Inefficient accumulation of low levels of monodispersed and feces-associated poliovirus in oysters.

The accumulation of low levels (0.002 to 0.18 PFU/ml) of both feces-associated and monodispersed poliovirus by oysters (Crassostrea virginica or C. gigas) and clams (Mercenaria mercenaria) was investigated. These levels were chosen to duplicate the conditions present in light to moderately polluted waters. Experiments were performed in both small- and large-scale flowing seawater systems, developed to mimic the natural marine habitats of shellfish. Under these experimental conditions, viral accumulation by physiologically active shellfish was only noted when water column concentrations exceeded approximately 0.01 PFU/ml. Bioaccumulation increased with increasing concentrations of both monodispersed and feces-associated viruses. At virus concentrations below this level, viruses were seldom detected in either clams or oysters. Evidence indicated that the lack of accumulation was not the result of inefficient extraction or detection methods. The modified Cat-Floc-beef extract procedure used in the experiment was found to be capable of detecting as few as 1.5 to 2.0 PFU per shellfish. Evidence is presented to indicate that an uptake-depuration equilibrium was present at virus exposure levels of 0.10 PFU/ml, but not at 0.01 PFU/ml. The results suggested that viral accumulation by shellfish may not be efficient at water column concentrations below congruent to 0.01 PFU/ml.     

Accumulation of sediment-associated viruses in shellfish.

The present study focused on the importance of contaminated sediments in shellfish accumulation of human viruses. Epifaunal (Crassostrea virginica) and infaunal (Mercenaria mercenaria) shellfish, placed on or in cores, were exposed to either resuspended or undisturbed sediments containing bound poliovirus type 1 (LSc 2ab). Consistent bioaccumulation by oysters (four of five trials) was only noted when sediment-bound viruses occurred in the water column. Virus accumulation was observed in a single instance where sediments remained in an undisturbed state. While the incidence of bioaccumulation was higher with resuspended rather than undisturbed contaminated sediment, the actual concentration of accumulated viruses was not significantly different. The accumulation of viruses from oysters residing on uninoculated sediments. When clams were exposed to undisturbed, virus-contaminated sediments, two of five shellfish pools yielded viral isolates. Bioaccumulation of undisturbed sediments by these bivalves was considered marginal when related to the concentration of virus in contaminated sediments; they would only represent a significant threat when suspended in the water column. Arguments were advanced for water-column sampling in the region of the water-sediment interface to provide an accurate determination of the virological quality of shellfish harvesting waters.     

Accumulation of sediment-associated viruses in shellfish

The present study focused on the importance of contaminated sediments in shellfish accumulation of human viruses. Epifaunal (Crassostrea virginica) and infaunal (Mercenaria mercenaria) shellfish, placed on or in cores, were exposed to either resuspended or undisturbed sediments containing bound poliovirus type 1 (LSc 2ab). Consistent bioaccumulation by oysters (four of five trials) was only noted when sediment-bound viruses occurred in the water column. Virus accumulation was observed in a single instance where sediments remained in an undisturbed state. While the incidence of bioaccumulation was higher with resuspended rather than undisturbed contaminated sediment, the actual concentration of accumulated viruses was not significantly different. The accumulation of viruses from oysters residing on uninoculated sediments. When clams were exposed to undisturbed, virus-contaminated sediments, two of five shellfish pools yielded viral isolates. Bioaccumulation of undisturbed sediments by these bivalves was considered marginal when related to the concentration of virus in contaminated sediments; they would only represent a significant threat when suspended in the water column. Arguments were advanced for water-column sampling in the region of the water-sediment interface to provide an accurate determination of the virological quality of shellfish harvesting waters.     

Methods for recovering poliovirus and rotavirus from oysters.

Polioviruses and rotaviruses are potential indicators of sewage pollution of water and shellfish. Several methods for detecting these viruses in oysters were assessed. Elution-precipitation involving Catfloc for clarification and skim milk for subsequent flocculation resulted in the recovery of an average of 79% of poliovirus type 1 and 37% of rotavirus SA-11 from oyster homogenates inoculated with low numbers of these viruses. Adsorption-elution-precipitation did not improve the recovery of poliovirus and was detrimental to the recovery of rotavirus. Ultrafiltration or ultracentrifugation resulted in improved recovery of rotavirus but also in higher toxicity of oyster extracts to cell cultures. We recommend the use of the described elution-precipitation method for detecting viral pollutants in sample of oysters.     

Methods for recovering poliovirus and rotavirus from oysters

Polioviruses and rotaviruses are potential indicators of sewage pollution of water and shellfish. Several methods for detecting these viruses in oysters were assessed. Elution-precipitation involving Catfloc for clarification and skim milk for subsequent flocculation resulted in the recovery of an average of 79% of poliovirus type 1 and 37% of rotavirus SA-11 from oyster homogenates inoculated with low numbers of these viruses. Adsorption-elution-precipitation did not improve the recovery of poliovirus and was detrimental to the recovery of rotavirus. Ultrafiltration or ultracentrifugation resulted in improved recovery of rotavirus but also in higher toxicity of oyster extracts to cell cultures. We recommend the use of the described elution-precipitation method for detecting viral pollutants in sample of oysters.     

