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PubMed2000

Detection of norwalk-like virus in shellfish implicated in illness.

In the 1990s, Norwalk-like viruses (NLVs) were identified in patient specimens as the primary pathogen associated with shellfish-borne gastroenteritis in the United States. Identification of these viruses from implicated shellfish has been difficult due to inefficient recovery of viruses, natural polymerase chain reaction (PCR) inhibitors in shellfish, and low virus contamination. Recent improvements to the method of detecting NLVs in shellfish include enhanced processing of virus and shellfish samples, application of nested PCR and nucleotide sequencing, and increased knowledge of NLV genetic diversity. Using a newly developed and sensitive method, an NLV G2 strain was identified in 2 oyster samples implicated in a 1998 California outbreak involving 171 cases. NLV capsid primers demonstrated a greater specificity of PCR detection than did polymerase primers. The 175-base viral capsid nucleotide sequences derived from oysters were 100% identical to those derived from a patient stool sample. This finding supports the epidemiologic associations indicating that contaminated shellfish serve as the vehicle for NLV transmission.

Animals↗
PubMed2006

First isolation of Shiga toxin 1d producing Escherichia coli variant strains in shellfish from coastal areas in France.

AIMS: This study was carried out to evaluate the presence of Shiga toxin-producing Escherichia coli (STEC) and E. coli O157:H7 in shellfish from French coastal environments. METHODS AND RESULTS: Shellfish were collected in six growing areas or natural beds (B category) and nonfarming areas (D category) from July 2002 to August 2004. PCR detection of stx genes was performed on homogenized whole shellfish and digestive gland tissues enrichments. STEC strains were detected by colony DNA hybridization using a stx-specific gene probe and E. coli O157 strains were additionally searched by immunomagnetic separation with O157-specific magnetic beads. Stx genes were detected in 40 of 144 (27.8%) sample enrichments from mussels, oysters or cockles, 32 of 130 enrichments (24.6%) were from B-category areas and eight of 14 (57.1%) from the D-category area. Five strains carrying stx(1) or stx(1d) genes and one stx negative, eae and ehxA positive E. coli O157:H7 were isolated from six of 40 stx-positive enrichments. No relation was found between the total E. coli counts in shellfish and the presence of STEC strains in the samples. CONCLUSIONS: The STEC strains of different serotypes and stx types are present in shellfish from French coastal environments. It is the first isolation of STEC stx1d strains in France. SIGNIFICANCE AND IMPACT OF THE STUDY: Shellfish collected in coastal environments can serve as a vehicle for STEC transmission.

Animals↗
PubMed1986

Abnormal metabolism of shellfish sterols in a patient with sitosterolemia and xanthomatosis.

Sitosterolemia and xanthomatosis together are a disease characterized by premature cardiovascular disease, and by elevated plasma concentrations of total sterols and of plant sterols, especially sitosterol which is hyperabsorbed. In order to determine whether this abnormal metabolism also involved other sterols, a patient with sitosterolemia was fed a diet high in shellfish that contain significant quantities of noncholesterol sterols, some of which are less well absorbed than cholesterol in humans. Compared with control subjects (n = 8), the sitosterolemic subject had an increased absorption of 22-dehydrocholesterol (71.5% vs. 43.8 +/- 11.4%, mean +/- SD), C-26 sterol (80.6% vs. 49.3 +/- 11.4%), brassicasterol (51.8% vs. 4.8 +/- 4.2%), and 24-methylene cholesterol (60.5% vs. 16.0 +/- 8.3%). This enhanced absorption was associated with an increased plasma total shellfish sterol level (13.1 mg/dl vs. 1.9 +/- 0.7 mg/dl in normals). In the sitosterolemic subject, as in normals, the shellfish sterols were not preferentially concentrated in any lipoprotein class, and 50-65% of these sterols were in the esterified form in plasma. Bile acids and neutral sterols were quantitated in bile obtained by duodenal aspiration. The bile acid composition did not differ significantly in the sitosterolemic subject compared with the normal controls. The sitosterolemic subject, though, was unable to concentrate normally the neutral shellfish sterols in bile. The normal controls concentrated the shellfish sterols in bile 6.3 +/- 1.7-fold relative to the plasma shellfish sterol concentration whereas the study subject was only able to concentrate them 2.1-fold. We propose that sitosterolemia and xanthomatosis occur from a generalized abnormality in the usual ability of the gut mucosa and other tissues of the body to discriminate among many different sterols. This has important implications for the understanding of the pathophysiology of this disease and for therapeutic recommendations.

