检测方法及作用机制等方面进行了综述,并对我国开展AZP研究的重点领域提出了建议。

Comparative study on in vitro transformation of paralytic shellfish poisoning (PSP) toxins in different shellfish tissues

Dissected tissues of three shellfish species, the Chinese scallop, Chlamys farreri, Manila clam, Ruditapes philippinarurn, and Razor shell, Solen strictu were evaluated for in vitro transformation of paralytic shellfish poisoning （PSP） toxins. Tissue homogenates were incubated with extraction from toxic algae Alexandriurn rninutura to determine toxin conversion. The effects of heating and addition of a natural reductant （glutathione） on toxin conversion were also assessed. The toxin profile was investigated through high performance liquid chromatography with fluorescence detection （HPLC-FLD）. The evident variations in the toxin content were observed only in Chinese scallop viscera homogenates. The concentration of GTX4 was reduced by 45% （approximately 0.8 μmol/dm^3） and 25% （approximately 1 μmol/dm^3） for GTX1, while GTX2 and GTX3 increased by six times （approximately 1 μmol/dm^3） and 3 times （approximately 0.3μmol/dm^3） respectively. Simultaneously, the total toxicity decreased by 38% during the 48 h incubation period, the final toxicity was 20.4 nmol STXeq/g. Furthermore, heated Chinese scallop viscera homogenates samples were compared with non-heated samples. The concentration of the GTX4 and GTX1 was clearly 28% （approximately 0.53 μmol/dm^3） and 17% （approximately 0.69μmol/dm^3） higher in heated samples, GTX2 and GTX3 were four times （0.66 μmol/dm^3） and two times （0.187 μmol/dm^3） lower respectively. GSH （＋） Chinese scallop viscera homogenates samples were compared with GSH （-） samples, the concentration in the GTX4 and GTX1 was 9% （approximately 0.12 μmol/dm^3） and 11% （approximately 0.36 μmol/dm^3） lower respectively, GTX2 and GTX3 was 17% （approximately 0.14 μmol/dm^3） and 19% （approximately 0.006 μmol/dm^3） higher respectively. In contrast,there was a little change in the concentration of PSP toxins of Manila clam and Razor shell tissue ho- mogenates. These observations on three shellfish tissues confirmed that there were species-specific differences in PSP toxins transformation. PSP toxins transformation was more pronounced in viscera tissue than in muscle tissue. PSP toxins was possibly interfered by some carbamoylase enzyme, and the activity in Chinese scallop viscera tissue is more remarkable than in the other two species.

A new simple screening method for the detection of paralytic shellfish poisoning toxins

The current testing for paralytic shellfish poisoning(PSP) in shellfish is based on the mouse bioassay(MBA).To alleviate animal welfare concerns,we evaluated the utility of using sublethal indicators of toxicity as an alternative to measuring time to death.Live mice were injected with a PSP congener and the changes in neurotransmitter levels were measured 60,90,and 120 min after injection.Acetylcholine(ACh) was the most sensitive marker for PSP toxicity.The changes in neurotransmitter levels were most pronounced in the blood.Thus,measurement of Ach levels in the blood may serve as a sensitive predictor for PSP that would not require sacrifice of the mice.This method was relatively simple,sensitive(less than 1 μg/kg weight,equivalent to 20 ng/mL),low maintenance,and rapid(less than 60 min).

Evaluation of mouse bioassay results in an inter-laboratory comparison for paralytic shellfish poisoning toxins

An inter-laboratory comparison of the AOAC mouse bioassay for paralytic shellfish poisoning (PSP) toxicity in shellfish was carried out among 25 Chinese laboratories to examine the overall performance for PSP testing in China, and to analyze the main factors affecting the performance of this method. The toxic scallop Patinopecten yessoensis collected from coast of Bohai Sea, China, was used as a test sample in the comparison study. The results were reported and evaluated using robust statistical methods. The z scores showed that 80%, 8%, and 12% of laboratories reported satisfactory results, unsatisfactory results, and questionable results, respectively. This evaluation demonstrates that the PSP mouse bioassay is an appropriate method for screening and testing PSP toxicity in shellfish. However, it was found that the experience and skill of technicians, as well as the body weight and health status of mice being used significantly affected the accuracy of the method.

