Tissue distribution of a coliphage and Escherichia coli in mussels after contamination and depuration.

Experiments were undertaken to determine the tissue distribution of Escherichia coli and a coliphage after contamination of the common mussel (Mytilus edulis). Mussels were contaminated with high levels of feces-associated E. coli and a 22-nm icosahedral coliphage over a 2-day period in a flowing-seawater facility. After contamination, individual tissues were carefully dissected and assayed for E. coli and the coliphage. Contaminated mussels were also analyzed to determine the tissue distribution of the contaminants after 24- and 48-h depuration periods. The majority of each contaminant was located in the digestive tract (94 and 89% of E. coli and coliphage, respectively). Decreasing concentrations were found in the gills and labial palps, foot and muscles, mantle lobes, and hemolymph. Our results indicate that contamination above levels in water occurred only in the digestive tract. Contaminated mussels were depurated in a commercial-scale recirculating UV depuration system over a 48-h period. The percent reductions of E. coli occurred in the following order: digestive tract, hemolymph, foot and muscles, mantle lobes, and gills and labial palps. The percent reductions of the coliphage were different, occurring in the following order: hemolymph, foot and muscles, gills and labial palps, mantle lobes, and digestive tract. Our results clearly demonstrate that E. coli and the coliphage are differentially eliminated from the digestive tract. The two microorganisms are eliminated at similar rates from the remaining tissues. Our results also clearly show that the most significant coliphage retention after depuration for 48 h is in the digestive tract. Thus, conventional depuration practices are inappropriate for efficient virus elimination from mussels.     

Behavior of Escherichia coli and male-specific bacteriophage in environmentally contaminated bivalve molluscs before and after depuration.

We monitored the differential reduction rates and elimination patterns of Escherichia coli and male-specific (F+) bacteriophage during UV depuration for 48 h in oysters (Crassostrea gigas) and mussels (Mytilus edulis) contaminated by short-term (1 to 3 weeks) and long-term (more than 6 months) exposure to sewage in the marine environment. The time taken to reduce levels of E. coli by 90% was 6.5 h or less in all cases. In contrast, the amounts of time needed to reduce levels of F+ bacteriophage by 90% were considerably longer: 47.3 and 41.3 h (after short- and long-term exposures, respectively) in mussels and 54.6 and 60.8 h (after short- and long-term exposures, respectively) in oysters. No differences in the rates of reduction of indicators of viral pollution following exposure of the shellfish to either short- or long-term sewage contamination were observed. Further experiments were conducted with mussels to determine the relative distributions of E. coli and F+ bacteriophage in tissue before and during depuration. Prior to depuration the majority of E. coli organisms (90.1%) and F+ bacteriophage (87.3%) were detected in the digestive tract (i.e., the digestive gland and intestine). E. coli and F+ bacteriophage were reduced in all tissues except the digestive gland to undetectable levels following depuration for 48 h. Within the digestive gland, levels of F+ bacteriophage were reduced to 30% of initial levels, whereas E. coli was reduced to undetectable levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential depuration of poliovirus, Escherichia coli, and a coliphage by the common mussel, Mytilus edulis

The elimination of sewage effluent-associated poliovirus, Escherichia coli, and a 22-nm icosahedral coliphage by the common mussel, Mytilus edulis, was studied. Both laboratory-and commercial-scale recirculating, UV depuration systems were used in this study. In the laboratory system, the logarithms of the poliovirus, E. coli, and coliphage levels were reduced by 1.86, 2.9, and 2.16, respectively, within 52 h of depuration. The relative patterns and rates of elimination of the three organisms suggest that they are eliminated from mussels by different mechanisms during depuration under suitable conditions. Poliovirus was not included in experiments undertaken in the commercial-scale depuration system. The differences in the relative rates and patterns of elimination were maintained for E. coli and coliphage in this system, with the logarithm of the E. coli levels being reduced by 3.18 and the logarithm of the coliphage levels being reduced by 0.87. The results from both depuration systems suggest that E. coli is an inappropriate indicator of the efficiency of virus elimination during depuration. The coliphage used appears to be a more representative indicator. Depuration under stressful conditions appeared to have a negligible affect on poliovirus and coliphage elimination rates from mussels. However, the rate and pattern of E. coli elimination were dramatically affected by these conditions. Therefore, monitoring E. coli counts might prove useful in ensuring that mussels are functioning well during depuration.     

