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Inefficient accumulation of low levels of monodispersed and feces-associated poliovirus in oysters.

The accumulation of low levels (0.002 to 0.18 PFU/ml) of both feces-associated and monodispersed poliovirus by oysters (Crassostrea virginica or C. gigas) and clams (Mercenaria mercenaria) was investigated. These levels were chosen to duplicate the conditions present in light to moderately polluted waters. Experiments were performed in both small- and large-scale flowing seawater systems, developed to mimic the natural marine habitats of shellfish. Under these experimental conditions, viral accumulation by physiologically active shellfish was only noted when water column concentrations exceeded approximately 0.01 PFU/ml. Bioaccumulation increased with increasing concentrations of both monodispersed and feces-associated viruses. At virus concentrations below this level, viruses were seldom detected in either clams or oysters. Evidence indicated that the lack of accumulation was not the result of inefficient extraction or detection methods. The modified Cat-Floc-beef extract procedure used in the experiment was found to be capable of detecting as few as 1.5 to 2.0 PFU per shellfish. Evidence is presented to indicate that an uptake-depuration equilibrium was present at virus exposure levels of 0.10 PFU/ml, but not at 0.01 PFU/ml. The results suggested that viral accumulation by shellfish may not be efficient at water column concentrations below congruent to 0.01 PFU/ml.

Animals↗

Chemosensory conditioning in molluscs: I. Failure of contextual conditioning in Hermissenda.

Aversive chemosensory conditioning alters Hermissenda's feeding behavior. But opposite behavioral changes have been reported, depending on whether discrete-trial or context-conditioning paradigms were used, raising questions about the roles of associative and nonassociative processes. We attempted to produce chemosensory contextual conditioning but failed to do so across a wide range of conditions. In Experiments 1-3, we observed large, nonspecific bite latency increases to shellfish extracts, regardless of whether they had signaled the presence or absence of shaking. In Experiment 4, we found that mere exposure to shellfish extract produced latency increases; vestibular stimulation was unnecessary. In a final experiment, using Y-maze choice tests, we failed to observe selective reductions in animals' preference for shellfish paired with shaking. Nonassociative processes stemming from prolonged exposure to concentrated shellfish extracts appear to be major factors in our failure to demonstrate associative chemosensory contextual conditioning.

Animals↗

[Marine toxins].

The consumption of seafood, which is contaminated by toxines of red tides, is a common cause of disease in tropic regions. The most important diseases, which are caused by red tides are Paralytic Shellfish Poisoning (PSP), Diarrhetic Shellfish Poisoning (DSP), Neurotoxic Shellfish Poisoning (NSP), Amnesic Shellfish Poisoning (ASP), Ciguatera Fish Poisoning (CFP).

Americas↗

Illness associated with seafood.

Seafood is a common vehicle for the transmission of infections and intoxications. The main concerns in the UK are scombroid fish poisoning, viral infections (associated with bivalve molluscs) and bacterial infections (associated with crustaceans). Paralytic shellfish poisoning (PSP) and red whelk poisoning occur sporadically, and ciguatera has started to appear associated with imported fish. The appearance in coastal waters of marine algae associated with diarrheic shellfish poisoning (DSP) has increased the likelihood of outbreaks of this toxic syndrome. The dinoflagellates causing neurotoxic shellfish poisoning and amnesic shellfish poisoning have not been detected in UK fishing waters but the fluctuating distribution of algal populations makes their appearance a possibility. The Food Hygiene Laboratory and the Torry research station of the Ministry of Agriculture, Fisheries and Food provide a reference service for scombrotoxin, ciguatera, DSP, PSP and red whelk poisoning in the UK.

Cross-Cultural Comparison↗

[Evaluation of the role of occupational and environmental exposure to inorganic arsenic in the urinary excretion of the metal: preliminary data].

