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The first evidence of paralytic shellfish toxins in the fresh water cyanobacterium Cylindrospermopsis raciborskii, isolated from Brazil.

The blooms of toxic cyanobacteria (blue-green algae) are causing problems in many countries. During a screening of toxic freshwater cyanobacteria in Brazil, three strains isolated from the State of Sao Paulo were found toxic by the mouse bioassay. They all were identified as Cylindrospermopsis raciborskii by a close morphological examination. Extracts of cultured cells caused acute death to mice when injected intraperitoneally after developing neurotoxic symptoms which resembled to those caused by paralytic shellfish toxins. The analysis of the sample by HPLC-FLD postcolumn derivatization method for paralytic shellfish toxins resulted in the detection of several saxitoxin analogs. To avoid being misled by false peaks, the sample was reanalyzed after purification and also under the different postcolumn derivatizing conditions. Finally, the newly developed LC-MS method for paralytic shellfish toxins was applied to unambiguously identify the toxins. One isolate produced neosaxitoxin predominantly with saxitoxin as a minor component. The other two showed identical toxin profiles containing saxitoxin and gonyautoxins 2/3 isomers in the ratio of 1:9. This is the first evidence of paralytic shellfish toxins in this species and also the occurrence of the toxin producing cyanobacterium in South American countries.

Animals↗

Presence of Salmonella spp. and Campylobacter spp. in shellfish.

Bivalve molluscs, (cockles, mussels, scallops and oysters) were examined according to EC shellfish bed classification regulations for faecal coliforms, Escherichia coli and salmonella, and for coliforms and campylobacter which are not specified by these regulations. Salmonella serotypes were detected in 8% of 433 molluscs. Seven salmonella isolations (2%) were made from category A beds, nominally suitable for immediate consumption according to E. coli counts. A higher percentage of salmonella isolates (6%) was detected in shellfish which require relaying or depuration prior to eating. In another survey, thermophilic Campylobacter spp. were found in 42% of 380 shellfish. These findings show bed classification on the basis of indicator organisms alone is not sufficient to assure the absence of bacterial, and no doubt viral, pathogens. Depuration and end product specifications which require the absence of salmonellae are an essential part of these regulations. Microbiologists may wish to consider whether tests for pathogens such as salmonella and campylobacter should be included when determining the suitability of shellfish for human consumption.

Campylobacter↗

Polybrominated diphenyl ethers in retail fish and shellfish samples purchased from Canadian markets.

Fish and shellfish retail samples (n = 122) were purchased from three Canadian cities in the winter of 2002 and analyzed for a total of 18 polybrominated diphenyl ether (PBDE) congeners. The samples (salmon, trout, tilapia, Arctic char, mussels, oysters, shrimp, and crab) represented the range of fish and shellfish commercially available to Canadian consumers at the time of purchase. Trout and salmon (geometric mean SigmaPBDE = 1600 and 1500 pg/g, wet weight, respectively) were found to contain significantly higher amounts of PBDEs than the mussel, tilapia, and shrimp groups (geometric mean SigmaPBDE = 260, 180, and 48 pg/g, wet weight, respectively). These differences in SigmaPBDE concentrations among fish and shellfish products were partly driven by differences in lipid content among the samples. Mean SigmaPBDE concentrations in domestic samples were also significantly greater than in imported samples, possibly reflecting global environmental distribution of PBDEs. These concentration differences will contribute to variations in dietary exposure to PBDEs when assorted fish and shellfish items from various origins are consumed.

Animals↗

Infectious outbreaks associated with bivalve shellfish consumption: a worldwide perspective.

Outbreaks of shellfish-associated infection have been reported for more than a century. Since the early 1970s, the global consumption of shellfish has increased considerably--and with it, the reports of outbreaks of infection. Most of these reports have originated from the United States, but Europe and, to a lesser extent, Asia and Australia have also been represented. The majority of outbreaks have been linked to oysters, followed by clams and mussels. Hepatitis A virus caused the largest ever shellfish-associated outbreak, but caliciviruses have caused the highest number of outbreaks; Vibrio species lead the list of bacterial pathogens. The prognosis of shellfish-associated infections is generally good, except for outbreaks of Vibrio vulnificus infection, which have a mortality rate of up to 50% in vulnerable people. Conventional and molecular techniques should be applied to better identify the causative agents, thereby enabling more-targeted control measures in growing, harvesting, and shipping bivalves.