Differential depuration of poliovirus, Escherichia coli, and a coliphage by the common mussel, Mytilus edulis.

The elimination of sewage effluent-associated poliovirus, Escherichia coli, and a 22-nm icosahedral coliphage by the common mussel, Mytilus edulis, was studied. Both laboratory-and commercial-scale recirculating, UV depuration systems were used in this study. In the laboratory system, the logarithms of the poliovirus, E. coli, and coliphage levels were reduced by 1.86, 2.9, and 2.16, respectively, within 52 h of depuration. The relative patterns and rates of elimination of the three organisms suggest that they are eliminated from mussels by different mechanisms during depuration under suitable conditions. Poliovirus was not included in experiments undertaken in the commercial-scale depuration system. The differences in the relative rates and patterns of elimination were maintained for E. coli and coliphage in this system, with the logarithm of the E. coli levels being reduced by 3.18 and the logarithm of the coliphage levels being reduced by 0.87. The results from both depuration systems suggest that E. coli is an inappropriate indicator of the efficiency of virus elimination during depuration. The coliphage used appears to be a more representative indicator. Depuration under stressful conditions appeared to have a negligible affect on poliovirus and coliphage elimination rates from mussels. However, the rate and pattern of E. coli elimination were dramatically affected by these conditions. Therefore, monitoring E. coli counts might prove useful in ensuring that mussels are functioning well during depuration.     

Characterization of intracytoplasmic prokaryote infections in Dreissena sp. (Bivalvia: Dreissenidae)

This study characterizes intracytoplasmic infections with prokaryote microorganisms in Dreissena sp. (near Dreissena polymorpha) from northeastern Greece and represents the first report of such infections in freshwater bivalves. Light microscope observations of stained tissues revealed basophilic, cytoplasmic inclusion bodies in 87.5% (28/32) of the mussels sectioned. Inclusions in epithelial cells and connective tissues were noted, respectively, in 34.4 and 71.9% of the sample, with 5 mussels (15.6%) having both tissue types infected. Epithelial cell infections were observed in histological sections only in digestive gland tubules and ducts; within tubules, inclusions were present more often in secretory than digestive cells. Connective tissue infections, however, were systemic; among the 32 mussels sectioned, inclusions were found in the gills (65.6%), foot (12.5%), mantle (9.4%), labial palps (6.3%), digestive gland (6.3%), stomach (6.3%), and gonads (3.1%). Cytoplasmic inclusions (maximum dimension, 138 microm) were prominent enough in the gills to be visible in 17.0% of the 247 mussels dissected. Ultrastructurally, prokaryote cells in gill connective tissues were clearly characteristic of Chlamydiales-like organisms, with each intracytoplasmic inclusion containing a loosely packed mixture of elementary, reticulate, intermediate bodies, and blebs. Prokaryote colonies in digestive gland epithelial cells exclusively contained 1 of 4 morphological cell types and were considered Rickettsiales-like. Hexagonal, virus-like particles were present in the cytoplasm of the largest of these Rickettsiales-like prokaryotes. Although host stress was evident from localized cell necrosis and dense hemocyte infiltration, overall infection was fairly benign, with no major, adverse impact on body condition evident among sectioned or dissected mussels. A possible negative effect was partial constriction of gill water tubes, but at the infection intensity observed (typical range 1 to 7 inclusion bodies per section), significant interference with respiration and other metabolic functions of the gills was highly unlikely.     

