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PubMed2002

Applied technique for increasing calicivirus detection in shellfish extracts.

AIMS: Optimal detection of enteric RNA viruses in clinical, environmental, and food products using reverse transcription-PCR (RT-PCR) when inhibitory substances in extracted sample materials are present. METHODS AND RESULTS: We adapted a device for detection of RNA viruses in plant tissues and insects to detect a calicivirus strain (San Miguel sea lion virus, serotype 17) in water and oyster tissue extracts. This single, compartmentalized tube-within-a-tube (TWT) device for RT-PCR-nested PCR was compared to a conventional protocol of RT-PCR-nested PCR. In the presence of 100 mg of shellfish tissue extract equivalent, this TWT device decreases the calicivirus assay detection limit 10-fold over that of conventional RT-PCR-nested PCR while maintaining an identical detection limit of viral nucleic acid suspended in water. Both the conventional and TWT methods estimated the total particle-to-infectious particle ratio for this strain of calicivirus at approximately 40 : 1. CONCLUSIONS: We believe that the TWT device with appropriate RT-PCR primers will decrease the detection limit for other calicivirus strains and RNA viruses in shellfish tissue extracts. SIGNIFICANCE AND IMPACT OF THE STUDY: We believe that the TWT approach is applicable to other situations where RT and/or PCR inhibitory materials are present or nucleic acid targets of bacteria or viruses are at low levels in extracts of food products or clinical specimens.

Animals↗

PubMed1998

Incidence of Listeria spp. in Breton live shellfish.

Live shellfish samples (120) were collected from nine littoral sites in Brittany (western France). They were screened for Listeria spp. and a count of faecal coliforms was carried out. Analysis of the results revealed Listeria spp. in 55% of samples, a much higher rate than the previous, infrequent, recorded data. Furthermore, the study demonstrated that the frequency of Listeria spp. in winter was more important than in summer (P < 0.001), and underlined a significant relationship between the occurrence of these bacteria and the concentration of faecal coliforms (P < 0.001). Finally, comparison of the official and Gen-Probe methods revealed the limits of the standardized technique in the search for L. monocytogenes in shellfish.

Incidence↗

PubMed1998

Rift Valley lake fish and shellfish provided brain-specific nutrition for early Homo.

An abundant, balanced dietary intake of long-chain polyunsaturated fatty acids is an absolute requirement for sustaining the very rapid expansion of the hominid cerebral cortex during the last one to two million years. The brain contains 600 g lipid/kg, with a long-chain polyunsaturated fatty acid profile containing approximately equal proportions of arachidonic acid and docosahexaenoic acid. Long-chain polyunsaturated fatty acid deficiency at any stage of fetal and/or infant development can result in irreversible failure to accomplish specific components of brain growth. For the past fifteen million years, the East African Rift Valley has been a unique geological environment which contains many enormous freshwater lakes. Paleoanthropological evidence clearly indicates that hominids evolved in East Africa, and that early Homo inhabited the Rift Valley lake shores. Although earlier hominid species migrated to Eurasia, modern Homo sapiens is believed to have originated in Africa between 100 and 200 thousand years ago, and subsequently migrated throughout the world. A shift in the hominid resource base towards more high-quality foods occurred approximately two million years ago; this was accompanied by an increase in relative brain size and a shift towards modern patterns of fetal and infant development. There is evidence for both meat and fish scavenging, although sophisticated tool industries and organized hunting had not yet developed. The earliest occurrences of modern H. sapiens and sophisticated tool technology are associated with aquatic resource bases. Tropical freshwater fish and shellfish have long-chain polyunsaturated lipid ratios more similar to that of the human brain than any other food source known. Consistent consumption of lacustrine foods could have provided a means of initiating and sustaining cerebral cortex growth without an attendant increase in body mass. A modest intake of fish and shellfish (6-12% total dietary energy intake) can provide more arachidonic acid and especially more docosahexaenoic acid than most diets contain today. Hence, 'brain-specific' nutrition had and still has significant potential to affect hominid brain evolution.

