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PubMed2000

Viral contamination of shellfish: evaluation of methods and analysis of bacteriophages and human viruses.

Viral outbreaks attributed to the consumption of contaminated shellfish have been clearly demonstrated. Thirty-five samples of mussels collected from areas with two different levels of faecal pollution were analysed for somatic coliphages, F-RNA phages and bacteriophages infecting Bacteroides fragilis HSP40 and RYC2056 following standardised protocols, and for enterovirus, human adenovirus and hepatitis A virus by nucleic acid amplification (Nested-PCR and RT-PCR). Four methods for viral recovery from shellfish have been compared. The first method is based on borate buffer at pH 9.5 as eluent, the second is based on glycine buffer at pH 10 as eluent, a third method is based on glycine buffer at pH 7.5 and changes in conductivity and the fourth method on nutritive broth with Tween 80 as eluent. The results obtained were analysed statistically and the method based in glycine buffer at pH 10 seems to be the most efficient and useful for the recovery of phages and human viruses. The results also show a different pattern in the proportions between the viral parameters when the source of the faecal pollution is close to or distant from the shellfish growing area.

Adenoviridae↗
PubMed2003

Detection of pathogenic bacteria in shellfish using multiplex PCR followed by CovaLink NH microwell plate sandwich hybridization.

Outbreak of diseases associated with consumption of raw shellfish especially oysters is a major concern to the seafood industry and public health agencies. A multiplex PCR amplification of targeted gene segments followed by DNA-DNA sandwich hybridization was optimized to detect the etiologic agents. First, a multiplex PCR amplification of hns, spvB, vvh, ctx and tl was developed enabling simultaneous detection of total Salmonella enterica serotype Typhimurium, Vibrio vulnificus, Vibrio cholerae and Vibrio parahaemolyticus from both pure cultures and seeded oysters. Amplicons were then subjected to a colorimetric CovaLink NH microwell plate sandwich hybridization using phosphorylated and biotinlylated oligonucleotide probes, the nucleotide sequences of which were located internal to the amplified DNA. The results from the hybridization with the multiplexed PCR amplified DNA exhibited a high signal/noise ratio ranging between 14.1 and 43.2 measured at 405 nm wavelength. The sensitivity of detection for each pathogen was 10(2) cells/g of oyster tissue homogenate. The results from this study showed that the combination of the multiplex PCR with a colorimetric microwell plate sandwich hybridization assay permits a specific, sensitive, and reproducible system for the detection of the microbial pathogens in shellfish, thereby improving the microbiological safety of shellfish to consumers.

Animals↗
PubMed2000

Viruses and bivalve shellfish.

The epidemiological data clearly demonstrates that filter feeding bivalve shellfish can, and do, act as efficient vehicles for the transmission of enteric viruses transmitted by the faecal-oral route. This identified hazard has been documented as a cause for concern by various international agencies and has a long history. Disease outbreaks can occur on an epidemic scale as graphically illustrated by an outbreak of Hepatitis A in Shanghai, China in 1988 involving about 300,000 cases. Improvement of harvesting area water quality offers the most sustainable route to improvement in the virological quality of bivalve shellfish sold live. However there is growing awareness, and concern, that current regulatory standards based on faecal coliform monitoring do not fully protect the shellfish consumer from viral infection. New viral test methods based on PCR, and the development of alternative more reliable faecal pollution indicators, offer new approaches for the further development of public health controls. However, further work is required to build a scientific consensus and to understand the implications of their introduction into legislation.

Adenoviridae↗
PubMed2002

Vancomycin-resistant enterococci in shellfish, unchlorinated waters, and chicken.

