Inactivation and elimination of human enteric viruses by Pacific oysters

Aims: To investigate the comparative elimination of three different human enterically transmitted viruses [i.e. hepatitis A virus (HAV), norovirus (NoV) and poliovirus (PV)] and inactivation of HAV and PV by Pacific oysters. Methods and Results: New Zealand grown Pacific oysters (Crassostrea gigas) were allowed to bioaccumulate HAV, NoV and PV. Samples of oyster gut, faeces and pseudofaeces were then analysed by using real‐time RT‐PCR to determine the amount of viral RNA and cell culture methods to identify changes in the number of plaque forming units. The results suggest that the majority of the PV present in the oyster gut and oyster faeces is noninfectious, while in contrast, most of the HAV detected in the oyster gut are infectious. Depuration experiments identified a large drop in the count of PV in the gut over a 23‐h cleansing period, whereas the levels of HAV and NoV did not significantly decrease. Conclusions: Human enterically transmitted viruses are eliminated and inactivated at different rates by Pacific oysters. Significance and Impact of Study: The research presented in this article has implications for risk management techniques that are used to improve the removal of infectious human enteric viruses from bivalve molluscs.     

Strain-dependent norovirus bioaccumulation in oysters

Noroviruses (NoVs) are the main agents of gastroenteritis in humans and the primary pathogens of shellfish-related outbreaks. Some NoV strains bind to shellfish tissues by using carbohydrate structures similar to their human ligands, leading to the hypothesis that such ligands may influence bioaccumulation. This study compares the bioaccumulation efficiencies and tissue distributions in oysters (Crassostrea gigas) of three strains from the two principal human norovirus genogroups. Clear differences between strains were observed. The GI.1 strain was the most efficiently concentrated strain. Bioaccumulation specifically occurred in digestive tissues in a dose-dependent manner, and its efficiency paralleled ligand expression, which was highest during the cold months. In comparison, the GII.4 strain was very poorly bioaccumulated and was recovered in almost all tissues without seasonal influence. The GII.3 strain presented an intermediate behavior, without seasonal effect and with less bioaccumulation efficiency than that of the GI.1 strain during the cold months. In addition, the GII.3 strain was transiently concentrated in gills and mantle before being almost specifically accumulated in digestive tissues. Carbohydrate ligand specificities of the strains at least partly explain the strain-dependent bioaccumulation characteristics. In particular, binding to the digestive-tube-specific ligand should contribute to bioaccumulation, whereas we hypothesize that binding to the sialic acid-containing ligand present in all tissues would contribute to retain virus particles in the gills or mantle and lead to rapid destruction.     

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.     

Susceptibility of juvenile Crassostrea gigas and resistance of Panope abrupta to Mikrocytos mackini

Samples from the field and laboratory exposure to Mikrocytos mackini (a tiny protistan parasite of unknown taxonomic affiliation) confirmed that juvenile Pacific oysters (Crassostrea gigas) are susceptible to infection and the resulting disease. In the laboratory bath exposure experiment, a prevalence of infection approaching 100% and mortalities were observed in the small oysters (about 18 mm in shell length). However, in the same laboratory exposure experiment, similar aged geoduck clams (Panope abrupta, about 8mm in shell length) were resistant to infection. The main route of infection in the oysters appeared to be via the digestive tract and possibly the gills where the parasite multiplied within host cells. Other tissues such as the adductor muscle and vesicular connective tissue were subsequently colonized. Although the infection resulted in the mortality of some oysters, others appeared to overcome the disease.     

Early developmental stages of a protozoan parasite, Marteilioides chungmuensis (Paramyxea), the causative agent of the ovary enlargement disease in the Pacific oyster, Crassostrea gigas

A paramyxea, Marteilioides chungmuensis, causes the irregular enlargement of the ovary in the Pacific oyster, Crassostrea gigas in Korea and Japan. The knowledge about the life cycle of the parasite has been limited to the sporulation stages within the oocyte of oysters. In this study, we used the parasite-specific DNA probes and electron microscopy to experimentally infected oysters in a field and successfully clarified early developmental stages of the parasite. The parasite invaded the oysters through the epithelial tissues of the gills, mantle and labial palps. Extrasporogony repeatedly occurred in the connective tissues by binary fusion. The inner cell of the extrasporogonic stage migrated into the gonadal epithelium, invaded the oocyte to start sporulation.     