Africa, Eastern↗

PubMed2003

Mercury, arsenic, lead and cadmium in fish and shellfish from the Adriatic Sea.

The aim was to measure concentrations of total mercury, total arsenic, lead and cadmium in common edible fresh fish and shellfish from various areas of the Adriatic Sea. Estimates of intake of these elements were made through seafood consumption by the general population. Samples were either wet digested for mercury and arsenic, or dry ashed for lead and cadmium analysis. Mercury was measured by cold vapour atomic absorption spectrometry (CV AAS) and arsenic, lead and cadmium by electrothermal atomic absorption spectrometry (ET AAS). Quality control procedures of analytical methods, which included analyses of dogfish muscle-certified reference material DORM-2, confirmed the acceptability of methods. The highest mercury and arsenic concentrations were found in hake (Merluccius merluccius) and the lowest in mackerel (Scomber scombrus). The respective values in hake were 0.373 +/- 0.075 and 23.3 +/- 3.6, and in mackerel 0.153 +/- 0.028 and 1.06 +/- 0.29 mg x kg(-1) fresh weight (mean +/- SD). Lead and cadmium concentrations were about 10 times higher in shellfish than in analysed fish. The highest lead and cadmium concentrations were found in mussel (Mytilus galloprovincialis) and the lowest in hake. Respective lead and cadmium values in mussel were 0.150 +/- 0.009 and 0.142 +/- 0.017, and in hake were 0.007 +/- 0.004 and 0.002 +/- 0.001 mg x kg(-1) fresh weight. The concentrations of analysed elements were below acceptable levels for human consumption set by the Croatian Ministry of Health, except for total arsenic. The estimated intake of those trace elements included in this study through seafood consumption by the general population did not exceed the provisional tolerable weekly intake recommended by the Joint FAO/WHO Expert Committee on Food Additives.

Animals↗

PubMed2005

Assessment of the quantitative determination of paralytic shellfish poisoning toxins by pre-column derivatization and elimination of interfering compounds by solid-phase extraction.

Monitoring programmes for paralytic shellfish poisoning toxins in bivalve molluscs still rely heavily on the use of mouse bioassays (MBA) for consumer protection. A high-performance liquid chromatography (HPLC) methodology (Lawrence method) was implemented in 1996 in the Portuguese monitoring programme as a complementary means of analysis. Comparison between MBA and HPLC was done at the time only by a qualitative approach due to the scarce number of positive samples tested. More quantitative data were obtained recently when studying toxin profiles in Moroccan shellfish, and the correlation found between these two methodologies is reported here for the first time. Two different matrices were studied: blue mussel and the giant cockle Acanthocardia tuberculatum. A good linear correlation was obtained for both matrices. However, a second-degree polynomial best fitted the data at both low and high extremes of toxicity. According to the HPLC quantitative results, 13% of false-negatives could be obtained by MBA due to an underestimation of toxicity near the limit of detection of the MBA. Difficulties on relying solely on HPLC for consumer protection have been aroused with uncommon matrices, such as imported clams or crustaceans, due to the presence of high concentrations of interfering compounds. The solid-phase extraction step of the Lawrence method was implemented to eliminate an unknown compound that could be mistaken for saxitoxin, and an 80% reduction of another common unknown compound eluting close to decarbamoylsaxitoxin. The implementation of the HPLC methodology achieved so far allows a high degree of consumer protection without the need to resource to animal sacrifice.

Animals↗

PubMedDate not supplied

Trace elements in shellfish on the Danish market.

Different kinds of shellfish have been investigated for their contents of the trace elements cadmium, lead, mercury, nickel, chromium, arsenic and selenium. The investigation included shrimps, mussels, lobster, langoustine, crayfish, crab claws and oyster. None of the Danish action levels for cadmium, lead and mercury respectively were exceeded, but the highest concentration of lead was very close to the Danish action levels. Shellfish do not contribute significantly to the intake of the trace elements investigated.