Vancomycin-resistant enterococci (VRE) have been a cause of increasing concern chiefly regarding the infection of hospital patients. There is suspicion, but limited evidence, that food and environmental spread may be important. Biomonitoring by examination of bivalve shellfish was used to assess the occurrence of VRE entering the environment. Using pre-enrichment and Lewisham and Slanetz and Bartley agars, 2/125 (1.6%) of shellfish were found to contain enterococci resistant to high levels of vancomycin. Lewisham agar allows relatively rapid identification of VRE. In a second phase of the work using pre-enrichment and Slanetz and Bartley agar, 4/151 (2.7%) shellfish and 5/27 (18.5%) raw chickens contained VRE. Using filtration and pre-enrichment, no VRE were found in 54 unchlorinated water samples. The study shows that environmental prevalence of VRE is low, and that raw chickens are frequently contaminated.

Agar↗
PubMed2003

Improved solid-phase extraction procedure in the analysis of paralytic shellfish poisoning toxins by liquid chromatography with fluorescence detection.

The analysis of shellfish extracts for the determination of paralytic shellfish poisoning (PSP) toxins by liquid chromatography with fluorescence detection repeatedly showed the presence of a compound suspected to interfere with gonyautoxin 4. The first aim of this study was to confirm by liquid chromatography coupled to tandem mass spectrometry that this compound was not gonyautoxin 4. The second part of this work was to improve a nonvolumetric C(18) solid-phase extraction (SPE) procedure to evaluate the removal of the interference associated with the recovery of PSP toxins. The cleanup procedure was modified into a volumetric SPE procedure and proved to efficiently and totally remove the interference while recovering from 78 to 85% of the PSP toxins available as commercial standards (saxitoxin, neosaxitoxin, gonyautoxins 1-4) and considered as major PSP toxins in human intoxication, with 85% recovery for gonyautoxin 4. The efficiency of this cleanup procedure was checked on shellfish extracts containing this interference and originating from France and Turkey.

Chromatography, Liquid↗
PubMed2001

Measurement of paralytic shellfish toxins in molluscan extracts: comparison of the microtitre plate saxiphilin and sodium channel radioreceptor assays with mouse bioassay, HPLC analysis and a commercially available cell culture assay.

This study compared five methods of measuring paralytic shellfish toxins (PSTs) including the long-used mouse lethality bioassay, a commercially available cell culture test (MIST Quantification kit), HPLC analysis, and two newly developed radioreceptor assays utilizing mammalian sodium channels and saxiphilin. Methods were challenged with toxic shellfish extracts prepared according to the AOAC official method. The best correlations between predicted toxicity values being 0.9 or better, were those between HPLC analysis when compared with both radioreceptor assays and the mouse lethality bioassay, as well as that between the saxiphilin and the sodium channel radioreceptor assays. In all cases, statistically significant correlations existed between the toxicity measurements of the same extracts. The ratios between some methods were not unitary as measured by the slopes of the regression lines used for correlation analyses. HPLC analysis predicted more toxicity than all of the bioassays. The saxiphilin assay underestimated toxicity relative to the mouse bioassay, the MIST kit determinations and the sodium channel assay. The sodium channel assay predicted there to be less toxicity than the mouse bioassay and the MIST kit. Of all of the techniques used, the MIST kit correlation with the mouse bioassay was nearest to one. Each method possesses differentt virtues and it may be that a multi-method approach would harness the benefits of each method for various aspects of a shellfish testing regime.

Amphibian Proteins↗
PubMed2004

Distribution of diarrhetic shellfish poisoning toxins in consignments of blue mussel.

Data describing the distribution of diarrhetic shellfish poisoning toxins in 13 consignments of Danish-produced blue mussels are reported. The content of diarrhetic shellfish poisoning toxins was measured by a liquid chromatography coupled with tandem mass spectrometry detection method, and mean levels in the 13 consignments varied from 58 to 243 microg kg(-1). The distributions of diarrhetic shellfish poisoning toxins in the consignments were relatively homogeneous as the relative standard deviation of the content varied from 7 to 19%. The results are discussed in relation to food safety, the uncertainty of sampling and analysis, and the newly introduced European Union maximum levels of marine biotoxins in seafood products.

Animals↗
PubMed1984

Organochlorine residues in shellfish from Maryland waters, 1976-1980.

