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Amnesic shellfish poisoning toxins in bivalve molluscs in Ireland.

In December 1999, domoic acid (DA) a potent neurotoxin, responsible for the syndrome Amnesic Shellfish Poisoning (ASP) was detected for the first time in shellfish harvested in Ireland. Two liquid chromatography (LC) methods were applied to quantify DA in shellfish after sample clean-up using solid-phase extraction (SPE) with strong anion exchange (SAX) cartridges. Toxin detection was achieved using photodiode array ultraviolet (LC-UV) and multiple tandem mass spectrometry (LC-MS(n)). DA was identified in four species of bivalve shellfish collected along the west and south coastal regions of the Republic of Ireland. The amount of DA that was present in three species was within EU guideline limits for sale of shellfish (20 microg DA/g); mussels (Mytilus edulis), <1.0 microg DA/g; oysters (Crassostrea edulis), <5.0 microg DA/g and razor clams (Ensis siliqua), <0.3 microg DA/g. However, king scallops (Pecten maximus) posed a significant human health hazard with levels up to 240 microg DA/g total tissues. Most scallop samples (55%) contained DA at levels greater than the regulatory limit. The DA levels in the digestive glands of some samples of scallops were among the highest that have ever been recorded (2,820 microg DA/g).

Animals↗

Fish, shellfish, and meat meals of the public in Singapore.

Understanding different patterns of fish consumption is an important component of the assessment of risk from contaminants in fish. While there have been extensive studies of fish consumption in Western cultures, less attention has been devoted to the role of fish and meat in the diets of people in other cultures. A survey of 212 people living in Singapore was conducted to examine the relative importance of fish, shellfish, and other meat in their diets and to ascertain whether there were differences as a function of age, income, education or gender. As expected, fish and shellfish played an important role in their daily diets. On average, people ate fish in about 10 meals a week, chicken for eight meals, and shrimp and pork for about six meals each. While nearly 8% never ate fish, 18% ate fish at all 21 meals a week and over 20% ate shellfish for all 21 meals. Income explained about 14% of the variation in the number of fish meals consumed, and age explained about 8% of the variation in number of chicken meals per week. There were no gender differences in the number of meals of each type. People less than 26 years old ate significantly more pork, chicken, and other meat meals and fewer shellfish meals than older people. People with higher incomes ate significantly more fish meals than those with lower incomes. Chinese individuals ate significantly more meals of pork, chicken, and other meat than other ethnic groups, and they ate only 26% of their meals at home, while others ate 33% of their meals at home. The data indicate a great deal of variation in the number of meals of fish, shellfish, and other meats eaten by the people interviewed, making dietary and risk assessments challenging.

Adolescent↗

Transmission of the paralytic shellfish poisoning toxins, from dinoflagellate to gastropod.

Purple clams, Hiatula diphos Linnaeus, are filter-feeding bivalves and maculated ivory shells. Babylonia areolata Link are carnivorous gastropods. Both shellfishes are popular seafood delicacies among the Taiwanese. Hiatula diphos were forced to contain gonyautoxins (GTXs) in this research by feeding them with cells of Alexandrium minutum Halim, a toxic dinoflagellate species responsible for the paralytic shellfish poisonings in Taiwan. The intoxicated purple clams of known toxicity and toxin composition were fed to B. areolata to observe the transmission and transformation of GTXs among this shellfish. It was found that the toxin composition in bivalve and gastropod were similar to that in dinoflagellate. Our data provide evidence for food-chain transmission of paralytic shellfish poisoning toxins, from dinoflagellate to gastropod through a filter-feeding bivalve. The transmitted GTX-I. -II. -III and -IV of A. minutum could only be found in the viscera of these shellfish. There was a notable degradation of GTX-I in the ivory shell that resulted in a decrease in toxicity while the total amount of toxins was accumulatively increasing.

Animals↗

Total mercury intake from fish and shellfish by Japanese people.