Detection of bacteriophage tracers in bivalve molluscan shellfish

Procedures to enumerate commonly used bacteriophage tracers in bivalve molluscan shellfish were evaluated. Bacteriophages specific to Serratia marcescens, Escherichia coli and Enterobacter cloacae can be recovered from shellfish flesh by chloroform treatment of homogenates and subsequent clarification of samples by centrifugation. Bacteriophages were enumerated using a soft agar overlay technique. Hard clams appeared to release toxic compounds during homogenization which dramatically reduced counts of Ent. clocae bacteriophage.     

Abundance of Vibrio cholerae,V. vulnificus,and V. parahaemolyticus in Oysters (Crassostrea virginica) and Clams (Mercenaria mercenaria) from Long Island Sound

Vibriosis is a leading cause of seafood-associated morbidity and mortality in the United States. Typically associated with consumption of raw or undercooked oysters, vibriosis associated with clam consumption is increasingly being reported. However, little is known about the prevalence of Vibrio spp. in clams. The objective of this study was to compare the levels of Vibrio cholerae, Vibrio vulnificus, and Vibrio parahaemolyticus in oysters and clams harvested concurrently from Long Island Sound (LIS). Most probable number (MPN)–real-time PCR methods were used for enumeration of total V. cholerae, V. vulnificus, V. parahaemolyticus, and pathogenic (tdh⁺ and/or trh⁺) V. parahaemolyticus. V. cholerae was detected in 8.8% and 3.3% of oyster (n = 68) and clam (n = 30) samples, with levels up to 1.48 and 0.48 log MPN/g in oysters and clams, respectively. V. vulnificus was detected in 97% and 90% of oyster and clam samples, with median levels of 0.97 and −0.08 log MPN/g, respectively. V. parahaemolyticus was detected in all samples, with median levels of 1.88 and 1.07 log MPN/g for oysters and clams, respectively. The differences between V. vulnificus and total and pathogenic V. parahaemolyticus levels in the two shellfish species were statistically significant (P < 0.001). These data indicate that V. vulnificus and total and pathogenic V. parahaemolyticus are more prevalent and are present at higher levels in oysters than in hard clams. Additionally, the data suggest differences in vibrio populations between shellfish harvested from different growing area waters within LIS. These results can be used to evaluate and refine illness mitigation strategies employed by risk managers and shellfish control authorities.     

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

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

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

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

Clams (Corbicula fluminea) as bioindicators of fecal contamination with Cryptosporidium and Giardia spp. in freshwater ecosystems in California

This study evaluated clams as bioindicators of fecal protozoan contamination using three approaches: (i) clam tissue spiking experiments to compare several detection techniques; (ii) clam tank exposure experiments to evaluate clams that had filtered Cryptosporidium oocysts from inoculated water under a range of simulated environmental conditions; (iii) sentinel clam outplanting to assess the distribution and magnitude of fecal contamination in three riverine systems in California. Our spiking and tank experiments showed that direct fluorescent antibody (DFA), immunomagnetic separation (IMS) in combination with DFA, and PCR techniques could be used to detect Cryptosporidium in clam tissues. The most analytically sensitive technique was IMS concentration with DFA detection of oocysts in clam digestive gland tissues, which detected 10 oocysts spiked into a clam digestive gland 83% of the time. In the tank experiment, oocyst dose and clam collection time were significant predictors for detecting Cryptosporidium parvum oocysts in clams. In the wild clam study, Cryptosporidium and Giardia were detected in clams from all three study regions by IMS-DFA analysis of clam digestive glands, with significant variation by sampling year and season. The presence of C. parvum DNA in clams from riverine ecosystems was confirmed with PCR and DNA sequence analysis.     

Concentration of poliovirus from tap water using positively charged microporous filters.

Microporous filters that are more electropositive than the negatively charged filters currently used for virus concentrations from water by filter adsorption-elution methods were evaluated for poliovirus recovery from tap water. Zeta Plus filters composed of diatomaceous earth-cellulose-"charge-modified" resin mixtures and having a net positive charge of up to pH 5 to 6 efficiently adsorbed poliovirus from tap water at ambient pH levels 7.0 to 7.5 without added multivalent cation salts. The adsorbed virus were eluted with glycine-NaOH, pH 9.5 to 11.5. Electropositive asbestos-cellulose filters efficiently adsorbed poliovirus from tap water without added multivalent cation salts between pH 3.5 and 9.0, and the absorbed viruses could be eluted with 3% beef extract, pH 9, but not with pH 9.5 to 11.5 glycine-NaOH. Under water quality conditions in which poliovirus recoveries from large volumes of water were less than 5% with conventional negatively charged filters and standard methods, recoveries with Zeta Plus filters averaged 64 and 22.5% for one- and two-stage concentration procedures, respectively. Electropositive filters appear to offer distinct advantages over conventional negatively charged filters for concentrating enteric viruses from water, and their behavior tends to confirm the importance of electrostatic forces in virus recovery from water by microporous filter adsorption-elution methods.     

