Detection and molecular characterization of betanodaviruses retrieved from bivalve molluscs.

Betanodaviruses are small ssRNA viruses responsible for viral encephalopathy and retinopathy, otherwise known as viral nervous necrosis, in marine fish worldwide. These viruses can be either horizontally or vertically transmitted and have been sporadically detected in invertebrates, which seem to be one of the possible viral sources. Twenty-eight new betanodavirus strains were retrieved in three molluscs species collected from different European countries between 2008 and 2015. The phylogenetic analyses revealed that strains retrieved from bivalve molluscs are closely related to viruses detected in finfish in Southern Europe in the period 2000-2009. Nevertheless, a new betanodavirus strain, markedly different from the other members of the RGNNV genotype, was detected. Such a massive and varied presence of betanodaviruses in bivalve molluscs greatly stresses the risks of transmission previously feared for other invertebrates. Bivalve molluscs reared in the same area as farmed and wild finfish could act as a reservoir of the virus. Furthermore, current European regulations allow relaying activities and the sale of live bivalve molluscs, which could pose a real risk of spreading betanodaviruses across different geographic regions. To our knowledge, this is the first study, which focuses on the detection and genetic characterization of betanodaviruses in bivalve molluscs.

Fate of redspotted grouper nervous necrosis virus (RGNNV) in experimentally challenged Manila clam Ruditapes philippinarum.

Redspotted grouper nervous necrosis virus (RGNNV), genus Betanodavirus, family Nodaviridae, is the causative agent of viral encephalopathy and retinopathy (otherwise known as viral nervous necrosis) and can infect several fish species worldwide. Betanodaviruses, including RGNNV, are very resilient in the aquatic environment, and their presence has already been reported in several wild marine species including invertebrates. In order to investigate the interaction between a bivalve mollusc (Manila clam Ruditapes philippinarum) and RGNNV, we optimised a culture-based method. The bioaccumulation of the pathogenic RGNNV by R. philippinarum and the potential shedding of viable RGNNV from RGNNV-exposed clams were evaluated through a culture-based method. R. philippinarum clearly accumulated viable RGNNV in their hepatopancreatic tissue and were able to release viable RGNNV via faecal matter and filtered water into the surrounding environment. The role of clams as bioaccumulators and shedders of viable RGGNV could put susceptible cohabiting cultured fish at risk. RGNNV-contaminated molluscs could behave as reservoirs for this virus and may modify the virus epidemiology.

Outbreak of mortality in Russian (Acipenser gueldenstaedtii) and Siberian (Acipenser baerii) sturgeons associated with sturgeon nucleo-cytoplasmatic large DNA virus.

Diseased outbreaks with high mortality in farmed sturgeon are a limiting factor to the success of this emerging aquaculture sector in Europe. Thorough investigations of outbreaks can determine the aetiological agents, identify important pathological and epidemiological pathways of infections and pave the way for effective control strategies. A thorough investigation of a mortality outbreak in Russian (Acipenser gueldenstaedtii) and Siberian (Acipenser baerii) sturgeons in Italy, demonstrated the primary involvement of a sturgeon nucleo-cytoplasmic large DNA virus (NCLDV). While, the taxonomy classification of this new virus is still uncertain, its involvement in sturgeon mortality outbreaks in Europe is, for the first time, fully investigated and described. Furthermore, the coinfection of bacteria such as motile Aeromonas spp. and Acinetobacter spp. was reported. Genetic characterisation showed the close relationship between the European sturgeon NCLDV with North American sturgeon NCLDVs. Similarly to the latter, the European sturgeon NCLDV persists in survivors. Furthermore, a systemic distribution of the European sturgeon NCLDV was evident in diseased A. baerii and A. gueldenstaedtii and in recovered A. gueldenstaedtii. These epidemiological and pathological findings will help in the identification of effective control strategies for sturgeon NCLDV infection, which afflicts an important and emerging European aquaculture sector.

Comparison of different biochemical and molecular methods for the identification of Vibrio parahaemolyticus.

AIMS: Multicentre evaluation of biochemical and molecular methods for the identification of Vibrio parahaemolyticus. METHODS AND RESULTS: For the biochemical identification methods, API 20E and API 20NE and Alsina's scheme were evaluated in intra- and interlaboratory tests in order to determine the accuracy and concordance of each method. Both in intra- and interlaboratory tests, the Alsina's scheme showed the highest sensitivity (86% of correct identifications in the interlaboratory test). False-positive results were obtained by all methods (specificity was 95% for API 20E, 73% for API 20NE and 84% for Alsina's scheme) and concordance varied from 65% of API 20NE to 84% of API 20E. For the molecular identifications, polymerase chain reaction (PCR) for the detection of toxR gene, tl gene and pR72H fragment were tested on 30 strains by two laboratories. The PCR for toxR showed the highest inclusivity (96%), exclusivity (100%) and concordance (97%). CONCLUSIONS: Among the biochemical identification methods tested, the Alsina's scheme gave more reliable results; however, in order to avoid false-positive results, all the biochemical identifications should be confirmed by means of molecular methods. SIGNIFICANCE AND IMPACT OF THE STUDY: Availability of an efficient identification method of Vibrio parahaemolyticus to use in official control of fisheries products.