Development of An ICR Mouse Bioassay for Toxicity Evaluation in Neurotoxic Poisoning Toxins-Contaminated Shellfish

Objective To develop an ICR （female） mouse bioassay （MBA） for toxicity confirmation and evaluation of neurotoxins （brevetoxins）-contaminated shellfish. Methods Brevetoxins （BTX-B） as a causative agent of neurotoxic shellfish poisoning （NSP） under different shellfish matrices were intraperitoneally injected at different doses into mice to study their toxic effects and to differentiate the range of lethal and sublethal dosages. Their sensitivity and specificity were analyzed with 2 competitive ELISA kits for quantitative determination of standard BTX-B and dihydroBTX-B under different shellfish matrix-diluent combinations. Detection rates of MBA and two antibody-based assays for BTX-B from field NSP-positive shellfish samples were compared. Results BTX-B could be detected in shellfish tissues at concentration of 50-400 μg/100 g under shellfish matrix-Tween-saline media, which were appropriate to identify toxic shellfish at or above the regulatory limit （80 μg/100 g shellfish tissues）. The LD 50 identified was 455 g/kg for BTX-B under general shellfish matrices （excluding oyster matrices） dissolved in Tween-saline. The presence of shellfish matrices, of oyster matrices in particular, retarded the occurrence of death and toxicity presentation in mice. Two antibody-based assays, even in the presence of different shellfish matrix-diluent combinations, showed acceptable results in quantifying BTX-B and dihydroBTX-B well below the regulatory limit. Conclusion The two ELISA analyses agree favorably （correlation coefficient, r 0.96; Student＇s t-tests, P〉0.05） with the developed bioassay.

Transfer of Paralytic Shellfish Toxins via Marine Food Chains:A Simulated Experiment

Objective To study the transfer of paralytic shellfish toxins （PST） using four simulated marine food chains： dinoflagellate Alexandrium tamarense→Artemia Artemia salina→Mysid shrimp Neomysis awatschensis; A. tamarense→N. awatschensis; A. tamarense→A, salina→Perch Lateolabrax japonicus; and A. tamarense→L, japonicus. Methods The ingestion of A. tamarense, a producer of PST, by L. japonicus, N. awatschensis, and A. salina was first confirmed by microscopic observation of A. tamarense cells in the intestine samples of the three different organisms, and by the analysis of Chl.a levels in the samples. Toxin accumulation in L. japonicus and N. awatschensis directly from the feeding on A. tamarense or indirectly through the vector of A. salina was then studied, The toxicity of samples was measured using the AOAC mouse bioassay method, and the toxin content and profile of A. tamarense were analyzed by the HPLC method. Results Both A. salina and N. awatschensis could ingest A. tamarense cells. However, the ingestion capability of A. salina exceeded that of N. awatschensis. After the exposure to the culture of A. tamarense （2 000 cells·mL^-1） for 70 minutes, the content of Chl.a in A. salina and N. awatschensis reached 0.87 and 0.024 μg.mg^-1, respectively. Besides, A. tamarense cells existed in the intestines of L. japonicus, N. awatschensis and A. salina by microscopic observation. Therefore, the three organisms could ingest A. tamarense cells directly. A. salina could accumulate high content of PST, and the toxicity of A. salina in samples collected on days 1, 4, and 5 of the experiment was 2.18, 2.6, and 2.1 MU.g^-1, respectively. All extracts from the samples could lead to death of tested mice within 7 minutes, and the toxin content in anemia sample collected on the 1st day was estimated to be 1.65×10 ^5 μg STX equal/individual. Toxin accumulation in L japonicus and N. awatschensis directly from the feeding on A. tamarense or indirectly from the vector ofA. salina was also studied. The mice injected with extracts from L japonicus and N. awatschensis samples that accumulated PST either directly or indirectly showed PST intoxication symptoms, indicating that low levels of PST existed in these samples. Conclusion Paralytic shellfish toxins can be transferred to L. japonicus, N. awatschensis, and A. salina from A. tamarense directly or indirectly via the food chains.