Adult↗
PubMed2006

Distribution of virulent and pandemic strains of Vibrio parahaemolyticus in three molluscan shellfish species (Meretrix meretrix, Perna viridis, and Anadara granosa) and their association with foodborne disease in southern Thailand.

Distribution of pandemic strains of Vibrio parahaemolyticus in seafood, particularly in molluscan shellfish, and their serological and molecular relationships to clinical strains were examined from Hat Yai City in southern Thailand. During 2000 to 2002, virulent strains (tdh+ or trh+) were isolated from 13 of 230 molluscan shellfish samples using alkaline peptone water enrichment followed by immunomagnetic separation. The isolates included 12 pandemic strains (tdh+, trh-, group-specific PCR positive) from five Oriental hard clam samples, five green mussel samples, and one bloody clam sample. Among the pandemic strains, eight belonged to serogroup O3:K6, three belonged to O1:K25, and one was O1:K untypeable. One hundred eighty-seven strains of V. parahaemolyticus were isolated from clinical specimens obtained from a hospital in this city during 2000 to 2001. The pandemic strains comprised 64 and 68% of the isolates in 2000 and 2001, respectively. Among the serotypes of the pandemic strains, O3:K6 was dominant at 73% in 2000 and 76% in 2001 followed by O1:K25 at 20% in 2000 and 13% in 2001. Pulsed-field gel electrophoresis profiles of the pandemic strains from molluscan shellfish were indistinguishable or very similar to those of patient isolates. Similarity of the serotype distribution and DNA fingerprints occurring between the molluscan shellfish strains and clinical strains suggests that molluscan shellfish may be an important source of pandemic V. parahaemolyticus infection in southern Thailand. For public health, proper cooking of molluscan shellfish in this area is strongly recommended.

Animals↗
PubMedDate not supplied

Determination of brevetoxins in shellfish by the neuroblastoma assay.

A neuroblastoma assay for determination of brevetoxins in shellfish was developed together with a method for sample cleanup that allows separation of brevetoxins from most of the components that cause matrix interference in the assay. This improved assay method was applied to a range of shellfish samples with different characteristics. Extracts of naturally contaminated and nontoxic shellfish together with extracts spiked with known amounts of toxin were tested. The results demonstrated that brevetoxins could be reliably detected in shellfish extracts at concentrations below the regulatory limit. Brevetoxin activity was detected in 15 of 23 samples from 5 separate toxicity incidents in which shellfish tested positive in the neurotoxic shellfish poisoning (NSP) mouse bioassay. Twelve of these positive NSP results came from 2 toxicity incidents. Yessotoxin was the major contributor to toxicity in 2 other incidents, although some samples contained both yessotoxin and brevetoxin. The sample from the remaining incident contained an unidentified toxin bioactivity, together with gymnodimine. In contrast to earlier versions of the neuroblastoma assay, gymnodimine was not detected by this modified method.

Animals↗
PubMed2005

[Study on rotavirus detection with single-tube seminested RT-PCR method in shellfish].

Being a foodborne virus, Rotavirus was often carried by shellfish. At the present, RT-PCR was the most effective method of Rotavirus detection in shellfish, but its sensitivity was low because of low levels of virus contamination and PCR inhibitors in shellfish. So contaminated shellfish experimentally in laboratory, and imitated the natural environment to concentrate Rotavirus, then detected by the developed single-tube seminested RT-PCR. Compared with ELISA and the conventional RT-PCR, the detection sensitivity is improved by 1000 times and 10 times respectively. In addition, the outer and inner contamination is reduced dramatically and the detection time is decreased from 6h to 4.5h. When the whole shellfish and only the digestive tissues of shellfish serve as samples, the detection ratio and sensitivity are more higher when sample is the latter.

Animals↗
PubMedDate not supplied

Variability of mouse bioassay for determination of paralytic shellfish poisoning toxins.

Toxic shellfish extracts and paralytic shellfish poison (PSP) standard solutions, tested over a range of pH levels, storage conditions, and temperatures, were monitored for toxin concentration, using the mouse bioassay and thin layer chromatography (TLC). A comparison of PSP toxin concentrations in toxic shellfish extracts and PSP standard solutions when dilution was varied suggests that other factors in the shellfish extracts contribute to the toxicity in mice; the closet agreement was at the death time range of 5-8 min. The toxicities of PSP standard solutions at pH levels ranging from 2 to 6 and held at 4 degrees C for various times were relatively constant; however, there was a gradual decrease in toxicity with pH 6 solutions. Also, standard solutions (pH 6) held at 4 degrees C for 28 days showed a 50% decrease in toxicity when the pH was adjusted to 2. TLC analyses of PSP standard solutions and toxic shellfish extracts revealed multiple spots at the Rf ranges of saxitoxin/neosaxitoxin and gonyaulax toxins I-IV. PSP standard solutions usually had a single spot in the saxitoxin/neosaxitoxin area. No attempt was made to confirm the identity of these compounds. Previously tested toxic shellfish extracts with subsequent pH adjustment to 1.5 and additional heat treatment (100 degrees C for 5 min) showed no appreciable difference in mouse toxicity. The use of antifoaming agents during the acid extraction step did not affect the final amounts of PSP obtained.