Asia↗

Effects of shellfish consumption on lipoproteins in normolipidemic men.

Eighteen normolipidemic males were fed six different species of shellfish; each shellfish was fed so that protein in shellfish equalled that in animal foods in the normal diet, with less than one-half of the amount of fat in animal foods allowed for preparation of the shellfish. Oyster, clam, crab, and mussel diets, low in cholesterol and high in n-3 fatty acids, lowered VLDL triglycerides and cholesterol and, except for the mussel diet, LDL and total cholesterol. Squid and shrimp diets, higher in cholesterol and lower in n-3 fatty acids, did not change the blood lipids. The ratio of LDL to HDL cholesterol was decreased on the oyster and mussel diets. Oyster, mussel, and squid diets increased HDL2 cholesterol. Cholesterol absorption was decreased on the oyster, clam, and mussel diets. When consumed with moderate dietary fat restriction, oysters, clams, mussels, and crab appear to be useful in hypolipidemic diets for normolipidemic men.

Adult↗

Shellfish and fish poisoning related to the toxic dinoflagellates.

At least four different species of the toxic dinoflagellates cause shellfish and fish poisoning in the United States: Ptychodiscus brevis, neurotoxic shellfish poisoning; Protogonyaulax catenella and P tamarensis, paralytic shellfish poisoning; and Gambierdiscus toxicus, ciguatera fish poisoning. These three disorders have similar clinical manifestations, primarily neurologic and alimentary. A complete history is essential; confirmation, while dependent on specific laboratory analysis, is usually based on a history of ingestion of fish or shellfish previously associated with these types of poisonings. The principal toxins affect sodium channels; Ptychodiscus brevis toxins and ciguatoxin by stimulating these channels and saxitoxin by blocking them. Since no antidote is known, treatment is symptomatic. Public health measures and public education are necessary to prevent this form of poisoning.

Animals↗

Detection of human enteric viruses in shellfish collected in Tunisia.

AIMS: The aim of this study was to detect the main pathogenic human RNA enteric viruses able to persist in the environment such as astrovirus, enterovirus, norovirus and hepatitis A virus (HAV) in shellfish collected from two locations in northern Tunisia. METHODS AND RESULTS: Viruses were eluted from digestive tissues and concentrated by polyethylene glycol precipitation before nucleic acid extraction and purification. After checking for inhibitors, all viruses were detected by reverse transcription-polymerase chain reaction (RT-PCR) and confirmed by hybridization. Overall, 83% of the samples were found positive for at least one virus. Astrovirus was detected in 61% of the samples, norovirus in 35% and HAV in 26%. Surprisingly, only one sample was found positive for enterovirus. CONCLUSIONS: The mean number of positive samples found in this study is in accordance with the data found in the literature, indicating that no real difference exists in this respect among countries studied. A notable exception is HAV, which reflects the epidemiological status of the population. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the interest to analyse shellfish samples from different production areas. These data will be helpful to understand virus circulation and to improve shellfish safety. The results, which confirm contamination, necessitate the development of appropriate studies and monitoring in all shellfish-producing countries.

Enterovirus↗

[Distribution of Vibrio vulnificus along the coastal area of Shimane Prefecture and contamination of retail fish and shellfish with V. vulnificus in Shimane Prefecture, Japan].

We studied distribution of Vibrio vulnificus in sea water, sediment, and shellfish along the coast and contamination of retail fish and shellfish with V. vulnificus in Shimane Prefecture, Japan between 2001 and 2004. V. vulnificus was isolated from 77 (68.1%) of 113 sea water samples with < or =10(5) MPN/L, 88 (70.4%) of 125 sediment samples with < or = 10(7) MPN/100g, 8 (17.2%) of 46 fish samples with < or = 10(6) CFU/10 g and 68 (43.8%) of 156 shellfish samples with < or = 10(6) MPN/10g. This organism was isolated from 64 (70.3%) of 91 shellfish samples, (short-necked clam, clam, hen clam, oyster and scallop, etc) with < or =10(6) MPN/10g and from 7 (43.7%) of 16 prawn samples with <10(2) MPN/10g. These isolates were divided into 11 serotypes with the most prevalent serotype being 04 followed by 01, 03 and 06. Serotype 012 strain (isolated from a patient from interior Shimane Prefecture in August 2004) was isolated from sea water and a sediment sample and from 3 samples of short-necked clam produced in Kumamoto Prefecture. These findings suggested potential transmission of V vulnificus through the water environment along the coast of the Japan Sea, that V. vulnificus is easily spread by coldtransport of fresh marine product, and that the occurrence of V. vulnificus infection is possible regardless of one's nearness to coastal areas where V. vulnificus is prevalent.

