One Perkinsus species may hide another: characterization of Perkinsus species present in clam production areas of France.

Although clam populations in France are known to be infected with protozoans of the genus Perkinsus, no molecular characterization was previously performed on these parasites. Considering that several members of this genus have been associated with mortalities of molluscs worldwide, a study was undertaken in order to characterize these parasites in France. For that purpose, clams, Ruditapes philippinarum and R. decussatus, collected from different production areas and found to be infected with Perkinsus sp. in thioglycolate culture medium, were selected for PCR-RFLP tests and sequencing. Perkinsus olseni was detected in all the investigated areas and results also suggested the presence of P. chesapeaki in Leucate, a lagoon on the Mediterranean coast and in Bonne Anse in Charente Maritime, on the Atlantic coast. Clonal cultures from both detected species were produced in order to describe and compare in vitro stages. Differences in size between both Perkinsus spp. were noticed especially for schizonts and zoosporangia. Lastly, in situ hybridization tests allowed confirmation of the presence of both species in the same R. decussatus population and even in same clams. This is the first detection of P. chesapeaki in Ruditapes species and outside North America, which questions its introduction into Europe.

Diagnosis of Ostreid herpesvirus 1 in fixed paraffin-embedded archival samples using PCR and in situ hybridisation.

In 1994, some of the high mortality episodes that affected oysters cultured in France were associated with herpesviral infections. Through histology analysis, however, viral presence could only be suspected and confirmation of histological diagnosis by transmission electron microscopy was performed in only a few cases. Subsequently, the characterisation and genome sequencing of Ostreid herpesvirus 1 (OsHV-1) made possible the development of specific molecular detection (PCR and in situ hybridisation (ISH)). Using both molecular tools, attempts were made to screen for OsHV-1 a number of fixed, paraffin-embedded oyster samples collected and processed in 1994. The aim was to compare these techniques and to estimate the accuracy of histology-based indication of viral infection. Existing DNA extraction protocols were adapted for oyster samples and two pairs of specific primers targeting small fragments (less than 200bp) were designed (C(9)/C(10) and B(4)/B(3)). The poor consistency observed between the results of PCR with both primer pairs was confirmed by statistical analysis. C(9)/C(10), which targets a repeated region of the OsHV-1 genome, appears to be the primer of choice for viral detection in archival samples. In situ hybridisation may furnish complementary information concerning the localisation of viral foci. Under certain conditions, retrospective examination of archival samples by molecular techniques may therefore provide valuable epidemiological data.

Ecological analysis of digestive cancer mortality related to contamination by diarrhetic shellfish poisoning toxins along the coasts of France.

Shellfish consumers are exposed to the risk of diarrhea from, among other contaminants, algae that produce diarrhetic shellfish poisoning (DSP) toxins, such as Dinophysis spp. These illnesses have been effectively prevented since 1984, when a phycotoxin monitoring network was set up along the coasts of France. There is nonetheless concern that residual levels of okadaic acid, a known tumor promoter that is the main toxin present in French coastal waters, might increase the risk of cancer among regular shellfish consumers. To test this hypothesis, we conducted an ecological study linking digestive cancer mortality rates with a proxy measure of contamination by DSP toxins in 59 coastal areas. Observed and expected numbers of deaths (using national rates as the reference) were computed by sex, cause of death, and area for two time periods: 1984-1988 and 1989-1993. The level of contamination in each area was estimated by the total number of weeks since monitoring began that production was shut down because of DSP toxin contamination. Using both Poisson regressions and test for trends of standardized mortality ratios across four exposure categories, we found some evidence of associations for several digestive cancer sites (esophagus, stomach, colon, liver, and total digestive cancers for men; stomach and pancreatic cancers for women). Among men, the only statistically significant result that remained after taking possible confounding by alcohol use into account involved colon cancer. The conclusions provided by this analysis are very tentative; they need to be reproduced and interpreted in the light of additional information on the potential long-term effects of DSP toxins. In the absence of human data, they provide some indication of a possible association between exposure to DSP toxins and digestive cancers.

[Magnitude of rainfall on viral contamination of the marine environment during gastroenteritis epidemics in human coastal population]. / Importance de la pluviométrie sur la contamination virale du milieu littoral lors de phénomènes épidémiques dans la population.

BACKGROUND: Sewage treatments are not efficient to eliminate enteric microorganisms. Viruses are able to persist and are discharged into the marine environment with treated effluents. Few data are now available on the magnitude and the contributive processes of marine viral contamination. This work evaluates the relationship between the magnitude of rainfall and the viral contamination of the marine environment during winter epidemics of gastroenteritis in human coastal populations. METHODS: A RT-PCR method was used to detect enterovirus, hepatitis A virus, Norwalk-like virus, astrovirus and rotavirus in shellfish, harvested monthly between August 1995 and July 1998. The frequency of virus detection in shellfish was expressed as an Index of Viral Contamination. Acute gastroenteritis in the population was estimated using the French Sentinel System for Monitoring of Communicable Diseases. Rainfall effects on the efficiency of sewage treatment were assessed using an estimated staying time of sewage effluents in the plant. RESULTS: The results indicate that the highest viral contamination occurs in winter. Maximal indexes of viral contamination were respectively 70% in January 1996, 100% in January 1997, but only 31% in January 1998. Viral contamination variations seemed to follow the pattern of the winter epidemic of acute gastroenteritis in the local population in 1996 and 1997. These observations should be linked to the winter rainfalls. Heavy rains on short periods of time could create an hydraulic overload in the sewage treatment plant, reducing the staying time of the sewage effluents and thus the efficiency of the disinfection process. CONCLUSION: The magnitude of the viral contamination of shellfish seems to result from the simultaneity between the winter epidemics of acute gastroenteritis in the coastal population and heavy rainfall. To prevent public health hazards associated with shellfish consumption, the monitoring of microbiological quality in shellfish harvesting areas should include accompagning survey of viral epidemic in the coastal population, and of sewage outputs in the coastal environment.

Three-year study to assess human enteric viruses in shellfish.

The main pathogenic enteric viruses able to persist in the environment, such as hepatitis A virus (HAV), Norwalk-like virus (NLV), enterovirus (EV), rotavirus (RV), and astrovirus (AV), were detected by reverse transcription-PCR and hybridization in shellfish during a 3-year study. Oyster samples (n = 108), occasionally containing bacteria, were less frequently contaminated, showing positivity for AV (17%), NLV (23%), EV (19%), and RV (27%), whereas mussel samples, collected in areas routinely impacted by human sewage, were more highly contaminated: AV (50%), HAV (13%), NLV (35%), EV (45%), and RV (52%). Sequences obtained from HAV and NLV amplicons showed a great variety of strains, especially for NLV (strains close to Mexico, Snow Mountain Agent, or Norwalk virus). Viral contamination was mainly observed during winter months, although there were some seasonal differences among the viruses. This first study of virus detection over a fairly long period of time suggests that routine analysis of shellfish by a molecular technique is feasible.