5-bromodeoxyuridine induction of hematopoietic neoplasia and retrovirus activation in the soft-shell clam, Mya arenaria

Hematopoietic neoplasia and virus replication were induced in shoft-shell clams, Mya arenaria, by 5-bromodeoxyuridine (5-BrdUrd). Eighty clams were found to be nonneoplastic by the in vivo bleeding method. These clams were randomly distributed into four aquaria and maintained in seawater at 1°C. 5-BrdUrd was added to aquaria in the following concentrations: 0, 20, 100, and 200 μg/ml. After 4 days of treatment, the aquaria were drained, rinsed, and fresh sea water without 5-BrdUrd was added. Sea water was changed on a weekly basis thereafter. The clams were diagnosed for neoplasia by the in vivo method at days 4, 9, 16, and 23 of the experiment. Results showed that on day 23, neoplasia was not found in the aquarium without 5-BrdUrd, but in the aquaria containing 20, 100, and 200 μg/ml of 5-BrdUrd the incidences of neoplasia were 37, 79, and 35%, respectively. 5-BrdUrd-induced neoplastic tissue was homogenized and subjected to differential ultracentrifugation. Retrovirus-like particles resembling ones previously shown to induce neoplasia were isolated from neoplastic clams which were induced by 5-BrdUrd. When these particles were inoculated into healthy clams, the clams developed neoplasia. Neoplastic hemocytes, cultured in sea water containing 50 μg/ml of 5-BrdUrd, formed pseudopodia after 4 days. Pseudopod formation is a trait of normal hemocytes. This indicates that differentiation of neoplastic hemocytes may be induced by the drug.     

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.     

Exposure to 2500 Lux Increases Serum Melatonin in Venezuelan Equine Encephalomyelitis

When mice infected with the Venezuelan equine encephalomyelitis (VEE) virus were exposed to 2500 lux with a 12 h light: 12 h dark photoperiod, the serum levels of melatonin (MLT) remained constantly elevated. In mice exposed to 400 lux low levels of serum MLT were detected during the day and high levels during the night. An increase in the survival rate of the infected mice from 6 to 13 days after virus inoculation was also observed. The significant increment in the concentration of serum MLT produced by the high intensity light could be responsible for the longer survival rate of mice infected with the VEE virus.     

Virio- and bacterioplankton of a small river: Influence that viruses have on the mortality of heterotrophic bacteria

The spatial distribution of virio- and bacterioplankton and the role that viruses play in heterotrophic bacteria elimination was studied in the small Il’d’ River (Rybinskoe Reservoir tributary, Upper Volga). The bacterioplankton abundance and biomass constituted 3.3–19.6 (8.5 on average) million cells/ml and 501–2353 mg/m³ (1054 mg/m³ on average), respectively. The highest abundance was registered for the shallow areas under anthropogenic and zoogenic load. The virus abundance varied from 7.4 to 66.9 million particles/ml, being 30.9 million particles/ml on average. The bacteria: virus ratio was in the range of 1.2–11.0 (4.0 on average); the maximal values were registered for the deepest parts of the river. One infected bacterial cell carried up to 45 virile phages. The virus-induced mortality (VIM) was in the range of 1.1–7.8% (3.7% on average) from daily bacterial production. These data evidenced a high abundance of bacterioplankton in the studied river and an insignificant impact of virus-mediated regulation on bacterial abundance and production.     

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.     

Perkinsosis in the Manila clam Ruditapes philippinarum affects responses to the harmful-alga, Prorocentrum minimum