Animals↗
PubMed1985

[Nutritive value of shellfish consumed in Chile].

The purpose of the present study was to determine the protein quality and digestibility of shellfish commonly consumed in Chile, and to estimate its contribution to the protein needs of the Chilean population. The shellfish studied were chorito (Mytilus edulis chilensis), macha (Mesodesma donacium), loco (Concholepas concholepas), cholga (Aulacomya ater), erizo (Loxechinus albus) and almeja (no specific variety). The NPU method was used to determine protein quality. The percentage of protein adequacy for adult rations was calculated according to FAO/WHO 1973. The contribution of shellfish to the protein availability according to the family income of the Santiago population, was also calculated. Most of the shellfish presented NPU values of about 70; the lowest values were found for loco (54.9) and macha (63.3). The apparent and true digestibility gave an average of 83.6 and 90.4, respectively. The percentage of protein adequacy of habitual rations ranged between 27% (erizo) and 58% (loco). The availability of shellfish protein in relation to total protein increased from 0.4 to 2.5% when income increased. It is concluded therefore, that shellfish protein is, in general, of good quality. Nevertheless, it might be considered of poor influence insofar as fulfilling the protein needs of the population studied, whatever its socioeconomic level.

Chile↗
PubMed1995

Microbial contamination of shellfish: prevalence, risk to human health, and control strategies.

There has been significant concern in recent times about the safety of molluscan shellfish for human consumption. Despite extensive efforts to assure a safe supply of molluscan shellfish, the number of cases of disease and death are still great enough to cause concern among the public. The number of cases of illness and death associated with the ingestion of shellfish falls in the lower end of the range of other similar microbial pathogen-related foodborne disease. Disease and deaths due to viruses and naturally occurring bacteria are now of greatest concern because they are the most often cited causative agents. The greatest risk of disease or death due to shellfish consumption is among the population with underlying health conditions who choose to consume raw shellfish. Control strategies to limit shellfish-borne disease should focus upon disease and death caused by viruses and naturally occurring bacteria among at-risk populations.

Animals↗
PubMed2000

Neurotoxic shellfish poisoning and brevetoxin metabolites: a case study from Florida.

In June of 1996, three family members were diagnosed as suffering from neurotoxic shellfish poisoning (NSP) as a result of eating shellfish harvested from Sarasota Bay, Florida. Urine from two of these patients and extracts of shellfish collected from the same location were analyzed by radioimmunoassay (RIA) and by receptor binding assay. Activity consistent with brevetoxins was present in both urine and shellfish extracts. High performance liquid chromatographic (HPLC) analysis of shellfish extracts demonstrated multiple fractions recognized by specific anti-brevetoxin antibodies, suggesting metabolic conversion of parent brevetoxins. Affinity-purification of these extracts yielded four major peaks of activity. One peak was identified by HPLC-mass spectroscopy (HPLC-MS) to be PbTx-3, which was likely produced metabolically from the dominant parent toxin PbTx-2. No PbTx-2, however, was detected. Other peaks of activity were determined to consist of compounds of apparent masses of [M + H]+ of 1018, 1034, and 1005. These higher masses are suggestive of conjugated metabolites, but their structures have yet to be determined. The material associated with these latter three peaks were recognized by both RIA and receptor binding assay, but they quantitated differently. This finding suggests that these metabolites react differently in the two assays, and this result may have important implications for seafood safety and regulation. We suggest these metabolites to be the true cause of NSP, and they should be taken into account during regulatory testing.

Animals↗
PubMed2003

A fluorimetric microplate assay for detection and quantitation of toxins causing paralytic shellfish poisoning.