Elevated mercury concentrations have been reported in fish in recent years. Japanese people eat a great deal of raw fishes and shellfishes as "Sashimi" and "Sushi". The action level of large predatory fish such as tuna with total mercury levels exceeding the Japanese maximum permitted limit of 0.4 ppm is exempted from regulation in Japan. Therefore, current total mercury intake from fish and shellfish of Japanese people is unknown. The purpose of this investigation was to estimate the total mercury intake from fish and shellfish. It was found that the mean total mercury concentration of 1.11 ppm in tuna of eatable base as Sashimi or Sushi was clearly higher than the normal level. The mean total mercury intake from fish and shellfish was 0.17 mg per capita per week. According to the hypothesis that 75% of total mercury in fish and shellfish is methylmercury, the weekly intake of 0.13 mg as methylmercury was corresponding amount to about 74% of provisional tolerable weekly intake 0.17 mg of methylmercury set by the Welfare Ministry of Japan.

Animals↗

Aquatic pollution: effects on the health of fish and shellfish.

As there is little evidence of pollution affecting the health of fish and shellfish on a global scale, this paper attempts to put into perspective the pollution/fish disease relationship by reviewing examples of studies and reports in the historic and current literature. Although there is no dispute that pollution can affect the aquatic organisms under laboratory conditions and may be responsible for the decline of populations of such animals in some inland waters and some estuaries, most of the evidence for pollution causing or increasing disease in fish in open waters is circumstantial. Historical data proves that almost all fish and shellfish diseases known today have been described since the end of the last century. However, it is also known that water pollution, especially in inland waters, has for the past 400-500 years been the result of urbanization and industrialization. This has resulted in some major rivers becoming devoid of or deficient in fish stocks. The concern that pollution may influence the health status of fish and shellfish stocks has increased over the past 20 years. Initial attention was paid to epidermal diseases, including fin-rot in demersal fish, and protozoan diseases in molluscs in the heavily polluted bays and estuaries in North America. As the interest in this subject spread, it became political, and often controversial, especially amongst the North Sea countries. The disagreements have largely been settled amongst scientists because international bodies, such as the International Council for Exploration of the Sea (ICES), established workshops to investigate sampling methods and disease-reporting techniques. Recommendations from those workshops have contributed to some form of standardization for field work and the subject, although largely subjective, has some objective approaches which are described. As there are variable, interacting biological and physical influences in the aquatic environment, it is difficult to establish the background prevalences of diseases in populations of fish and shellfish. Examples of the influences of climatic changes are presented, and these show that short-term catastrophes can be directly related. However, a more long-term problem is water acidification resulting largely from anthropogenic activities. In parts of Scandinavia this has, and is, leading to decimation of fish stocks in inland waters. In general, diseases in fish and shellfish are very localized, but there is concern amongst scientists that certain cancers, especially liver tumours, occurring in demersal fish inhabiting polluted estuarine and coastal waters, are related to the release of chemicals, e.g. hydrocarbons, pesticides and heavy metals.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Studies on heat inactivation of hepatitis A virus with special reference to shellfish. Part 1. Procedures for infection and recovery of virus from laboratory-maintained cockles.

The consumption of bi-valve molluscan shellfish has been associated with outbreaks of viral gastroenteritis and hepatitis A. Investigations were undertaken to determine the heat inactivation conditions necessary to render shellfish such as cockles safe for the consumer. Conditions for the laboratory maintenance of live cockles are described. In preliminary experiments either poliovirus (10(6) TCID50/ml seawater) or hepatitis A virus (HAV) (approx. 10(4) RFU/ml seawater) was introduced into the shellfish tank. Following 48 h filter feeding, virus was recovered from cockles using an adsorption-elution extraction procedure. Titres of virus recovered ranged from 10(4) to 10(5) TCID50/ml of shellfish extract for poliovirus and from 10(3) to 10(5) RFU/ml of shellfish extract for HAV. Active ingestion of the virus from the seawater was demonstrated by recovering virus from within cockle guts. To quantify recovered HAV, end-point dilutions and an adaptation of a radioimmunofocus assay (RIFA) were compared. The tests were of similar sensitivity but the RIFA has the advantage of being relatively rapid, shortening the time taken to complete an experiment by as much as 4 weeks.