A Transcriptome Analysis of Neural Tissue of Litopenaeus vannamei After Acute Exposure to Alexandrium pacificum

Alexandrium pacificum(A.pacificum)is a typical paralytic shellfish poisonous dinoflagellate.Harmful algal blooms(HABs)caused by this species can bring serious environmental problems and economic losses to the aquaculture industry.In this study,transcriptome sequencing and analyses were performed on the neural tissue of Litopenaeus vannamei(L.vannamei)after acute exposure to A.pacificum disrupted solution for 72 h,and differentially expressed genes(DEGs)were identified.The results showed that,compared with the control samples,300 DEGs were identified in the experimental group,of which 194 were up-regulated,and 106 down-regulated.The gene ontology(GO)functional enrichment analysis showed that DEGs were significantly enriched in the cortical cytoskeleton organization,troponin complex,amylo-alpha-1,6-glucosidase and thymidine phosphorylase.Kyoto encyclopedia of genes and genomes(KEGG)enrichment analysis found that DEGs were mainly enriched in the oxidative phosphorylation process,intercellular tight junctions and mitophagy.The results showed that the proteoglycans,signaling pathways,and various metabolic processes that regulate cell proliferation,differentiation,and apoptosis all played an essential role in the response of L.vannamei to A.pacificum toxins.

Reporter Gene Assay for Detection of Shellfish Toxins

Objective To explore the potential reporter gene assay for the detection of sodium channel-specific toxins in shellfish as an alternative for screening harmful algal bloom (HAB) toxins, considering the fact that the existing methods including HPLC and bioassay are inappropriate for identifying HAB toxins which poses a serious problem on human health and shellfish industry. Methods A reporter plasmid pEGFP-c-fos containing c-fos promoter and EGFP was constructed and transfected into T24 cells using LipofectAMINE 2000. Positive transfcctants were screened by G418 to produce a pEGFP-c-fos-T24 cell line. After addition of increasing neurotoxic shellfish poison (NSP) or GTX2,3, primary components of paralytic shellfish poison (PSP), changes in expression of EGFP in the cell line were observed under a laser scanning confocal microscope and quantified with Image-pro Plus software. Results Dose-dependent changes in the intensity of green fluorescence were observed for NSP in a range from 0 to 10 ng/mL and for GTX2,3 from 0 to 16 ng/mL. Conclusion pEGFP-c-fos-T24 can be applied in detecting HAB toxins, and cell-based assay can be used as an alternative for screening sodium channel-specific HAB toxins.

天津市售海产贝类腹泻性毒素污染状况分析

为掌握天津市售海产贝类腹泻性贝类毒素(diarrhetic shellfish poison,DSP)的污染状况,保障消费者的食用安全。2019—2023年每年4—8月在天津市大型水产批发农贸市场采集10种共354个海产贝类样品,采用酶联免疫(ELISA)法进行检测,并对检测结果进行数据分析。检测结果共有253个样品检出阳性,检出率为71%,检出样品DSP的含量范围为10.40~154.47μg/kg,均值为51.44μg/kg,并未有超过食用安全限量值160μg/kg的样品。研究表明,在每年4—8月染毒高发期间,天津市售海产贝类DSP毒素含量整体较低,在采集的10种贝类中,牡蛎、扇贝、毛蚶的检出率、检出平均含量均位于前列,DSP含量5月最低,8月最高。通过本次研究发现,至2023年4—8月高发期,天津市售海产贝类腹泻性贝类毒素的检出率已达到100%,说明养殖环境中已存在产毒藻类繁殖,尽管含量普遍较低,应坚持在高发期对染毒情况进行监测,并建议河北省、山东省、辽宁省等天津周边养殖区域持续对养殖环境进行监测。