Functional responses associated with pallial organ variations in the Pacific oyster Crassostrea gigas (Thunberg, 1793)

The plasticity and function of the pallial organs were studied in the Pacific oyster Crassostrea gigas from three sites of Bourgneuf Bay (French Atlantic coast, 46-47°N, 1-2°W) characterized by different turbidity conditions. Labial palp area was closely and positively related to the turbidity gradient. No clear pattern was established between the gill area and the gradient of suspended particulate matter (SPM). The functional responses induced by these morphological variations were investigated in the laboratory by means of ecophysiological experiments and endoscopic observations. Oysters with different pallial organ areas were supplied with mixed suspensions of heat-killed Tetraselmis suecica and living Skeletonema costatum added to different concentrations of kaolinite to simulate low (SPM = 8.5 ± 0.4 mg l^− 1) and high (SPM = 48.3 ± 1.4 mg l^− 1) turbidity conditions. At each SPM concentration, heat-killed T. suecica were preferentially rejected in pseudofaeces compared to S. costatum, indicating a preingestive particle selection. At low seston load, clearance rate (CR) was closely and positively related to gill area and particle selection occurred only on the gills, between the ventral grooves and dorsal tracts. At higher seston load, palps exhibited a particle-sorting capacity dependent on gill area. Indeed, with small gills, an increase in selection efficiency (SE) and CR was positively related to palp area. On the other hand, large gills processed the particles without an effect of palps but with a decrease in CR. The functional responses associated with pallial organ variations clearly showed that the preingestive particle processing in oysters is an integrated mechanism dependant on the gill and labial palp areas.     

Detection of white spot syndrome virus (WSSV) in the Pacific oyster Crassostrea gigas

Oysters Crassostrea gigas were placed at water supply canals of three shrimp farms in Guasave, Mexico where WSSV outbreaks occur. Animals were sampled through April-August and September-December to detect WSSV DNA.By using three different PCR protocols, only oysters from a farm undergoing a WSSV outbreak were found WSSV-positive in gills and digestive gland. Two WSSV amplicons were sequenced and they corresponded over 99% to WSSV genome segments. Results showed that oysters can capture WSSV particles suspended in water. Susceptibility of oysters to WSSV infection and their role as a carrier remain to be determined.     

Tulane Virus as a Potential Surrogate To Mimic Norovirus Behavior in Oysters

Oyster contamination by noroviruses is an important health and economic problem. The present study aimed to compare the behaviors of Norwalk virus (the prototype genogroup I norovirus) and two culturable viruses: Tulane virus and mengovirus. After bioaccumulation, tissue distributions were quite similar for Norwalk virus and Tulane virus, with the majority of viral particles detected in digestive tissues, while mengovirus was detected in large amounts in the gills and mantle as well as in digestive tissues. The levels of persistence of all three viruses over 8 days were comparable, but clear differences were observed over longer periods, with Norwalk and Tulane viruses displaying rather similar half-lives, unlike mengovirus, which was cleared more rapidly. These results indicate that Tulane virus may be a good surrogate for studying norovirus behavior in oysters, and they confirm the prolonged persistence of Norwalk virus in oyster tissues.     

