First description of a mortality event in adult Pacific oysters in Italy associated with infection by a Tenacibaculum soleae strain.

Summer mortality episodes in adult Pacific oysters have been described since the 1950s in various farming areas. Starting in 2012, a recrudescence of mortalities in commercial-sized oysters was first observed in France and then in Italy, with seasonality extension and translation later in the year. Moribund individuals collected during an event in Italy in December 2014 showed yellowish lesions of the mantle and adductor muscle. Histological examination revealed filamentous bacteria associated with necrotic areas. Quantitative PCRs targeting OsHV-1 and Vibrio aestuarianus detected only high loads of the pathogenic bacteria in tissues of symptomatic individuals. A lower diversity of the hemolymph microbiota was also evidenced in moribund individuals, with a predominance of Vibrio and Arcobacter species. A strain of Flavobacteriaceae was isolated from all the symptomatic individuals. Sequence analysis of the 16S rRNA gene identified the strain as Tenacibaculum soleae. When strain pathogenicity was tested by injection in adult individuals, it induced mortality rates of up to 45%, even in the absence of V. aestuarianus. As mortality occurred only 11 days post-infection, further investigation is needed to determine its effective virulence in natural conditions. This is the first description of a Tenacibaculum strain associated with bivalve mortalities.

Mycobacteriosis caused by Mycobacterium marinum in reared mullets: first evidence from Sardinia (Italy).

Mycobacterium marinum is a slow-growing non-tuberculous mycobacterium, and it is considered the most common aetiologic agent of mycobacteriosis in wild and cultured fish. The diagnosis is principally made by histology when positive Ziehl-Neelsen stain granulomas are detected. The aim of this study was to investigate the occurrence of mycobacteriosis in extensively cultured Mugilidae of two lagoons (Cabras and San Teodoro) from Sardinia by the use of histology, microbiology, PCR and DNA sequencing. Nine of 106 mullets examined were affected by mycobacteriosis, and the spleen was the most affected organ. The histology detected higher rate (100%) of infection in spleen than the culture and PCR (75% and 62.5%, respectively). The sequencing of hsp65 gene identified M. marinum as the primary cause of mycobacteriosis in the mullets examined. Mullets affected by mycobacteriosis were mainly fished in the San Teodoro lagoon characterized by critical environmental conditions. Histology remains the most common method in detecting fish affected by mycobacteriosis, and PCR-based methods are essential for species identification. Our finding are worthy of attention because mycobacteriosis caused by M. marinum in reared mullets was evidenced for the first time in Sardinia, suggesting that this disease may be underestimated also in other cultured fish species.

Molecular identification of iridoviruses infecting various sturgeon species in Europe.

Iridoviridae are known to cause disease in sturgeons in North America. Here, histological and molecular methods were used to screen for this family of virus in sturgeons from various European farms with low-to-high morbidity. Some histological samples revealed basophilic cells in the gill and labial epithelia, strongly suggesting the accumulation of iridovirus particles. Newly developed generic PCR tests targeting the major capsid protein (MCP) gene of sturgeon iridoviruses identified in North America, namely the white sturgeon iridovirus and the Namao virus (NV), produced positive signals in most samples from four sturgeon species: Russian (Acipenser gueldenstaedtii), Siberian (A. baerii), Adriatic (A. naccarii) and beluga (Huso huso). The sequences of the PCR products were generally highly similar one another, with nucleotide identities greater than 98%. They were also related to (74-88%), although distinct from, American sturgeon iridoviruses. These European viruses were thus considered variants of a single new virus, provisionally named Acipenser iridovirus-European (AcIV-E). Moreover, three samples infected with AcIV-E showed genetic heterogeneity, with the co-existence of two sequences differing by five nucleotides. One of our European samples carried a virus distinct from AcIV-E, but closely related to NV identified in Canada (95%). This study demonstrates the presence of two distinct sturgeon iridoviruses in Europe: a new genotype AcIV-E and an NV-related virus.

Ostreid herpesvirus type 1 genomic diversity in wild populations of Pacific oyster Crassostrea gigas from Italian coasts.