Animals↗

Feline calicivirus as a model system for heat inactivation studies of small round structured viruses in shellfish.

Commercial heat treatment procedures for molluscan shellfish are based on data obtained for the inactivation of hepatitis A virus (HAV) in cockles. However, the most frequently reported illness associated with consumption of bivalve molluscs is gastroenteritis caused by small round structured viruses (SRSVs) of the Norwalk group. Conditions for inactivation of SRSVs are unknown. In this study a feline calicivirus was used as a model for the SRSV group and conditions for its heat inactivation determined. Experiments showed that feline calicivirus is more readily inactivated in shellfish than HAV, and confirmed that current heating recommendations to the UK shellfish industry are adequate. A reverse transcription polymerase chain reaction (RT-PCR) assay for the detection of calicivirus in shellfish was developed and results compared with isolation in cell culture. The RT-PCR detected virus in some samples that failed to yield virus on culture. This has important implications if molecular virology techniques are to be used in the design and monitoring of shellfish treatment procedures and for routine testing of food samples.

Animals↗

Basis for a new procedure to eliminate diarrheic shellfish toxins from a contaminated matrix.

The natural contamination of shellfish with diarrheic shellfish toxins (DSP) has important public health implications. To avoid the economic effects of toxic episodes on shellfish farmers and the related industry, research on artificial methods alternative to the natural detoxification of shellfish is needed. Because the usual thermal processes are not efficient, alternative technologies have to be studied. Here preliminary results are presented about the lability of the DSP toxin okadaic acid in a supercritical atmosphere of carbon dioxide with acetic acid. Most of the toxin is eliminated (up to 90%), and the biological activity against its target enzyme is also severely affected (up to 70% reduction). Detoxification of contaminated shellfish requires a partial dehydration, and the detoxification yield is lower than that obtained with free toxin. Mass spectrometry experiments suggest that acetylation of the toxin molecule is not the basis of the inactivating mechanism, but a conformational change is suggested. This is the first report of the use of supercritical fluids to inactivate toxins.

Acetic Acid↗

Characterization of vitamin B12 compounds from edible shellfish, clam, oyster, and mussel.

Vitamin B12 content of various edible shellfish was determined by both Lactobacillus leichmannii ATCC 7830 microbiological and intrinsic factor-chemiluminescence methods. The values determined by the microbiological method were 1.2-19.8 (M/C ratio) fold greater in the shellfish than the values determined by the chemiluminescence method. Vitamin B12 compounds were purified from most eaten shellfish, oyster (M/C, 1.5), mussel (M/C, 1.2), and short-necked clam (M/C, 2.7), and partially characterized. TLC and HPLC patterns of each red-colored vitamin B12 compound (M/C, 1.0-1.2) purified from these shellfish were identical to those of authentic vitamin B12. Although the higher values in the determination of vitamin B12 by the microbiological method may be due to the occurrence of vitamin B12-substitutive compounds, the edible shellfish would be excellent vitamin B12 sources judging from the values (> or = 6 micrograms/100 g) determined by the chemiluminescence method.

Animals↗

Vibrio parahaemolyticus in shellfish and clinical samples during two large epidemics of diarrhoea in southern Chile.

Large epidemics of diarrhoea associated with seafood consumption and Vibrio parahaemolyticus occurred during the austral summers of 2004 and 2005 in the environs of Puerto Montt, Chile (41 degrees 29'S 72 degrees 24'W). There are no reports of V. parahaemolyticus infections before 2004 in this region, their absence being explained by the low ocean temperatures which seldom reach 16 degrees C. We analysed V. parahaemolyticus obtained from shellfish and clinical samples during epidemics. Isolates were examined using conventional protocols and an improved method for restriction enzyme analysis using total bacterial DNA which permits direct genome restriction enzyme analysis by conventional gel electrophoresis (DGREA) with a similar discrimination index as restriction fragment length polymorphism-pulsed field gel electrophoresis (RFLP-PFGE). Analysis of clinical samples showed that the epidemics were caused by the V. parahaemolyticus O3:K6 pandemic clonal group. On the other hand, analysis of shellfish samples during both epidemics showed that 53% contained V. parahaemolyticus (3-93 g(-1)). Detailed analysis of 50 positive shellfish samples showed that only three contained detectable levels of the pandemic clone. Most V. parahaemolyticus isolates obtained from shellfish corresponded to non-pandemic clones differentiated into 14 groups by DGREA. In summary, the causative agent during epidemics was only a minor component of a small but diverse population of V. parahaemolyticus in shellfish.