Carbon isotope signatures reveal that diet is related to the relative sizes of the gills and palps in bivalves

In marine bivalves, the relative sizes of the gills and palps appear to be a useful functional trait that reflect feeding mode, i.e. suspension feeders have relatively larger gills than palps for pumping, whereas deposit feeders have relatively larger palps than gills for sorting. Also, within a species, the relative sizes of the gills and palps are related to changes in local food conditions. However, there is still no firm evidence showing that differences in the relative gill and palp sizes between species are related to diet selection. Based on the knowledge that carbon and nitrogen isotope signatures of an animals tissues reflect past diet, we compared the relative gill and palp sizes of bivalves from Roebuck Bay, northwestern Australia with their carbon and nitrogen isotope signatures. The carbon isotope signatures distinguished clear differences in diet between bivalves along a gradient from suspension to deposit feeding, and strikingly this pattern was closely followed by the relative sizes of the gills and palps of the bivalves. This study confirms that relative gill and palp sizes in bivalves are a functional trait that can be used to compare resource use between species. Furthermore, these data may suggest that morphospace occupation, as determined by relative gill and palp sizes of bivalves, could reflect a gradient of resource use between species.     

Sublethal effects of experimental exposure to mercury in European flat oyster Ostrea edulis: Cell alterations and quantitative analysis of metal

Oysters display a diversity of uptake mechanisms for metallic elements and distribution in the target organs, namely gills and the digestive gland. Various tissues of the flat oyster, Ostrea edulis, were studied following experimental exposure to 0.025 m (5 g l) of mercury, for up to 34 days. All animals survived the treatment. Data indicate Hg accumulation in gill tissue with a maximum concentration of 38.76 g g dry weight after 25 days of exposure. Hg levels were lower in remaining tissues, in which the maximum concentration (18.47 mg g-1 dry weight) was reached after 18 days of exposure. After these times, concentration in both tissues decreased. Results show that oysters can accumulate Hg from the environment, without their survival being affected during the experimental period. Structural alteration of epithelial tissues of gill and digestive gland of flat oyster was comparable with effects described for other metallic elements in bivalve molluscs. Interstitial tissue was disorganized in the digestive gland, and ultrastructural changes in intracellular endomembranes were detected in epithelial cells of the digestive gland after 18 days of treatment. After 25 days, absorptive epithelial cells of gills showed highly dilated, swollen microvilli. These intracellular alterations are parameters of the incipient response to the accumulation of mercury.     

Detection and phylogenetic analysis of norovirus in Corbicula fluminea in a freshwater river in Japan

To study the molecular epidemiology of noroviruses (NoVs) in bivalves residing in freshwater rivers, we detected, quantified and phylogenetically analyzed the NoV genome in purified concentrates obtained from the gills and digestive diverticula of Corbicula fluminea in a freshwater river in Gunma Prefecture, Japan. We detected the NoV genome in 35 of the 58 C. fluminea samples. Based on our phylogenetic analysis, the NoV genome detected in the samples was classified into 4 genotypes (GI/1, GI/2, GI/3 and GI/4) in genogroup I and 5 genotypes (GII/3, GII/4, GII/5, GII/8 and GII/12) in genogroup II. The phylogenetic tree showed wide genetic diversity among the genogroups. In addition, more than 10(4) copies of the NoV genome were detected in 2 of 35 samples. These results suggest that the freshwater bivalve C. fluminea is a reservoir for NoVs, similar to seawater bivalves such as oysters.     

Detection of genogroup IV norovirus in wastewater and river water in Japan

Aims:  To test wastewater and river water in Japan for genogroup IV norovirus (GIV NoV).
Methods and Results:  Influent and effluent samples from a wastewater treatment plant and the Tamagawa River water samples were collected monthly for a year. The water samples were concentrated by the adsorption–elution method, using an HA electronegative filter with acid rinse procedure, followed by quantitative detection of GIV NoV using TaqMan-based real-time RT-PCR. Both wastewater and river water samples showed a high positive ratio of GIV NoV during winter and spring. The highest concentration in wastewater and river water was 6·9 × 10⁴ and 1·5 × 10⁴ copies l⁻¹, respectively.
Conclusions:  Presence of GIV NoV in the environments demonstrates that not only GI and GII NoVs but also GIV strains are circulating and that routine monitoring of GIV NoV in water environments is recommended to understand its epidemics, environmental distribution and potential health risks.
Significance and Impact of the Study:  This is the first study providing quantitative data on the occurrence of GIV NoV in environmental water over a 1-year period.     

