Identification of tetrodotoxin and fish species in a dried dressed fish fillet implicated in food poisoning

There were five victims of neurotoxic food poisoning from a dried dressed fish fillet in Changhua County, Taiwan, in February 2000. The toxicity of the dried dressed fish fillets was 243 mouse units per g according to a tetrodotoxin bioassay. The partially purified toxin was identified as tetrodotoxin and anhydrotetrodotoxin. The sequence of the 376-nucleotide region in the cytochrome b gene of the mitochondrial DNA exhibited the same genotype as that of the toxic puffer fish Lagocephalus lunaris. The same single restriction site for Hinfl was found in the polymerase chain reaction (PCR) products from the dried dressed fish fillet and the muscle of L. lunaris, yielding two DNA fragments of 170 and 206 bp. However, no restriction site for Hinfl was found in the PCR products from other toxic puffer fishes, including Takifugu niphobles, Takifugu oblongus, and Takifugu rubripes. Therefore, the species of the dried dressed fish fillet was identified as L. lunaris and its causative agent was identified as tetrodotoxin.     

Identification of gadoid fish species using DNA-based techniques

The polymerase chain reaction (PCR) technique was employed to obtain a 464 bp amplicon from the mitochondrial cytochrome b gene from gadoid species to study its ability to differentiate them. The sequences of this fragment from 16 species were analysed using a genetic distance method, and polymorphic sites were determined. The fragment was shown to be moderately polymorphic (151 sites), and this permitted the differentiation of most of the species. A phylogenetic tree construction using Tamura-Nei distances was employed to allow the identification of Gadidae species, each species resulted in a well-differentiated clade, with the exception of Gadus ogac and Gadus macrocephalus, which could not be differentiated. Based on the sequences obtained, three restriction enzymes, Dde I, Hinc II and Nla III, were selected to provide specific restriction profiles, which allowed the differentiation of 15 species of gadoids in a faster and less expensive way than sequencing. The PCR-restriction fragment length polymorphism methodology was also tested using commercial samples.     

Liquid chromatography-tandem mass spectrometry determination of the toxicity and identification of fish species in a suspected tetrodotoxin fish poisoning

Suspected tetrodotoxin (TTX) poisoning was associated with eating unknown fish in April 2009 in Taiwan. After ingestion of the fish, symptoms of the victim included perioral paresthesia, nausea, vomiting, ataxia, weakness of all limbs, respiration failure, and death within several hours. The toxicity in the remaining fish was determined, with the mice exhibiting symptoms of neurotoxin poisoning. The implicated fish and deceased victim tissues were analyzed for TTX by liquid chromatography-tandem mass spectrometry. The urine, bile, cerebrospinal fluid (spinal cord), pleural effusion, and pericardial effusion of the victim contained TTX. In addition, the partial cytochrome b gene of the implicated fish was determined by PCR. The DNA sequence in the partial 465-bp cytochrome b gene identified the implicated fish as Chelonodon patoca (puffer fish). These results indicate that people should avoid eating unknown fish species from fish markets where harvested fish may include toxic species.     

DNA检测技术在鳕鱼及其制品鉴定中的应用

鳕鱼是经济价值高和营养价值高的重要食用鱼类,但由于市场上鱼类俗名混乱问题,以及不同鱼种之间价值的巨大差异,错误标识鳕鱼产品或以次充好的现象时有发生,有可能因为含有过敏原成分或有毒成分造成食品安全风险。因此,需要快速可靠的鳕鱼成分鉴定方法和来保障市场监管。目前,鳕鱼及其制品的鉴定方法主要为DNA检测技术。本文主要介绍了DNA指纹图谱PCR-RFLP技术、DNA条形码技术、环介导等温扩增技术和实时荧光定量PCR技术的原理、优缺点、在鳕鱼及其制品鉴定中的应用,最后讨论了鳕鱼及其制品鉴定方法的发展趋势和监管建议。     

Challenges in the identification of species of canned fish

The identification of fish species becomes a problem when the usual identifying characteristics are removed on processing and only a portion of flesh is available. When the flesh is raw or cooked under normal conditions, the species is readily established by electrophoresis of the muscle proteins. The procedure cannot be used for heat-sterilised canned fish as the proteins are severely denatured. DNA is also degraded but techniques are now available for targeting and amplifying species-specific fragments. The amplified products can then be analysed by a range of techniques some of which are suitable for food control laboratories.     