The dinoflagellate Prorocentrum minimum is increasingly recognized as a harmful algal bloom (HAB) species that affects filter-feeding shellfish. An experiment was done to investigate possible interactions between parasitic diseases and exposure to P. minimum in Manila clams, Ruditapes philippinarum. Manila clams, with variable levels of infection with Perkinsus olseni, were exposed for three or six days to the benign phytoplankton species Chaetoceros neogracile or a mixed diet of C. neogracile and P. minimum. After three or six days of exposure, clams were assessed individually for condition index, parasite status, and plasma and hemocyte parameters (morphological and functional) using flow-cytometry. Histological evaluation was also performed on individual clams to assess prevalence and intensity of parasitic infection, as well as other pathological conditions.Prorocentrum minimum caused several changes in Manila clams, especially after six days of exposure, such as decreased hemocyte phagocytosis and size and clam condition index. Pathological conditions observed in Manila clams exposed to P. minimum were hemocyte infiltration in the intestine and gonad follicles, myopathy, and necrosis of the intestine epithelial cells. The parasite P. olseni alone had no significant effect on Manila clams, nor did it modulate the hemocyte variables in clams exposed to P. minimum; however, the parasite did affect the pathological status of Manila clams exposed to the P. minimum culture, by causing atrophy and degeneration of residual ova in the gonadal follicles and hyaline degeneration of the muscle fibers, indicating synergistic effects of both stressors on the host over a short period of time. Additionally, an in vitro experiment also demonstrated detrimental effects of P. minimum and exudates upon P. olseni cells, thus suggesting HAB antagonistic suppression of transmission and proliferation of the parasite in the natural environment over a longer period of time. The results of this experiment demonstrate the complexity of interactions between host, parasite, and HAB.     

Chlorophyllin protects HEp-2 cells from nuclear fragmentation induced by poliovirus

AIMS: Chlorophyllin (CHLN) is a synthetic derivative of chlorophyll that possesses antimutagenic activity against several environmental contaminants. In the present study, CHLN was assayed for its capacity to prevent nuclear fragmentation (NF) in HEp-2 cells infected with poliovirus. METHODS AND RESULTS: CHLN was assayed at concentrations of 0.5 and 2.5 microg ml(-1), and NF was monitored using the comet assay and acridine orange staining. We demonstrated that CHLN reduced the percentage of NF in poliovirus-infected HEp-2 cells, when cells were treated with drug before infection or exposed continuously to drug. However, the highest degree of protection was achieved when the virus was exposed to CHLN before infection followed by protocol where infected cultures were continuously exposed to the drug after infection. CONCLUSIONS: It is suggested that CHLN primarily binds to the virus which inhibits cell penetration, thereby maintaining nuclear integrity. SIGNIFICANCE AND IMPACT OF THE STUDY: Considering that CHLN has several beneficial properties and no significant toxic effects in humans and animals, it would be an ideal candidate drug to test for antiviral activity.     

Quantitative Determination of Gymnodimine-A by High Performance Liquid Chromatography in Contaminated Clams from Tunisia Coastline

Quantitative determination by high performance liquid chromatography (HPLC) was performed for gymnodimine-A (GYM-A), a phycotoxin responsible for the contamination of Tunisian clams. This study demonstrates a rapid and reproducible HPLC-ultraviolet (UV) method for extraction, detection and quantification of GYM-A in toxic clams. The extraction of GYM-A from the digestive gland of clams in acetone, subsequent clean-up with diethyl ether and extraction with dichloromethane is the more valid protocol. Chromatography analyses were performed using a gradient of acetonitrile–water (10:90 to 90:10), containing trifluoroacetic acid (0.1%) for 20 min at 1 mL/min rate with a C18 column. Recovery rates exceeded 96%, and limits of detection and quantification were 5 ng/mL and 8 ng/g digestive gland, respectively. Repeatability and reproducibility were tested for various samples containing different levels of GYM-A. A significant correlation was observed between toxicity level of samples and the determined amount of GYM-A. Also, the persistence of GYM-A in contaminated clams from Boughrara lagoon was demonstrated. The kinetics discharge study of GYM-A in controlled medium, during 1 month, showed that the process of depuration was biphasic with an exponential discharge of 75% of the total amount of sequestered GYM-A during the first 12 days followed by a slow discharge (>10%) for the subsequent days up to the seventeenth day. This is the first time that a quantitative study of GYM-A in clams from Tunisian coasts is performed through the development of a new method for detection and quantify of this phycotoxin. We found HPLC-UV a reliable and suitable alternative to the mouse bioassay.     