Chile↗

Evaluation of two viral extraction methods for the detection of human noroviruses in shellfish with conventional and real-time reverse transcriptase PCR.

AIMS: Comparison of two viral extraction methods in order to establish a sensitive and simple detection method for human noroviruses (NV) in bivalve shellfish. METHODS AND RESULTS: A direct RNA extraction method and an alkaline virus elution-concentration method were tested on artificially contaminated mussels. The latter used an alkaline buffer and polyethylene glycol (PEG) to isolate and concentrate the virus particles from shellfish. In both methods Trizol was used to release RNA. The final RNA extracts were amplified and detected with conventional and real-time reverse transcriptase PCR. The direct RNA extraction method was not able to detect low inoculation levels. However, the virus elution-concentration method was more sensitive. CONCLUSIONS: The alkaline elution-PEG concentration method followed by Trizol effectively removed inhibitory components and fulfilled the demands to be a useful tool for routine testing of shellfish for NV detection. SIGNIFICANCE AND IMPACT OF THE STUDY: Because of the lack of standardized methods to detect NV in shellfish, many 'in-house' extraction methods are used in practice. A comparison of these methods aims to determine a simple, rapid and sensitive method that could be a candidate method for screening suspected shellfish.

Animals↗

Concentration and detection of hepatitis A virus and rotavirus from shellfish by hybridization tests.

A modified polyethylene glycol precipitation method for concentration of virus followed by a new method to recover nucleic acid was used to detect hepatitis A virus (HAV) and rotavirus (SA11) in shellfish (oysters and hard-shell clams) by hybridization tests. Infectious virus, seeded into relatively large quantities of shellfish, was recovered consistently, with greater than 90% efficiency as measured by either in situ hybridization (HAV) or plaque assay (rotavirus SA11). Viral nucleic acid for dot blot hybridization assays was extracted and purified from virus-containing polyethylene glycol concentrates. Separation of shellfish polysaccharides from nucleic acid was necessary before viral RNA could be detected by dot blot hybridization. Removal of shellfish polysaccharides was accomplished by using the cationic detergent cetyltrimethylammonium bromide (CTAB). Use of CTAB reduced background interference with hybridization signals, which resulted in increased hybridization test sensitivity. After polysaccharide removal, dot blot hybridization assays could detect approximately 10(6) physical particles (corresponding to approximately 10(3) infectious particles) of HAV and 10(4) PFU of SA11 rotavirus present in 20-g samples of oyster and clam meats. These studies show continuing promise for the development of uniform methods to directly detect human viral pathogens in different types of shellfish. However, practical applications of such methods to detect noncultivatable human viral pathogens of public health interest will require additional improvements in test sensitivity.

Animals↗

Detection of hepatitis A virus, rotavirus, and enterovirus in naturally contaminated shellfish and sediment by reverse transcription-seminested PCR.

A reverse transcription-PCR method was developed to detect enterovirus (EV), hepatitis A virus (HAV), and rotavirus (RV) RNAs in shellfish and sediment. The method was first tested under experimental conditions by using virus-spiked shellfish to evaluate assay sensitivity. The use of CC41 cellulose was found to be efficient for removing inhibitors of RV detection. For sediment samples, a Sephadex column was used to allow the detection of EV and HAV RNAs. The specificity of amplified products was controlled by hybridization with digoxigenin-labeled oligoprobes. The method was then applied to naturally contaminated shellfish and sediments. EV, HAV, and RV RNAs were detected in 22, 14, and 20% of the shellfish samples, respectively. No relationship between viral contamination and bacterial contamination was found. When viral RNAs (HAV or EV) were detected in sediments, they were also detected in shellfish.