渤海海域唐山贝类养殖区腹泻性和麻痹性贝类毒素的监测与风险评估

为监测渤海海域唐山贝类养殖区贝类毒素的污染情况,防止食用贝类中毒事件发生,于2019年10月—2020年9月间,每月持续在渤海海域唐山贝类养殖区采集四角蛤(Mactra veneriformis)、菲律宾蛤仔(Ruditapes philippinarum)、脉红螺(Rapana venosa)、牡蛎(Crassostrea gigas)、青蛤(Cyclina sinensis)、文蛤(Meretrix meretrix)和硬壳蛤(Mercenaria mercenaria)7种经济贝类样品,采用高效液相色谱–串联质谱(HPLC-MS/MS)法测试了5种腹泻性贝类毒素(diarrhetic shellfish poisoning,DSP)和14种麻痹性贝类毒素(paralytic shellfish poisoning,PSP)。结果显示,在7种经济贝类样品中均未检出DSP。检出的PSP成分包括石房蛤毒素(Saxitoxin,STX)、膝沟藻毒素1(Gonyautoxin 1,GTX 1)、膝沟藻毒素2(Gonyautoxin 2,GTX 2)和脱氨甲酰基膝沟藻毒素3(Ddecarbamoy l gonyautoxin 3,dcGTX 3),其中,GTX 1含量最高且最高值为537.95μg/kg。不同季节贝类毒素蓄积含量有一定差异,PSP主要集中在4月检出。菲律宾蛤仔、牡蛎、文蛤和硬壳蛤中PSP的检出率分别为11.76%、47.06%、5.90%和8.82%,其他贝类均未检出。PSP总量均低于欧盟及中国的食用安全限量标准800μg STXeq/kg。应用风险熵值法和点评估法进行食用安全风险评估,显示风险熵值和暴露风险指数均在安全范围内,结果表明,渤海海域唐山贝类养殖区7种经济贝类不存在食用安全风险。

秦皇岛近岸海域脂溶性藻毒素污染状况与来源分析

2016年以来,秦皇岛地区多次出现麻痹性贝类毒素导致的中毒事件,但对于该海域脂溶性藻毒素的污染状况仍不够了解。2020—2021年,本研究通过对秦皇岛近岸海域为期1年的现场采样调查,应用高效液相色谱-质谱联用手段检测了浓缩浮游植物和贻贝(Mytilus galloprovincialis)、牡蛎(Crassostrea gigas)和扇贝(Chlamys farreri)3种贝类中脂溶性藻毒素的污染状况,并通过高通量测序手段对浮游植物样品中的潜在有毒藻种进行了分析。结果表明,在秦皇岛近海浮游植物样品和贝类中均可检出扇贝毒素-2(pectenotoxin-2,PTX2),其中2021年4—5月浓缩浮游植物样品中的PTX2毒素含量达到最高值1.36 ng/L,贝类中的PTX2毒素也主要在该时段检出,4月26日3种贝类中均可检出PTX2,含量最高达到1.10μg/kg。对浮游植物样品的高通量测序结果表明,样品中检测到的鳍藻序列数与毒素含量具有显著线性相关性,推测PTX2毒素主要来自样品中的渐尖鳍藻(Dinophysis accuminata)和倒卵形鳍藻(D.fortii)等。综合一年的调查结果表明,秦皇岛近岸海域的脂溶性藻毒素主要为PTX2,其污染状况在春季最高,由于贝类中PTX2毒素含量很低,引发脂溶性藻毒素中毒事件的风险较小。本文调查结果可为秦皇岛近岸海域有害藻华和藻毒素的监测研究工作提供依据。

利玛原甲藻产毒差异及影响因素研究进展

利玛原甲藻(Prorocentrum lima,P.lima)是全球广泛分布的有害赤潮藻类,也是腹泻性贝毒(Diarrhetic Shellfish Toxins,DSTs)的主要产生藻之一,其产生的DSTs给海洋环境、渔业及人类健康带来了严重的危害。研究发现,全球范围内不同P.lima藻株的产毒能力具有显著差异,多种环境因子如营养盐、盐度、光照等因素均会对P.lima的产毒能力产生影响,从而决定了不同区域贝类中DSTs的风险。本文比较分析了全球范围内P.lima的产毒差异,并重点解析了环境因子对其产毒能力的影响,对于全球范围内P.lima产毒能力差异及影响因素形成了较为系统的科学认知,对未来开展P.lima海洋生态学及食品安全研究具有重要的借鉴意义。