Cadmium Accumulation and Metallothionein Biosynthesis in Cadmium-Treated Freshwater Mussel Anodonta woodiana

This study investigated the distribution of cadmium (Cd) and the protein level of metallothionein (MT) and examined the relationship of Cd accumulation and the MT concentration in different tissues of freshwater mussel Anodonta woodiana following Cd treatment. The mussels were exposed to Cd (4.21, 8.43, 16.86, 33.72 and 67.45 mg L^-1) for 24, 48, 72 and 96 h, respectively. After Cd treatment, the gills, mantle, foot, visceral mass and digestive gland tissues were collected for analysis. We found that, in the controls, Cd distributed in all tissues in the concentration order of gills>mantle>foot>visceral mass>digestive gland. Upon Cd treatment, Cd concentration significantly increased in all tissues. The highest Cd accumulation was found in the digestive gland, which was 0.142 mg g^-1 (P<0.05). MT levels in the gills and mantle of the mussels increased significantly (P<0.05), which were in positive correlation with Cd accumulation in the tissues (P<0.05). In conclusion, our results demonstrated a correlation between Cd accumulation and MT up-regulation in gills and mantle of the mussels after Cd treatment. It is suggested that the protein level of MT in gills and mantle of Anodonta woodiana is a good biomarker for Cd contamination.     

Characterization of a Monoclonal Antibody Directed against Mytilus spp Larvae Reveals an Antigen Involved in Shell Biomineralization

The M22.8 monoclonal antibody (mAb) developed against an antigen expressed at the mussel larval and postlarval stages of Mytilus galloprovincialis was studied on adult samples. Antigenic characterization by Western blot showed that the antigen MSP22.8 has a restricted distribution that includes mantle edge tissue, extrapallial fluid, extrapallial fluid hemocytes, and the shell organic matrix of adult samples. Other tissues such as central mantle, gonadal tissue, digestive gland, labial palps, foot, and byssal retractor muscle did not express the antigen. Immunohistochemistry assays identified MSP22.8 in cells located in the outer fold epithelium of the mantle edge up to the pallial line. Flow cytometry analysis showed that hemocytes from the extrapallial fluid also contain the antigen intracellularly. Furthermore, hemocytes from hemolymph have the ability to internalize the antigen when exposed to a cell-free extrapallial fluid solution. Our findings indicate that hemocytes could play an important role in the biomineralization process and, as a consequence, they have been included in a model of shell formation. This is the first report concerning a protein secreted by the mantle edge into the extrapallial space and how it becomes part of the shell matrix framework in M. galloprovincialis mussels.     

Histological Characterization and Glucose Incorporation into Glycogen of the Pacific Oyster Crassostrea gigas Storage Cells

In order to investigate glycogen metabolism in the oyster Crassostrea gigas, the distribution of storage cells in the whole animal was studied before histological and biochemical characterization. These cells were found mainly in the labial palps, the mantle, and gonadal area and also in gills and the digestive area. Storage cells from palps, mantle, and gonad presented the same morphological features and the same seasonal glycogen variations. Storage cells were isolated from the labial palps and the mantle plus gonadal area of the oyster by enzymatic dispersion and centrifugation through discontinuous Percoll gradient. These cells have a modal density of 1.043 g/ml. An ultrastructural study confirmed that glycogen is present in the cytoplasm either as fine particles or sequestered within vesicles. Glucose incorporation into glycogen was evaluated in vitro using [U-¹⁴C]glucose: the incorporation in isolated cells increased linearly for at least 8 hours, was proportional to the cell concentration, and showed saturation kinetics with respect to the exogenous glucose concentration. Received March 18, 1999; accepted September 27, 1999.     

