Paralytic shellfish poisoning toxins in green mussels (Perna viridis) from the Gulf of Paria, Trinidad.

Paralytic shellfish poisoning (PSP) toxins were determined in green mussels (Perna viridis) collected from one collection site in the Gulf of Paria in Trinidad in 1999 and 2000. Aqueous extracts of PSP were purified by passage through C-18 SPE cartridges, oxidized with peroxide and periodate, respectively, then analyzed by HPLC with fluorescence detection. This procedure provided rapid and highly sensitive screening of samples for PSP toxins. Further purification of PSP-containing extracts using COOH SPE cartridges resulted in the separation and identification of individual PSP toxins. The method of analysis was validated by spike and recovery experiments, with 85-103% recoveries of mixed toxins. PSP toxins determined in our samples in both years were GTX2,3, dcGTX2,3, STX, and dcSTX, while GTX1,4 and NeoSTX were only identified in 1999 and 2000, respectively. In 1999, GTX1,4, GTX2,3 and dcGTX2,3 predominated, as compared to NeoSTX, GTX2,3 and dcGTX2,3 in 2000. However, mussel samples in 2000 contained higher total concentrations of detected PSP toxins than those of 1999. These results represent the first identification of specific PSP toxins in local shellfish and provide a basis for effective monitoring and control of these toxins in Trinidad.     

一起鸭沙门菌引起食物中毒调查

目的：查明本起群体性食物中毒的原因,为今后预防和控制类似事件提供借鉴。方法：根据流行病学调查、临床表现、实验室检验和食物中毒诊断标准进行分析。结果：在10份患者肛拭样品及1份剩余食品中检出生物学形状一致的鸭沙门菌4株。结论：这是一起鸭沙门菌引起的食物中毒。     

罗山县某酒店猪伤寒沙门氏菌引起食物中毒的调查报告

目的：了解此次食物中毒的基本情况和引起食物中毒原因。方法对聚集性食物中毒患者和就餐酒店,进行流行病学调查,采集样本病原学检测及分析。结果在一份卤制品和3个粪便标本中检出了共同血清型为O6,7：Hc的猪沙门氏菌。结论确定了此次食物中毒的原因为食品受猪伤寒沙门氏菌污染。     

Paralytic toxicity in the ribbon worm Cephalothrix species (Nemertea) in Hiroshima Bay, Hiroshima Prefecture, Japan and the isolation of tetrodotoxin as a main component of its toxins.

Paralytic toxicity of ribbon worms ("himomushi" in Japanese), identified as undescribed species of the genus Cephalothrix, found on the surface of the shells of cultured oysters in Hiroshima Bay, Hiroshima Prefecture was examined between April 1998 and December 2001. The toxicity study showed that all of specimens were found to contain toxins with strong paralytic action in mice; the highest toxicity (as tetrodotoxin, TTX) was 25,590 mouse units (MU) per gram for whole body throughout the monitoring period.The main toxic component of this himomushi toxin (HMT) was isolated from a pooled specimen (390 g; total toxicity 2,897,000MU) by a method that consisted of treatment with activated charcoal, chromatography on Bio-Gel P-2 and Bio-Rex 70 (H+ form), and finally crystallization from an acidified methanolic solution. The recrystallized toxin showed a specific toxicity of 3520MU/mg. This toxin showed (M+H)+ and (M+H-H(2)O)+ ion peaks at m/z 320 and 302, respectively, by electrospray ionization-mass spectrometry (ESI-MS). The absorption band at 3353, 3235, 1666, 1612 and 1076 cm(-1) were observed in infrared spectrum of this toxin. This spectrum was indistinguishable from that of TTX. The 1H-NMR spectrum for the recrystallized toxin was the same as that for TTX. The pair of doublets centered at 2.33 (J=10.0Hz) and 5.48 ppm (J=10.0Hz) which are characteristic of TTX, were shown to be coupled by double irradiation. Furthermore, by gas chromatography-mass spectrometry (GC-MS) of the alkali-hydrolyzate of this toxin indicated the presence of quinazoline skeleton (C9-base) specific to TTX.     