Identification of causative agents and species in shrimp implicated in a food poisoning case in Taiwan

The possible causative agent and shrimp species involved in a bait shrimp poisoning case that occurred in northern Taiwan was determined. Because the patient's symptoms were similar to those caused by boric acid and slightly similar to those caused by sulfite, the concentrations of boric acid and sulfite (as sulfur dioxide) in the patient's vomitus and in shrimp collected from bait stores and markets were analyzed. The concentration of boric acid was 36.8 to 37.1 mg/g in the patient's vomitus, 1.4 to 3.8 mg/g in shrimp meats obtained from bait stores, and not detectable (less than 0.001 mg/g) in shrimp meat obtained from commercial markets. No significant differences in sulfur dioxide concentrations (0.067 to 0.088 mg/g) were found in patient's vomitus and the shrimp meat from both bait stores and commercial markets. A fragment of the cytochrome b gene (～406 bp) was amplified by PCR using a pair of primers (UCYTB151F and UCYTB270R) from shrimp meat of two species and the vomitus. The vomited shrimp was identified as Parapenaeus fissuroides on the basis of gene sequencing and restriction fragment length polymorphism patterns after treatment with endonuclease Alu I. Based on the patient's symptoms and analytical data, we concluded that boric acid at toxic levels had been illegally added to the bait shrimp P. fissuroides.     

Histamine level and species identification of billfish meats implicated in two food-borne poisonings

Two incidents of food-borne poisonings, causing illness in 59 and 43 victims due to ingestion of billfish meats, occurred in May 2004, in Pingtung, southern Taiwan and in December 2004, Taichung, central Taiwan, respectively. One fried billfish fillet and five frozen billfish fillet samples collected, respectively, from the suspected restaurants in Pingtung and Taichung, respectively, were tested to determine the histamine levels and identify fish species. Analyses of histamine showed that the suspected billfish samples in two food poisonings contained more than 150 mg/100 g of histamine, which is higher than the hazard action level of 50 mg/100 g. Judging from the allergy-like symptoms of the victims and the high histamine levels in the suspected billfish samples, both food-borne poisonings were strongly suspected to be caused by histamine intoxication. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used to identify the species of the suspected billfish samples in both food poisonings. The 348 bp amplified fragment of the mitochondrial cytochrome b gene by PCR was digested with BsaJI, Cac8I and HpaII enzymes to distinguish the species of the suspected billfish samples. Consequently, the species of Pingtung and Taichung billfish samples implicated in food poisonings were identified as Makaira nigricans and Xiphias gladius, respectively.     

Applicability of DNA barcode for identification of fish species

DNA barcoding is a species identification technique, which uses a very short DNA sequence from a region of approximately 650 base-pairs in the 5'-end of the mitochondrial cytochrome c oxidase subunit I gene as a marker to identify species of mammals and fishes. The applicability of DNA barcoding for identification of fish species consumed in Japan was studied. Among thirty-one fresh or processed fishes were obtained from the market, two samples could not be identified due to lack of data in the Barcode of Life Data (BOLD) database. However, BLAST-search of 16S rRNA genes in the National Center for Biotechnology Information (NCBI) database and the PCR-RFLP method published by the Food and Agricultural Materials Inspection Center (FAMIC) were found to be applicable to identify these 2 fishes. The results show that the DNA barcoding technique is potentially useful as a tool for confirming the proper labeling of fish species in the Japanese market.     

Analysis of toxin profiles in three different fish species causing ciguatera fish poisoning in Guadeloupe, French West Indies.

A grey snapper (Lutjanus griseus), a grouper (Serranidae) and a black jack (Caranx lugubris) were implicated in three different ciguatera poisonings in Guadeloupe, French West Indies. A mouse bioassay indicated toxicity for each specimens: 0.5-1, > or = 1 and > 1 MUg g(-1), respectively. After purification by gel filtration chromatography, the samples were analysed by high-performance liquid chromatography coupled to mass spectrometry (LC-MS). The toxin profiles differ from one fish to another. C-CTX-1 was detected at 0.24, 0.90 and 13.8 ng g(-1) flesh in the snapper, grouper and jack, respectively. It contributed only to part of the whole toxicity determined by the mouse bioassay. Other toxins identified were C-CTX-2 (a C-CTX-1 epimer), three additional isomers of C-CTX-1 or-2, and five ciguatoxin congeners (C-CTX-1127, C-CTX-1143 and its isomer C-CTX-1143a, and C-CTX-1157 and its isomer C-CTX-1157b). Putative hydroxy-polyether-like compounds were also detected in the flesh of the grouper with [M+ + H]+ ions at m/z 851.51, 857.50, 875.51, 875.49 and 895.54 Da. Some of these compounds have the same mass range as some known dinoflagellate toxins. In conclusion, this study confirms the usefulness of LC-MS analysis to determine the ciguatoxins levels and the toxin profile in fish flesh hazardous to humans.     