Localization of norovirus and poliovirus in Pacific oysters

Aims:  To examine the uptake and tissue distribution of norovirus (NoV) and poliovirus (PV) experimentally bioaccumulated in feeding Pacific oysters ( Crassostrea gigas ).
Methods and Results:  Pacific oysters were allowed to bioaccumulated either PV or NoV under tidally synchronized feeding conditions in laboratory tanks. Oysters were then either fixed and paraffin wax embedded prior to localizing virus within tissues by immunohistochemistry (IHC), or they were dissected into digestive tract (stomach, intestine and digestive diverticula), gill and labial palp tissues, and the viral load determined by quantitative RT-PCR. Both PV and NoV immunoreactivities were predominantly found in the lumen and within cells of the digestive tract tissues; however, PV was also found within cells of nondigestive tract tissues, and in the gills and labial palp. Quantitative RT-PCR of tissue extracts corroborate the immunohistochemical data in that the major site for virus localization is the gut, but significant amounts of viral RNA were identified in the gills and labial palp.
Conclusions:  The human enteric viruses, PV and NoV, are readily bioaccumulated by feeding Pacific oysters and that some of the virus is internalized within cells of both digestive and nondigestive tissues.
Significance and Impact of the Study:  Oysters that have been virally contaminated even after depuration (cleaning) in uncontaminated seawater could pose a human health risk if consumed.     

Ciliary ultrastructure of protobranchs (Mollusca, Bivalvia)

Abstract. The external epithelial cilia and other surface structures of the nuculoid protobranchs Nuculana pernula and Nucula nitidosa were studied. The gill lamellae and labial palps are partly covered with very long cilia. These have a modified slender distal portion, an ordinary metazoan-type basal body, a basal foot. and a single, long cross-striated rootlet. In cilia on the gills of N. nitidosa , the basal foot is thick and attaches to the next basal body directly behind. Unciliated surface areas on the gills, labial palps, and foot are covered with a dense brushborder of microvilli. We observed no specific homologies between the cilia of the protobranchs studied and the epidermal cilia of the enigmatic Xenoturbella bocki , hence the recent hypothesis of a close connection of the latter to the protobranch bivalves is questioned.     

Persistence of Vibrio vulnificus in tissues of Gulf Coast oysters, Crassostrea virginica, exposed to seawater disinfected with UV light.

Vibrio vulnificus is an estuarine bacterium which can cause opportunistic infections in humans consuming raw Gulf Coast oysters, Crassostrea virginica. Although V. vulnificus is known as a ubiquitous organism in the Gulf of Mexico, its ecological relationship with C. virginica has not been adequately defined. The objective of the present study was to test the hypothesis that V. vulnificus is a persistent microbial flora of oysters and unamenable to traditional methods of controlled purification, such as UV light depuration. Experimental depuration systems consisted of aquaria containing temperature-controlled seawater treated with UV light and 0.2-microns-pore-size filtration. V. vulnificus was enumerated in seawater, oyster shell biofilms, homogenates of whole oyster meats, and tissues including the hemolymph, digestive region, gills, mantle, and adductor muscle. Results showed that depuration systems conducted at temperatures greater than 23 degrees C caused V. vulnificus counts to increase in oysters, especially in the hemolymph, adductor muscle, and mantle. Throughout the process, depuration water contained high concentrations of V. vulnificus, indicating that the disinfection properties of UV radiation and 0.2-microns-pore-size filtration were less than the rate at which V. vulnificus was released into seawater. Approximately 10(5) to 10(6) V. vulnificus organisms were released from each oyster per hour, with 0.05 to 35% originating from shell surfaces. These surfaces contained greater than 10(3) V. vulnificus organisms per cm2. In contrast, when depuration seawater was maintained at 15 degrees C, V. vulnificus was not detected in seawater and multiplication in oyster tissues was inhibited.     

Persistence of Vibrio vulnificus in tissues of Gulf Coast oysters, Crassostrea virginica, exposed to seawater disinfected with UV light.

