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.     

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.     

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)     

Removal of faecal indicator bacteria and bacteriophages from the common mussel (Mytilus edulis) under artificial depuration conditions

Artificial self-purification (depuration) of mussels (Mytilus deulis) was undertaken at three temperatures, under conditions similar to those likely to be experienced in the commercial shellfish industry of the UK. During a 72 h depuration period, samples of mussel flesh were examined for three faecal indicator bacteria, Escherichia coli, Group D faecal streptococci and sulphite-reducing Clostridium spores, and two types of bacteriophage. There was a statistically significant difference in the elimination rate of faecal indicator bacteria compared with the slower rate for both bacteriophages.     

Effects of depuration of molluscs experimentally contaminated with Escherichia coli, Vibrio cholerae 01 and Vibrio parahaemolyticus

AIMS: The aim of the present study was to investigate the behaviour of two pathogenic vibrios (Vibrio cholerae O1 and Vibrio parahaemolyticus) during depuration and to compare it with that of Escherichia coli, used as an indicator of suitability for consumption. METHODS AND RESULTS: Samples of Mytilus galloprovincialis were experimentally contaminated with E. coli, V. cholerae O1 and V. parahaemolyticus, depurated in a pilot plant using ozone and analysed at selected intervals. Numbers of E. coli and vibrios were estimated using an MPN method. The presence of vibrios was confirmed by the use of PCR. The target genes used were ctx for V. cholerae O1 and the restriction fragment pR72H for V. parahaemolyticus. There was a substantially smaller reduction in the numbers of both vibrios (approximately 1 log) during the depuration process than of E. coli (approximately 3 log). CONCLUSIONS: The results confirm the inadequacy of E. coli as an indicator that molluscs have been cleansed of other microbiological agents. SIGNIFICANCE AND IMPACT OF THE STUDY: The study confirms the risk associated with the consumption of mussels and the need to correctly conserve and cook them prior to consumption.     

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.     

Accumulation and Elimination of Coliphage S-13 by the Hard Clam, Mercenaria mercenaria

Accumulation and elimination of viral particles by hard clams, Mercenaria mercenaria, were studied with the coliphage S-13 as a working model. Escherichia coli uptake and elimination were simultaneously monitored. Clams were exposed to low levels of S-13 (7 particles/ml) in running seawater for several days, achieving titers in tissues from 2 to more than 1,000 times the levels to which they had been exposed. Bacterial accumulation (previously established by other workers) was comparable. Upon exposure to virus-free running water, clams polluted to relatively low levels (100 plaque-forming units/ml) eliminated most of their bacterial contaminants in 24 to 48 hr. Viral contaminants, however, persisted for several days to weeks even under ideal conditions for clam activity, provided that the temperature remained below the inactivation threshold for the virus. Most of the accumulated virus appeared to be sequestered in the digestive gland. These sequestered particles are refractory to those mechanisms responsible for elimination of bacterial contaminants. This discrepancy points out the need for caution in evaluating the efficiency of shellfish depuration processes, especially if only a bacterial criterion is used as a monitoring system.     

The effect of size and age on depuration rates of diarrhetic shellfish toxins (DST) in mussels (Mytilus edulis L.)

Depuration or elimination of diarrhetic shellfish toxins (DST) was followed for 73 days in 1- and 2-year-old mussels. The age groups also differed in size, providing a broad approach to studying the effect of the differences in physiology accompanying the differences in size. Content of DST was analysed both on groups and individual mussels. Environmental variables were measured to evaluate their effect on depuration.We found no significant differences in elimination rate of DST between 1- and 2-year-old mussels under natural conditions. This suggests that size and age do not affect the elimination rate of the DST. The present study is the first study on the effect of age and size on the elimination rate of algal toxins in bivalves. The natural variations in food levels and temperature were not found to affect the elimination rate of DST.The digestive gland weights in the 1-year-old mussels increased four times while the DST content per individual decreased eight times. This demonstrated that dilution of toxins due to tissue growth could have an important contribution to declines in toxin concentrations. Changes in tissue mass are affected by environmental variables via growth or starvation, and when such changes lead to concentration or dilution of toxins this does not reflect the accumulation or removal of toxins from the tissues. We hence suggest that when evaluating the actual elimination capacity of the mussels, as in the present study, the total content of toxins per individual should be used, rather than toxin concentrations.The 1-year-old mussels had faster growth compared to the 2-year-old mussels in both total soft tissue and digestive glands. The mechanism of DST elimination is still unknown. If this process involves metabolism of the toxins, one could expect the rates of elimination to follow overall metabolic rates. However, the results from the present study suggest that large differences in growth rates, which also include difference in feeding and metabolic rates, do not affect the elimination rate of DST. Our results support the assumption that the depuration rates cannot be accelerated, even in artificial systems, as a cost-effective way to solve the problem with toxic mussels for the industry.     

