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The family Oceanospirillaceae (Garrity et al., 2005) includes 18 genera, 11 of which were described in the last 10 years (Arahal et al., 2007). Members of this family are motile and exclusive from aquatic environments. As part of a study on the microbiota associated to Great scallop (Pecten maximus) and its rearing system performed in a hatchery in Bergen (Norway), seven strains: 3CM4T(= CECT 8267T; = CAIM 1884T) 3CH4, 2CH8, 2SH3, 1CH5, 1CH6 and 1SH4 (= CECT 8268; = CAIM 1885) were isolated from broodstock gonads. These strains are aerobic, oxidase and catalase positive and they do not grow in TCBS. Isolates form small (less than 1 mm) pale brown pigmentated colonies on marine agar. They are halophiles and their temperature range for growth is 4 to 20ºC. Cells are Gram-negative, non-motile long rods, measuring 0.2-0.4 μm in diameter and 5-20 μm in length. With oxidative metabolism, the isolates do not ferment carbohydrates. The results of analysis of DNA-DNA hybridization confirmed that these isolates constitute a new and differentiated cluster within the genus Sinobacterium. The predominant fatty acids were C16: 0, C16: 1 ω7c/C16: 1 ω6c and C18: 1 ω7c. The G + C content of the new isolates is 52.2 ± 1 mol%. Based on phenotypic and phylogenetic characteristics, the seven isolates represent a new species for which the name Sinobacterium norvegicum sp. nov. is proposed, with strain 3CM4T (= CECT 8267T = CAIM 1884T) as the type strain.

Sapovirus (SaV), etiologic agent of human gastroenteritis, is a RNA virus from the Caliciviridae family, mostly detected in Asia. It has been classified by the U.S. Environmental Protection Agency as an emergent human pathogen with risk for public health in aquatic environments. Bivalves, due to their filtrating feeding, concentrate viral particles presented in seawater, and it has been demonstrated that the bioaccumulated viruses can resist depuration treatments to which bivalves are submitted. In addition, the traditional way of bivalve consumption increases the probability of infection, making them a high risk food [1]. The main objective of this work was to detect and quantify SaV in bivalves from Galicia, Northwestern Spain. A total of 168 samples, including mussels (Mytilus galloprovincialis), clams (Venerupis philippinarum and V. decussata) and cockles (Cerastoderma edule) were obtained during a period of 18 months from two Galician Rías (Ría of Ares-Betanzos and Ría of Vigo, respectively at North and South of the region). Samples consisted into 10 or 20 individuals, which were dissected under aseptic conditions to obtain their digestive tissue. The viral RNA was extracted using Nucleospin RNA Virus Kit (Macherey-Nagel; Dürem, Germany). Subsequently, viral detection and quantification by real time RT-PCR was performed using Platinum Quantitative RT-PCR Thermoscript One-Step System Kit (Invitrogen, Saint Aubin, France), according to the recently developed standard method ISO/TS 15216:2013. Primers SaV124F and SaV124R, and probe SaV124TP designed to amplify human genogroups I, II and IV were used [2]. Both through viral RNA extraction and viral detection and quantification, internal controls were used to avoid false positive or negative results, and the possible presence of inhibitors. SaV was detected in 30 of the 168 samples (17,85%). There was no significant difference between the two Rías in terms of detections, although it was observed a seasonality increment of positive samples from November 2011 until April 2012 in both Rías due to the rainy season and the decrease of seawater temperature. Total quantification ranged between 103 and 105 copies of viral RNA/g of digestive tissue (c/g), being mussels the specie with lower main quantification (3,1 x 104 c/g) and clams the species with higher medium rates (2,0 x 105 c/g). This represents the first study out of Japan in which human Sapovirus was detected and quantified into human food intended for consumption.