基于近红外光谱和多层感知机的贻贝中腹泻性贝毒快速无损检测

以腹泻性贝毒(diarrheal shellfish poison,DSP)污染和未污染良好贻贝为研究对象,利用近红外光谱仪采集950~1700 nm波长内各120个样本的光谱数据;分析确定适合贻贝近红外光谱(near-infrared spectroscopy,NIS)的最佳预处理方法来消除环境光的影响;构建多层感知机(multi-layer perceptron,MLP)模型作为检测DSP污染贻贝的分类器。将240个样本构成的数据集按照7∶3随机划分为训练集和测试集,将运行50次模型得到的准确率的平均值作为最终评价指标,检测DSP污染贻贝的准确率达到99.94%。该研究所构建的基于NIS的MLP模型对DSP的检出限为35μg/kg。对于实际应用中存在的数据集中训练集所占比重不同、小样本和类别不均衡等问题,分析了MLP模型的检测性能。实验结果表明,基于一阶导数光谱预处理的MLP模型对以上3种问题不敏感,说明该分类器泛化能力及鲁棒性较强。因此,将NIS与MLP分类器结合是一种可行的贝毒无损鉴别的新方法。

贝类毒素大田软海绵酸的免疫分析检测技术研究进展

大田软海绵酸(okadaicacid,OA)是一种广泛存在于贝类等生物中的海洋生物毒素,可引起人或动物的急性中毒,对食品安全和海产养殖具有严重危害。因此建立快速、可靠、灵敏的OA检测技术具有重要意义。免疫分析检测技术基于抗原抗体的结合,特异性强、灵敏度高、应用范围广,是当前检测贝类毒素OA的主要手段。本文综述了近年来针对贝类毒素OA的免疫分析检测技术,其中包括酶联免疫吸附检测、免疫层析检测、时间分辨荧光免疫检测和基于免疫传感器的检测技术等。本文着重阐述了不同免疫分析技术的原理及其在OA检测中的实际应用,同时探讨了免疫分析技术在贝类毒素OA检测方面的挑战和发展趋势,以期为开发性能更加优异的OA免疫检测技术提供研究思路。

间隔影响分析波长选择算法在近红外光谱鉴别贝类毒素中的应用

该文采用近红外光谱技术与化学计量学方法结合实现贝类毒素无损鉴别。该研究以新鲜翡翠贻贝为研究对象,使用近红外光谱仪采集健康贻贝和感染腹泻性毒素贻贝的反射光谱数据,利用Savitzky-Golay卷积平滑求导结合标准正态变量变换光谱预处理方式消除光谱中的干扰因素,采用间隔影响分析(margin influence analysis,MIA)结合连续投影算法(successive projections algorithm,SPA)对数据进行降维处理,应用偏最小二乘线性判别分析(partial least squares linear discriminant analysis,PLS-LDA)方法构建贝类毒素鉴别模型,并与支持向量机和随机森林分析模型进行比较。结果表明,采用MIA-SPA-PLS-LDA方法,可实现贝类毒素的100%鉴别。为此,利用MIA-SPA-PLS-LDA方法可建立准确的贝类毒素鉴别模型,为贝类毒素的快速鉴别提供了新途径,也为后续各种贝类水产品的毒素鉴别分析提供了参考。

基于LDA降维方法的腹泻性贝类毒素检测研究

提出应用近红外(NIR)技术结合线性判别分析(LDA)方法和机器学习算法,对被腹泻性毒素污染贝类进行快速无损检测的新方法。首先,采集贻贝样本NIR光谱;然后,采用LDA对NIR光谱数据降维;最后,分别应用K近邻(KNN)、随机森林(RF)、极端梯度提升和逻辑回归(LR)4种算法实现腹泻性毒素污染贻贝分类检测。被腹泻性毒素污染的贻贝和健康贻贝的混合近红外光谱数据集经LDA降维后,使用4种分类器进行检测,准确率均达到100.00%。