Animals↗

Use of sodium dodecyl sulfate-polymyxin B-sucrose medium for isolation of Vibrio vulnificus from shellfish.

The differential and selective sodium dodecyl sulfate-polymyxin B-sucrose medium (SPS) of Kitaura et al. (T. Kitaura, S. Doke, I. Azuma, M. Imaida, K. Miyano, K. Harada, and E. Yabuuchii, FEMS Microbiol. Lett. 17:205-209, 1983), which highlights alkylsulfatase activity, was evaluated for its potential use in the direct isolation and enumeration of Vibrio vulnificus from shellfish. V. vulnificus was detected by this method in six of nine shellfish samples collected from diverse geographic locales during the summer of 1986. Direct enumeration of V. vulnificus at 7.0 X 10(2) to 2.2 X 10(4) CFU/g of shellfish was achieved on SPS agar. All sample results were confirmed in parallel examinations by using conventional glucose-salt-Teepol (Shell Oil Co.) broth and alkaline peptone water enrichment with plating onto thiosulfate-citrate-bile salts-sucrose agar. Additionally, alkylsulfatase activity was evaluated in vitro for 97 strains representing 14 Vibrio spp. V. vulnificus and Vibrio cholerae-01 were the only species consistently found to possess this activity. The range of plating efficiencies for random V. vulnificus strains analyzed on SPS was 11 to 74% (mean, 39%). The use of SPS shows great promise for the study of shellfish and other environmental sources for V. vulnificus.

Animals↗

Biotransformations of paralytic shellfish toxins by bacteria isolated from bivalve molluscs.

Due to the possibility that bacteria could be involved in the clearance of paralytic shellfish toxins (PST) from bivalve molluscs, investigations into which, if any, bacteria were able to grow at the expense of PST focused on several common shellfish species. These species were blue mussels, oysters, razor fish, cockles, and queen and king scallops. Bacteria associated with these shellfish were isolated on marine agar 2216 and characterized by their carbon utilization profiles (BIOLOG). Selected isolates from groups demonstrating 90% similarity were screened for their ability to metabolize a range of PST (gonyautoxins 1 and 4 [GTX 1/4], GTX 2/3, GTX 5, saxitoxin, and neosaxitoxin) using a novel screening method and confirming its results by high-performance liquid chromatography. Results suggest that molluscan bacteria have different capacities to utilize and transform PST analogues. For example, isolates M12 and R65 were able to reductively transform GTX 1/4 with concomitant production of GTX 2/3, while isolate Q5 apparently degraded GTX 1/4 without the appearance of other GTXs. Other observed possible mechanisms of PST transformations include decarbamoylation by isolate M12 and sulfation of GTXs by isolates Q5, R65, M12, and C3. These findings raise questions as to the possible role of bacteria resident in the shellfish food transport system. Some researchers have suggested that the microflora play a role in supplying nutritional requirements of the host. This study demonstrates that bacteria may also be involved in PST transformation and elimination in molluscan species.

Animals↗

Detection of pathogenic Vibrio spp. in shellfish by using multiplex PCR and DNA microarrays.

This study describes the development of a gene-specific DNA microarray coupled with multiplex PCR for the comprehensive detection of pathogenic vibrios that are natural inhabitants of warm coastal waters and shellfish. Multiplex PCR with vvh and viuB for Vibrio vulnificus, with ompU, toxR, tcpI, and hlyA for V. cholerae, and with tlh, tdh, trh, and open reading frame 8 for V. parahaemolyticus helped to ensure that total and pathogenic strains, including subtypes of the three Vibrio spp., could be detected and discriminated. For DNA microarrays, oligonucleotide probes for these targeted genes were deposited onto epoxysilane-derivatized, 12-well, Teflon-masked slides by using a MicroGrid II arrayer. Amplified PCR products were hybridized to arrays at 50 degrees C and detected by using tyramide signal amplification with Alexa Fluor 546 fluorescent dye. Slides were imaged by using an arrayWoRx scanner. The detection sensitivity for pure cultures without enrichment was 10(2) to 10(3) CFU/ml, and the specificity was 100%. However, 5 h of sample enrichment followed by DNA extraction with Instagene matrix and multiplex PCR with microarray hybridization resulted in the detection of 1 CFU in 1 g of oyster tissue homogenate. Thus, enrichment of the bacterial pathogens permitted higher sensitivity in compliance with the Interstate Shellfish Sanitation Conference guideline. Application of the DNA microarray methodology to natural oysters revealed the presence of V. vulnificus (100%) and V. parahaemolyticus (83%). However, V. cholerae was not detected in natural oysters. An assay involving a combination of multiplex PCR and DNA microarray hybridization would help to ensure rapid and accurate detection of pathogenic vibrios in shellfish, thereby improving the microbiological safety of shellfish for consumers.