Variety of PSP toxins in four culture strains of Alexandrium minutum collected from southern Taiwan.

Paralytic shellfish poisoning (PSP) toxin profiles were compared among four culture strains of Alexandrium minutum. GTX-1, 2, 3 and 4 are the PSP toxins that occur in A. minutum, and other PSP toxins were not detected. When comparing the toxin profile of four A. minutum strains, GTX1 and 4 were the major toxins in Amtk1, Amtk2, and Amtk4, but in Amtk7, GTX3 and 2 were the major toxins. The results indicate that strains with various toxin profiles exist in southern Taiwan, and suggest that the comparison of the toxin profiles between strains at different localities is difficult. Additionally, the toxin profiles of A. minutum strains cultured in the same environment were different, suggesting that it was owing to the intrinsic nature of toxic algae.     

Paralytic shellfish poisoning (PSP) toxin- and/or tetrodotoxin-contaminated crabs and food poisoning by them

Outbreaks of human intoxication by toxic crabs have been reported in various parts of the world. Sporadic outbreaks of intoxication in humans and domestic animals due to ingestion of the toxic crab have especially occurred in the Ryukyu Islands, Japan. Using a screening test for toxic crabs, 1000 specimens of 72 species in the family Xanthidae were tested for toxicity and Zosimus aeneus, Atergatis floridus and Platypodia granulosa were found to be toxic. The toxin was confirmed to be identical to saxitoxin (STX). It was later confirmed that there are great individual and regional variations in toxicity and the toxin varies with exposure of the toxic crab to Paralytic Shellfish Poisoning (PSP) toxin and/or tetrodotoxin (TTX). Edible crabs “Togekurigani” (Telmessus acutidens) and “Ishigani” (Charybdis japonica) were contaminated with PSP for a short time in 2003, in Japan. The mechanism of PSP intoxication was shown to be the food chain by feeding PSP-contaminated bivalves. However, in the case of toxic xanthid crabs, which are not plankton feeders, the conclusions were uncertain. This review describes the occurrence, causative agents and intoxication mechanisms for food poisoning by toxic crabs containing PSP or TTX or other marine toxins, either alone or mixed.     

护理程序在群体急性食物中毒患者急救中的应用

目的探讨群体急性食物中毒患者抢救的管理方法与效果。方法在2009年1月至2010年12月期问,对6宗共223例急性食物中毒的患者应用：评估一诊断一计划一实施一评价等五个步骤,进行抢救。结果使抢救工作忙而不乱,患者能在短时间内得到妥善安置,无一例患者死亡。结论合理应用护理程序对群体急性食物中毒患者进行抢救,规范了抢救流程,是抢救成功的关键所在。     

Comparison of paralytic shellfish poisoning toxin between carnivorous crabs (Telmessus acutidens and Charybdis japonica) and their prey mussel (Mytilus galloprovincialis) in an inshore food chain.

Paralytic shellfish poisoning toxin in two shore crab species, Telmessus acutidens and Charybdis japonica, were compared with the toxin in the prey mussel Mytilus galloprovincialis and causative dinoflagellates Alexandrium tamarense, all having been collected at Onahama, Fukushima Prefecture, in the northern part of Japan. When the toxicities were detected in mussels by mouse bioassays, 73.7% of the sampled T. acutidens were toxic in the hepatopancreas. T. acutidens has been found to become toxic for three years, therefore, it can be concluded that the crab commonly and repeatedly accumulate the toxins via the food chain at Onahama. C. japonica was also expected to be a possible vector species, because small quantities of the toxins were detected in eight specimens of the crab by HPLC analysis. By the comparison of the toxin profiles in the dinoflagellates, mussels and the crab T. acutidens, reductive conversions of GTX1 and GTX4 were observed when the toxins passed through the three species in the food chain. But increases of STX and neoSTX by further reductive process were not observed in the crab. The absence of the STX group toxins in the crab suggests that the crab eliminates the toxin before such reductive process occur.     