Denmark↗

PubMed2000

Alkyltins in farmed fish and shellfish.

A study to update dibutyltin (DBT) and tributyltin (TBT) residues in farmed fish and shellfish (Mytilus edulis) was carried out 4 years after the adopting of the restricting regulation of the antifouling uses of organotins in Italy. DBT and TBT were simultaneously extracted from farmed fish and shellfish (M. edulis) and from free living specimens, used as control, purchased from retail stores or fishermen in the province of Naples (Italy), and detected using a capillary gas chromatograph equipped with a flame photometric detector (GC-FPD). Dosable amounts of DBT were found in the 10% of the farmed fish analyzed, ranging from 1 to 26 micrograms kg-1 wet wt (mean 10 micrograms kg-1) and in the 23% of the free living fish at an average level of 2 micrograms kg-1 wet wt (range 1-4 micrograms kg-1 wet wt). TBT was detected in 85% of the farmed fish, in concentrations varying from 2 to 260 micrograms kg-1 wet wt (mean 28 micrograms kg-1 wet wt) and in 46% of the free living specimens (mean 39 micrograms kg-1 wet wt; range 1-93 micrograms kg-1). All the mussel samples analyzed were polluted by both DBT and TBT. In the farmed mussels the average amounts of DBT and TBT were, respectively, 4 and 2 micrograms kg-1 wet wt; in the free living they were 4 and 5 micrograms kg-1 wet wt, respectively. The results indicate that the DBT and TBT contamination is as highly diffuse in farmed as in free living fish and mussels on sale in retail markets in Naples province even if the levels of the contamination are meanly quite low.

Animals↗

PubMed1980

Gas chromatography combined with mass spectrometry for the identification of organic sulfur compounds in shellfish and fish.

The authors determined that the organic sulfur compounds usually contained in crude oil can be used as a marker of oil pollution in shellfish and fish. Short-necked clams (Tapes Angdala phillipinarum A. et Reeve) and eels were maintained in a controlled laboratory environment in water with suspension of crude oil. Mass spectra and mass chromatograms of short-necked clam extract showed the presence of organic sulfur compounds of alkyl- (from C1 to C10) benzothiophenes, dibenzothiophene, alkyl- (C1 to C8) dibenzothiophene, naphthalene, and alkyl- (C1 to C6) naphthalene. Alkyl benzothiophenes and alkyl dibenzothiophenes of low carbon numbers were more easily transferred to short-necked clams than those of higher carbon numbers. Capillary column gas chromatography-mass chromatograms of crude oil and extract from the soft body of a short-necked clam showed the presence of organic sulfur compounds of alkyl benzothiophene (from C2-C6), dibenzothiophene, and alkyl dibenzothiophene (C1-C4). Besides sulfur components, various other compounds were contained in crude oil and short-necked clam, namely alkyl benzene (C3-C9), naphthalene, alkyl naphthalene (C1-C5), phenanthrene, alkyl phenanthrene (C1-C6), n-paraffin (C12-C21), and probably pyrene and alkyl pyrene (C1-C2). Also, mass chromatograms of crude oil and the extract from eel flesh showed the presence of alkyl benzothiophene (C1-C5), dibenzothiophene, naphthalene, and alkyl naphthalene (C1-C5). Data indicated that the organic sulfur compounds and polyaromatic compounds could serve as markers of oil pollution in shellfish and fish. for the identification of sulfur compounds by capillary gas chromatography/mass spectrometry, a new intensity matching method was devised.

Animals↗

PubMed2003

Food poisoning associated with biotoxins in fish and shellfish.