Shellfish samples, including the American oyster (Craessostrea virginica), the soft shell clam (Mya arenaria), the hard shell clam (Mercenaria mercenaria) and the blue crab (callinectes sapidus), were taken from the Maryland section of the Chesapeake Bay and its tributaries over a five year period (1976-80) and analyzed to determine residue levels of various organochlorine pesticides. Qualitative and quantitative information was obtained for a variety of polychlorinated hydrocarbons. The purpose of this study includes the establishment of baseline values for levels of shellfish contamination, utilization of the data as an indicator of probable levels of water and sediment contamination in the vicinity of the sampling site and ensurance that shellfish harvested for human consumption are within U.S. Food and Drug Administration guidelines for organochlorine contamination. All mean values and virtually all individual values were within FDA guidelines.

Animals↗
PubMed2004

When enough is not enough: shorebirds and shellfishing.

In a number of extensive coastal areas in northwest Europe, large numbers of long-lived migrant birds eat shellfish that are also commercially harvested. Competition between birds and people for this resource often leads to conflicts between commercial and conservation interests. One policy to prevent shellfishing from harming birds is to ensure that enough food remains after harvesting to meet most or all of their energy demands. Using simulations with behaviour-based models of five areas, we show here that even leaving enough shellfish to meet 100% of the birds' demands may fail to ensure that birds survive in good condition. Up to almost eight times this amount is needed to protect them from being harmed by the shellfishery, even when the birds can consume other kinds of non-harvested prey.

Animals↗
PubMed2005

Loop-mediated isothermal amplification: an emerging technology for detection of fish and shellfish pathogens.

Fish and shellfish diseases are a constant threat to the sustainability and economic viability of aquaculture. Early diagnosis plays a vital role in management of fish and shellfish diseases. Traditionally, various biochemical and serological tests have been used for fish disease diagnosis. However, the time and expertise required for such diagnoses makes it difficult for aquaculturists to easily adopt them under production conditions. Polymerase chain reaction and probe-based nucleic acid detection have become increasingly popular in fish and shellfish diagnostics. Recently, a novel technique called loop-mediated isothermal amplification (LAMP) has been developed, which is highly sensitive and rapid. LAMP has been used for the detection of bacterial, viral, fungal and parasitic diseases in both animal and plants. In aquaculture, LAMP-based detection of pathogens like Edwardsiella tarda, E. ictaluri, Nocardia seriolae, Tetracapsuloides bryosalmonae, white spot syndrome virus and infectious haematopoietic necrosis virus have been reported. In this review, the application of LAMP for the detection of aquaculture-associated pathogens is discussed.

Animals↗
PubMed1990

[Evaluation of recovery methods of Shigella species from fresh marine fish and shellfishes].

Recovery experiments of Shigella strains from fresh marine fish and shellfishes, including fresh sea urchin, which have been artificially contaminated with the strains, were performed using the improved Shigella broth-enrichment method and the culture method reported by Mehlman et al. All of the 43 Shigella stock cultures strains tested were recovered easily by the enrichment method from sea urchin individuals inoculated with a small number of viable cells of each strain. That is, a total of 24 strains (56%) were recovered from sea urchin individuals inoculated with less than 10 viable cells per one individual, and the other 19 strains were also recovered when 10 to 1,000 cells of each strain were inoculated. Recovery of Shigella strains from fish and shellfishes by the enrichment method was hardly affected by the number of contaminated bacteria (SPC, standard plate counts) in these materials. In order to confirm reliability of the enrichment method, similar experiments were performed using S. flexneri strain B as the inoculum and more fish and shellfishes as the samples (24 specimens of fresh sea urchins, 11 specimens of fresh oysters and 5 other specimens including prawns). Except for one oyster specimen which showed an especially high SPC value, the inoculum was able to be recovered from most of the materials inoculated with less than 10 viable cells, and all of the tested samples became Shigella positive when they were inoculated with up to 1,000 viable cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1981

Sediment bacterial indicators in an urban shellfishing subestuary of the lower Chesapeake Bay.