Removal of faecal indicator bacteria and bacteriophages from the common mussel (Mytilus edulis) under artificial depuration conditions

Artificial self-purification (depuration) of mussels ( Mytilus edulis ) was undertaken at three temperatures, under conditions similar to those likely to be experienced in the commercial shellfish industry of the UK. During a 72 h depuration period, samples of mussel flesh were examined for three faecal indicator bacteria, Escherichia coli , Group D faecal streptococci and sulphite-reducing Clostridium spores, and two types of bacteriophage. There was a statistically significant difference in the elimination rate of faecal indicator bacteria compared with the slower rate for both bacteriophages.     

Inactivation of hepatitis A virus and indicator organisms in water by free chlorine residuals.

Hepatitis A virus (HAV) and selected indicator organisms were mixed together in chlorine-demand-free buffers at pH 6, 8, or 10 and exposed to free chlorine residuals, and the survival kinetics of individual organisms were compared. HAV was enumerated by a most-probable-number dilution assay, using PLC/PRF/5 liver cells for propagation of the virus and radioimmunoassay for its detection. At all pH levels, HAV was more sensitive than Mycobacterium fortuitum, coliphage V1 (representing a type of phage common in some sewage-polluted waters), and poliovirus type 2. Under certain conditions, HAV was more resistant than Escherichia coli, Streptococcus faecalis, coliphage MS2, and reovirus type 3. It was always more resistant than SA-11 rotavirus. Evidence is presented that conditions generally specified for the chlorine disinfection of drinking-water supplies will also successfully inactivate HAV and that HAV inactivation by free chlorine residuals can reliably be monitored by practical indicator systems consisting of appropriate combinations of suitable indicators such as coliform and acid-fast bacteria, coliphages, the standard plate count, and fecal streptococci.     

Inactivation of hepatitis A virus and indicator organisms in water by free chlorine residuals

Hepatitis A virus (HAV) and selected indicator organisms were mixed together in chlorine-demand-free buffers at pH 6, 8, or 10 and exposed to free chlorine residuals, and the survival kinetics of individual organisms were compared. HAV was enumerated by a most-probable-number dilution assay, using PLC/PRF/5 liver cells for propagation of the virus and radioimmunoassay for its detection. At all pH levels, HAV was more sensitive than Mycobacterium fortuitum, coliphage V1 (representing a type of phage common in some sewage-polluted waters), and poliovirus type 2. Under certain conditions, HAV was more resistant than Escherichia coli, Streptococcus faecalis, coliphage MS2, and reovirus type 3. It was always more resistant than SA-11 rotavirus. Evidence is presented that conditions generally specified for the chlorine disinfection of drinking-water supplies will also successfully inactivate HAV and that HAV inactivation by free chlorine residuals can reliably be monitored by practical indicator systems consisting of appropriate combinations of suitable indicators such as coliform and acid-fast bacteria, coliphages, the standard plate count, and fecal streptococci.     

Inactivation of indicator micro-organisms from various sources of faecal contamination in seawater and freshwater

AIM: The survival of indicator micro-organisms in aquatic systems is affected by both biotic and abiotic factors. Much of the past research on this topic has been conducted using laboratory-generated cultures of indicator bacteria. For this study, we used natural sources of faecal contamination as inoculants into environmental water samples, thereby representing the wide diversity of organisms likely to be found in faecal contamination. METHODS AND RESULTS: Rates of inactivation of water quality indicators, total coliforms (TC), Escherichia coli, enterococci (EC) and F+-specific coliphage were studied in three experiments using inoculants of sewage influent, sewage effluent and urban storm drain run-off. Effects of temperature, nutrients, total suspended solids, bacterial load and solar irradiation were studied in fresh and seawater matrices. Results demonstrated that temperature and solar irradiation had significant effects upon rates of inactivation (anova, P < 0.001). Inactivation rates were similar, regardless of the inoculant type. EC degraded the slowest in the dark with T90s of 115-121 and 144-177 h at 20 and 14 degrees C, respectively. When incubated in sunlight, EC was inactivated significantly more rapidly than either E. coli or F+-specific coliphage (P < 0.001). CONCLUSIONS: Inactivation of indicator bacteria is not dependent upon the original source of contamination. Inactivation rates of indicator bacteria were similar in fresh and seawater matrices. However, EC degraded more rapidly in sunlight than E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that the source of faecal contamination is not an important factor to inactivation rates of indicator bacteria. However, rates of inactivation of indicator bacteria are likely system specific.     