Paralytic toxins in a ribbon worm Cephalothrix species (Nemertean) adherent to cultured oysters in Hiroshima Bay, Hiroshima Prefecture, Japan.

In 1998, during the surveillance of the toxicity of various marine fouling organisms in Hiroshima Bay, Hiroshima Prefecture, Japan, specimens of the ribbon worm, "himomushi" Cephalothrix sp. (Nemertean) adherent to the shells of cultured oysters hanging onto floating culture rafts were found to contain toxins which showed strong paralytic action in mice throughout the survey period, February to May. The maximum toxicity (as tetrodotoxin, TTX) was 14,734 MU/g whole body. Attempts were made to identify the paralytic toxins in this worm. The "himomushi" toxin (HMT) was extracted from the worm with 80% methanol acidified with acetic acid and the extract defatted with dichloromethane. The aqueous layer was chromatographed on activated charcoal and the unbound and bound toxic fractions were analyzed by high-performance liquid chromatography and gas chromatography-mass spectrometry. It was rather unexpectedly revealed from these results that HMT was comprised of TTX, 4-epiTTX, anhydroTTX and three unidentified toxins. To our knowledge, this is the first report of the occurrence of toxic organisms, containing a high concentration of TTX, adherent to cultured bivalves such as oysters.     

Development of standardized methodology for identifying toxins in clinical samples and fish species associated with tetrodotoxin-borne poisoning incidents

Tetrodotoxin (TTX) is a naturally occurring toxin in food, especially in puffer fish. TTX poisoning is observed frequently in South East Asian regions. In TTX-derived food poisoning outbreaks, the amount of TTX recovered from suspicious fish samples or leftovers, and residual levels from biological fluids of victims are typically trace. However, liquid chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry methods have been demonstrated to qualitatively and quantitatively determine TTX in clinical samples from victims. Identification and validation of the TTX-originating seafood species responsible for a food poisoning incident is needed. A polymerase chain reaction-based method on mitochondrial DNA analysis is useful for identification of fish species. This review aims to collect pertinent information available on TTX-borne food poisoning incidents with a special emphasis on the analytical methods employed for TTX detection in clinical laboratories as well as for the identification of TTX-bearing species.     

Anatomical distribution of diarrhetic shellfish poisoning (DSP) toxins in the mussel Mytilus galloprovincialis

The aim of this work was to shed light on the anatomical distribution of diarrhetic shellfish poisoning (DSP) toxins in the mussel Mytilus galloprovincialis and to determine any possible changes undergone during the depuration process. To this end, the distribution of two DSP toxins—okadaic acid and DTX2—and some of their derivatives were studied by means of HPLC/MS at different stages of the depuration process. Mussels were collected from mussel farms located in the Galician Rías and they were collected under three types of circumstances: (a) while ingesting toxic phytoplankton cells; (b) 1 week after the toxic cells had disappeared from the water; and (c) after ca. 2 months of depuration. Additionally, in case (b), the distribution among tissues was checked every week over a depuration period of 35 days in the laboratory. DSP toxins were only detected in non-visceral tissues when the extracts were concentrated 20-fold and, even in these cases, the concentrations found were very low. When the maximum possible contribution of non-visceral tissues was computed, taking into account the technique's detection limits and tissue weight, no relevant contribution to the toxin burden of non-visceral tissues was found at any stage of depuration, with the maximum possible contributions usually below 7%. The concentrated samples analysed showed that the actual contribution in all the cases studied was, in fact, less than 1% of the total toxin burden. These findings suggest that (1) when analytical methods are used to monitor DSP toxic mussels, non-visceral tissues should be assumed to be free of toxins in order to precisely compute the toxin concentration of the whole mass of edible tissues and (2) when studying the accumulation kinetics of DSP toxins, transference from the digestive gland to other tissues should not be taken into account, as the other tissues do not contain relevant amounts of DSP toxins.     

