DNA-based identification of fish species implicated in Puffer fish poisoning

A method for identification of fish species using three different mitochondrial DNA regions, 16S rRNA, cytochrome b and cytochrome c gene fragments, was investigated. The combined use of all three regions enabled reliable species identification in not only raw fish, but also dried, seasoned and boiled fish, products. Furthermore, the method was applicable even to vomitus from a patient involved in a puffer fish poisoning incident. However, further improvement is necessary to discriminate between closely related species such as Takifugu rubripes and T. chinensis, because they showed close similarity in the nucleotide sequences in the three gene fragments analyzed in this study.     

Photobacterium phosphoreum caused a histamine fish poisoning incident

An incident of histamine fish poisoning (HFP) occurred due to the consumption of iwashi maruboshi (dried sardine) in Osaka, Japan in March 2002. A histamine-producing bacterial strain, YS4-7, was isolated from iwashi maruboshi that contained 1700 mg of histamine per kilogram. This strain was identified as Photobacterium phosphoreum by biochemical examinations and partial sequencing of 16S rDNA. P. phosphoreum YS4-7 showed greater capability as a histamine producer at 4 and 12 degrees C than Morganella morganii JCM 1672. Strain YS4-7 produced 546 mg of histamine per kilogram in a sardine homogenate stored for 12 h at 20 degrees C. M. morganii, Raoultella planticola and Hafnia alvei have been isolated from fish implicated in HFP incidents, whereas this is the first report of P. phosphoreum being the causative bacterium in a sporadic case of histamine food poisoning.     

Histamine contents of fermented fish products in Taiwan and isolation of histamine-forming bacteria

Twenty-seven imported fermented fish products from Southeast Asian countries and sold in the supermarkets in Taiwan, including fish sauce, fish paste and shrimp paste, were tested to determine the occurrence of histamine and histamine-forming bacteria. The levels of pH, salt content, total volatile basic nitrogen, trimethylamine, and aerobic plate count in all samples ranged from 4.8% to 6.5%, 16.2% to 45.3%, 51 to 275 mg/100 g, 5.4 to 53.9 mg/100 g and 1.0 to 4.2 log CFU/g, respectively. The average content for each of eight different biogenic amines in all samples was less than 90 ppm, except for histamine which has an average content of 394 ppm in fish sauce, 263 ppm in fish paste, and 382 ppm in shrimp paste. Most of the tested fermented fish products (92.6%) had histamine levels greater than the FDA guideline of 50 ppm, while seven of them (25.9%) contained >500 ppm of histamine. Although Bacillus coagulans and Bacillus megaterium were identified as the two histamine-producing bacteria capable of producing 13.7 and 8.1 ppm of histamine, respectively, in trypticase soy broth broth supplemented with 1.0% l-histidine, they were not determined to be the main contributors to histamine accumulation in these fermented fish products.     

鱼露前期发酵过程中组胺变化规律研究

鱼露是我国沿海地区以及东南亚各国的传统调味品.鱼露在前期发酵过程中,会产生各种胺类物质,其中以组胺为主.文章采用不同种类的海鱼(金枪鱼、缇鱼、巴浪鱼、鲒鱼)、不同发酵温度(室温,35℃,45℃)、不同腐败程度、以及不同NaCl含量(15%,25%,35%,45%)四种条件下发酵鱼露,定期检测其中组胺的含量,得出组胺变化规律图.结果表明,随着鱼露发酵时间的延长,组胺含量先升高后降低,但不同的发酵条件组胺的含量及变化规律有所不同.     

一起食用养殖鲢鱼引起的克伦特罗食物中毒

对安徽省广德县一起食物中毒事件进行了流行病学调查，经临床表现分析及实验室检验，确认中毒食品为人工养殖的鲢鱼，致病物质为鱼体生物富集的盐酸克伦特罗。食谱分析表明73人分别在5个不同饮食场所共同进餐，其中50人食用了该批鲢鱼，50人均中毒，未食用不中毒。50名中毒病人中进食鲢鱼量为：100至400g不等，折合一次性食入盐酸克伦特罗最小量为0．0059mg。中毒病人的主要临床表现为头痛、头晕、心悸、恶心、四肢乏力、手足震颤等，少数病人伴有呕吐，无腹泻，部分病人血压下降，多数病人心率较快，严重血钾偏低。在剩余鲢鱼、病人呕吐物和养鱼塘的鲜活鱼中均检出盐酸克伦特罗，从而形成了科学的证据链。本起鱼体受盐酸克伦特罗污染的原因可能是养鱼户使用或误用了添加盐酸克伦特罗的动物饲料喂鱼所致。     

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.     