Paralytic shellfish poisoning is one of the most severe forms of food poisoning. The toxins responsible for this type of poisoning are metabolic products of dinoflagellates, which block neuronal transmission by binding to the voltage-gated Na(+) channel. Accumulation of paralytic toxins in shellfish is an unpredictable phenomenon that necessitates the implementation of a widespread and thorough monitoring program for mollusk toxicity. All of these programs require periodical collection and analysis of a wide range of shellfish. Therefore, development of accurate analytical protocols for the rapid determination of toxicity levels would streamline this process. Our laboratory has developed a fluorimetric microplate bioassay that rapidly and specifically determines the presence of paralytic shellfish toxins in many seafood samples. This method is based on the pharmacological activity of toxins and involves several steps: (i) Incubation of excitable cells in 96 well microtiter plates with the fluorescent dye, bis-oxonol, the distribution of which across the membrane is potential-dependent. (ii) Cell depolarization with veratridine, a sodium channel-activating toxin. (iii) Dose-dependent inhibition of depolarization with saxitoxin or natural samples containing paralytic shellfish toxins. Measuring toxin-induced changes in membrane potential allowed for quantification and estimation of the toxic potency of the samples. This new approach offers significant advantages over classical methods and can be easily automated.

Animals↗
PubMed2002

Determination of azaspiracids in shellfish using liquid chromatography/tandem electrospray mass spectrometry.

Azaspiracid (AZA1), a recently discovered marine toxin, is responsible for the new human toxic syndrome, azaspiracid poisoning (AZP), which is caused by the consumption of contaminated shellfish. A new, sensitive liquid chromatography/mass spectrometry (LC/MS) method has been developed for the determination of AZA1 and its analogues, 8-methylazaspiracid (AZA2) and 22-demethylazaspiracid (AZA3). Separation of these toxins was achieved using reversed-phase LC and coupled, via an electrospray ionisation (ESI) source, to an ion-trap mass spectrometer. Spectra showed the protonated molecules, [M + H]+, and their major product ions, due to the sequential loss of two water molecules, [M + H - H2O]+, [M + H - 2H2O]+, in addition to fragment ions that are characteristic of these cyclic polyethers. A highly specific and sensitive LC/MS(3) analytical method was developed and, using shellfish extracts containing AZA1, the detection limit (S/N = 3) was 4 pg on-column, corresponding to 0.8 ng/mL. Using the protocol presented here, this is equivalent to 0.37 ng/g shellfish tissue and good linear calibrations were obtained for AZA1 in shellfish extracts (average r2 = 0.9988). Good reproducibility was achieved with % RSD values (N = 5) ranging from 1.5% (0.75 microg/mL) to 4.2% (0.05 microg/mL). An efficient procedure for the extraction of toxins from shellfish aided the development of a rapid protocol for the determination of the three predominant azaspiracids.

Animals↗
PubMed1992

Detection of the marine toxins okadaic acid and domoic acid in shellfish and phytoplankton in the Gulf of Mexico.

Liquid chromatographic analyses of extracts from shellfish and phytoplankton from the Gulf of Mexico indicated the presence of the marine toxins okadaic acid (0.162 microgram/g shellfish) and domoic acid (2.1 pg/cell phytoplankter). These toxins are causative agents of diarrhetic shellfish poisoning (DSP) and amnesic shellfish poisoning (ASP), respectively. The presence of DSP and ASP toxins in a region with no previous record of outbreaks may indicate a potential for human poisoning under conditions appropriate for accumulation of these toxins in shellfish.

Animals↗
PubMed1989

A method for the enumeration of poliovirus in selected molluscan shellfish.

A virus extraction procedure was developed and evaluated on five commercially important molluscan shellfish species: Crassostrea virginica (Eastern oyster), Mya arenaria (softshell clam), Mytilus edulis (blue mussel), Mercenaria mercenaria (hardshell clam), and Crassostrea gigas, (Pacific oyster). Shellfish tissue homogenates were spiked with poliovirus, extracted, and plaque assayed. Mean virus recoveries were: C. virginica, 63.8%; M. arenaria, 42.1%; M. edulis, 67.3%; M. mercenaria, 48.3%; and C. gigas 10.1%. Shellfish were also allowed to accumulate poliovirus from spiked seawater (10 to 20 PFU/ml of water) over 48 to 72 h. The results indicate that poliovirus could be extracted from four shellfish species exposed to near environmental levels of virus. Virus recoveries per gram of tissue were: M. arenaria, 11.7 PFU; M. mercenaria, 26.0 PFU; M. edulis, 21.5 PFU; and C. virginica, 2.0 PFU. The results of this study indicate that the procedure is effective in extracting ingested viruses from several shellfish species. This procedure may have practical application for enumeration of enteric viruses in environmental samples.

Animals↗
PubMed2004

Fish and shellfish as dietary sources of methylmercury and the omega-3 fatty acids, eicosahexaenoic acid and docosahexaenoic acid: risks and benefits.