Accumulation and depuration of okadaic acid esters in the European green crab (Carcinus maenas) during a feeding study.

Soft shell crab is a seafood delicacy in many parts of the world. In Denmark, it has been investigated whether a commercial production of soft shell European green crabs (Carcinus maenas) would be feasible. In relation to this, a feeding study was performed to examine if occurrence of DSP toxins in the product could be a food safety problem. The crabs were fed with mussels containing DSP toxins (2500 microg total okadaic acid equivalents/kg) for 17 days and then fasted for 19 days. The content of total okadaic acid equivalents in the digestive organs was on average 27 times higher than the corresponding content in the body meat. The highest level of total okadaic acid equivalents measured was 12 microg/kg in body meat and 503 microg/kg in digestive organs. The results show that the content of DSP toxins in a commercial product of soft shell European green crab (without digestive organs) could be regarded as negligible.     

Comparative determination of paralytic shellfish toxins (PSTs) using five different toxin detection methods in shellfish species collected in the Aleutian Islands, Alaska

Paralytic shellfish poisoning (PSP), a human illness caused by the ingestion of shellfish contaminated with paralytic shellfish toxins (PSTs), has been reported in Alaska for decades. These poisoning incidents have resulted in losses to local economies due to shellfish harvest closures. Thus the development of an effective biotoxin monitoring program designed specifically for the remote regions of Alaska would provide protection for public health and allow for a viable shellfish industry. The present study provides data useful for the development of an effective toxin screening protocol by comparing PST levels quantified in shellfish by many of the currently available PST detection techniques. Seven bivalve species were collected along beaches of the Aleutian Islands from June 2006 to September 2007. The concentration of PSTs was quantified and compared using five different analytical methods: the mouse bioassay, high performance liquid chromatography (HPLC), receptor-binding assay, the commercially available Jellett Rapid PSP Test strips, and an enzyme linked immunosorbent assay technique. The Association of Official Analytical Chemists (AOAC)-approved HPLC method proved to be valuable for characterizing the suite of individual PSTs in each species for research purposes, but was not considered practical for rapid toxin screening in remote Alaskan regions due to its time-consuming nature and requirement of expensive equipment and considerable expertise. In the present study, Jellett test strips were shown to be an effective tool for rapid screening, however due to the high percentage of false positives, subsequent validation via AOAC-approved methods would be required to prevent unnecessary closures.     

Paralytic shellfish poisoning: post-mortem analysis of tissue and body fluid samples from human victims in the Patagonia fjords.

In July 5, 2002 fishermen working in harvesting sea urchin (Loxechinus albus) in the Patagonia Chilean fjords were intoxicated by consumption of filter-feeder bivalve Aulacomya ater. After the ingestion of 7-9 ribbed mussel, two fishermen died 3-4 h after shellfish consumption. The forensic examination in both victims did not show pathological abnormalities with the exception of the lungs conditions, crackling to the touch, pulmonary congestion and edema. The toxic mussel sample showed a toxicity measured by mouse bioassay of 8575 microg of STX (saxitoxin) equivalent by 100 g of shellfish meat. Using post-column derivatization HPLC method with fluorescent on line detection was possible to measure mass amount of each paralytic shellfish poisoning (PSP) toxin yielding individual toxin concentrations. These PSP toxins were identified in the gastric content, body fluids (urine, bile and cerebrospinal fluid) and tissue samples (liver, kidney, lung, stomach, spleen, heart, brain, adrenal glands, pancreas and thyroids glands). The toxin profiles of each body fluid and tissue samples and the amount of each PSP toxin detected are reported. The PSP toxins found in the gastric content, were STX and the gonyautoxins (GTX4, GTX1, GTX5, GTX3 and GTX2) which showed to be the major amount of PSP toxins found in the victims biological samples. The PSP toxin composition in urine and bile showed as major PSP toxins neoSaxitoxin (neoSTX) and GTX4/GTX1 epimers, both STX analogues with an hydroxyl group (-OH) in the N(1) of the tetrahydropurine nucleus. The neoSTX was not present in the gastric content sample, indicating that the oxidation of N(1) in the STX tetrahydropurine nucleus resulted neoSTX, in a similar way that GTX3/GTX2 epimers were transformed in GTX4/GTX1 epimers. Beside this metabolic transformation, also the hydrolysis of carbamoyl group from STX to form its decarbomoyl analogue decarbamoylsaxitoxin was detected in liver, kidney and lung. These two findings show that PSP toxins went under metabolic transformation during the 3-4 h of human intoxication period, in which PSP toxins showed enzymatic oxidation of N(1) in the tetrahydropurine nucleus, producing neoSTX and GTX4/GTX1 epimers starting from STX and GTX3/GTX2 epimers, respectively. This study conclude, that PSP toxins are metabolically transformed by humans and that they are removed from the body by excretion in the urine and feces like any other xenobiotic compound.     