Animals↗

Development of a method for detection of enteroviruses in shellfish by PCR with poliovirus as a model.

The application of the PCR to complex samples is hindered by amplification inhibitors. We describe a reverse transcription-PCR-based method capable of inhibitor removal for the detection of enteroviruses in shellfish. Initial virus extraction stages based on a modified polyethylene glycol precipitation technique (G.D. Lewis and T.G. Metcalf, Appl. Environ. Microbiol. 54:1983-1988, 1988) were followed by virus purification with 1,1,2-trichloro,2,2,1-trifluoroethane and concentration by ultrafiltration. A guanidine isothiocyanate-glass powder extraction system was utilized for sample lysis, RNase protection, and nucleic acid purification. Removal of PCR inhibitors and method sensitivity were quantified in shellfish (oysters and mussels) seeded with poliovirus. PCR sample tolerance exceeded 4 g for depurated shellfish; however, polluted field samples were more inhibitory. Virus recoveries of 31% for oyster extracts and 17% for mussel extracts and nucleic acid extraction reverse transcription-PCR detection limits down to 1 PFU yielded an overall sensitivity limit of < 10 PFU of poliovirus in up to 5 g of shellfish. PCR-positive results were obtained from a variety of polluted field samples naturally contaminated with human enteroviruses. The methods developed for virus recovery and PCR inhibitor removal should be equally applicable to detection of other RNA viruses such as hepatitis A virus, Norwalk virus, and other small round-structured viruses in shellfish.

Base Sequence↗

Detection of small round structured viruses in shellfish by reverse transcription-PCR.

We describe the application of a previously developed sample extraction procedure to the detection of small round structured viruses (SRSVs) in shellfish. Initial seeding experiments showed that PCR inhibitor removal and virus recoveries were comparable to those in previous studies with poliovirus. Shellfish from a range of sewage-contaminated sites were then tested for the presence of SRSVs by using broadly reactive PCR primers followed by Southern blotting with internal probe sites. Positive results were obtained from 5 of 31 field samples tested. Four of these positive samples were from highly polluted sites. PCR product sequence analysis confirmed their identity as SRSV and showed sequence diversity compared with virus controls, suggesting that the results were not a consequence of PCR cross-contamination. Finally, shellfish associated with four separate outbreaks of viral gastroenteritis were tested by PCR and Southern blot for the presence of SRSVs. All outbreak samples tested gave positive results. As far as we are aware, this is the first demonstration of the detection in environmentally contaminated shellfish of the SRSVs responsible for human gastroenteritis. This development may help contribute to the further development of public health controls for molluscan shellfish.

Amino Acid Sequence↗

Distribution of human virus contamination in shellfish from different growing areas in Greece, Spain, Sweden, and the United Kingdom.

Viral pollution in shellfish has been analyzed simultaneously across a wide range of geographical regions, with emphasis on the concomitant variations in physicochemical characteristics and social features. The methods for sample treatment and for the detection of human enteric viruses were optimized by the participating laboratories. The second part of this study involves the selection of a protocol for virus detection, which was validated by analyzing the distribution and concentration of human viral pathogens under diverse conditions during an 18-month period in four European countries. Shellfish-growing areas from diverse countries in the north and south of Europe were defined and studied, and the microbiological quality of the shellfish was analyzed. Human adenovirus, Norwalk-like virus, and enterovirus were identified as contaminants of shellfish in all the participating countries. Hepatitis A virus was also isolated in all areas except Sweden. The seasonal distribution of viral contamination was also described. Norwalk-like virus appeared to be the only group of viruses that demonstrated seasonal variation, with lower concentrations occurring during warm months. The depuration treatments currently applied were shown to be adequate for reducing Escherichia coli levels but ineffective for the elimination of viral particles. The human adenoviruses detected by PCR correlate with the presence of other human viruses and could be useful as a molecular index of viral contamination in shellfish.