二倍体和三倍体太平洋牡蛎鳃扫描电镜的比较

利用扫描电子显微镜技术对二倍体和三倍体太平洋牡蛎(Crassostrea gigas)鳃的表面结构进行了观察和比较。结果显示：三倍体牡蛎鳃丝的宽度、鳃丝间的距离较二倍体大;鳃丝的微细结构比二倍体更致密;鳃丝间通过丝问连接形成的孔洞大于二倍体。这些不同表明二倍体和三倍体呼吸及摄食可能存在差异。     

Bioavailability and toxicity of sediment-bound lead to a filter-feeder bivalve Crassostrea gigas (Thunberg)

Two different approaches were used to study the bioavailability of sediment-bound lead. In vitro techniques simulating the potential metal desorption under conditions prevailing in the digestive tract were assayed on a contaminated sediment. An experimental model of a food chain was designed to determine the retention of lead in the soft tissues of oysters according to the environmental source of the metal (water or sediment). Neither enzymatic action nor leaching at low pH (both aspects of digestion) induce the release of important lead amounts from particles. Therefore, after 3 weeks of exposure, the retention of lead from the trophic source is lower (1%) compared with direct contamination (5%). Lysosomes are the major intracellular structures responsible for lead storage in the gills, digestive tract and digestive gland. The abundance of lysosomes and their lead amount vary according to the gross concentrations in the soft tissues. The cytopathological data are in agreement with the results about lead accumulation: in oysters exposed to sediment-bound lead, impairments are not so marked as in individuals contaminated directly from water but the same organelles are concerned. Mitochondrial impairments may be related to the effect of lead on cellular respiration processes and changes involving the granular endoplasmic reticulum may have an effect on the level of protein synthesis. Cellular extrusions carrying away numerous lysosomes loaded with lead could account for the balancing of lead incorporation between 2 and 3 weeks of exposure.     

Systemic dissemination of H5N1 influenza A viruses in ferrets and hamsters after direct intragastric inoculation

Although oral exposure to H5N1 highly pathogenic avian influenza viruses is a risk factor for infection in humans, it is unclear how oral exposure to these virus results in lethal respiratory infections. To address this issue, we inoculated ferrets and hamsters with two highly pathogenic H5N1 strains. These viruses, inoculated directly into the stomach, were isolated from the large intestine and the mesenteric lymph nodes within 1 day of inoculation and subsequently spread to multiple tissues, including lung, liver, and brain. Histopathologic analysis of ferrets infected with virus via direct intragastric inoculation revealed lymph folliculitis in the digestive tract and mesenteric lymph nodes and focal interstitial pneumonia. Comparable results were obtained with the hamster model. We conclude that, in mammals, ingested H5N1 influenza viruses can disseminate to nondigestive organs, possibly through the lymphatic system of the gastrointestinal tract.     

Anti-poliovirus activity of Baccharis dracunculifolia and propolis by cell viability determination and real-time PCR

Aims:  The aim of this work was to evaluate the antiviral activities of Baccharis dracunculifolia (extract and essential oil), propolis and some isolated compounds (caffeic and cinnamic acids) against poliovirus type 1 (PV1) replication in HEp-2 cells.
Method:  Three different protocols (pre-, simultaneous and post-treatments) were used to verify the effect of addition time of the variables on PV1 replication by crystal violet method and relative viral RNA quantification by real-time PCR for analysing in which step of virus replication the variables could interfere.
Conclusions:  Data revealed that the B. dracunculifolia showed the best antiviral activity percentage in the simultaneous treatment, as well as lower relative viral quantification by real-time PCR. Variables might block partially the viral entry within cells, affect the steps of viral cycle replication into cells, or lead to RNA degradation before the virus entry into cells or after their release to the supernatant.
Significance and Impact of the Study:  Baccharis dracunculifolia is the most important botanical source of the south-eastern Brazilian propolis, and its potential for the development of new phytotherapeutic medicines has been investigated. Propolis is commonly used for its antimicrobial and immunomodulatory activities. Nevertheless, B. dracunculifolia and propolis effects on PV1 have not been investigated yet.