INTRODUCTION: Urinary inorganic arsenic is an expression of occupational exposure to the metal, provided that there is no history of ingestion of foods containing high concentrations of inorganic and/or organic arsenic. The present study was conducted to assess the contribution of professional and environmental exposure to inorganic arsenic on urinary excretion of the metal. MATERIALS AND METHODS: We examined 195 workers at a steelfoundry in Taranto, exposed to very low concentrations of inorganic arsenic and two control groups consisting of 105 subjects resident near the factory and 144 subjects resident approximately 20 Km away. All participants were administered a questionnaire enquiring about general characteristics, lifestyle, occupational and extra occupational exposure to arsenic. Urinary arsenic was determined by atomic-absorption spectrophotometry. RESULTS: Exposed and non exposed subjects were similar as regards general characteristics and lifestyle. The environmental concentration of arsenic for the foundryworkers was invariably lower than 0.1 microg/m3. Urinary excretion of arsenic was higher in the subjects in all three groups, examined singly and together, if they had eaten crustaceans and/or shellfish in the three days before urine collection. There was a significant positive correlation with the consumption of shellfish and a significant negative correlation with the number of days since the last crustacean/shellfish meal. Multiple regression analysis showed a dependence of urinary elimination of arsenic on the days since the last crustacean/shellfish meal. DISCUSSION: The absence of occupational exposure to arsenic allowed us to attribute the higher urinary elimination of arsenic to ingestion of crustaceans and/or shellfish in the three days before collection of the urine, both in subjects exposed to inorganic arsenic and in the two groups belonging to the general population. Our results support the hypothesis that inorganic arsenic, determined by atomic-absorption spectrophotometry, may derive from the catabolism of organic arsenic compounds contained in crustaceans and/or shellfish included in the diet.

Arsenic↗

Development of a protein phosphatase-based assay for the detection of phosphatase inhibitors in crude whole cell and animal extracts.

Diarrhetic shellfish poisoning (DSP) is a serious and globally widespread phytoplankton-related seafood illness. Although DSP is rarely life-threatening, it causes incapacitating diarrhea and vomiting with no known medical treatments. In addition, phytoplankton producing DSP toxins have been identified in temperate coastal waters worldwide, and their numbers may be increasing as a result of coastal eutrophication. The toxic effects of the major DSP toxins, okadaic acid and dinophysistoxin-1 (35-methylokadaic acid), appear to originate from their inhibitory activity against a family of structurally related serine/threonine protein phosphatases (PSPases). In particular, the inhibition of essential PSPases (e.g. PP1 and PP2A) has catastrophic consequences in most eukaryonic cells. Exploiting the potent inhibitory property of the DSP toxins, we have developed an enzyme-based assay (PP2A assay) capable of detecting both okadaic acid and dinophysistoxin-1 in nanogram amounts. The assay employs purified PP2A, which has an extremely high affinity for both DSP toxins. This provides the PP2A assay with a level of sensitivity comparable to, or surpassing, that of most monoclonal antibody probes. To evaluate the PP2A assay as a means of detecting contaminated shellfish, a series of spike recovery experiments was conducted. The findings from these studies suggest that the PP2A assay has the potential for development into a rapid and relatively simple method for detecting PSPase inhibitors in crude extracts produced from shellfish.

Animal Population Groups↗

Analysis of domoic acid and related compounds by mass spectrometry and gas chromatography/mass spectrometry as N-trifluoroacetyl-O-silyl derivatives.

A method is presented for the analysis of shellfish tissue for domoic acid, a neurotoxic amino acid responsible for cases of amnesic shellfish poisoning. Tissue extracts are first taken through a two-stage solid-phase extraction clean-up, using reversed-phase and strong cation exchange cartridges. A two-stage derivatization, using N-methyl-bis-trifluoroacetamide followed by either N-methyl-tert-butyldimethylsilyltrifluoroacetamide or N, O-bis-trimethylsilyltrifluoroacetamide, is then used to produce an N-trifluoroacetyl-O-silyl derivative which can be analyzed by mass spectrometry with introduction via direct inlet probe, moving-belt liquid chromatograph/mass spectrometer interface, or capillary column gas chromatography. The N-trifluoroacetyl-O-tert-butyldimethylsilyl derivative, which has good stability towards hydrolysis, provides a spectrum well suited to gas chromatography/mass spectrometry (GC/MS) using selected ion recording. GC/MS data for two related compounds, kainic acid and dihydrokainic acid, are also reported. The latter is used as an internal standard for quantification of domoic acid, although the method reported is intended primarily for confirmation of the toxin and related compounds in shellfish tissue.

Chromatography, Gas↗

Typhoid fever in the Neapolitan area: a case-control study.