基于DNA的分型方法有助于食源性病原体的鉴定

<正> 分型方法的进步增强了对食源性疾病的监测,但是将分型信息与流行病学数据进行整合以便迅速找到疾病大爆发的原因,仍然存在挑战。在1999年出版的一项研究中,我们已经评估了每年在美国引起大约7600万例食源性疾病的致病微生物,这其中包括325000例住院事件和5000例死亡事件(Mead等,1999)。为了能够更     

线粒体细胞色素b基因在鱼唇制品物种鉴定中适用性的研究

目的 探讨线粒体细胞色素b基因(cytochrome b, Cyt b)作为DNA条形码在鱼唇制品物种鉴定中的适用性。方法 对全国31个城市购买的252份鱼唇样品进行聚合酶链式反应(polymerase chain reaction, PCR)测序,同源基因比较分析,构建系统发育树,鉴定制作鱼唇产品的鱼种,并对其进行濒危评价分析。结果 成功鉴定250个样品,一致性物种基因序列相似性在99%以上,涉及8个鲨鱼物种,最多样品为大青鲨(Prionace glauca),占样品65.5%,其余还有镰形真鲨(Carcharhinus falciformis)、路氏双髻鲨(Sphyrna lewini)、锤头双髻鲨(Sphyrna zygaena)等7类鲨鱼物种。结论 Cyt b可以作为对鲨鱼物种进行鉴定的一种DNA条形码,在对鲨鱼种鉴定时可以使用Cyt b基因及细胞色素氧化酶亚基I基因联合鉴定条形码,为深加工海产品物种鉴定提供更多的技术支撑。     

Histamine fish poisoning revisited

Histamine (or scombroid) fish poisoning (HFP) is reviewed in a risk-assessment framework in an attempt to arrive at an informed characterisation of risk. Histamine is the main toxin involved in HFP, but the disease is not uncomplicated histamine poisoning. Although it is generally associated with high levels of histamine (> or =50 mg/100 g) in bacterially contaminated fish of particular species, the pathogenesis of HFP has not been clearly elucidated. Various hypotheses have been put forward to explain why histamine consumed in spoiled fish is more toxic than pure histamine taken orally, but none has proved totally satisfactory. Urocanic acid, like histamine, an imidazole compound derived from histidine in spoiling fish, may be the "missing factor" in HFP. cis-Urocanic acid has recently been recognised as a mast cell degranulator, and endogenous histamine from mast cell degranulation may augment the exogenous histamine consumed in spoiled fish. HFP is a mild disease, but is important in relation to food safety and international trade. Consumers are becoming more demanding, and litigation following food poisoning incidents is becoming more common. Producers, distributors and restaurants are increasingly held liable for the quality of the products they handle and sell. Many countries have set guidelines for maximum permitted levels of histamine in fish. However, histamine concentrations within a spoiled fish are extremely variable, as is the threshold toxic dose. Until the identity, levels and potency of possible potentiators and/or mast-cell-degranulating factors are elucidated, it is difficult to establish regulatory limits for histamine in foods on the basis of potential health hazard. Histidine decarboxylating bacteria produce histamine from free histidine in spoiling fish. Although some are present in the normal microbial flora of live fish, most seem to be derived from post-catching contamination on board fishing vessels, at the processing plant or in the distribution system, or in restaurants or homes. The key to keeping bacterial numbers and histamine levels low is the rapid cooling of fish after catching and the maintenance of adequate refrigeration during handling and storage. Despite the huge expansion in trade in recent years, great progress has been made in ensuring the quality and safety of fish products. This is largely the result of the introduction of international standards of food hygiene and the application of risk analysis and hazard analysis and critical control point (HACCP) principles.     

Mitochondrial cytochrome b DNA sequence variations: an approach to fish species identification in processed fish products

The identification of fish species in food products is problematic because morphological features of the fish are partially or completely lost during processing. It is important to determine fish origin because of the increasing international seafood trade and because European Community Regulation 104/2000 requires that the products be labeled correctly. Sequence analysis of PCR products from a conserved region of the cytochrome b gene was used to identity fish species belonging to the families Gadidae and Merluccidae in 18 different processed fish products. This method allowed the identification of fish species in all samples. Fish in all of the examined products belonged to these two families, with the exception of one sample of smoked baccalà (salt cod), which was not included in the Gadidae cluster.     