The objectives of this study were to document the spatial and temporal distributions and compositions of bacteria in the sediments and overlying waters of an important urban shellfishing area in the lower Chesapeake Bay region, the Lynnhaven Estuary. Marked fluctuations were observed in the date of many of the physicochemical parameters and the indicator bacteria. The higher-salinity water and coarser sediment of the inlet site showed lower overall bacterial densities than did the headwater sites, where freshwater runoff and decreased tidal action were characteristic. Densities of benthic indicator bacteria, when expressed on a volumetric basis, were significantly greater than counts in the overlying waters. These counts were indicative of a fecally polluted system and were well above the safe maximum limits for shellfish-growing waters. Significantly fewer total and fecal bacteria were observed in both the water and the sediment during the warm months of May, July, and August. The primary sources of the Lynnhaven's bacterial pollution appeared to be typical of urban and agricultural runoff, although failure of septic tank systems was suspected as a problem in the Lynnhaven's western branch. These results illustrated that sediments in shellfishing areas could serve as a reservoir for high densities of indicator bacteria and that, potentially, pathogens could pose a health hazard.

Enterobacteriaceae↗
PubMed1983

Relationship among fecal coliforms, Escherichia coli, and Salmonella spp. in shellfish.

The relationship of fecal coliforms, Escherichia coli, and Salmonella spp. was examined in freshly harvested and stored shellfish. In 16 of 40 freshly collected oyster samples, fecal coliform levels were above the recommended wholesale level suggested by the National Shellfish Sanitation Program (less than or equal to 230/100 g), and Salmonella spp. were present in three of these samples. Salmonella spp. were not, however, present in any sample containing less than 230 fecal coliforms per 100 g. Analysis of the data suggests that low fecal coliform levels in both fresh and stored oysters are good indicators of the absence of Salmonella spp., but that high levels of fecal coliforms are somewhat limited in predicting the presence of Salmonella spp. E. coli levels correlated very strongly with fecal coliform levels in both fresh and stored oysters and clams, suggesting that there is no advantage in replacing fecal coliforms with E. coli as an indicator of shellfish quality.

Animals↗
PubMed1983

Effects of storage on microbial loads of two commercially important shellfish species, Crassostrea virginica and Mercenaria campechiensis.

The effects of storage on the microbial load in two commercially important species of shellfish were examined. Oysters (Crassostrea virginica) were stored as shellstock, shucked meats, and fully processed meats at four temperatures for up to 21 days, and clams (Mercenaria campechiensis) were stored only as shellstock. The concentrations of most microbiological groups of organisms increased with the duration and temperature of storage in both shellfish species, although the increases were significantly lower in claims. Concentrations of Vibrio cholerae rose by approximately 1 log in oysters stored as shellstock after 7 days at 2 degrees C, and Lac+ vibrios increased 2 logs at 8 degrees C. Total counts of bacteria, fungi, coliforms, fecal streptococci, Aeromonas hydrophila, and clostridia were significantly higher in shucked oysters than in those stored as shellstock. Fecal coliforms were statistically the same, but V. cholerae, Vibrio parahaemolyticus, and the Lac+ vibrios were higher in oysters stored as shellstock. The concentrations of all microbial groups were higher in fully processed oysters than in shucked meats, with the exception of the vibrios, which showed no significant difference among the treatments. The results showed that although traditional methods of storing shellfish resulted in an overall increase in the microbial load, vibrio levels increased only in oysters stored as shellstock. Although fecal coliform and total bacterial counts did not correlate with those for vibrios in fresh oysters, strong correlations were observed in oysters stored for 7 days, suggesting that these indicators may be useful in monitoring oyster quality when meats are stored for a limited time as shellstock.

Aeromonas↗
PubMed1992

Survival and replication of male-specific bacteriophages in molluscan shellfish.

