Ann-Sofi Rehnstam-Holm

Environmental Factors Influencing Human Viral Pathogens and Their Potential Indicator Organisms in the Blue Mussel, Mytilus edulis: the First Scandinavian Report

ABSTRACT 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.

Improving marine water quality by mussel farming : a profitable solution for Swedish society

Abstract Eutrophication of coastal waters is a serious environmental problem with high costs for society globally. In eastern Skagerrak, reductions in eutrophication are planned through reduction of nitrogen inputs, but it is unclear how this can be achieved. One possible method is the cultivation of filter-feeding organisms, such as blue mussels, which remove nitrogen while generating seafood, fodder and agricultural fertilizer, thus recycling nutrients from sea to land. The expected effect of mussel farming on nitrogen cycling was modeled for the Gullmar Fjord on the Swedish west coast and it is shown that the net transport of nitrogen (sum of dissolved and particulate) at the fjord mouth was reduced by 20%. Existing commercial mussel farms already perform this service for free, but the benefits to society could be far greater. We suggest that rather than paying mussel farmers for their work that nutrient trading systems are introduced to improve coastal waters. In this context an alternative to nitrogen reduction in the sewage treatment plant in Lysekil community through mussel farming is presented. Accumulation of bio-toxins has been identified as the largest impediment to further expansion of commercial mussel farming in Sweden, but the problem seems to be manageable through new techniques and management strategies. On the basis of existing and potential regulations and payments, possible win-win solutions are suggested.

Parvilucifera infectans norén et moestrup gen. et sp. nov. (perkinsozoa phylum nov.): a parasitic flagellate capable of killing toxic microalgae

Summary The toxic dinoflagellate Dinophysis, collected on the Swedish West Coast, was found to contain round bodies previously interpreted as the result of sexual reproduction. After two weeks of darkness in the refrigerator, all Dinophysis had died, however, and round bodies were present. These proved to be sporangia of a parasitic protist, here named Parvilucifera infectans gen. et sp. nov. Its identity was examined by LM, EM, and DNA sequencing. It is related to Perkinsus, an oyster-killing protist, and Colpodella, a phagocytic protist. Perkinsus has been indicated by 18S rRNA sequencing to be related to dinoflagellates, and the opportunity was taken to examine the ultrastructure of the flagellar apparatus of Parvilucifera in detail. Parvilucifera and its allies, known as perkinsids, share features with both dinoflagellates and apicomplexans. They do not fit readily into any of these groups but appear to form a missing link between them. They are described as a taxon on level with the other alvelolate phyla, as Perkinsozoa phylum nov. Infection studies showed that Parvilucifera infectans infects several other dinoflagellates, notably Alexandrium spp. which are responsible for PSP (paralytic shellfish poisoning). A discussion of the ecological role, in terms of biocontrol of harmful algal blooms, is included.

Polymerase Chain Reaction in Detection of Gymnodinium mikimotoi and Alexandrium minutum in Field Samples from Southwest India

Abstract: Polymerase chain reaction (PCR) primers were constructed for the detection of two toxic dinoflagellate species, Gymnodinium mikimotoi and Alexandrium minutum. The primers amplified a product of expected size from cultured cells of G. mikimotoi and A. minutum. The species-specific primers targeting G. mikimotoi did not yield any product with a wide range of other cultured algae used as negative controls. Primers designed for A. minutum were species-group-specific since it PCR yielded a product from the closely related species A. ostenfeldii and A. andersonii, but not from other species of this genus tested. The confirmation of PCR products was performed by digestion of the products with restriction enzymes. Sensitivity analyses of the primers on DNA template from cultured cells was positive by PCR at a DNA template concentration of 1.5 × 10−4 ng/μl (0.3 cells/L) for A. minutum, and at a DNA concentration of 2.5 × 10−2 ng/μl (697 cells/L) for G. mikimotoi. The PCR method for detection of G. mikimotoi and A. minutum was applied on field samples collected with a plankton net. Gymnodinium mikimotoi could be detected in 11 field samples by microscopy, and all these field samples were positive by PCR. The cell counts of G. mikimotoi in simultaneously collected water samples ranged from 306 to 2077/L. Alexandrium minutum could be detected by microscopy in 3 different field samples. The cell counts in water samples collected at the same time as the net samples ranged from 115 to 1115 cells/L. Alexandrium minutum was detected by PCR in these field samples, with the exception of the sample displaying the lowest cell count (115 cells/L). Plankton samples that were negative by microscopy for any of the two target species were also negative by PCR. All the PCR products from field samples were confirmed by restriction enzyme digestion. The application of PCR-based detection of harmful algal bloom species for aquaculture and monitoring purposes in natural field samples is discussed.