Vibrio vulnificus is an estuarine bacterium which can cause opportunistic infections in humans consuming raw Gulf Coast oysters, Crassostrea virginica. Although V. vulnificus is known as a ubiquitous organism in the Gulf of Mexico, its ecological relationship with C. virginica has not been adequately defined. The objective of the present study was to test the hypothesis that V. vulnificus is a persistent microbial flora of oysters and unamenable to traditional methods of controlled purification, such as UV light depuration. Experimental depuration systems consisted of aquaria containing temperature-controlled seawater treated with UV light and 0.2-microns-pore-size filtration. V. vulnificus was enumerated in seawater, oyster shell biofilms, homogenates of whole oyster meats, and tissues including the hemolymph, digestive region, gills, mantle, and adductor muscle. Results showed that depuration systems conducted at temperatures greater than 23 degrees C caused V. vulnificus counts to increase in oysters, especially in the hemolymph, adductor muscle, and mantle. Throughout the process, depuration water contained high concentrations of V. vulnificus, indicating that the disinfection properties of UV radiation and 0.2-microns-pore-size filtration were less than the rate at which V. vulnificus was released into seawater. Approximately 10(5) to 10(6) V. vulnificus organisms were released from each oyster per hour, with 0.05 to 35% originating from shell surfaces. These surfaces contained greater than 10(3) V. vulnificus organisms per cm2. In contrast, when depuration seawater was maintained at 15 degrees C, V. vulnificus was not detected in seawater and multiplication in oyster tissues was inhibited.     

Uptake and retention of Vibrio cholerae non-O1, Salmonella typhi, Escherichia coli and Vibrio harvey by mussels in seawater.

Vibrio cholerae non O1 is known to persist in estuarine and freshwater environments. Experiments evaluated the amount of microorganisms accumulated in mussels maintained in static seawater, contaminated with 10(4) to 10(6) cells/ml and the depuration time required in circulating water. Accumulation and retention times were compared with those for Escherichia coli, Salmonella typhi and Vibrio harvey. E. coli and S. typhi accumulated to a greater extent and were released from mussels more quickly than vibrios which became undetectable 2 to 3 days later than E. coli. Seasonal seawater temperatures (14 to 21 degrees C) had a limited influence on depuration but vibrios appear to be retained with more efficacy over 16 degrees C while E. coli and S. typhi were eliminated to a greater extent. When mussels were contaminated with mixed culture, vibrios appeared to predominate on E. coli, while no interference was observed between E. coli and S. typhi.     

Expression of recombinant goldfish glutamic acid decarboxylase 65 and evidence for differential pH and PLP responsiveness compared to the human enzyme

Variability of taurine (2-aminoethane sulfonic acid) was studied as a function of size in the mussel Mytilus galloprovincialis and tissue specificity. Isometric and/or allometric relationships were established with regard to total soft mass of the mussels between 20 and 60 mm shell length. Relative amounts of taurine dropped significantly with increasing mass of whole soft tissues with an allometric coefficient value of -0.15. The inverse relationship between taurine and increasing size of mussels was confirmed for gill epithelium and labial palp (allometric coefficient values of -0.16 and -0.10, respectively), tissues that, in turn, represented isometric functions with regard to total soft mass. Although relative amounts of taurine were significantly different in digestive gland, mantle and foot, relationships with increasing size of mussels remained unchanged in these tissues. Gill area of mussels was related to soft mass with an allometric coefficient of 0.70 by 2D Image Analysis, but increased to 0.85 when introducing a third dimension, i.e. gill thickness. Results are discussed according to gill structure analysis and taurine functionality.     

Morphological development of glochidia in artificial media through early juvenile of freshwater pearl mussel,Hyriopsis (Hyriopsis) bialatus Simpson, 1900

The morphological development and the sequence of organogenesis from glochidium to the early juvenile stage of the freshwater pearl mussel, Hyriopsis bialatus, were observed. Larvae of H. bialatus were cultured in standard tissue culture medium (M199) supplemented with common carp (Cyprinus carpio) plasma and they showed transformation within 10 days. Larval samples were collected every 2 days during glochidia development and subjected to histological processing. Three types of cell masses were developed during this period: the ventral plate (the foot rudiment), lateral pits (the gill rudiment), and the oral plate or endodermic sac (the origin of the digestive tract). The ventral plate gave rise to two foot lobes which fused into a single lobe. The gills were developed from the lateral pits next to the ventral plate, forming a pair of gill buds that became elongated and turned into gill bars. The digestive tract began with the formation of mouth by invagination of the oral plate (or endodermic sac) and formation of a tube underneath the growing foot. Several controversial aspects of organogenesis have been inferred, e.g., de novo formation of the anterior and posterior juvenile adductors, the fate of the mushroom body structure, and foot lobe formation from two separate precursor lobes. A mushroom body protruded into the mantle cavity and remained there throughout the transformation period. Moreover, the evidence of a supporting band (mucoid structure) in the mature glochidium of H. bialatus has never been reported in other freshwater mussel species, and its function and composition need to be further investigated.     

