Multilocus genetic analysis of Cryptosporidium in naturally contaminated bivalve molluscs

AIMS: To evaluate the application of discriminatory multilocus PCR procedures for the characterization of Cryptosporidium in samples of naturally contaminated bivalve molluscan shellfish. METHODS AND RESULTS: Nucleic acid was extracted from 22 shellfish previously identified as contaminated with Cryptosporidium spp. and subjected to PCR-based analysis for two independent fragments of the Cryptosporidium oocyst wall protein (COWP) gene, three microsatellite markers (ML 1, GP 15 and MS 5) and an extra-chromosomal small double-stranded RNA (dsRNA). Overall, at least one COWP gene fragment was amplified from all 22 samples, 21 amplified the dsRNA and 14 at least one of the three microsatellite loci. More than one dsRNA or microsatellite allele was detected in 50% of samples. The majority of samples were contaminated with Cryptosporidium parvum types circulating in both humans and livestock. A novel dsRNA element was identified in one sample, which did not amplify any of the three microsatellite loci investigated. CONCLUSIONS: Multilocus analysis of Cryptosporidium can be applied to DNA extracted from naturally contaminated shellfish. SIGNIFICANCE AND IMPACT OF STUDY: This multilocus genetic analysis highlights that filter feeder molluscs are a potential source of cryptosporidial oocysts, which may be infectious to humans.     

A tentative national reference procedure for isolation and enumeration of Escherichia coli from bivalve molluscan shellfish by most probable number method

In the UK several quantitative methods exist for the examination of bivalve molluscan shellfish for sewage contamination. These methods include roll tubes, pour plates and most probable number (MPN) techniques, but there is no national standard method. A comparative study was made of the most commonly used methods for detection of Escherichia coli in bivalve shellfish. Schemes employing solid media, such as the roll tube and pour plate methods, underestimated faecal contamination in shellfish tissue compared with a liquid MPN multiple test-tube method using minerals-modified-glutamate broth (MMGB) as primary enrichment medium. The composition of MMGB apparently permits repair of sublethally injured cells of E. coli. Incorporation of resuscitation stages into the pour plate technique did not yield higher counts. A standardized MPN technique for examination of bivalve molluscan shellfish for E. coli content is proposed as a possible national reference procedure pending further collaborative assessment.     

A tentative national reference procedure for isolation and enumeration of Escherichia coli from bivalve molluscan shellfish by most probable number method

In the UK several quantitative methods exist for the examination of bivalve molluscan shellfish for sewage contamination. These methods include roll tubes, pour plates and most probable number (MPN) techniques, but there is no national standard method. A comparative study was made of the most commonly used methods for detection of Escherichia coli in bivalve shellfish. Schemes employing solid media, such as the roll tube and pour plate methods, underestimated faecal contamination in shellfish tissue compared with a liquid MPN multiple test-tube method using minerals-modified-glutamate broth (MMGB) as primary enrichment medium. The composition of MMGB apparently permits repair of sublethally injured cells of E. coli. Incorporation of resuscitation stages into the pour plate technique did not yield higher counts. A standardized MPN technique for examination of bivalve molluscan shellfish for E. coli content is proposed as a possible national reference procedure pending further collaborative assessment.     

Recent advances in analytical procedures for the detection of diarrhetic phycotoxins: a review

Okadaic acid and dinophysistoxins are produced by some marine unicellular algae from the plankton and also benthic microalgae and may accumulate in shellfish. These phycotoxins are involved in a gastrointestinal syndrome called diarrhetic shellfish poisoning (DSP), which occurs in humans after consumption of bivalve molluscs. Thousands cases of human poisonings in Europe were caused by consumption of toxic shellfish during the past decade. The rapid detection and the reliable determination of the main phycotoxins implicated in DSP are a major concern for governmental institutions in charge of the sanitary control of seafood safety. Analytical procedures for the detection and determination of DSP toxins can be classified as: bioassays, biochemical methods including immunoassays, or physicochemical methods. Although a large number of methods have been developed, none have been officially validated. A complete panel of tools for DSP toxin analysis should include screening, investigation, and confirmation methods. This paper presents a compilation of recent developments and optimisations of these methods. This revised version was published online in June 2006 with corrections to the Cover Date.     