Typhoid fever is endemic in the Neapolitan area, where its yearly incidence rate largely exceeds the corresponding national figure. During the period from January to June, 1990, a matched case-control study was carried out in order to identify risk factors of the disease in this area; 51 subjects (mean age 27.2 years) with typhoid fever were compared with 102 controls matched with respect to age, sex and educational level. Consumption of raw shellfish was reported by 76.5% of the cases, as opposed to 19.6% of the controls (P < 0.01). Subjects who had eaten this food item had a 13.3-fold risk (C.I. 95% = 5.5 - 32.8) of contracting typhoid fever. In contrast, no risk was found to be associated with consumption of cooked shellfish, raw vegetables, ice-cream, non-potable water, or unpasteurized milk. The risk factor identified in this study shows that hazardous dietary habits and inadequate sewage treatment facilities, combined with lack of sanitation in the harvesting and marketing of shellfish, play a major role in the endemicity of typhoid fever in the Neapolitan area.

Case-Control Studies↗

Comparison of liquid-junction and coaxial interfaces for capillary electrophoresis-mass spectrometry with application to compounds of concern to the aquaculture industry.

The application of capillary electrophoresis-mass spectrometry (CE-MS) to the analysis of compounds of concern to the aquaculture industry is reported. Two different approaches to coupling the CE column to an IonSpray atmospheric pressure ionization (API) interface, viz., a liquid-junction and a coaxial arrangement, are describe and compared with regard to ruggedness, ease of use, sensitivity and electrophoretic performance. The different injection modes used in three commercial capillary electrophoresis systems were also evaluated for their applicability to CE-MS. The use of CE-MS for the analysis of a variety of classes of antibiotics used in the fish aquaculture industry, such as the sulfonamides and their potentiators (e.g., trimethoprim), is demonstrated and was used to confirm the presence of these components in shellfish extracts at the low ppm level. CE-MS was also applied to the analysis of marine toxins such as saxitoxin and its analogues which are associated with paralytic shellfish poisoning, and also the toxins responsible for amnesic and diarrheic shellfish poisoning. Tandem mass spectrometry (MS-MS) was used to provide structural information on these analytes, and the ability to distinguish isomeric compounds based on their different migration and fragmentation characteristics using CE-MS-MS is demonstrated.

Anti-Bacterial Agents↗

Urinary elimination of saxitoxin after intravenous injection.

Paralytic shellfish poisoning is a serious public health concern throughout the world. An analytical method with diagnostic potential was used to isolate and measure saxitoxin, the most potent and studied paralytic shellfish poisoning toxin, in the urine of rats injected i.v. with sublethal doses (2 micrograms/kg) of saxitoxin. Urine was collected at intervals between 4 and 144 hr after injection. Saxitoxin was isolated from urine with an ion-exchange procedure, identified, and measured with a precolumn-oxidation-HPLC procedure coupled with fluorescence detection. The identity of oxidized saxitoxin was confirmed with electrospray ionization mass spectrometry. Four hours after injection, approximately 19% of the injected saxitoxin dose was excreted. By 24 hr, approximately 58% of the administered dose was excreted. Average total urinary excretion of administered saxitoxin was approximately 68% for the full study period. These results demonstrate that small quantities of unmetabolized saxitoxin can be detected in rat urine up to 144 hr after i.v. administration, and that the analytical method may have diagnostic potential for saxitoxin intoxication and paralytic shellfish poisoning.

Animals↗

Transfer of environmental plutonium and americium across the human gut: a second study.

A follow-up to our previous study is described on human gut transfer factors for plutonium and americium in winkles harvested from the shoreline near the British Nuclear Fuels plc Sellafield reprocessing plant. Six male and two female adult volunteers ate single quantities of shellfish; samples of their urine before and after consumption were analysed. As in the previous study, the plutonium and americium excreted were difficult to detect, and counting times of approximately 8 weeks were employed. Results are presented in detail to allow further interpretation, but preliminary assessment has been carried out on the basis of published data for excretion following transfer to blood. The mean and range obtained for the gut transfer for plutonium was 1.3 (0.2-3.2) x 10(-4); for americium the result was 0.8 (0.4-1.7) x 10(-4). Combined with the results of the previous study when interpreted on the same basis, the results were 1.7 (0.2-4.9) x 10(-4) and 0.9 (0.3-2.8) x 10(-4), respectively. No significant evidence was revealed in this study for differences in gut transfer between males and females, nor for seasonal differences, e.g. due to differences in form of incorporation of actinides in shellfish. For realistic assessments of doses due to shellfish consumption near Sellafield, to assess compliance with criteria recommended by the International Commission on Radiological Protection (ICRP), a suitable value for the gut transfer factor of both plutonium and americium might be 2 x 10(-4). A value of 5 x 10(-4) would appear to be cautious.