The survival and replication of male-specific bacteriophages in hard-shelled clams (Mercenaria mercenaria) and their homogenates were examined to further assess their potential utility as indicator organisms. Trials were conducted in the presence and absence of a suitable bacterial host, Escherichia coli HS[pFamp]R. Results of this study demonstrated that male-specific bacteriophages were unable to replicate in hard-shelled clams, with or without added host cells. In addition, the densities of these bacteriophages were stable for up to 7 days in shellfish held at ambient seawater temperatures (less than 25 degrees C). Evidence of replication, although not observed in live shellfish, was found to occur in temperature-abused shellfish homogenates and supernatants, but only when a suitable bacterial host was present.

Animals↗
PubMed1993

Use of genomic probes to detect hepatitis A virus and enterovirus RNAs in wild shellfish and relationship of viral contamination to bacterial contamination.

Genomic probes were used to investigate hepatitis A virus (HAV) and enterovirus RNAs in two types of shellfish from natural beds (Atlantic coast, France). After elution concentration, nucleic acid extracted by proteinase K and purified by phenol-chloroform and ethanol precipitation was assayed by dot blot hybridization. The probes used were a specific HAV probe corresponding to the 3' end (3D polymerase coding region) and an enterovirus probe corresponding to the 5' noncoding region. The method was first tested under experimental conditions by using virus-spiked shellfish before being applied under field conditions. Our results show that shellfish were highly contaminated: enterovirus and HAV RNAs were found in 63 and 67%, respectively, of samples examined with the riboprobes. On the same site, viral (HAV and enterovirus) RNAs were found in a larger fraction of cockles than mussels. Statistical tests of dependence showed no relationship between viral contamination and bacterial contamination (evaluated by fecal coliform counts).

Animals↗
PubMed1995

Detection of Norwalk virus and hepatitis A virus in shellfish tissues with the PCR.

A method for the detection of Norwalk virus and hepatitis A virus from shellfish tissues by PCR was developed. Virus was added to the stomach and hepatopancreatic tissues of oysters or hard-shell clams, and viral nucleic acids were purified by a modification of a previously described method (R.L. Atmar, T.G. Metcalf, F.H. Neill, and M.K. Estes, Appl. Environ. Microbiol. 59:631-635, 1993). The new method had the following advantages compared with the previously described method: (i) more rapid sample processing; (ii) increased test sensitivity; (iii) decreased sample-associated interference with reverse transcription-PCR; and (iv) use of chloroform-butanol in place of the chlorofluorocarbon trichlorotrifluoroethane. In addition, internal standards for both Norwalk virus and hepatitis A virus were made which demonstrated when inhibitors to reverse transcription-PCR were present and allowed quantitation of the viral nucleic acids present in samples. This assay can be used to investigate shellfish-associated gastroenteritis outbreaks and to study factors involved in virus persistence in shellfish.

Animals↗
PubMed2001

Rapid and efficient extraction method for reverse transcription-PCR detection of hepatitis A and Norwalk-like viruses in shellfish.

As part of an effort to develop a broadly applicable test for Norwalk-like viruses and hepatitis A virus (HAV) in shellfish, a rapid extraction method that is suitable for use with one-step reverse transcription (RT)-PCR-based detection methods was developed. The method involves virus extraction using a pH 9.5 glycine buffer, polyethylene glycol (PEG) precipitation, Tri-reagent, and purification of viral poly(A) RNA by using magnetic poly(dT) beads. This glycine-PEG-Tri-reagent-poly(dT) method can be performed in less than 8 h on hard-shell clams (Mercenaria mercenaria) and Eastern oysters (Crassostrea virginica) and, when coupled with RT-PCR-based detection, can yield results within 24 h. Observed sensitivities for seeded shellfish extracts are as low as 0.015 PFU of HAV and 22.4 RT-PCR50 units for Norwalk virus. Detection of HAV in live oysters experimentally exposed to contaminated seawater is also demonstrated. An adaptation of this method was used to identify HAV in imported clams (tentatively identified as Ruditapes philippinarum) implicated in an outbreak of food-borne viral illness. All of the required reagents are commercially available. This method should facilitate the implementation of RT-PCR testing of commercial shellfish.

Animals↗