Environmental Transmission of Human Noroviruses in Shellfish Waters

Human noroviruses (NoV) are the most common cause of epidemic gastroenteritis following consumption of bivalve shellfish contaminated with fecal matter. NoV levels can be effectively reduced by some sewage treatment processes such as activated sludge and membrane bioreactors. However, tertiary sewage treatment and substantial sewage dilution are usually required to achieve low concentrations of virus in shellfish. Most outbreaks have been associated with shellfish harvested from waters affected by untreated sewage from, for example, storm overflows or overboard disposal of feces from boats. In coastal waters, NoV can remain in suspension or associate with organic and inorganic matter and be accumulated by shellfish. Shellfish take considerably longer to purge NoV than fecal indicator bacteria when transferred from sewage-polluted estuarine waters to uncontaminated waters. The abundance and distribution of NoV in shellfish waters are influenced by the levels of sewage treatment, proximity of shellfish beds to sewage sources, rainfall, river flows, salinity, and water temperature. Detailed site-specific information on these factors is required to design measures to control the viral risk.     

Comparison of hemolytic activities among strains of Heterocapsa circularisquama isolated in various localities in Japan

Heterocapsa circularisquama (Dinophyceae), a red tide dinoflagellate, is toxic to bivalve molluscs such as the pearl oyster (Pinctada fucata), but no harmful effects of this alga on fish have been observed so far. We found that 7 strains of H. circularisquama showed hemolytic activities toward rabbit erythrocytes in a cell-density dependent manner, but to quite different extents. The strains which are known to be highly toxic to bivalves tend to show stronger hemolytic activities and vice versa, suggesting that the hemolytic activity is parallel with the shellfish toxicity of H. circularisquama. Since the hemolytic assay is simple, semiquantitative, and reproducible, this assay is useful not only to search for certain toxins responsible for the shellfish toxicity of H. circularisquama but also to estimate the potential toxicity of a newly appeared strain of H. circularisquama.     

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.     

Effects of vertebrate growth factors on digestive gland cells from the mollusc Pecten maximus L.: an in vitro study

In relation with the digestive cycle, the digestive gland cells of bivalve molluscs undergo a sequence of cytological changes which is controlled by external and internal effectors such as putative gastrointestinal hormones and growth differentiation factors. A tissue dissociation method was developed to investigate the in vitro effect of the vertebrate growth and differentiation factors: insulin, insulin growth factor I (IGF-I), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) on the digestive gland cells of the scallop Pecten maximus. All these vertebrate peptides induced a dose-dependent increased incorporation of ³H-leucine and ¹⁴C-uridine in whole digestive gland cell suspensions. However, after Percoll density gradient purification of the digestive cells, only stem and undifferentiated enriched cell fractions were responsive to the different peptides. In addition, insulin and IGF-I, but not EGF and bFGF, stimulated ³H-leucine incorporation in control dispersed mantle edge cells. These results suggest that insulin-related peptides could work as general growth promoting factors in molluscs. On the other hand, EGF and bFGF, or at least their molluscan counterparts, may be efficient growth differentiation factors in the regenerative processes occurring in the digestive gland of molluscs. Accepted: 26 September 1997     

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.     

The role of bivalve molluscs as tools in estuarine sediment toxicity testing: a review

Estuarine sediments frequently are repositories and therefore potential sources of anthropogenic contaminants. Many organic and metallic chemical compounds released into aquatic systems bind to particulates and so accumulate in the sediments, thus, sediments become repositories of contaminants in estuaries. These may also cause contamination through diffusion of porewater, resuspension of particulates and dispersal of benthic fauna. There is a need to assess the biological affects of these anthropogenic contaminants because they may be toxic to infauna and bottomfish. Sediment toxicity bioassays are a means for carrying out such an assessment and primarily provide data on toxicity by measuring the effects on the test organism. Existing sediment toxicity bioassays rely on a battery of aquatic toxicity tests, which are based on the extraction of pore water, and elutriate from sediments and then subjecting these sediment phases to toxicity testing regimes. Two estuarine bivalve molluscs, Scrobicularia plana and Tapes semidecussatus were used to assess the ecotoxicity of field-collected sediments from estuarine and coastal areas around the Irish and English Coast over a 3-year study period. A variety of endpoints were measured during the study including survival in air, behaviour, animal condition, biochemistry, soft tissue metal concentrations, lysosomal membrane integrity and histopathology. Of these endpoints, the most sensitive were survival, survival in air, lysosomal membrane integrity, behaviour and histopathology.     