Whole bowel irrigation (WBI) in acute iron poisoning — case report

Objective: In theory, WBI (whole bowel irrigation) is indicated in iron poisoning, but the existing data are still insufficient to support or exclude its efficacy. Case report: A 46 year-old female admitted to the Toxicology Department due to suicidal ingestion of 5000 miligrams of elemental iron (57 mg/kg) in sustained-release preparation. WBI was instituted at 2 hours after ingestion, because abdominal X-ray showed mass of tablet material inside stomach and past pylorus. After 3-hour WBI subsequent X-ray revealed residual opacities in small intestine. Levels of serum iron were increasing 289 μg/dL on admission, (1,5 h post ingestion) 408 μg/dL (6 h after ingestion), 424 μg/dL (after 12 h). WBI was continued within 7 hours, until there was no radiographic evidence of toxin remaining in the gastrointestinal tract and clear rectal effluent was apparent. Subsequent serial serum iron concentrations were as follows: 389 μg/dL (after 14 h), 239 μg/dL (after 21 h), 119 μg/dL (after 36 h). Since the patient was asymptomatic did not undergo deferoxamine therapy. Patient recovered without sequelae. Conclusion: WBI seems to be beneficial in early stage of iron intoxication ensuring rapid and effective cleansing of gut, and decreasing toxin absorption. Authors believe that the use of early WBI allows to provide supportive care and avoid deferoxamine treatment.     

Immunoenzymatic visualization of tetrodotoxin (TTX) in Cephalothrix species (Nemertea: Anopla: Palaeonemertea: Cephalotrichidae) and Planocera reticulata (Platyhelminthes: Turbellaria: Polycladida: Planoceridae).

Tetrodotoxin (TTX) was localized as brown color in different tissues of an undescribed species of the nemertean genus Cephalothrix (phylum Nemertea) and a turbellarian Planocera reticulata (phylum Platyhelminthes) on light microscopy by means of a monoclonal anti-TTX antibody. In the Cephalothrix sp., TTX was recognized in the vesicles apically arranged in the bacillary cells in the epidermis, basal lamina, the granular cells in the proboscis epithelium, rhynchocoel epithelium, and the vesicles in the basal portion of the intestinal wall near the blood vessels and rhynchocoel. The excretory system and the ovum also showed positive reaction of TTX antigen-antibody. On the other hand, the hermaphrodite flatworm P. reticulata exhibited TTX antigen-antibody complex only in their ovum. To our knowledge, this is the first experimental effort on micro-distribution of TTX in invertebrates.     

Determination of paralytic shellfish poisoning toxins in Norwegian shellfish by liquid chromatography with fluorescence and tandem mass spectrometry detection.

