Molecular Characterization of an Endozoicomonas-Like Organism Causing Infection in the King Scallop (Pecten maximus L.).

One of the fastest growing fisheries in the UK is the king scallop (Pecten maximus L.), also currently rated as the second most valuable fishery. Mass mortality events in scallops have been reported worldwide, often with the causative agent(s) remaining uncharacterized. In May 2013 and 2014, two mass mortality events affecting king scallops were recorded in the Lyme Bay marine protected area (MPA) in Southwest England. Histopathological examination showed gill epithelial tissues infected with intracellular microcolonies (IMCs) of bacteria resembling Rickettsia-like organisms (RLOs), often with bacteria released in vascular spaces. Large colonies were associated with cellular and tissue disruption of the gills. Ultrastructural examination confirmed the intracellular location of these organisms in affected epithelial cells. The 16S rRNA gene sequences of the putative IMCs obtained from infected king scallop gill samples, collected from both mortality events, were identical and had a 99.4% identity to 16S rRNA gene sequences obtained from "Candidatus Endonucleobacter bathymodioli" and 95% with Endozoicomonas species. In situ hybridization assays using 16S rRNA gene probes confirmed the presence of the sequenced IMC gene in the gill tissues. Additional DNA sequences of the bacterium were obtained using high-throughput (Illumina) sequencing, and bioinformatic analysis identified over 1,000 genes with high similarity to protein sequences from Endozoicomonas spp. (ranging from 77 to 87% identity). Specific PCR assays were developed and applied to screen for the presence of IMC 16S rRNA gene sequences in king scallop gill tissues collected at the Lyme Bay MPA during 2015 and 2016. There was 100% prevalence of the IMCs in these gill tissues, and the 16S rRNA gene sequences identified were identical to the sequence found during the previous mortality event.IMPORTANCE Molluscan mass mortalities associated with IMCs have been reported worldwide for many years; however, apart from histological and ultrastructural characterization, characterization of the etiological agents is limited. In the present work, we provide detailed molecular characterization of an Endozoicomonas-like organism (ELO) associated with an important commercial scallop species.

Arcobacter lekithochrous sp. nov., isolated from a molluscan hatchery.

Four bacterial strains, LFT 1.7T, LT2C 2.5, LT4C 2.8 and TM 4.6, were isolated from great scallop (Pecten maximus) larvae and tank seawater in a Norwegian hatchery and characterized by a polyphasic approach including determination of phenotypic, chemotaxonomic and genomic traits. All were Gram-stain-negative, motile rods, oxidase- and catalase-positive and required sea salts for growth. Major fatty acids present were summed feature 3 (C16 : 1ω7c/C16 : 1ω6c), summed feature 8 (C18 : 1ω7c or C18 : 1ω6c), C16 : 0, C14 : 0, summed feature 2 (C14 : 0 3-OH/iso-C16 : 1 I), C12 : 0 3-OH and C12 : 0. Strain LFT 1.7T contained menaquinone MK-6 as the sole respiratory quinone. Phylogenetic analysis based on 16S rRNA gene sequences indicated that all strains formed a distinct lineage within the genus Arcobacter with a low similarity to known species (94.77-95.32 %). The DNA G+C content was 28.7 mol%. Results of in silico DNA-DNA hybridization and average nucleotide identity confirmed that the isolates constitute a novel species of Arcobacter, for which the name Arcobacter lekithochrous sp. nov. is proposed. The type strain is LFT 1.7T (=CECT 8942T=DSM 100870T).

Neptuniibacter pectenicola sp. nov. and Neptuniibacter marinus sp. nov., two novel species isolated from a Great scallop (Pecten maximus) hatchery in Norway and emended description of the genus Neptuniibacter.

Nine isolates obtained from a great scallop hatchery in Norway were characterized using a polyphasic approach. Strains were Gram-negative, aerobic and motile rods with oxidative metabolism. Phylogenetic analysis based on the sequences of 16S rRNA and rpoB genes showed that these strains formed two different groups associated with members of the genus Neptuniibacter. DNA-DNA hybridization (DDH) and Average Nucleotide Identity (ANI) demonstrated that the isolates constituted two novel species of this genus, which can be phenotypically differentiated from their closest relatives. The names Neptuniibacter marinus sp. nov. and Neptuniibacter pectenicola sp. nov are proposed, with ATR 1.1T (=CECT 8938T=DSM 100783T) and LFT 1.8T (=CECT 8936T=DSM 100781T) as respective type strains.

Characterization of the microbiota associated to Pecten maximus gonads using 454-pyrosequencing.

A next-generation sequencing (NGS) approach was used to study the microbiota associated to Pecten maximus broodstock, applying pyrosequencing of PCR-amplified V1-V4 16S rRNA gene regions. We analysed the resident bacterial communities in female and male scallop gonads before and after spawning. DNA samples were amplified and quality-filtered reads were assigned to family and genus taxonomic levels using the Ribosomal Database Project classifier. A total of 18,520 sequences were detected, belonging to 13 phyla, including Proteobacteria (55%), Bacteroidetes (11,7%), Firmicutes (3%), Actinobacteria (2%) and Spirochaetes (1,2%), and 110 genera. The major fraction of the sequences detected corresponded to Proteobacteria, Beta- and Gammaprotebacteria being the most abundant classes. The microbiota of P. maximus gonad harbour a wide diversity, however differences on male and female samples were observed, where female gonad samples show a larger number of genera and families. The dominant bacterial genera appeared to be Delftia, Acinetobacter, Hydrotalea, Aquabacterium, Bacillus, Sediminibacterium, Sphingomonas, and Pseudomonas that were present among the four analysed samples. This next generation sequencing technique, applied for the first time in P. maximus (great scallop) gonads was useful for the study of the bacterial communities in this mollusc, unravelling the great bacterial diversity in its microbiota. [Int Microbiol 19(2): 93-99(2016)].