Environmental contaminants and the prevalence of hemic neoplasia (leukemia) in the common mussel (Mytilus edulis complex) from Puget Sound, Washington, U.S.A

The relationship between hemic neoplasia, a blood cell disorder in bivalve molluscs, and chemical contaminants was evaluated in the common mussel (Mytilus edulis complex). Hemic neoplasia (HN) is endemic to mussel populations in Puget Sound. The prevalence of hemic neoplasia ranged from 0 to 30% in mussels from nine sites in Puget Sound, Washington. Organic chemical contamination in sediment from these sites range from 0.1 to 64.0 ppm of polycyclic aromatic hydrocarbons (PAHs) and 0.07 to 0.50 ppm chlorinated hydrocarbons. No relationship between the body burden of environmental contaminants and the prevalence of HN in mussels was identified. To evaluate the short-term ability of chemical contaminants to induce HN in mussels, mussels, from a site where mussels were previously determined to be HN free, were fed microencapsulated PAHs (composed of a mixture of phenanthrene, flouranthene, and benzo[a]pyrene) or PCBs (Aroclor 1254) and the prevalence of HN was assessed after 30 days of exposure. Although an apparent increase in HN prevalence (20 to 30%) was observed in all treatments groups except the untreated controls, no significant difference in the prevalence of HN was observed between the control group of mussels fed corn oil (vehicle) and mussels fed either PAHs or PCBs in corn oil. A long-term (180-day) exposure study was conducted to evaluate the influence of PAHs or PCBs in modulating the prevalence of HN in a mussel population already exhibiting a moderate HN prevalence. Mussels, from a site where mussels were previously determined to exhibit a background prevalence of HN, fed microencapsulated PAHs, PCBs, and corn oil (vehicle) over a long time period (180 days), revealed an apparent increased prevalence of HN (30 to 40%) above the low levels (20%) initially present. However, no significant difference in the prevalence of HN was observed between the control group of mussels fed corn oil (vehicle) and mussels fed either PAHs or PCBs in corn oil. Although chemical contaminants have been proposed as a modulating factor in the development and promotion of HN in bivalve molluscs from environmentally stressed and degraded habitats, we find no evidence that chemical contaminants induce or promote the development of HN in the mussel M. edulis complex.     

香港淡水及海产贝类感染吸虫幼虫期的调查研究

作者于1983年4月及1986年4月二次在香港检查该地区20个村庄11种淡水螺(共11680粒),及14个海区包括红树林地带和无红树林的海滩中的22种海产贝类(共12580粒)。查获26种吸虫幼虫期,其中12种见于淡水螺(5种〕,8种寄生于红树林地带的海螺(7种),5种寄生于无红树林海区的贝类(6种),1种见于在红树林地带和无红树林海滩生存的海螺(5种)。寄生淡水螺的吸虫幼虫期分隶于Cortrematidae;Maseniidae;Schistosomatidae;Notocotylidae;Strigeidae;Paramphi-stomidae;Plagiorchidae;Philophthalmidaes;Microphallidae及Heterophyidae等科。寄生于海产贝类的吸虫幼虫期分隶于Philophthalmidae; Heterophyidae;Fellodistomidae; Cyathoco-tylidae;Echinostomatidae;Opecoelidae等科及Plagiorchioidea总科。     