A novel extraction and clean-up method has been developed for the determination of paralytic shellfish poisoning (PSP) toxins in shellfish samples. Raw shellfish material was extracted with an acidic acetonitrile/water (80:20, v/v) solution, whilst being homogenised. During the homogenisation the sample extraction solution was cooled with ice water. Subsequently, the extract was frozen at -20 degrees C for at least 4h. During freezing, two layers were formed, only the lower predominantly aqueous layer was used for the determination. The final extract solution was cleaned-up using a combination of Oasis HLB and Carbograph activated carbon SPE columns. The developed extraction and clean-up methods combined with gradient elution liquid chromatography (LC)-mass spectrometry/mass spectrometry (MS/MS) has resulted in a method which can determine the analogs GTX 1-5, C1-2, DcGTX 2-3, DcSTX, Neo, STX in a single analysis with an overall detection limit of 313mug STXdiHCL-eq./kg shellfish meat. The use of the developed extraction method with post-column high performance liquid chromatography (HPLC) with fluorescence detection (FLD) method provided an overall limit of detection of 89mug STXdiHCL-eq./kg shellfish meat for the same toxins. Both post-column HPLC-FLD and LC-MS/MS was used to investigate the Norwegian PSP toxin profile. It was found that the PSP toxins could be detected in shellfish samples from the Norwegian coastline for 10 months of the year, from March till December. The toxin profile consisted mainly of the carbamate toxins, GTX 1-4, Neo and STX, in terms of both concentrations and contribution to the overall toxicity. In addition, several of the n-sulfo-carbamoyl toxins were either detected in the samples at relatively low concentrations or their presence in the samples were indicated but could not be confirmed by the post-column HPLC-FLD and LC-MS/MS analyses.     

A repeated dose study of the toxicity of inhaled 2-chlorobenzylidene malononitrile (CS) aerosol in three species of laboratory animal

The repeated dose inhalation toxicity of 2-chlorobenzylidene maloninitrile (CS) was investigated in male mice, rats, and guinea-pigs. Exposure was 1 h · day^–1 for 120 days surviving animals being killed approximately 1 year after the start of exposure. Excess mortality was noted in the high dose groups of all three species but at exposure concentrations below 30 g · 1^–1 mortality varied little between the control and test groups. Death during the experiment was related to concentration of exposure rather than total dose (Ct). No case of a dose-response relationship between tumours in a particular site, and total dose of CS was noted.     

Fly agaric (Amanita muscaria) poisoning, case report and review.

Gathering and eating mushrooms and other plants containing psychoactive substances has become increasingly popular among young people experimenting with drugs. Dried fly agaric Amanita muscaria fruiting bodies were eaten by five young persons (18-21 years of age) at a party in order to evoke hallucinations. Visual and auditory hallucinations occurred in four of them, whereas a 18-year-old girl lost consciousness. The following morning, she went to the Clinic of Toxicology. Due to the fact that not all the active substances present in the fly agaric have been identified, and some of them have an effect after a period of latency, the patient was admitted for several days of observation during which check-up examinations were performed. After four days without any problems, she was discharged. The poisoning regressed with no organ complications. The remaining persons who had eaten the fly agaric were free from any complaints.     

Azaspiracid poisoning (AZP) toxins in shellfish: Toxicological and health considerations

It has been almost a decade since a previously unknown human toxic syndrome, azaspiracid poisoning (AZP), emerged as the cause of severe gastrointestinal illness in humans after the consumption of mussels (Mytilus edulis). Structural studies indicated that these toxins, azaspiracids, were of a new unprecedented class containing novel structural features. It is now known that the prevalent azaspiracids in mussels are AZA1, AZA2 and AZA3, which differ from each other in their degree of methylation. Several hydroxylated and carboxylated analogues of the main azaspiracids have also been identified, presumed to be metabolites of the main toxins. Since its first discovery in Irish mussels, the development of facile sensitive and selective LC-MS/MS methods has resulted in the discovery of AZA in other countries and in other species. Mice studies indicate that this toxin class can cause serious tissue injury, especially to the small intestine, and chronic exposure may increase the likelihood of the development of lung tumours. Studies also show that tissue recovery is very slow following exposure. These observations suggest that AZA is more dangerous than the other known classes of shellfish toxins. Consequently, in order to protect human consumers, proper risk assessment and regulatory control of shellfish and other affected species is of the utmost importance.