Assessment of haemic neoplasia in different soft shell clam Mya arenaria populations from eastern Canada by flow cytometry

Diagnosis of haemic neoplasia (HN) in the soft shell clam, Mya arenaria, is often achieved by hematocytology and histology. Since neoplastic cells display tetraploid DNA contents, haemocyte cell cycle analysis was developed for use as a diagnosis tool. The aim of this study was to assess the application of a flow cytometry procedure of cell cycle analysis established for the common cockle, to clams and to evaluate different thresholds of value for the percentage of tetraploid cells for establishing HN disease status of individual clams and clam populations. HN status of six clam populations from eastern Canada was determined. Results of the present study demonstrate a flow cytometry procedure to be useful for HN diagnosis in clams. Individual clams were considered to be affected by HN when presenting at least 20% of haemocytes in S-4N phase; and negative when presenting less that 5% of haemocytes in S-4N phase. As discussed in this paper, intermediate cases represent uncertain diagnoses including either false-negative or false-positive clams, which are difficult to discriminate. At a population level, an additional threshold of 15% for the mean intensity of the disease is proposed, which means having in the population several individual clams presenting more than 20% of their haemocytes in S-4N phase. Based on these thresholds of value, only one population was considered as free of HN disease, and one population was unequivocally affected by HN. For the four other clam populations, further investigations are needed toward development and use of specific and objective biomarkers of HN.     

Host specificity of Pisidium coreanum (Bivalvia: Sphaeriidae) to larval infection with a human intestinal fluke Echinostoma cinetorchis (Trematoda: Echinostomatidae) in Korea

The fingernail clam, Pisidium coreanum, has been traditionally consumed raw as a so-called drug therapy by patients with bone fractures in Korea. The present study was designed to determine the possible occurrence and, if present, the prevalence of Echinostoma cinetorchis in P. coreanum collected at a local site, and to determine the susceptibility of the clams in the laboratory to infection with miracidia and cercariae of E. cinetorchis. No cercariae or metacercariae of E. cinetorchis were observed in field-collected P. coreanum clams. In susceptibility experiments with laboratory-reared clams, individuals exposed to miracidia of E. cinetorchis did not release cercariae by 20 days after exposure; necropsy of exposed clams failed to show development of any sporocysts or rediae. To confirm the possibility of these clams serving as an experimental second intermediate host of E. cinetorchis, 20 of them were exposed to E. cinetorchis cercariae from experimentally infected Segmentina hemisphaerula that had been previously exposed to miracidia of E. cinetorchis; all exposed clams became infected. Metacercariae from clams at 14 days postinfection were fed to rats, and adult worms were recovered from the ileocecal regions. This is the first report of P. coreanum serving as second intermediate host of E. cinetorchis.     

Delayed gametogenesis of Mya arenaria in the Saguenay fjord (Canada): a consequence of endocrine disruptors?

The Saguenay fjord located in Canada on the north coast of the Saint-Lawrence Estuary, is well known for its multiple contaminations following a linear distribution upstream to downstream. Mya arenaria is well established in the fjord and potentially exposed to persistent sediment contamination as an endobenthic bivalve. From May to October 1997, clams energy storage and utilization in the gonad was shown to be closely linked with reproduction. Wherever the sampling site location in the fjord, a seasonal pattern is evident for lipid and glycogen levels in the gonad, this, reflecting the vitellogenic process. Nevertheless, in comparison with energy status and gametogenesis of clams collected on the north coast of the Saint-Lawrence Estuary, clams located in the fjord have to face limited nutritive conditions resulting in a single and shorter reproductive period. Although environmental factors could explain differences in physiological condition and reproductive status observed between clams from the fjord and the estuary, our results can discriminate clams from the upper part of the fjord, according to a delayed gametogenesis, concomitant with significant higher gonad glycogen concentrations observed in June 1996 and 1997. A persistent dysfunction of a vitellogenic process is suspected to be due to the exposure to anti-estrogenic contaminants.     