Animals↗

Rapid and sensitive detection of noroviruses by using TaqMan-based one-step reverse transcription-PCR assays and application to naturally contaminated shellfish samples.

Noroviruses (NoV), which are members of the family Caliciviridae, are the most important cause of outbreaks of acute gastroenteritis worldwide and are commonly found in shellfish grown in polluted waters. In the present study, we developed broadly reactive one-step TaqMan reverse transcription (RT)-PCR assays for the detection of genogroup I (GI) and GII NoV in fecal samples, as well as shellfish samples. The specificity and sensitivity of all steps of the assays were systematically evaluated, and in the final format, the monoplex assays were validated by using RNA extracted from a panel of 84 stool specimens, which included NoV strains representing 19 different genotypes (7 GI, 11 GII, and 1 GIV strains). The assays were further validated with 38 shellfish cDNA extracts previously tested by nested PCR. Comparison with a recently described real-time assay showed that our assay had significantly higher sensitivity and was at least as sensitive as the nested PCR. For stool specimens, a one-step duplex TaqMan RT-PCR assay performed as well as individual genogroup-specific monoplex assays. All other enteric viruses examined were negative, and no cross-reaction between genogroups was observed. These TaqMan RT-PCR assays provide rapid (less than 90 min), sensitive, and reliable detection of NoV and should prove to be useful for routine monitoring of both clinical and shellfish samples.

Animals↗

Molecular and immunological characterization of shellfish allergens.

Shellfish (crustaceans and mollusks) have long been known as a common cause of allergic reactions to food. Like other food allergies, the allergic reactions to shellfish involve IgE-mediated Type I hypersensitivity. Biochemical and molecular studies have documented the major shrimp allergen is the muscle protein tropomyosin. Subsequent molecular cloning studies on lobsters and crabs have characterized this protein as the common allergen in crustaceans. There has also been strong immunological evidence that tropomyosin is a cross-reactive allergen among crustaceans and mollusks. This is further confirmed by recent studies on the identification of allergens in squid and abalone. The advances in the characterization of shellfish allergens will not only enhance our understanding on the physiological basis of shellfish allergy but also lay the groundwork for the development of diagnostic and therapeutic design in food allergies.

Allergens↗

Simultaneous presence of paralytic and diarrheic shellfish poisoning toxins in Mytilus chilensis samples collected in the Chiloe Island, Austral Chilean fjords.

The study shown here provides the first indisputable evidence that shellfish can be contaminated with Paralytic Shellfish Poisoning (PSP) and Diarrheic Shellfish Poisoning (DSP) toxins during the summer season in the Southern Chilean fjords. Quantitative analysis of the simultaneous presence of PSP and DSP toxins in Mytilus chilensis samples collected in the Chiloe Island are shown. The High Performance Liquid Chromatography (HPLC) analysis with pre-column derivatization method for DSP toxins and the post-column derivatization methods for PSP toxins, both with fluorescent on-line detections, showed that both type of toxins were concentrated by the filter bivalve Mytilus chilensis in amounts above the international safe limits. The phytoplankton analysis showed the presence of both Alexandrium catenella and Dinophysis acuta in the water column. The data shows stratification of the toxic dinoflagellates in the water column, since the lowest amount of both DSP and PSP toxins were measured in the superficial and deeper levels of the water column. Moreover, the highest toxicities of both types of toxins were shown by the shellfish samples collected at a depth of 6 meters with 190 nanograms of DTX-1 / gram of digestive gland and 709.8 microg of PSP toxins / 100 grams of mussel meat.

Animals↗

Distribution of Norwalk virus within shellfish following bioaccumulation and subsequent depuration by detection using RT-PCR.