43 Testeria gen. nov. (dinophyceae) from manatee cay belize, Central America

Saxitoxin and domoic acid sequestration by bivalve molluscs occurs periodically along the Oregon coast, presumably as a result of harmful algal blooms (HABs). Since 1958 and more continuously since 1979, the Oregon Shellfish Program (OSP) has assayed toxin levels in these molluscs as part of a monitoring program for paralytic (PSP) and amnesic (ASP) shellfish poisoning. We have created a working data base for all PSP sampling by the OSP between 1958 and 2001 and have examined the data for spatial and temporal trends in the appearance of toxin in shellfish, amount of toxin, and apparent duration of toxic events. In this report, we examine the data from the five stations with the longest record of continuous sampling (1979–2000) for evidence of correlation with El Niño events, upwelling, and/or a pattern of increasing frequency or intensity of toxic events. We also compare the pattern of appearance of toxin at open coast stations with the timing of first appearance of toxin in shellfish at adjacent estuarine stations. This is an important analysis because, in Oregon, shellfish closures due to PSP occur frequently in mussel beds on the open coast and the source of toxin-producing organisms is unknown.     

41 Incidence of paralytic shellfish toxin in bivalve mollusc tissue from the oregon coast

Saxitoxin and domoic acid sequestration by bivalve molluscs occurs periodically along the Oregon coast, presumably as a result of harmful algal blooms (HABs). Since 1958 and more continuously since 1979, the Oregon Shellfish Program (OSP) has assayed toxin levels in these molluscs as part of a monitoring program for paralytic (PSP) and amnesic (ASP) shellfish poisoning. We have created a working data base for all PSP sampling by the OSP between 1958 and 2001 and have examined the data for spatial and temporal trends in the appearance of toxin in shellfish, amount of toxin, and apparent duration of toxic events. In this report, we examine the data from the five stations with the longest record of continuous sampling (1979–2000) for evidence of correlation with El Niño events, upwelling, and/or a pattern of increasing frequency or intensity of toxic events. We also compare the pattern of appearance of toxin at open coast stations with the timing of first appearance of toxin in shellfish at adjacent estuarine stations. This is an important analysis because, in Oregon, shellfish closures due to PSP occur frequently in mussel beds on the open coast and the source of toxin‐producing organisms is unknown.     

The relationship of bioenergetics and the environment to the field growth of cultured bivalves

The field culture of bivalve molluscs is dependent on the production and supply of phytoplankton and other food sources, its consumption by these suspension feeders, and its transformation into bivalve tissue. An understanding of the processes by which food is captured and utilized is fundamental to prediction of bivalve growth and management of shellfish aquaculture. The following perspective considers the role of food quality and quantity in the ingestion and absorption of food particles, as well as the influence of temperature, water circulation and other environmental variables on bivalve energy budgets and growth rate. A particular focus is the role of simulation modelling in quantifying the influence of these variables on scope for growth. Seston depletion is a major influence on cultured animals such that bivalves are limited by both food quality and quantity. Seston depletion can be managed by taking advantage of particle maxima (e.g. resuspension), matching culture species to turbidity gradients, and arranging hanging culture to take advantage of seston renewal in oblique flow. Food limitation interacts with temperature in cold temperate areas where high primary production occurs at low temperatures. Model simulations of mussel growth demonstrate the importance of temperature acclimation of filtration and its relationship to the timing and magnitude of the spring bloom. In addition to the energetics of individual animals, energy flow through culture ecosystems is considered in terms of bivalve processing of organic matter and competition with Zooplankton and fish. Because bioenergetics is at the root of all of these issues, simulation modelling of scope for growth and related components of the ecosystem is a vital tool in prediction and management of commercial bivalve aquaculture.     