Adult↗

Incidence of Listeria spp. in tropical fish.

The incidence of Listeria spp. in tropical fish and shellfish was studied. The isolation protocol included a pre-enrichment, followed by two selective enrichment steps and plating on three selective agars. Listeria monocytogenes could be detected in 17.2% of finfish and 12.1% of shellfish. L. innocua was the most common species encountered. In 6.9% finfish and 5.6% shellfish, both L. monocytogenes and L. innocua were detected. Polymerase chain reaction (PCR)-based amplification of internal fragments of the iap gene was found to be useful in differentiation of L. monocytogenes from L. innocua.

Animals↗

Diversity of enterovirus sequences detected in oysters by RT-heminested PCR.

Oysters harvested in western France, from five sites associated with outbreaks of food-borne norovirus gastroenteritis between February 2000 and March 2001, were assayed for enterovirus RNA by reverse transcriptase-heminested polymerase chain reaction (RT-heminested PCR). Forty percent (21/52) of shellfish samples (pool of seven oysters) were contaminated by enteroviruses. Infectious coxsackieviruses serotype A21 were isolated from three of these positive samples. Amplicons corresponding to 65 base sequences in the 5' untranslated region of the enteroviral genome were analyzed by direct sequencing. Interpretable results were obtained from 18 amplicons, but mixtures of sequences confused the results from 3 samples. Sequences isolated from samples from the different sites were different but similarities were observed between sequences detected in shellfish from two sites at different dates. Sequences were also compared to sequences of human, bovine and porcine enteroviruses. Both human and animal origins of enterovirus contamination of shellfish seemed likely.

Animals↗

Pectenotoxin-2 in single-cell isolates of Dinophysis caudata and Dinophysis acuta from the Galician Rías (NW Spain).

Dinophysis acuta and Dinophysis caudata are seasonal components of the dinoflagellate community in the Galician Rías Bajas (NW Spain). These species can be the main contributors to the occurrence of Lipophilic Shellfish Toxins (LST) in September-October, leading to prohibition of harvesting in an area of intensive mariculture (250 x 10(3)t of cultured mussels, 60 x 10(3)t of other shellfish in natural banks, per year). Previous analyses of okadaic acid (OA) and related toxins in these two species by HPLC revealed significant amounts of OA and DTX2 in D. acuta, but only trace amounts of OA in D. caudata cells, and led to the erroneous conclusion that the contribution of the latter species to autumn LST events was negligible. Recent analyses by LC-MS/MS of individually picked cells of D. acuta and D. caudata have shown that both species may have high levels of PTX2 (up to 30 pg and 130 pg cell(-1), respectively) and that this toxin can be the dominant toxin during toxic outbreaks associated with Dinophysis spp. Although the quick conversion of PTX2 to PTX-2SA in mussels may reduce the risks for human health, these results have important implications for monitoring programmes. The complex toxin profile of shellfish exposed to Dinophysis spp. populations should be taken into account when making decisions on the toxin control methods to be used as the basis of the programme. Conclusive toxicological studies are required to elucidate the public health relevance of the different PTXs derivatives and to provide the scientific basis for regulations.

Animals↗

Esterification of DSP toxins by Portuguese bivalves from the Northwest coast determined by LC-MS--a widespread phenomenon.

Okadaic acid (OA) and dinophysistoxin-2 (DTX2) were confirmed by liquid chromatography with mass spectrometry detection both in extracts of digestive glands and edible parts of Portuguese shellfish. No dinophysistoxin-1 was found even in highly contaminated samples examined. However, only in blue mussel (Mytilus edulis) were these two parent toxins commonly found in a free form. Usually they were found largely esterified in all remaining shellfish species-common cockle (Cerastoderma edule), peppery furrow shell (Scrobicularia plana), carpet shell (Venerupis pullastra), oyster (Crassostrea japonica), razor clam (Ensisspp.), and clam (Ruditapes decussata). Oysters were the least toxic. In mussels esterified OA did not surpass 50% of the total OA found in edible parts, while DTX2 esterification rates were usually much lower. In remaining shellfish species usually more than 95% of the total OA was found esterified, while free DTX2 was rarely found. Also ratios of total DTX2/total OA were higher in mussels than in the remaining species examined. From all these species commercially exploited at the northern coast, mussels and cockles contained the highest levels of DSP toxins, thus representing the highest theoretical health risk. The previous association of DTX2 with the dinoflagellate Dinophysis acuta was confirmed with selective MS detection; while OA was the only parent diarrhoeic toxin found associated with Dinophysis acuminata.