Consumption of raw bivalve mollusks contaminated with pathogens from human feces continues to present a human health risk. The purpose of this study was to monitor the uptake, localization, and removal of Norwalk virus (NV) in shellfish (oyster and clam) tissues by analyzing virus distribution in selected dissected tissues. Live shellfish were allowed to bioaccumulate different input titers of NV for time periods from 4 to 24 h. In some experiments, depuration by shellfish that bioaccumulated NV and Escherichia coli bacteria was allowed to proceed for 24 or 48 hours. Dissected stomach (St), digestive diverticula (DD), adductor muscle (AM), and hemolymph cells (HC) tissues were assayed for NV by the reverse transcription polymerase chain reaction (RT-PCR) method. An internal RNA standard control was added to the RT-PCR to identify the presence of inhibitors to RT-PCR. NV titers in DD tissues before and after depuration were estimated using quantitative RT-PCR end-point dilution. NV was found in the alimentary tract (DD or St) at all concentrations of input virus, but was present more frequently after exposure to higher levels of virus. NV was detected in AM and HC only following exposure to higher levels of virus. In experiments where depuration by oysters was continued for 48 h, depuration of bacteria was efficient (95% reduction of bacteria), but minimal (7%) reduction of NV titers from DD tissues was detected. These findings indicate that NV can localize both within and outside the alimentary tract of shellfish, and NV is poorly depurated using conditions favorable for E. coli depuration.

Animals↗

Detection of hepatitis A virus in shellfish (Mytilus galloprovincialis) with RT-PCR.

A PCR assay for the detection of hepatitis A virus (HAV) in shellfish is described. The procedure involves the concentration of viral particles with the use of polyethylene glycol (PEG), followed by viral RNA extraction and purification with oligo(dT) cellulose. Reverse transcriptase-PCR detection was accomplished in a single step with the use of primers specific for the VP3-VP1 region of the genome. The procedure detected one 50% tissue culture infective dose (0.6 PFU) per 25 g of shellfish homogenate. Heminested PCR was then carried out to verify the specificity of the PCR products. The method was used to detect HAV in shellfish samples from EU categories B and C and to evaluate the quality of shellfish in routine monitoring for HAV in view of the relevant public health implications of this foodborne disease.

Animals↗

Polyclonal antibodies to domoic acid, and their use in immunoassays for domoic acid in sea water and shellfish.

Ovine antibodies raised against conjugates linked through the secondary amino group of domoic acid (1) were used, together with activated-ester-derived conjugates of domoic acid (DA) as the plate coater, to develop a robust indirect competitive enzyme-linked immunosorbent assay (cELISA) for DA in shellfish and seawater. The ELISA was used to analyze shellfish samples for DA, and was compatible with several extraction procedures. The ELISA had a detection limit below 0.01 ng ml(-1), a limit of quantitation (LOQ) of 0.15 ng ml(-1) and a working range of 0.15-15 ng ml(-1) DA. The LOQ is equivalent to 38 ng g(-1) DA in shellfish flesh, assuming a 250-fold dilution during extraction. This is more than 500 times lower than the maximum permitted level (20 microg g(-1) flesh). The ELISA is designed for use alongside regulatory analyses, and, following formal validation, should be available for pre-screening of regulatory shellfish flesh samples. The ELISA was also shown to be appropriate for analysis of DA in algal cultures and in samples of seawater, and thus has the potential to provide early warning of developing algal blooms.

Animals↗

Proficiency testing of eight French laboratories in using the AOAC mouse bioassay for paralytic shellfish poisoning: interlaboratory collaborative study.

In an interlaboratory study, 8 French laboratories were tested for their proficiency in using the AOAC mouse bioassay for paralytic shellfish poisoning (PSP). Each laboratory received 1 saxitoxin (STX) standard solution, 1 STX acidified water solution for determination of the titer, 1 noncontaminated shellfish sample, 1 naturally contaminated shellfish sample, and 2 shellfish samples spiked, respectively, at low (152.8 microg STX/100 g meat) and moderate (334.7 microg STX/100 g meat) levels. All samples were analyzed in duplicate. Mean recoveries were 35.1% for the low level and 46.6% for the moderate level. Relative standard deviations (RSD) for within-laboratory variations (repeatability) ranged from 5.4 to 9.8%; RSD for between-laboratory variations (reproducibility) varied from 7.8 to 39.6%, depending on STX level. On the basis of overall performance, all 8 participating laboratories were proficient in their use of the AOAC mouse bioassay.

Animals↗