Levels of male-specific RNA bacteriophage and Escherichia coli in molluscan bivalve shellfish from commercial harvesting areas

AIMS: Current measures for controlling the public health risks associated with bivalve molluscan shellfish consumption rely on the use of Escherichia coli to indicate the sanitary quality of shellfish harvesting areas. However, it has been demonstrated that E. coli is an inadequate indicator of the viral risk associated with shellfish. An alternative indicator organism, male-specific RNA (FRNA) bacteriophage has been proposed for this role. This study compared the distribution of E. coli and FRNA bacteriophage in shellfish harvesting areas. METHODS AND RESULTS: A total of 608 shellfish samples from 49 shellfish harvesting areas were analysed for E. coli and FRNA bacteriophage using standard published methods. The geometric mean concentration of FRNA bacteriophage in all samples was over three times greater than that of E. coli (1800 and 538 counts/100 g for FRNA bacteriophage and E. coli, respectively). In contrast to E. coli, FRNA bacteriophage concentrations were strongly influenced by season with a geometric mean count of 4503 PFU/100 g in the winter (October-March) compared with 910 PFU/100 g in the summer (April-September). CONCLUSIONS: FRNA bacteriophage were present in shellfish at higher concentrations than E. coli. Elevated levels of FRNA bacteriophage observed in the winter concur with the known increased viral risk associated with shellfish harvested at that time of year in the UK. Levels of FRNA bacteriophage found in many shellfish from category B harvesting areas would not be eliminated by conventional treatment processes. SIGNIFICANCE AND IMPACT OF THE STUDY: Data from this study will inform future proposals to introduce FRNA bacteriophage as an indicator of the viral risk associated with shellfish.     

Calibration of the impedance method for rapid quantitative estimation of Escherichia coli in live marine bivalve molluscs

AIM: Calibration of impedance measurement was performed vs the Association Fran?oise de Normalisation (AFNOR) MPN method with a view to rapid enumeration of Escherichia coli in live marine bivalve molluscs. METHODS AND RESULTS: Linear regression models between log10 MPN and detection time (DT) were adjusted for several shellfish types, growth media, and impedance instruments (BacTrac and Malthus systems). Escherichia coli concentrations could be estimated from DT using a single regression line for BacTrac 4100 with M1 medium (R2 = 87.8%) and Malthus with M2 medium (R2 = 86.7%), and two regression lines for BacTrac 4110 with M2 medium (R2 = 86.4 and 88.2%). The uncertainty of the predicted bacterial concentration was around +/-0.43 log unit for duplicate sample analysis. The impedance signal was attributable to E. coli in 99% of cases. All cultures containing E. coli produced an impedance signal with BacTrac 4100 and BacTrac 4110, whereas 5.6% did not exhibit a signal with Malthus. CONCLUSIONS: Impedance measurement is a possible alternative to the MPN method for rapid quantitative estimation of E. coli in live bivalve shellfish. SIGNIFICANCE AND IMPACT OF THE STUDY: The impedance method reduces analysis handling time considerably and is much easier to use than the MPN method. Moreover, results can be obtained within 5-10 h, allowing rapid intervention to ensure public health protection in case of shellfish contamination.     

Biochemical identification and numerical taxonomy of Aeromonas spp. isolated from environmental and clinical samples in Spain

AIMS: To study the phenotypic characteristics of Aeromonas spp. from environmental and clinical samples in Spain and to cluster these strains by numerical taxonomy. METHODS AND RESULTS: A collection of 202 Aeromonas strains isolated from bivalve molluscs, water and clinical samples was tested for 64 phenotypic properties; 91% of these isolates were identified at species level. Aeromonas caviae was predominant in bivalve molluscs and Aerom. bestiarum in freshwater samples. Cluster analyses revealed eight different phena: three containing more than one DNA-DNA hybridization group but including strains that belong to the same phenospecies complex (Aerom. hydrophila, Aerom. sobria and Aerom. caviae), Aerom. encheleia, Aerom. trota and three containing unidentified Aeromonas strains isolated from bivalve molluscs. CONCLUSIONS:Aeromonas spp. are widely distributed in environmental and clinical sources. A selection of 16 of the phenotypical tests chosen allowed the identification of most isolates (91%), although some strains remain unidentified, mainly isolates from bivalve molluscs, suggesting the presence of new Aeromonas species. Numerical taxonomy was not in total concordance with the identification of the studied strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Numerical taxonomy of Aeromonas strains isolated from different sources revealed the presence of potentially pathogenic Aeromonas spp., especially in bivalve molluscs, and phena with unidentified strains that suggest new Aeromonas species.