Chemical and biochemical parameters of cultured diatoms and bacteria from the Adriatic Sea as possible biomarkers of mucilage production.

Bacteria and diatom strains from the Adriatic Sea were investigated, under standard and altered environmental conditions, for carbohydrate production and for the presence of specific biomarkers. Algae from P-depleted cultures showed an increase in extracellular carbohydrate production, a significantly lower chlorophyll a content and unchanged total lipid levels. However, the fatty acid composition of algal cultures was severely affected by low P levels, in that, total saturated and monounsaturated fatty acids increased and total polyunsaturated fatty acids decreased. Marine heterotrophic bacteria resulted enriched by 4 to 6 orders of magnitude in mucilage samples respect to surrounding seawater, unlike other groups of bacteria such as the non-halophylic heterotrophs. The major fatty acids detected in bacteria were 16:0 and 18:1n-7; the uneven fatty acids 17:0i, 17:0 and 17:1 also constituted an important component of various strains and, as a result, the total monounsaturated fraction represented the main component of total fatty acids. All the mucilage samples analysed shared the same general fatty acid composition features with a high amount of saturated components, especially 16:0; typical marine polyunsaturated fatty acids, such as 20:5n-3 and 22:6n-3, were found at very low levels. With regard to the sterol composition, the analysed algal species and bacteria showed that different compounds prevailed in the different species, and under P-deprivation sterol distribution resulted differently affected in the various algal species. In mucilage samples an overall prevalence of cholesterol was observed and, among 4alpha-methylsterols, constantly present, dinosterol prevailed in all samples. Vibrational IR spectroscopic analyses confirmed the main results obtained with the GC analysis: a higher unsaturation degree in nutrient replete diatom cultures than in P-depleted ones, a lower amount of P-containing compounds in the latter, bacterial lipid profiles with a high amount of free carboxylic acids and/or ketones and a low unsaturation degree and, finally, mucilage samples with a very low unsaturation degree. All these results allowed some speculations on the involvement of the various microbial and phytoplankton components in mucilage genesis.

Detection of Vibrionaceae in mussels and in their seawater growing area.

AIMS: The seasonal trend and frequency of detection of Vibrionaceae in seawater samples and in molluscs collected in the Adriatic Sea was measured. METHODS AND RESULTS: Over a 2-year period, 726 bacterial strains were isolated, of which 46.9% belonged to the Vibrio genus, 29.8% to the Aeromonas genus and the remaining 23.3% was made up of the Pseudomonas, Flavobacterium, Pasteurella, Agrobacterium and Ochrobacterium genera. Many of the isolated strains were shown to produce toxins. CONCLUSION: The Vibrio genus, which was isolated more often than the other genera, was particularly prevalent in summer (54.4% of the total number of bacteria isolated during this season), while it was scarce in the winter months.

HDL-cholesterol-raising effect of orange juice in subjects with hypercholesterolemia.

BACKGROUND: Orange juice-a rich source of vitamin C, folate, and flavonoids such as hesperidin-induces hypocholesterolemic responses in animals. OBJECTIVE: We determined whether orange juice beneficially altered blood lipids in subjects with moderate hypercholesterolemia. DESIGN: The sample consisted of 16 healthy men and 9 healthy women with elevated plasma total and LDL-cholesterol and normal plasma triacylglycerol concentrations. Participants incorporated 1, 2, or 3 cups (250 mL each) of orange juice sequentially into their diets, each dose over a period of 4 wk. This was followed by a 5-wk washout period. Plasma lipid, folate, homocyst(e)ine, and vitamin C (a compliance marker) concentrations were measured at baseline, after each treatment, and after the washout period. RESULTS: Consumption of 750 mL but not of 250 or 500 mL orange juice daily increased HDL-cholesterol concentrations by 21% (P: < 0.001), triacylglycerol concentrations by 30% (from 1.56 +/- 0.72 to 2.03 +/- 0.91 mmol/L; P: < 0.02), and folate concentrations by 18% (P: < 0.01); decreased the LDL-HDL cholesterol ratio by 16% (P: < 0.005); and did not affect homocyst(e)ine concentrations. Plasma vitamin C concentrations increased significantly during each dietary period (2.1, 3.1, and 3.8 times, respectively). CONCLUSIONS: Orange juice (750 mL/d) improved blood lipid profiles in hypercholesterolemic subjects, confirming recommendations to consume >/=5-10 servings of fruit and vegetables daily.