Airborne Stability of Tailless Bacterial Viruses S-13 and MS-2

The effect of relative humidity (RH) on the airborne stability of two small bacterial viruses, S-13 and MS-2, was studied. Poorest recovery of S-13 was obtained at 50% RH. Humidification prior to aerosol sampling significantly increased the recovery of S-13 at RH deleterious to the airborne virus. A commercial preparation of MS-2 suspended in a buffered saline solution showed a rapid loss of viability at RH above 30%, whereas a laboratory preparation containing 1.3% tryptone showed high recoveries at all RH studied. Dilution of the commercial MS-2 into tryptone broth conferred stability on the airborne virus. Humidification prior to sampling significantly reduced the viable recovery from aerosols of commercial MS-2, whereas the laboratory preparation was unaffected.     

Environmental Cadmium Exposure Impacts Physiological Responses in Manila clams

The physiological responses of marine bivalves to chronic cadmium (Cd) exposure at sub-lethal concentrations have been well documented. As of now, few studies have examined the effect of Cd exposure and subsequent recovery period at environmentally realistic concentrations. In this study, environmentally, Cd exposures were performed to assess the physiological responses of the Manila clam Ruditapes philippinarum. The clams were exposed to waterborne Cd at two environmentally realistic concentrations (4 and 40 μg L^?1) for 35 days and then allowed to recover for another 35 days. The accumulation and elimination of Cd in R. philippinarum were tissue-specific and dose- and time-dependent. Cd accumulation increased sharply in the digestive gland, and Cd elimination was rapid in the gill. Major physiological responses, including clearance rate, absorption efficiency, respiration rate, excretion rate, oxygen to nitrogen ratio, and scope for growth, were significantly affected by Cd exposure. Yet, the clams exposed to 4-μg L^?1 Cd were able to quickly recover their normal physiological processes and clearly exhibited catch-up growth once they were transferred to clean seawater. Hence, R. philippinarum can exhibit good physiological plasticity when confronted with moderately environmental Cd exposure. All physiological responses measured exhibited a highly significant and generally predictable correlation with tissue Cd concentration, which in turn, reflected environmentally realistic exposure conditions. Our results further confirm that the measurement of physiological responses is a sensitive method for assessing stress at environmentally realistic metal concentrations.     

Photodynamic treatment of herpes simplex virus during its replicative cycle.

Photodynamic treatment of herpes simplex virus type 1-infected hamster embryo fibroblasts (LSH strain) with a low concentration of proflavine (0.08 mug/10(5) cells per ml), a 3-9-diamine acridine dye, inhibited production not only of infectious progeny but also of virion particles. However, there was no appreciable inhibition of viral or cellular DNA synthesis, even when the infected cells were repeatedly exposed to this low concentration of dye and light during the replication cycle of the virus. It thus appears that photodynamic treatment of infected cells interferes with the processes involved in virus maturation.     

Ultrastructure of Measles Virus in Cultures of Hamster Cerebellum

Replication of Edmonston strain of measles virus in cultures of hamster central nervous system tissue was studied by electron microscopy of ultrathin sections. Infected cultures were fixed from 3 hr to 39 days postinoculation (PI). Measles nucleocapsid was first seen within the cytoplasm of giant cells, the latter appearing 5 to 6 days PI. Measles virus particles were most abundant at 10 days PI and appeared to bud off from areas of the cell membrane along which nucleocapsid was aligned. Intranuclear nucleocapsid was more abundant at later stages, and by 39 days PI entire nuclei were seen to be occupied. By this time, the cytoplasmic formations, which had been sequestered by membranes, appeared to lose their regular structure. Budding viral particles at 39 days PI were of a much simplified structure and did not involve the alignment of nucleocapsid about their periphery.     