Adenoviruses, Human↗

Chemical risks associated with consumption of shellfish harvested on the north shore of the St. Lawrence River's lower estuary.

Shellfish have the capacity to accumulate chemical contaminants found in their biotope and therefore present a potential risk for consumers. This study was conducted to assess the chemical risks associated with consumption of shellfish harvested on the north shore of the St. Lawrence River's lower estuary. A survey was carried out on 162 recreational harvesters, and shellfish were sampled for chemical contaminant analysis. We quantified 10 metals, 22 polycyclic aromatic hydrocarbons (PAHs), 14 polychlorinated biphenyls (PCBs), and 10 chlorinated pesticides. We subsequently evaluated cancer and noncancer risks for four consumption scenarios based on our survey results and published results. Soft-shell clams (Mya arenaria) were by far the most consumed shellfish species. Of the 56 selected contaminants, 36 were detected in the 23 homogenates of soft-shell clam meat. None of the contaminants found in the soft-shell clams were associated with intakes that exceed the main exposure limit recommendations proposed to prevent noncancer effects. However, several limits must be considered before drawing conclusions about the relative safety of shellfish consumption regarding this end point. Furthermore, inorganic arsenic and PCBs were present in sufficient concentrations to lead to cancer risks exceeding the level often considered acceptable for environmental exposure (1 x 10 (-4) to 1 x 10(-6)) in each of the four scenarios, even for the lowest observed scenario of 15 meals of soft-shell clams per year.

Adult↗

The seasonality of fecal coliform bacteria pollution and its influence on closures of shellfish harvesting areas in Mississippi Sound.

Runoff from agricultural lands and farm animal feedlots is one of the major sources of fecal coliforms in surface waters, and fecal coliform (FC) bacteria concentrations tend to vary with season because of seasonal variations in climatic factors. However, El Niño--Southern Oscillation (ENSO) events may affect the extent and patterns of seasonality in FC levels in coastal waters. Water quality monitoring data for shellfish management collected during El Niño (1990, 1992, 1997), and La Niña (1999, 2000) years were analyzed to evaluate the extent to which these events influenced Pearl River stage, and bacterial levels, water temperature, and salinity in the western part of Mississippi Sound. Models to predict FC levels in relation to various environmental factors were also developed. In 1990, 1992 and 1997, FC geometric mean counts peaked in late winter (January/February) reaching 120 MPN (February 1990), 165 MPN (January 1992), and 86 MPN (January 1997), and then decreased considerably during spring and summer (1.2 - 19 MPN). Thereafter, FC abundance increased slightly in fall and early winter (1.9 - 24 MPN). Fecal coliform abundance during the 2000 La Niña year was much lower (1.0 -10.3 MPN) than in 1992 (1.2 - 165 MPN), and showed no seasonal pattern from January to August, perhaps due to the relative scarcity of rainfall in 2000. In 1995 (ENSO neutral year), peak geometric mean FC count (46 MPN) was lower than during El Niño years and occurred in early spring (March). The seasonal and between year variations in FC levels determined the number of days during which the conditionally approved shellfish growing area was opened for harvesting shellfish. For example, from January to April 1997, the area was not opened for shellfish harvesting, whereas in 2000, the number of days during which the area was opened ranged from 6 - 27 (January to April) to 24 - 26 (October to December). ENSO events thus influenced the extent and timing of the peak levels of fecal coliforms in Mississippi Sound. Models consisting of one or more of the variables: Pearl River stage, water temperature, and salinity were developed to predict FC concentrations in the Sound. The model parameter(s) explained 56 to 91% of the variations in FC counts. Management of shellfish in Mississippi Sound can be improved by utilizing information on the forecasted three to seven years occurrence of ENSO events. In addition, since Pearl River stage was the most important variable predicting FC concentration in the Sound, a study of the levels and sources of FC bacteria in the river, especially the middle and lower sections, is needed for developing a management plan for reducing FC bacteria pollution in the Sound.

Colony Count, Microbial↗