Enteric viruses contaminating the environment represent a danger for public health. They are present quite frequently in wastewater and transmitted to humans by the fecal-oral route. The aim of our study was to detect hepatitis A and E viruses (HAV and HEV) in wastewater samples collected from 6 different Tunisian sewage treatment plants during thirteen months. A total of 325 samples were analyzed by real time reverse transcriptase-polymerase chain reaction (RT-qPCR). Results showed the presence of HAV contamination in 36% of the samples. Msaken (58.9%) and SidiBouzid (51.85%) regions showed the highest contamination, followed by Ouerdanine (35.2%), Kasserine (38.9%), Sbitla (23.6%) and El Jem (7.7%) regions. Contamination with HEV was significantly lower (4.9%), being detected HEV positive samples in SidiBouzid (9.25%), El Jem (5.76%), Kasserine (3.7%), Ouerdanine (3.7%), Sbitla (3.63%) and Msaken (3.6%) regions. On the another hand, our research found that, in entry points, 54% of wastewater samples were contaminated with HAV, whereas, in exit points a percentage of 17.9% was found. HAV values were also analyzed by plant showing percentages of positive samples in entry points between 15.38% and 85.18%, while in exit points values ranged from 3.57% to 57.14%. HAV was detected in exit points of all plants except El Jem region. Concerning HEV, the average percentage of contaminated wastewater at entry was 6.75% which was reduced until 3.1% at exit. By plant, HEV positive samples were detected only in three plants, Kasserine, El Jem and Sidi Bouzid at rates of 3.7% 3.84% and 11.11%, respectively. These results support previous findings on the stability of enteric viruses in wastewater and the inefficiency of the procedures employed in the sewage treatment plants. On the other hand, molecular procedures proved to be very sensitive to detect viral genomes in this type of samples. In conclusion, wastewater treatments employed in Tunisian plants can reduce the number of viral particles of hepatitis A and E but do not remove all of them, which constitute a potential risk for human health.

Bivalve molluscs, due to its filter-feeding mechanism, have an abundant associated bacterial microbiota composed of a large number of species of different genera. The knowledge about the composition of the microbiota associated with bivalve molluscs is based, mostly, on techniques of cultivation of microorganisms, but also on DGGE methods. However, it is estimated that less than 1% of the bacteria are culturable with current methods, which lead to an underestimation of the microbial diversity. On the other hand, DGGE methodology has a limited resolution due to the low number of DNA fragments obtained as representative of a microbial community. The introduction of new molecular approaches allowed the improvement of the knowledge about bacterial communities present in a sample [1]. In recent years, new molecular techniques known as Next Generation Sequencing (NGS) have been developed, based on high- throughput sequencing [2]. In this study, we present the analysis of the microbiota associated to reared scallop gonads before and after spawning by pyrosequencing of the 16S rRNA gene. After the DNA extraction of the samples, amplification was performed using a set of degenerate primers with low stringency conditions. Pyrosequencing of samples was carried out using the sequencer 454 GLX Titanium Plus (Roche). The data obtained was further subjected to bioinformatics analysis using Galaxy and UCHIME program and the Ribosomal Database Project. Rarefaction curves were obtained, the composition of the bacterial community was analysed after the taxonomic assignment, and the total composition of the bacterial communities of the samples were compared using the principal coordinates analysis (PCoA) by FastUnifrac tool [3]. Pyrosequencing of the samples resulted in a total of 18520 sequences (3000 per sample, approximately) with an average length of 325 bp (base pairs). The taxonomic assignment of sequences allowed the identification to the genus level, being observed a large bacterial diversity with over 110 genera. The most prevalent genera in the samples were Hydrotalea, Acinetobacter, Delftia, Sediminibacter and Pseudomonas, among others. Differences in the microbial communities were observed among the samples, and the PCoA analysis allowed their separation by means on their gender and if they proceed from sampling before or after the spawning. Nevertheless, the rarefaction curves obtained for each sample failed to reach a saturation phase, indicating that more sequencing effort would be necessary.

From a virological standpoint, shellfish safety continues to be a sanitary challenge. Bivalves are one of the most common vehicles of viral illness and the adoption of viral standards into European Union legislation is being considered. In this study, a 18-months survey were conducted in ten harvesting areas from two estuaries in Galicia (NW of Spain), the most important bivalve production area in Europe. Hepatitis A virus (HAV) and human norovirus (NoV), including genogroups I (GI) and II (GII) were quantified by reverse transcription-real time PCR (RT-qPCR), according to the recently developed standard method ISO/TS 15216:2013. Four bivalve species were studied, including wild and cultured mussels (Mytilus galloprovincialis), clams (Venerupis philippinarum and V. decussata) and cockles (Cerastoderma edule). Overall, 54.8% of the analysed samples were contaminated by at least one of the studied viruses, being detected the simultaneous presence of two or three viruses in 11.3% of the samples. NoV GI was the most prevalent virus (32.1%) followed by NoV GII (25.6%) and HAV (9.5%). The results showed the presence of viral contamination throughout the year in both areas and all species of molluscs. However, diverse patterns of prevalence and seasonality were observed among different viruses. HAV was detected only between March and June 2012. NoV GI was detected intermittently throughout the study period, with significant peaks in the spring and summer of 2011. NoV GII showed a clear seasonality to during the cold months by 2011; however, a significant peak was detected in the spring of 2012.