Particle transport in the zebra mussel, Dreissena polymorpha (Pallas)

The capture, transport, and sorting of particles by the gills and labial palps of the freshwater mussel Dreissena polymorpha were examined by endoscopy and video image analysis. More specifically, the morphology of the feeding organs in living zebra mussels was described; the mode and speeds of particle transport on the feeding organs was measured; and the sites of particle selection in the pallial cavity were identified. Particle velocities (outer demibranch lamellae, 90 microm s(-1); inner demibranch lamellae, 129 microm s(-1); marginal food groove of inner demibranchs, 156 microm s(-1); dorsal ciliated tracts, 152 microm s(-1)), as well as the movement of particles on the ctenidia and labial palps of D. polymorpha, are consistent with mucociliary, rather than hydrodynamic, transport. Particles can be sorted on the ctenidia of zebra mussels, resulting in a two-layer transport at the marginal food groove of the inner demibranch. That is: preferred particles are transported inside the marginal groove proper, whereas particles destined for rejection are carried superficially in a string of mucus. Sorting also occurs at the ventral margin of the outer demibranch; desirable particles are retained on the outer demibranch, whereas unacceptable particles are transferred to the inner demibranch and ultimately excluded from ingestion. We suggest that the structure of homorhabdic ctenidia does not preclude particle sorting, and that some ecosystem modifications attributed to zebra mussels may ultimately be due to ctenidial sorting mechanisms.     

The kinetics of accumulation and excretion of ferric hydroxide in Mytilus edulis (I.) and its distribution in the tissues

Iron, which occurs in sea water as particulate ferric hydroxide, is accumulated to high concentrations by the common mussel, Mytilus edulis (L.). The kinetics of the accumulation and excretion of iron in Mytilus has been studied using ⁵⁹Fe-labelled ferric hydroxide and the tissue distribution and identification at the sub-cellular level determined by analytical electron microscopy. Iron-59 accumulates in linear proportion to the sea-water concentration and is found in all tissues; the concentration factors for viscera, kidneys, gills, muscle = mantle is 25: 6: 4: 1, respectively. The particulate iron is taken up by pinocytosis by special epithelial cells in the gills, gut, kidney and possibly labial palps and held in membrane-bound vesicles, unaccompanied by mucus in the case of gills and kidney, but with mucus present in the digestive diverticulum and mid-gut cells. There is no free iron within the cytoplasm. Approximately 30 % of the iron presented to the gut is not absorbed being voided with the faeces. The absorbed iron is exocytosed and then passed on to amoebocytes in the haemolymph for transport to other tissues, a major portion being excreted by transfer to the byssal threads.     

The Escherichia coli Content of Mytilus edulis from Analysis of Whole Tissue or Digestive Tract

The dissected digestive tract of edible mussels from two sites was examined for Escherichia coli and the counts compared with those from the total tissue of parallel mussel samples. The method using 4 x 1 ml pour plates was preferred to the 1 x 4 ml pour plate or to the 2 x 0.2 ml spread plates. Counts from the digestive tract were up to sixfold higher than parallel counts using total tissue. Digestive tract inoculum also produced amplified Esch. coli detection when lightly polluted mussels were examined by the semi-quantitative percentage-clean technique. It was concluded that dissection of the bacteriologically rich digestive tract significantly increases the sensitivity of Esch. coli detection and has application in environmental assay procedures using mussels or other filter-feeding molluscs. Significant differences in the frequency of Esch. coli biotypes from the two mussel locations could be related to small differences in the pollution regimes at these two sites.