Concentration of poliovirus from tap water using positively charged microporous filters.

Microporous filters that are more electropositive than the negatively charged filters currently used for virus concentrations from water by filter adsorption-elution methods were evaluated for poliovirus recovery from tap water. Zeta Plus filters composed of diatomaceous earth-cellulose-"charge-modified" resin mixtures and having a net positive charge of up to pH 5 to 6 efficiently adsorbed poliovirus from tap water at ambient pH levels 7.0 to 7.5 without added multivalent cation salts. The adsorbed virus were eluted with glycine-NaOH, pH 9.5 to 11.5. Electropositive asbestos-cellulose filters efficiently adsorbed poliovirus from tap water without added multivalent cation salts between pH 3.5 and 9.0, and the absorbed viruses could be eluted with 3% beef extract, pH 9, but not with pH 9.5 to 11.5 glycine-NaOH. Under water quality conditions in which poliovirus recoveries from large volumes of water were less than 5% with conventional negatively charged filters and standard methods, recoveries with Zeta Plus filters averaged 64 and 22.5% for one- and two-stage concentration procedures, respectively. Electropositive filters appear to offer distinct advantages over conventional negatively charged filters for concentrating enteric viruses from water, and their behavior tends to confirm the importance of electrostatic forces in virus recovery from water by microporous filter adsorption-elution methods.     

Enzymatic transformation of PSP toxins in the littleneck clam (Protothacastaminea)

Stemming from investigations into the relationship between toxins produced by Gonyaulax sp. and accumulated in shellfish, we wish to report enzymatic transformations of the PSP toxins to decarbamoyl derivatives in the littleneck clam (Protothaca staminea). No toxin transformations were observed in either mussels (Mytilus edulis) or in butter clams (Saxidomus giganteus). In addition, littleneck clam samples from the natural environment contained predominantly the decarbamoyl derivatives, while other shellfish species collected from the same vicinity contained the previously reported PSP toxins.     

DIET AND FORAGING BEHAVIOR OF SEA OTTERS IN SOUTHEAST ALASKA

Abstract: Direct observations of feeding sea otters ( Enhydra lutris ) at 11 sites in southeast Alaska showed infaunal clams to be the primary prey utilized by otters throughout the region. Foraging dive times associated with clam and sea urchin prey were significantly longer than those for more easily captured prey (crabs and mussels). Dive times and surface intervals were also generally correlated with water depth or apparent difficulty in obtaining buried prey. Male otters, which fed more extensively on clams than females, made significantly longer foraging dives than females. Foraging success remained high, even at sites where prey numbers were found to be very low during a related study. The very deeply burrowing geoduck clam ( Panope abrupta ), while common at several otter feeding sites, was rarely captured by otters. These results, combined with those of a companion study on prey numbers, indicate that butter clams ( Saxidomus giganteus ) account for the majority of the sea otter diet in southeast Alaska, and that sea urchins may represent relatively short-term prey in comparison to infaunal bivalves in regions where both prey types co-exist. Furthermore, the importance of butter clams in the sea otter diet and the tendency for this bivalve to retain chronically high levels of paralytic shellfish poisoning toxins in southeast Alaska increases the probability that toxic phytoplankton blooms influence sea otter distribution in this region.     

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.     

Filter-feeding bivalves can remove avian influenza viruses from water and reduce infectivity

Avian influenza (AI) viruses are believed to be transmitted within wild aquatic bird populations through an indirect faecal–oral route involving contaminated water. This study examined the influence of filter-feeding bivalves, Corbicula fluminea, on the infectivity of AI virus in water. Clams were placed into individual flasks with distilled water inoculated 1:100 with a low pathogenic (LP) AI virus (A/Mallard/MN/190/99 (H3N8)). Viral titres in water with clams were significantly lower at 24 and 48 h post-inoculation compared to LPAI-infected water without clams. To determine whether clams affected the infectivity of AI viruses, 18 wood ducks (Aix sponsa) were divided into test groups and inoculated with a variety of treatments of clam supernatants, whole clams and water exposed to a high pathogenic (HP) AI (A/whooper swan/Mongolia/244/05 (H5N1)). None of the wood ducks inoculated with HPAI-infected water that was filtered by clams or that was inoculated with or fed tissue from these clams exhibited morbidity or mortality. All wood ducks exposed to either HPAI-infected water without clams or the original viral inoculum died. These results indicate that filter-feeding bivalves can remove and reduce the infectivity of AI viruses in water and demonstrate the need to examine biotic environmental factors that can influence AI virus transmission.     

Uptake and Elimination of Poliovirus by West Coast Oysters

Accumulation of poliovirus Lsc-2ab by West Coast oysters was determined by using a stationary seawater system, and depuration was determined by using both stationary and free-flow systems. Results indicate that these shellfish have the same pattern of accumulation and localization of viruses as do East Coast species. However, uptake appeared to occur more rapidly than described for East Coast shellfish. There appeared to be a gradual diffusion of virus from the digestive area into the body. Depuration was found to occur more rapidly and completely under free-flow conditions than in a stationary system.