PURPOSE OF REVIEW: In recent times the number of blooms of algae that produce toxins has increased in frequency, intensity and geographical distribution. This review describes some of the illnesses caused by fish and shellfish contaminated with toxins produced by marine algae and by bacteria. RECENT FINDINGS: The increase in toxic algal blooms may be a result of increased awareness, aquaculture, eutrophication, or transport of cysts in ship ballast. Improved chemical methods for the detection of algal toxins are now being developed, and so the number of toxins recognized is increasing. Toxicological data on some of these algal toxins are lacking. Despite the increase in occurrence of algal toxins, scombrotoxic poisoning remains the most common cause of food poisoning associated with the consumption of fish and shellfish. This may be real or it may be a reflection of lack of suitable tests for algal toxins or under-recognition by workers in health care. SUMMARY: The major problem worldwide in this field is the lack of pure toxins for use in developing and standardizing chemical methods for toxin detection. Such methods would permit increased testing of both food and clinical specimens, and hence would prevent the entry of toxic food into the food chain and increase laboratory confirmation of incidents of illness.

Animals↗

PubMed1985

Bacteria in bivalve shellfish with special reference to the oyster.

The bacterial flora of the Pacific oyster Crassostrea gigas, the sea mussel Perna viridis and the arkshell clam Scapharca cornea differed considerably from that of seawater in both numbers and generic composition. The numbers of heterotrophic bacteria in the bivalve shellfish, including the anaerobes and spore-forming bacteria, were greater than that in the surrounding water. Pseudomonas spp. were the dominant organisms, comprising over one third of the 321 strains characterized after isolation from the bivalves and seawater. Other bacteria isolated from the shellfish included Vibrio, Acinetobacter, and Aeromonas spp., whereas the seawater flora consisted mainly of coliform organisms, coryneform bacteria and Flavobacterium/Cytophaga spp. Bacteria associated with the deposit-feeding clams were higher in density and more distinct in generic composition as compared with those in the suspension-feeding oysters and mussels. Over 90% of the coliform and heterotrophic bacteria in oysters were found in organs associated with the digestive tract. Coliforms were mainly found in the stomach while heterotrophs were present in both stomach and the lower intestine. The results suggest that the stomach flora of oysters are mainly derived from the external environment and, through a process of selection and multiplication, that it may be gradually replaced by a more indigenous population which dominates the lower digestive tract.

Animals↗

PubMed1996

Rapid estimation of Escherichia coli in live marine bivalve shellfish using automated conductance measurement.

Conductance measurement for quantitative estimations of Escherichia coli in live bivalve shellfish was evaluated as an alternative to the conventional most probable number (MPN) method used in France. The sensitivity of the two techniques was comparable. A single regression line (r = -0.968, P < 10(-6)) between log10 MPN and detection time (DT) was used to estimate E. coli concentrations for all shellfish examined. Estimation accuracy was +/- 0.92 log unit. Repeatability was better for DT than the log10 of MPN estimations (average coefficients of variation 2.7 and 9.3%, respectively). The conductance signal was attributable to E. coli in 96% of cases, and only 0.7% of E. coli cultures failed to exhibit a signal within 20 h. The conductance method reduces analysis handling time and is much easier to use than the MPN method. Moreover, results can be obtained within 5-9 h compared to 3 d for the MPN method.

Animals↗

PubMed2005

LENTICULE discs provide a homogenous format for external quality assessment samples: a comparison with freeze-dried samples for shellfish microbiology.

AIMS: The aim was to compare the variability in Escherichia coli enumeration data and detection of Salmonella spp. between four samples of LENTICULE discs and freeze-dried samples for the Health Protection Agency's External Quality Assessment (EQA) scheme for shellfish microbiology. METHODS AND RESULTS: Four samples of known but undisclosed microbiological content were dispatched in both freeze-dried and LENTICULE disc formats to 57 participating laboratories in 20 countries. Participants examined samples using their routine methods for the most probable number (MPN) of E. coli per 100 g and the presence/absence of Salmonella spp. There was no significant difference between the Food and Environmental Proficiency Testing Unit and participating laboratories for E. coli and Salmonella spp. results. There were significantly less outlying results using the LENTICULE discs than freeze-dried sample format and equivalent or less variance for the former for E. coli MPN. There was no significant difference between LENTICULE discs and freeze-dried samples for the presence/absence of Salmonella spp. CONCLUSIONS: Overall the results indicated that there was equivalent or less variance in results for the LENTICULE discs than for freeze-dried samples, therefore LENTICULE discs are a homogenous and stable matrix for EQA samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides validation data for the replacement of freeze-dried samples by LENTICULE discs for the Health Protection Agency Shellfish EQA Scheme.