Histamine levels in commercially important fresh and processed fish of Oman with reference to international standards

Histamine was investigated in fresh (378), frozen (441), canned (290) and dried (24) fish samples of scombroid and non-scombroid species of Oman using a high performance liquid chromatography with a fluorescence detector. Of the 1133 fish samples tested, histamine was detected in 551 samples with a detection rate of about 41.8% among fresh fish, 61.0% of frozen fish, 78.9% of canned fish and 91.6% of dried fish samples and the mean histamine levels were 2.6, 5.8, 3.1 and 104 mg kg⁻¹, respectively. A total of 3.7% and 0.79% of the total samples exceeded the FDA and EU regulatory limits for histamine. Imported dried anchovies contained high histamine levels. The study confirms that post catching and commercialisation practices of seafood are adequate, warranting good quality fish and may not cause histamine risk to consumer in terms of human diet. While necessary monitoring may be done for imported dried fish products.     

Histamine formation by Tetragenococcus muriaticus, a halophilic lactic acid bacterium isolated from fish sauce.

We examined histamine formation in cultures of Tetragenococcus muriaticus, a halophilic lactic acid bacterium isolated from fish sauce. T. muriaticus formed histamine in low acidity (pH 5.8), O2 limiting conditions with optimal NaCl and glucose concentrations of 5-7% (w/v) and above 1%, respectively. Histamine formation could not be prevented even at 20% (w/v) NaCl, indicating that NaCl could not prevent histamine formation by this bacterium. A conspicuous amount of histamine accumulated only during the late stationary phase regardless of the growth conditions. Studies of cell suspension experiments confirmed the results obtained from cultured cells.     

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.     

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.     

一起雪卡毒素中毒事件中裸胸鳝毒性的研究

目的研究厦门一起引发食物中毒的裸胸鳝毒性及种类,以预防雪卡毒素中毒。方法对中毒事件进行流行病学调查,采集裸胸鳝样品共5份,采用小鼠生物法、雪卡毒素免疫膜检测试剂盒检测毒性大小,高效液相色谱-串联质谱法测定太平洋雪卡毒素(P-CTX-1),同时提取中毒鱼肉组织中的mtDNA,PCR技术扩增细胞色素b(Ctyb)后测序鉴定中毒鱼种。结果小鼠生物法试验检测5份样品,鱼肉毒性为无毒性(ND)～0.052 MU/g,内脏毒性为ND～0.23 MU/g;高效液相色谱-串联质谱法未检出P-CTX-1。5种鱼种经鉴定均为裸胸鳝属(Gymnothorax),其中中毒鱼种经基因测序为波纹裸胸鳝(Gymnothorax undulates)。结论引发该起食物中毒的是一种含有雪卡毒素的波纹裸胸鳝。     

Screening Test for Histamine in Fish

A rapid screening method is described that can detect histamine in fish. The method uses a two–step sequential enzyme system. First, diamine oxidase catalyzes the breakdown of histamine with formation of hydrogen peroxide. Hydrogen peroxide is then detected by formation of crystal violet from the leuco base in the presence of peroxidase. The two reactions can be combined and peroxidase can be used to control the tightness of the screening. The method can be used for raw or heat-processed fish.     

Histamine Formation in Fish Sauce Production

ABSTRACT: The formation of histamine in fish sauce made from Stolephorus sp was studied. In the first experiment, fish were iced on board and mixed with salt at the factory, while in the second experiment fish were mixed with salt on board collection vessels. Eight batches were fermented for 12 mo. Histamine levels increased during fermentation to 22 to 159 and 589 to 686 ppm in the first and second experiments respectively. Good correlation between histamine levels in raw material and final products was found. It was concluded that histamine was formed both in the raw material and during fermentation. It was speculated that histidine decarboxylase enzymes formed prior to fermentation produced histamine during fermentation.     

Rapid screening of tetrodotoxin in urine and plasma of patients with puffer fish poisoning by HPLC with creatinine correction

A rapid and simple detection method for tetrodotoxin (TTX) in urine and plasma of patients with puffer fish poisoning was developed using commercially pre-packed solid-phase extraction (SPE) cartridges (C18 and weak cation exchange columns) and subsequent analyses by HPLC with UV detection. The detection limit of the standard TTX, TTX-spiked urine and plasma samples were all 10 ng/ml and the average TTX recovery in urine and plasma samples after SPE were 90.3 ± 4.0 and 87.1 ± 2.9%, respectively. It was noticed that the creatinine-adjusted urinary TTX levels obtained within the first 24 h of presentation apparently correlated much better with the severity of poisoning than the urinary TTX concentration without adjusting for variations in concomitant creatinine excretion.     

Skin-lightening products revisited

Skin colour typology depends on the amount and location of its chromophores. Among them, eumelanins derived from 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and 5,6-dihydroxyindole (DHI), and phaeomelanins are of utmost importance. These biomolecules result from the multi-step enzymatic and non-enzymatic conversion of tyrosine into melanins. Pigmentation disorders are multiple and depend on alterations in the density in active melanocytes, and on specific abnormalities of any of the complex melanogenesis mechanisms. This review presents some of the main skin-lightening agents with respect to their mechanisms of action and side-effects. Some of the novel compounds may lead to new perspectives in the fields of dermatology and cosmetology. The methods commonly used to assess efficacy of skin-lightening products rely on in vitro models including cell-free enzymatic assays, melanocyte cultures and reconstructed epidermis bioassays. Animal models have little relevance. By contrast, human testing with the support of instrumental evaluations is the most informative.