Fish and shellfish supply the human diet with not only complex nutrients including the omega-3 fatty acids, but also highly toxic chemicals including methylmercury. The dietary essential fatty acids are linoleic and alpha-linolenic acid. Two omega-3 fatty acids with longer carbon chains, eicosahexaenoic acid (EPA) and docosahexaenoic acid (DHA), can be synthesized in humans from alpha-linolenic precursors. Though not required in the diet per se, EPA and DHA have important roles in metabolism. The almost exclusive source of preformed dietary DHA is fish and shellfish. These foods are also an important source of EPA. In marked contrast to the benefits of fish and shellfish as sources of preformed omega-3 fatty acids, fish and shellfish are almost exclusively the dietary source of methylmercury. Fortunately, these chemicals are not uniformly distributed across many species of fish and shellfish. The purpose of this article is to provide information on the comparative distribution of these chemicals and nutrients to help groups formulating dietary recommendations.

Animals↗
PubMed2006

Detection of noroviruses in shellfish in the Netherlands.

Shellfish from oyster farms in the Netherlands and imported from other European countries were examined for viral contamination. A method that allows sequence matching between noroviruses from human cases and shellfish was used. The samples of shellfish (n = 42) were analyzed using a semi-nested RT-PCR that had been optimized for detection of norovirus in shellfish (SR primer sets). In addition, a different genome region was targeted using a second primer set which is routinely used for diagnosis of norovirus infection in humans (JV12Y/JV13I). To improve the detection limit for this RT-PCR a semi-nested test format was developed (NV primer sets). One of 21 oyster samples (4.8%) from Dutch farms was norovirus positive, whereas norovirus was detected in 1 out of 8 oyster samples (12.5%) and 5 out of 13 mussel samples (38.5%) collected directly after importation in the Netherlands. RNA from samples associated with an outbreak of gastro-enteritis in the Netherlands in 2001 was re-analyzed using the NV primer sets. At least one identical sequence (142/142 nt) was found in three fecal and in two oyster samples related to this outbreak. Further surveillance of norovirus by detection and typing of viruses from patients with gastroenteritis and shellfish is warranted to clarify the causes of future outbreaks.

Animals↗
PubMed2005

Nested multiplex PCR assay for detection of human enteric viruses in shellfish and sewage.

Environmental samples and contaminated shellfish present frequently low concentrations of more than one viral species. For this reason, a nested multiplex RT-PCR was developed for the detection of adenoviruses, enteroviruses and hepatitis A viruses in different environmental samples such as urban sewage and shellfish. This assay will save time and cost for detection of these enteric viruses with a smaller sample volume, which otherwise can be a limiting factor in routine analysis. The limit of detection was approximately 1 copy for adenovirus and 10 copies for enterovirus and hepatitis A virus per PCR reaction using titrated cell-cultured viruses as template material. In shellfish and environmental samples, this multiplex PCR was optimized to detect all three viruses simultaneously when the concentration of each virus was equal or lower than 1000 copies per PCR reaction. This is the level found predominantly in the environment and in shellfish when the numbers of fecal bacterial and phage indicators are low. The detection of human adenoviruses by PCR has been suggested as a molecular index of fecal contamination of human origin in the environment and food and the multiplex assay developed may be a tool for evaluating the presence of viral contamination in shellfish and water and to expand microbiological control to include viral markers.

Adenoviruses, Human↗
PubMed2004

Is there a risk of human poisoning by azaspiracids from shellfish harvested at the Portuguese coast?

Azaspiracid poisoning (AZP), the most recently discovered human gastrointestinal illness resulting from consumption of contaminated shellfish, so far has been found in coastal areas of northern Europe. This is the first report of a survey carried out for contamination of shellfish harvested in costal areas of Portugal for the presence of azaspiracids. The study design covered the commercial species usually more contaminated by toxins from dinoflagellates (blue mussel, common cockle, donax clam) in coastal areas representative of the NW, SW and south coasts. A method based on liquid chromatography-mass spectrometry was setup for the first time for this purpose. No azaspiracids were found on 300 samples tested between 2002 and 2003. On at least three samples a peak with a retention time matching that of AZA2 was found, never surpassing one tenth of the current EU limit. Unambiguous identification of any known AZA did not occur yet. The risk for human outbreaks of AZP seems to be very low, comparatively with amnesic shellfish poisoning (ASP), where levels close to the allowance level are found sparsely, or to diarrhetic shellfish poisoning (DSP) and paralytic shellfish poisoning (PSP), where high levels and registered human outbreaks have been found in recent years.

Animals↗