Characterization of the microbiota associated to Pecten maximus gonads using 454-pyrosequencing

A next-generation sequencing (NGS) approach was used to study the microbiota associated to Pecten maximus broodstock, applying pyrosequencing of PCR-amplified V1-V4 16S rRNA gene regions. We analysed the resident bacterial communities in female and male scallop gonads before and after spawning. DNA samples were amplified and quality-filtered reads were assigned to family and genus taxonomic levels using the Ribosomal Database Project classifier. A total of 18,520 sequences were detected, belonging to 13 phyla, including Proteobacteria (55%), Bacteroidetes (11,7%), Firmicutes (3%), Actinobacteria (2%) and Spirochaetes (1,2%), and 110 genera. The major fraction of the sequences detected corresponded to Proteobacteria, Beta- and Gammaprotebacteria being the most abundant classes. The microbiota of P. maximus gonad harbour a wide diversity, however differences on male and female samples were observed, where female gonad samples show a larger number of genera and families. The dominant bacterial genera appeared to be Delftia, Acinetobacter, Hydrotalea, Aquabacterium, Bacillus, Sediminibacterium, Sphingomonas, and Pseudomonas that were present among the four analysed samples. This next generation sequencing technique, applied for the first time in P. maximus (great scallop) gonads was useful for the study of the bacterial communities in this mollusc, unravelling the great bacterial diversity in its microbiota (AU)

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Sinobacterium norvegicum sp. nov., isolated from great scallop (Pecten maximus) broodstock and emended description of Sinobacterium caligoides.

Six isolates were recovered from great scallop (Pecten maximus) broodstock in a hatchery in Bergen, Norway. The strains were thoroughly characterized by a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences indicated that these strains are related to the genus Sinobacterium, showing sequence similarities between 96.97 and 97.63 % with the only species of the genus, Sinobacterium caligoides. Phenotypic characterization showed that the strains are typical marine halophiles, Gram negative, aerobic chemoorganotrophs, and allowed their differentiation from the closely related taxa. The G+C content of the novel strains was 52.2 ± 1 mol% and the predominant fatty acids were C16:0, C16:1 ω7c/C16:1 ω6c and C18:1 ω7c. The value for DNA-DNA relatedness between strain 3CM4(T) and the S. caligoides type strain LMG 25705(T) was 46  %. Hybridization values between strain 3CM4(T) and the other scallop isolates ranged from 82 to 93.6 %. Based on all data collected, the six scallop strains represent a novel species of the genus Sinobacterium, for which the name Sinobacterium norvegicum sp. nov., is proposed with strain 3CM4(T) (=CECT 8267(T); =CAIM 1884(T)) as type strain.

Sources of variation of Escherichia coli concentrations in bivalve molluscs.

Bivalve molluscs can concentrate contaminants, including pathogenic microorganisms, from the water column during their normal filter-feeding activity. In the European Union, the risk of human and animal faecal contamination in bivalves is estimated by determining the concentration of Escherichia coli in time-series samples from production areas. A structured field study was undertaken to determine the extent to which such concentrations varied between sites, sampling occasions and shellfish species and to determine the residual variability of the method. E. coli was enumerated in three species of bivalve mollusc (Crassostrea gigas, Mytilus spp. and Pecten maximus) co-located in each of three geographically separate commercial shellfisheries. The data were subjected to analysis of variance (ANOVA). This showed that the effects of site, sampling occasion, species and site/sampling occasion interaction were all significant. The proportion of variation due to site was markedly greater than that due to other factors. Post-ANOVA analysis showed that the concentration of E. coli in P. maximus was significantly higher than in the other two species. Mytilus spp. and C. gigas exhibited comparable levels of E. coli. The observed standard deviation of the most probable number method in the study was 0.33 log(10).

Immunohistochemistry of great scallop Pecten maximus larvae experimentally challenged with pathogenic bacteria.

Three challenge experiments were carried out on larvae of the great scallop Pecten maximus. Larvae were bath-challenged with Vibrio pectenicida and 5 strains resembling Vibrio splendidus and one Pseudoalteromonas sp. Unchallenged larvae were used as negative controls. The challenge protocol was based on the use of a multidish system, where the scallop larvae (10, 13 and 15 d post-hatching in the 3 experiments, respectively) were distributed to 2 ml wells with stagnant seawater and exposed to the bacterial cultures by bath challenge. Presence of the challenge bacteria in the wells was verified by polymerase chain reaction (PCR). A significantly increased mortality was found between 24 and 48 h in most groups challenged with V. pectenicida or V. splendidus-like strains. The exception was found in larval groups challenged with a Pseudoalteromonas sp. LT 13, in which the mortality rate fell in 2 of the 3 challenge experiments. Larvae from the challenge experiments were studied by immunohistochemistry protocol. Examinations of larval groups challenged with V. pectenicida revealed no bacterial cells, despite a high degree of positive immunostaining. In contrast, intact bacterial cells were found in larvae challenged with V. splendidus. In the case of larvae challenged with the Pseudoalteromonas sp., positive immuno-staining was limited to visible bacteria inside the digestive area and cells of the mucosa. The experiments confirm that V. splendidus and V. pectenicida are pathogenic to scallop larvae, and that the Pseudoalteromonas strain is probably not a primary pathogen, although it cannot be ruled out as a secondary pathogen.