Animals↗

PubMed2006

Detection of pandemic Vibrio parahaemolyticus O3:K6 serovar in Gulf of Mexico water and shellfish using real-time PCR with Taqman fluorescent probes.

We describe a real-time multiplexed PCR method using Taqman probes for the detection of total and pandemic Vibrio parahaemolyticus O3:K6 serovar in oysters and Gulf of Mexico water (gulf water). The specificity of these primers and probes was tested for amplification of a 450 bp thermolabile hemolysin (tlh) and a 369 bp ORF8 amplicon representing all V. parahaemolyticus and post-1996 clinical isolates of pandemic serovar O3:K6, respectively. The sensitivity of detection was 10 pg purified DNA or 10(3) CFU in 1 mL pure culture. Enrichment of this pathogen in oyster tissue homogenate or gulf water for 5 or 8 h resulted in the detection of an initial inoculum of 1 CFU in 1 mL or 1 g of samples. Application of the Taqman PCR assay on natural oysters exhibited a positive detection of V. parahaemolyticus, ranging from 16% to 100% of the samples collected primarily during the summer months. None of the samples exhibited a positive detection of O3:K6 serovar. Rapid and sensitive detection of this pathogen will help shellfish industry and Interstate Shellfish Sanitation Conference (ISSC) undertake appropriate measures to monitor this pathogen in oysters and oyster-growing waters, thereby preventing disease outbreaks and consequently protecting consumer health.

Animals↗

PubMed1980

Improved method and test strategy for recovery of enteric viruses from shellfish.

An improved recovery method and testing strategy were devised for recovery of low numbers of enteric viruses from each of three commercially important shellfish species. Effective recovery of virus depended as much upon details of the test strategy adopted for use of the improved method with each species as on the method itself. The most important test details involved sample composition, pool size, and method of use of cell cultures. Recovery sensitivity measured permitted detection of 25 to 3 plaque-forming units of enteroviruses and 100 to 27 plaque-forming units of reovirus through their recovery in cell culture, with effectivenesses averaging 64 and 46%, respectively. Test samples prepared by the improved recovery method were virtually cytotoxicity free. Optimal recovery of virus on 45-cm2 cell culture monolayers was obtained with 1-ml inocula adsorbed for 2 h. The most effective recovery of virus from shellfish samples was made by a sequential adsorption procedure which allowed equal exposure of an entire sample to each of two or more cell cultures. Removal of nonviral contaminants from test samples by antibiotic treatment was preferable to the use of ether or membrane filtration procedures.

Animals↗

PubMed1996

Collaborative evaluation of a method for the detection of Norwalk virus in shellfish tissues by PCR.

A multicenter, collaborative trial was performed to evaluate the reliability and reproducibility of a previously described method for the detection of Norwalk virus in shellfish tissues with the PCR (R.L. Atmar, F. H. Neill, J. L. Romalde, F. Le Guyader, C. M. Woodley, T. G. Metcalf, and M. K. Estes, Appl. Environ. Microbiol. 61:3014-3018, 1995). Virus was added to the stomachs and hepatopancreatic tissues of oysters or hard-shell clams in the control laboratory, the samples were shipped to the participating laboratories, and viral nucleic acids were extracted and then detected by reverse transcription-PCR. The sensitivity and specificity of the assay were 85 and 91%, respectively, when results were determined by visual inspection of ethidium bromide-stained agarose gels; the test sensitivity and specificity improved to 87 and 100%, respectively, after confirmation by hybridization with a digoxigenin-labeled, virus-specific probe. We have demonstrated that this method can be implemented successfully by several laboratories to detect Norwalk virus in shellfish tissues.