牡蛎基体麻痹性贝类毒素GTX1-4标准样品的研制

贝类食品安全是重大公共卫生问题,然而贝类毒素标准品和标准样品均很难获得。将天然产生麻痹性贝类毒素膝沟藻毒素1-4(Gonyaulax toxin 1-4,GTX1-4)的链状裸甲藻培养液与牡蛎肉均质混匀,冷冻干燥脱水后,反复过筛分处理制备成松散状细粉。采用液相色谱-串联质谱法(Liquid chromatography-tandem mass spectrometry,LC-MS/MS)测定牡蛎基体中GTX1-4含量,并比较不同制备工艺对特性值分散度的影响。样品分装成500瓶,进行均匀性、稳定性、赋值及不确定度评估。结果表明,标准样品均匀性、长期稳定性和模拟运输条件下的短期稳定性良好,特性参考值可靠,达到标准样品的技术要求,为有效开展实验室质量控制和能力评价活动,保护消费者安全具有重要意义。

大亚湾和大鹏湾麻痹性贝类毒素动态分析

于 1 997～ 1 999年在大亚湾和大鹏湾的主要贝类养殖区设立了四个采样点 ,以华贵栉孔扇贝和翡翠贻贝为对象逐月采样 ,用美国分析化学家协会推荐的生物测定法进行麻痹性贝类毒素测定。结果显示 ,1 999年 1月～ 1 999年 6月大亚湾东山海域的扇贝消化腺毒素含量为 1 2 63～ 1 0 5Mu/g ,剔除消化腺后的其他软组织毒素含量为 1 3 4至低于 1 7Mu/g ,消化腺和其他软组织毒素含量的加权平均值为 7 7～ 1 1 7Mu/g ,高于联合国粮农组织制定的贝类安全食用标准的限定值 4Mu/g ,尤其是 1月份 ,超过限定值近 30倍。同期该样点的贻贝消化腺毒素含量为 0～ 567Mu/g ,剔除消化腺后的其他软组织毒素含量为 0～ 1 4 2Mu/g ,消化腺和其他软组织毒素含量的加权平均值为 0～ 72 8Mu/g ,仅在 1月份贻贝毒素含量超标。总体上贝毒含量表现为由冬春季高峰期逐月波动下降的趋势。大亚湾澳头海域的两种贝类毒素含量都不高 ,扇贝长期含有较低的毒素 ,贻贝在大部分时间内不含毒素 ,仅在 1 998年 5月间 ,出现一低于 3 5Mu/g的高峰期。大鹏湾南澳海域的两种贝类毒素含量不高 ,贻贝毒素含量均低于生物测定法极限值 ,而扇贝消化腺毒素含量仅 1 998～ 1 999年冬季略有超标。本文还就麻痹性贝毒的分布与贝类种类、时空间的关系进行?

广东沿海麻痹性贝类毒素的研究

于1990－1992年间在广东沿海采集24种贝类，用麻痹性贝类毒素小白鼠生物测定法对所采样品进行毒素分析研究。结果表明，所检测的大部分生物体均不同程度的积累了麻痹性毒素；大亚湾和大鹏湾2种贝体内存在着12或13种麻痹性毒素的成分；贝体中麻痹性毒素存在着一定的季节差异。还对贝体中的毒素和有毒藻类的出现进行了简单的讨论。

长江以北沿海经济贝类中的麻痹性贝毒

应用小白鼠检测方法，对我国长江以北沿岸的经济贝类进行了麻痹性贝毒检测。采样时间为１９９７ 年９ 月２３ 日～１２ 月８ 日。从鸭绿江口到长江口共调查２０ 个站位，检测经济贝类１４ 种，样品３８ 份，其中只有１ 份样品检测出麻痹性贝毒（６４ Ｍｕ／ｇ） 。