Fish species identification by fish muscle dry powder as reference material

A method for preparing dry powders from fish muscles is described. The muscle dry powders (MDP) were extracted with low ionic strength buffer and compared with sarcoplasmic extracts of raw muscles by isoelectric focusing on ultra-thin layer (100 μm) polyacrylamide gels. The protein patterns of MDP extracts corresponded to the patterns of the acidic proteins of raw muscle extracts. Because of the species specificity of the patterns, MDP can be used as reference material for fish species identification. The acidic proteins remained extractable after storage of MDP for several months at room temperature.     

Formation of histamine and biogenic amines in cold-smoked tuna: an investigation of psychrotolerant bacteria from samples implicated in cases of histamine fish poisoning

Two outbreaks and a single case of histamine fish poisoning associated with cold-smoked tuna (CST) were reported in Denmark during 2004. The bacteria most likely responsible for histamine formation in CST implicated in histamine fish poisoning was identified for the first time in this study. Product characteristics and profiles of biogenic amines in the implicated products were also recorded. In the single poisoning case, psychrotolerant Morganella morganii-like bacteria most likely was responsible for the histamine production in CST with 2.2% +/- 0.6% NaCl in the water phase (WPS). In outbreak 1, Photobacterium phosphoreum most likely formed the histamine in CST with 1.3% +/- 0.1% WPS. In outbreak 2, which involved 10 persons, the bacteria responsible for histamine formation could not be determined. The measured concentrations of WPS were very low compared with those of randomly collected commercial samples of CST and cold-smoked blue marlin (4.1 to 12.7% WPS). Challenge tests at 5 degrees C with psychrotolerant M. morganii and P. phosphoreum in CST with 4.4% WPS revealed growth and toxic histamine formation by the psychrotolerant M. morganii-like bacteria but not by P. phosphoreum. In a storage trial with naturally contaminated CST containing 6.9% WPS, lactic acid bacteria dominated the microbiota, and no significant histamine formation was observed during the shelf life of about 40 days at 5 degrees C and of about 16 days at 10 degrees C. To prevent toxic histamine formation, CST should be produced with >5% WPS and distributed with a declared 5 degrees C shelf life of 3 to 4 weeks or less.     

Caribbean ciguatoxin profile in raw and cooked fish implicated in ciguatera

A cooked meal remnant and uncooked portion of a Caribbean barracuda suspected in ciguatera fish poisoning were examined for the presence of ciguatoxins (CTX). Samples were analysed using a tiered method of CTX analysis consisting of in vitro cell (N2a) assay to assess composite toxicity and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for structural confirmation. Meal remnant and uncooked fish extracts were cytotoxic by N2a cell assay and Caribbean ciguatoxin congener C-CTX-1 was structurally confirmed. Sample extracts were fractionated by LC and fractions analysed by the cell assay. The cytotoxicity profiles of cooked meal remnant and uncooked fish were similar. Cytotoxicity-guided LC-MS/MS analyses identified several CTX congeners contributing to the composite toxicity of the samples. C-CTX-1 was a major contributor, supporting its utility as a biomarker of Caribbean ciguatoxic fish.     

Tetrodotoxin in gastropods (snails) implicated in food poisoning in Northern Taiwan

The toxin in the gastropods (snails) Zeuxis sufflatus and Niotha clathrata implicated in a food poisoning incident in northern Taiwan in April 2001 was studied. The symptoms exhibited by four victims were general paresthesia, paralysis of the phalanges and the extremities, paralysis, coma, vomiting, and aphasia. The remaining gastropods were assayed for toxicity in the form of tetrodotoxin (TTX). The ranges of specimen toxicity were 345 to 1,640 mouse units (MU) for Z sufflatus and 190 to 643 MU for N. clathrata. The toxicities of the digestive gland and for other parts of the gastropod were 1,120 +/- 477 MU and 497 +/- 238 MU, respectively, for Z sufflatus and 683 +/- 113 MU and 289 +/- 169 MU, respectively, for N. clathrata. The toxin from the methanolic extract of the gastropods was partially purified by ultrafiltration and Bio-Gel P-2 column chromatography. Cellulose acetate membrane electrophoresis, thin-layer chromatography, high-performance liquid chromatography, and gas chromatography-mass spectrometry analyses demonstrated that the toxin consisted of TTX. It was concluded that the causative agent of the food poisoning in question was TTX.