Serological Diagnosis of Classical Swine Fever

Antibody levels in post-infection sera from a pig inoculated with a low virulent strain of classical swine fever virus (Hannover 62) and in sera from two pigs inoculated with another low virulent strain (Spielbach 66) and from an in-contact pig were assayed by complement fixation and immunofluorescence using classical swine fever virus (ALD strain) and bovine virus diarrhoea virus (UG 59 strain) as antigens. The complement fixation test used was modified by addition of a preparation of porcine Glq to the complement and by mercaptoethanol treatment of the immune serum before use. The mercaptoethanol treatment of the immune serum resulted in complete elimination of a haemolytic prozone often seen with porcine immune sera. In the sera from the inoculated animals complement-fixing antibodies appeared earlier than neutralizing antibodies. A few weeks after inoculation there was a correlation between the presence of complement-fixing and neutralizing antibodies. During the entire observation period of 13 weeks it was not possible to demonstrate complement-fixing or neutralizing antibodies in serum from a pig exposed to infection by contact with the two pigs inoculated with the Spièlbach 66 strain of classical swine fever virus.     

At the tipping point: Differential influences of warming and deoxygenation on the survival,emergence, and respiration of cosmopolitan clams

Although warming and low dissolved oxygen (DO) levels are co‐occurring significant climatic stressors in the ocean, the combined effects of these stressors on marine benthic animals have not been well established. Here, we tested the effects of elevated temperatures and low dissolved oxygen levels on the survival, emerging behavior from sediment, and the respiration of juvenile cosmopolitan Manila clams (Venerupis philippinarum) by exposing them to two temperatures (20 and 23.5°C) and DO levels (3.5 and 6–7 mg/L). Although within previously described tolerable ranges of temperature and DO, this 3.5°C increase in temperature combined with a 50% decrease in DO had a devastating effect on the survival of clams (85% mortality after 8 days). The mortality of clams under normoxia at 23.5°C appeared to be higher than under the low DO condition at 20°C. On the other hand, more clams emerged from sediment under the low DO condition at 20°C than under any other conditions. Oxygen consumption rates were not significantly affected by different conditions. Our results suggest temperature elevation combined with low oxygen additively increases stress on Manila clams and that warming is at least as stressful as low DO in terms of mortality. However, low DO poses another threat as it may induce emergence from sediment, and, thus increase predation risk. This is the first evidence that a combination of warming and deoxygenation stressors should reduce population survival of clams much more so than changes in a single stressor.     

Bioaccumulation and depuration of enteroviruses by the soft-shelled clam, Mya arenaria.

Low levels of feces-associated natural virus, simulating virus numbers estimated to exist in moderately polluted shellfish-growing waters, were used to evaluate the effectiveness of depuration as a virus depletion procedure in soft-shell clams. Depuration effectiveness depended upon the numbers of virus bioaccumulated and whether virus was solids associated. Virus uptake was greatest when viruses were solids associated and pollution levels were equivalent or greater than those likely to be found in grossly polluted growing waters. Virtually all bioaccumulated feces-associated natural virus was deposited within either the hepatopancreas or siphon tissues. Viruses usually were eliminated within a 24- to 48-h depuration period. Dependence upon depuration of clams to elimate health hazards of virus etiology involved a risk factor not measureable in the study. The greatest reduction of health risks would come from the routine depuration of clams harvested from growing waters of good sanitary quality.     

Bioaccumulation and depuration of enteroviruses by the soft-shelled clam, Mya arenaria.

Low levels of feces-associated natural virus, simulating virus numbers estimated to exist in moderately polluted shellfish-growing waters, were used to evaluate the effectiveness of depuration as a virus depletion procedure in soft-shell clams. Depuration effectiveness depended upon the numbers of virus bioaccumulated and whether virus was solids associated. Virus uptake was greatest when viruses were solids associated and pollution levels were equivalent or greater than those likely to be found in grossly polluted growing waters. Virtually all bioaccumulated feces-associated natural virus was deposited within either the hepatopancreas or siphon tissues. Viruses usually were eliminated within a 24- to 48-h depuration period. Dependence upon depuration of clams to elimate health hazards of virus etiology involved a risk factor not measureable in the study. The greatest reduction of health risks would come from the routine depuration of clams harvested from growing waters of good sanitary quality.