Comparative study of the bioaccumulation and elimination of trace metals (Cd,Pb, Zn,Mn and Fe) in the digestive gland,gills and muscle of bivalve Pinna nobilis during a field transplant experiment

The purpose of this study was to investigate the long-term bioaccumulation and elimination of Cd, Pb, Mn, Zn and Fe by Pinna nobilis tissues after their 90 day-transplantation period at Téboulba fishing harbor. During the transplantation period, the Cd, Pb, Mn, Zn and Fe concentrations in the different tissues of the mussels were measured before and after exposure period. Metal (Cd, Pb, Mn, Zn and Fe) accumulation in P. nobilis mussels varied depending on the analyzed tissue and the caging times. Notable differences in Cd, Pb, Mn, Zn and Fe accumulation patterns within the digestive gland, gills and muscle were found and may be due to the ability of each tissue to accumulate metals. During the depuration phase, the elimination of Cd, Pb, Mn, Zn and Fe depended on the target tissue and the metal speciation. Cd, Pb, Mn and Fe were eliminated rapidly from one organ and increased in other when compared to those of 90 day transplanted mussels. The increase of metal loads during the elimination phase is not clear and particularly what kind of processes is responsible for such response. However, it is reasonable to assume that metals increase is related to the existence of an accumulation/detoxification mechanism, which involves the transport of metals from an organ to another. The data obtained indicate that because of the significantly high quantities of Cd, Pb, Mn, Zn and Fe accumulated during the exposure phase, the transplanted mussels are suitable bioindicators for monitoring trace metals in marine ecosystem.     

Comparative inactivation of poliovirus type 3 and MS2 coliphage in demand-free phosphate buffer by using ozone.

MS2 coliphage (ATCC 15597-B1) has been proposed by the U.S. Environmental Protection Agency as a surrogate for enteric viruses to determine the engineering requirements of chemical disinfection systems on the basis of previous experience with chlorine. The objective of this study was to determine whether MS2 coliphage was a suitable indicator for the inactivation of enteric viruses when ozone disinfection systems were used. Bench-scale experiments were conducted in 2-liter-batch shrinking reactors containing ozone demand-free 0.05 M phosphate buffer (pH 6.9) at 22 degrees C. Ozone was added as a side stream from a concentrated stock solution. It was found that an ozone residual of less than 40 micrograms/liter at the end of 20 s inactivated greater than 99.99% of MS2 coliphage in the demand-free buffer. When MS2 was compared directly with poliovirus type 3 in paired experiments, 1.6 log units more inactivation was observed with MS2 coliphage than with poliovirus type 3. It was concluded that the use of MS2 coliphage as a surrogate organism for studies of enteric virus with ozone disinfection systems overestimated the inactivation of enteric viruses. It is recommended that the regulatory agencies evaluate their recommendations for using MS2 coliphage as an indicator of enteric viruses.     

Comparative inactivation of poliovirus type 3 and MS2 coliphage in demand-free phosphate buffer by using ozone.

MS2 coliphage (ATCC 15597-B1) has been proposed by the U.S. Environmental Protection Agency as a surrogate for enteric viruses to determine the engineering requirements of chemical disinfection systems on the basis of previous experience with chlorine. The objective of this study was to determine whether MS2 coliphage was a suitable indicator for the inactivation of enteric viruses when ozone disinfection systems were used. Bench-scale experiments were conducted in 2-liter-batch shrinking reactors containing ozone demand-free 0.05 M phosphate buffer (pH 6.9) at 22 degrees C. Ozone was added as a side stream from a concentrated stock solution. It was found that an ozone residual of less than 40 micrograms/liter at the end of 20 s inactivated greater than 99.99% of MS2 coliphage in the demand-free buffer. When MS2 was compared directly with poliovirus type 3 in paired experiments, 1.6 log units more inactivation was observed with MS2 coliphage than with poliovirus type 3. It was concluded that the use of MS2 coliphage as a surrogate organism for studies of enteric virus with ozone disinfection systems overestimated the inactivation of enteric viruses. It is recommended that the regulatory agencies evaluate their recommendations for using MS2 coliphage as an indicator of enteric viruses.     