Animals↗

Comparison of two methods for the detection of hepatitis A virus in clam samples (Tapes spp.) by reverse transcription-nested PCR.

The detection of hepatitis A virus in shellfish by reverse transcription-nested polymerase chain reaction (RT-nested PCR) is hampered mainly by low levels of virus contamination and PCR inhibitors in shellfish. In this study, we focused on getting a rapid and sensitive processing procedure for the detection of HAV by RT-nested PCR in clam samples (Tapes spp.). Two previously developed processing methods for virus concentration in shellfish have been improved upon and compared. The first method involves acid adsorption, elution, polyethylene glycol (PEG) precipitation, chloroform extraction and PEG precipitation. The second method is based on elution with a glycine buffer at pH 10, chloroform extraction and concentration by ultracentrifugation. Final clam concentrates were processed by RNA extraction or immunomagnetic capture of viruses (IMC) before the RT-nested PCR reaction. Both methods of sample processing combined with the RNA extraction from the concentrates were very efficient when they were assayed in seeded and naturally contaminated samples. The results show that the first method was more effective in removal inhibitors and the second was simpler and faster. The IMC of HAV from clam concentrates processed by method 1 was revealed to be a very effective method of simultaneously removing residual PCR inhibitors and of concentrating the virus.

Animals↗

Vibrio vulnificus.

V vulnificus is a halophilic or salt-requiring vibrio that has been isolated repeatedly from seawater and shellfish in coastal waters. This vibrio, first described by Hollis et al in 1976, can be differentiated from other similar vibrios by its ability to ferment lactose and by its lower tolerance for sodium chloride. V vulnificus, unlike most other vibrios, has seldom been incriminated as a cause of gastroenteritis but is a particularly virulent organism that causes severe wound infections in mostly healthy persons, or causes primary septicemia in persons with an underlying chronic disease, particularly chronic liver disease. Wound infections may range from relatively mild to severe and rapidly progressive cellulitis and myositis. Approximately 50% of patients with wound infections have some type of chronic underlying disease and the mortality rate is in the range of 15%. Wound infections are almost always associated with contact with seawater or the handling or cleaning of shellfish. Patients with primary septicemia have fever, chills, and prostration, and rapidly become hypotensive. Over 70% have distinctive bullous skin lesions that can strongly suggest the diagnosis in a patient with the appropriate history. The mortality rate is over 50%. There is a striking association between eating raw oysters and primary septicemia, with patients usually reporting having eaten raw oysters (or other shellfish) 24 to 48 hours before onset of symptoms.(ABSTRACT TRUNCATED AT 250 WORDS)

Food Microbiology↗

Essential and toxic elements in seafood available in poland from different geographical regions.

The concentrations of 15 elements (Cu, Fe, Mn, Zn, Co, Ni, Cr, Se, Cd, Pb, Hg, Ca, Na, K, and Mg) were determined in the edible parts of shellfish on sale in the local market in Gdańsk. The samples consisted of three groups--crustaceans, molluscs, and surimi--that are processed to different degrees. For the purposes of this analysis, they were dried, homogenized, and digested in an automatic microwave system. The samples were analyzed quantitatively for Cu, Zn, Fe, Mn, Co, Ni, Cr, Mg, Na, K, and Ca (F-AAS), Cd and Pb (GF-AAS), Se (HG-AAS), and Hg (CV-AAS). The elemental levels detected in shellfish were compared to those in cod, herring, pork, beef, chicken, and eggs. The recommended dietary allowance (RDA) of essential elements and the provisional tolerable weekly intake (PTWI) of toxic elements were estimated. With factor analysis of the data, taxonomically different groups of raw and processed shellfish could be distinguished.

Animals↗