Occurrence and potential pathogenesis of Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus on the South Coast of Sweden

During the summer of 2006, several wound infections - of which three were fatal - caused by Vibrio cholerae were reported from patients who had been exposed to water from the Baltic Sea. Before these reports, we initiated a sampling project investigating the occurrence of potential human pathogenic V. cholerae, Vibrio vulnificus and Vibrio parahaemolyticus in The Sound between Sweden and Denmark. The Blue mussel (Mytilus edulis) was used as an indicator to follow the occurrence of vibrios over time. Molecular analyses showed high frequencies of the most potent human pathogenic Vibrio spp.; 53% of mussel samples were positive for V. cholerae (although none were positive for the cholera toxin gene), 63% for V. vulnificus and 79% for V. parahaemolyticus (of which 47% were tdh(+) and/or trh(+)). Viable vibrios were also isolated from the mussel meat and screened for virulence by PCR. The mortality of eukaryotic cells when exposed to bacteria was tested in vivo, with results showing that the Vibrio strains, independent of species and origin, were harmful to the cells. Despite severe infections and several deaths, no report on potential human pathogenic vibrios in this area had been published before this study.

PCR detection of dinoflagellate cysts in field sediment samples from tropic and temperate environments

Species-specific primers were constructed for Scrippsiella trochoidea, Protoceratium reticulatum and Lingulodinium polyedrum, which all are common cosmopolitan cyst forming dinoflagellates. The designed primers amplified a product of expected size from cultured planktonic cells of the three species, and did not yield any product with a wide range of other algal species used as negative controls. The PCR method for detection and identification of dinoflagellate cysts from the three species was applied on field samples. Undisturbed surface sediment was collected along the southwest coast of India and the west coast of Sweden. DNA extract from sediment including DNA from dinoflagellate cysts could be obtained after repeated grinding with mortar and pestle under liquid nitrogen followed by microwave boiling. All sediment samples that contained any of the target species as confirmed by microscopy, were also positive for PCR. Field samples negative for any of the target species by microscopy, were also negative by PCR. Restriction enzyme digestion and/or DNA sequencing confirmed the specificity of all the PCR products from field samples. The yield of DNA from sediment extraction was low, and therefore nested PCR was necessary for accurate species-specific detection of the three species in most of the field samples.

Characteristics of potentially pathogenic vibrios from subtropical Mozambique compared with isolates from tropical India and boreal Sweden.

Reported outbreaks of Vibrio parahaemolyticus have increased worldwide, particularly in regions of high seafood consumption. In Mozambique, seafood constitutes an important food resource and diarrheal diseases are common among its inhabitants. Edible clams were collected in Maputo Bay during both the dry and rainy seasons, with the results showing the number of viable counts of vibrios in clams to peak during the latter. Vibrioxa0parahaemolyticus was the predominant species identified among the isolated strains. Although only one of 109 total strains carried the tdh virulence gene, 69% of isolates showed evidence of hemolytic capacity when subjected to a functional test. Similar virulence patterns and biochemical properties were found in strains isolated from Indian and Swedish marine waters. Antibiotic resistance was, however, more pronounced in strains isolated from these latter two environments.