Environmental factors influencing human viral pathogens and their potential indicator organisms in the blue mussel,Mytilus edulis: the first Scandinavian report

This study was carried out in order to investigate human enteric virus contaminants in mussels from three sites on the west coast of Sweden, representing a gradient of anthropogenic influence. Mussels were sampled monthly during the period from February 2000 to July 2001 and analyzed for adeno-, entero-, Norwalk-like, and hepatitis A viruses as well as the potential viral indicator organisms somatic coliphages, F-specific RNA bacteriophages, bacteriophages infecting Bacteroides fragilis, and Escherichia coli. The influence of environmental factors such as water temperature, salinity, and land runoff on the occurrence of these microbes was also included in this study. Enteric viruses were found in 50 to 60% of the mussel samples, and there were no pronounced differences between the samples from the three sites. E. coli counts exceeded the limit for category A for shellfish sanitary safety in 40% of the samples from the sites situated in fjords. However, at the site in the outer archipelago, this limit was exceeded only once, in March 2001, when extremely high levels of atypical indole-negative strains of E. coli were registered at all three sites. The environmental factors influenced the occurrence of viruses and phages differently, and therefore, it was hard to find a coexistence between them. This study shows that, for risk assessment, separate modeling should be done for every specific area, with special emphasis on environmental factors such as temperature and land runoff. The present standard for human fecal contamination, E. coli, seems to be an acceptable indicator of only local sanitary contamination; it is not a reliable indicator of viral contaminants in mussels. To protect consumers and get verification of "clean" mussels, it seems necessary to analyze for viruses as well. The use of a molecular index of the human contamination of Swedish shellfish underscores the need for reference laboratories with high-technology facilities.     

Behavior of pathogenic bacteria in the oyster, Crassostrea commercialis, during depuration, re-laying, and storage

Oysters (Crassostrea commercials) harvested from major cultivation areas within the state of New South Wales, Australia, were commonly contaminated with low levels of the food-poisoning organisms Bacillus cereus, Clostridium perfringens, and Vibrio parahaemolyticus. Salmonella was found in oysters on only one occasion. These bacteria were cleansed from oysters during oyster purification by re-laying in a non-polluted waterway. Oysters were laboratory contaminated to levels in excess 1,000 cells per g with either B. cereus, C. perfringens, V. parahaemolyticus, Salmonella typhimurium, or S. senftenberg. These species were cleansed from such oysters during purification in a laboratory depuration unit that used ultraviolet light for sterilizing the depuration water. Escherichia coli was also cleansed from oysters under the same re-laying or depuration conditions so that its measurement alone could be used to indicate the cleansing of the above pathogenic species. The levels of these bacteria were also measured during the storage of oysters under conditions that occur during marketing. While B. cereus counts remained relatively stable during storage, the Salmonella spp. gradually decreased in numbers and C. perfringens rapidly died off. V. parahaemolyticus counts increased slightly during the first 4 days of storage, after which decreases occurred.     

Behavior of pathogenic bacteria in the oyster, Crassostrea commercialis, during depuration, re-laying, and storage.

Oysters (Crassostrea commercials) harvested from major cultivation areas within the state of New South Wales, Australia, were commonly contaminated with low levels of the food-poisoning organisms Bacillus cereus, Clostridium perfringens, and Vibrio parahaemolyticus. Salmonella was found in oysters on only one occasion. These bacteria were cleansed from oysters during oyster purification by re-laying in a non-polluted waterway. Oysters were laboratory contaminated to levels in excess 1,000 cells per g with either B. cereus, C. perfringens, V. parahaemolyticus, Salmonella typhimurium, or S. senftenberg. These species were cleansed from such oysters during purification in a laboratory depuration unit that used ultraviolet light for sterilizing the depuration water. Escherichia coli was also cleansed from oysters under the same re-laying or depuration conditions so that its measurement alone could be used to indicate the cleansing of the above pathogenic species. The levels of these bacteria were also measured during the storage of oysters under conditions that occur during marketing. While B. cereus counts remained relatively stable during storage, the Salmonella spp. gradually decreased in numbers and C. perfringens rapidly died off. V. parahaemolyticus counts increased slightly during the first 4 days of storage, after which decreases occurred.