Animals↗

PubMed1998

Viral pollution in the environment and in shellfish: human adenovirus detection by PCR as an index of human viruses.

A study of the presence of human viruses (adenoviruses, enteroviruses, and hepatitis A viruses [HAVs]) in environmental and shellfish samples was carried out by applying DNA and cDNA amplification techniques by PCR. The detection of human adenoviruses by PCR was also examined as a potential molecular test to monitor viral pollution. The samples studied were urban and slaughterhouse sewage, river water, seawater, and shellfish. Enteroviruses were quantified by PFU in Buffalo green monkey kidney cells and fecal coliforms and phages of Bacteroides fragilis HSP40 were also evaluated in some of the samples. The amplification of viral DNA and cDNA has shown a high prevalence of human viruses that would not be detected by the use of classical techniques, such as the quantification of PFU in cell lines. The results of the analysis of slaughterhouse sewage samples together with the test of farm animal feces indicate that the adenoviruses and the HAVs detected in the environment are mostly of human origin. A significative correlation between the detection of human viruses by PCR and the values of bacteriophages of B. fragilis HSP40 in urban raw sewage was observed. Human adenoviruses were the viruses most frequently detected throughout the year, and all the samples that were positive for enteroviruses or HAVs were also positive for human adenoviruses. The results suggest that the detection of adenoviruses by PCR could be used as an index of the presence of human viruses in the environment where a molecular index is acceptable.

Abattoirs↗

PubMed2000

Selective accumulation may account for shellfish-associated viral illness.

From 1991 through 1998, 1,266 cases of shellfish-related illnesses were attributed to Norwalk-like viruses. Seventy-eight percent of these illnesses occurred following consumption of oysters harvested from the Gulf Coast during the months of November through January. This study investigated the ability of eastern oysters (Crassostrea virginica) to accumulate indicator microorganisms (i.e., fecal coliforms, Escherichia coli, Clostridium perfringens, and F(+) coliphage) from estuarine water. One-week trials over a 1-year period were used to determine if these indicator organisms could provide insight into the seasonal occurrence of these gastrointestinal illnesses. The results demonstrate that oysters preferentially accumulated F(+) coliphage, an enteric viral surrogate, to their greatest levels from late November through January, with a concentration factor of up to 99-fold. However, similar increases in accumulation of the other indicator microorganisms were not observed. These findings suggest that the seasonal occurrence of shellfish-related illnesses by enteric viruses is, in part, the result of seasonal physiological changes undergone by the oysters that affect their ability to accumulate viral particles from estuarine waters.

Animals↗

PubMed2000

Three-year study to assess human enteric viruses in shellfish.

The main pathogenic enteric viruses able to persist in the environment, such as hepatitis A virus (HAV), Norwalk-like virus (NLV), enterovirus (EV), rotavirus (RV), and astrovirus (AV), were detected by reverse transcription-PCR and hybridization in shellfish during a 3-year study. Oyster samples (n = 108), occasionally containing bacteria, were less frequently contaminated, showing positivity for AV (17%), NLV (23%), EV (19%), and RV (27%), whereas mussel samples, collected in areas routinely impacted by human sewage, were more highly contaminated: AV (50%), HAV (13%), NLV (35%), EV (45%), and RV (52%). Sequences obtained from HAV and NLV amplicons showed a great variety of strains, especially for NLV (strains close to Mexico, Snow Mountain Agent, or Norwalk virus). Viral contamination was mainly observed during winter months, although there were some seasonal differences among the viruses. This first study of virus detection over a fairly long period of time suggests that routine analysis of shellfish by a molecular technique is feasible.

Animals↗