Ostreid herpesvirus 1 (OsHV-1) is a significant pathogen affecting the young Pacific oyster Crassostrea gigas, worldwide. A new variant, OsHV-1 µVar, has been associated with recurrent mortality events in Europe since 2008. Epidemiological data collection is key for global risk assessment; however little is known about health status and genotypes present in European wild oyster beds. Most studies to date have involved only cultivated individuals during mortality events, and reported low genotype diversity. With this study, conducted along the Italian coasts, we investigated for the first time the presence of OsHV-1 in European natural oyster beds. Analysis of three genomic regions revealed the presence of at least nine different genotypes, including two variants close to the OsHV-1 reference, known since the early 1990s but with no European record reported since 2010, and highlights relevant genotype diversity in natural environment. Phylogenetic analysis distinguished two distinct clusters and geographical distribution of genotypes, with the exception of a variant very closely related to the µVar, which appeared the single genotype present in all the Adriatic sites. Interestingly, these wild symptom free populations could represent, in Europe, an accessible alternative to the import of OsHV-1-resistant oyster strains from the East Pacific, the native area of C. gigas, avoiding the high-risk of non-native marine species and new pathogen introductions.

PCDD/Fs, DL-PCBs and NDL-PCBs in European catfish from a northern Italian lake: the contribution of an alien species to human exposure.

PCDDs/Fs, DL-PCBs and NDL-PCBs are environmentally persistent substances that have been associated with adverse effects on human health. Humans are mainly exposed to these pollutants through ingestion of contaminated fish and fishery products; the consumption of fatty fish such as European catfish can contribute considerably to the intake of dioxins and PCBs. Samples of fish muscle of the top-of-the-food-chain predator Silurus glanis from the northern Italian Lake Varese were analyzed to detect the levels of 17 PCDD/F and 18 PCB congeners. All samples presented detectable levels of PCDD/Fs and PCBs, with concentrations ranging from 0.001 to 1.310pgg(-1) wet weight (w.w.) for Σ PCDD/Fs, 0.031 to 21.000pgg(-1) w. w. for Σ DL-PCBs, and 0.397 to 117.910ngg(-1) w. w. for Σ NDL-PCBs. One sample exceeded the maximum levels of 6.5pgg(-1) w. w. for the sum of PCDD/Fs and DL-PCBs set by European regulations for fish (1259/2011 EU), while other samples exceeded the action levels of 0.75pgg(-1) w. w. for the sum of PCDD/Fs, and 2pgg(-1) w. w. for the sum of DL-PCBs (277/2012 EU). Consuming contaminated catfish may pose a risk for human health, especially for the subpopulation traditionally accustomed to eating this fish, as well as sensitive individuals, such as children and pregnant women.

Oxidative stress related to chlorpyrifos exposure in rainbow trout: Acute and medium term effects on genetic biomarkers.

Organophosphates (OPs) are derivatives of phosphoric acid widely used in agriculture as pesticides. Chlorpyrifos (CPF) is an OP that is extremely toxic to aquatic organisms. Rainbow trout (Oncorhynchus mykiss) is considered as a sentinel model species for ecotoxicology assessment in freshwater ecosystems. An exposure study was carried out on rainbow trout to investigate genetic responses to CPF-induced oxidative stress by Real-Time PCR, and to determine the accumulation dynamics of CPF and toxic metabolite chlorpyrifos-oxon (CPF-ox) in edible parts, by HPLC-MS/MS. Among the genes considered to be related to oxidative stress, a significant increase in HSP70 mRNA levels was observed in liver samples up to 14 days after CPF exposure (0.05 mg/L). CPF concentrations in muscle samples reach mean values of 285.25 ng/g within 96 hours of exposure, while CPF-ox concentrations were always under the limit of quantification (LOQ) of the applied method. Our findings lead us to consider HSP70 as a suitable genetic marker in rainbow trout for acute and medium-term monitoring of CPF exposure, complementary to analytical determinations.

Association of a specific major histocompatibility complex class IIß single nucleotide polymorphism with resistance to lactococcosis in rainbow trout, Oncorhynchus mykiss (Walbaum).

Major histocompatibility complex (MHC) loci encode glycoproteins that bind to foreign peptides and initiate immune responses through their interaction with T cells. MHC class II molecules are heterodimers consisting of α and ß chains encoded by extremely variable genes; variation in exon 2 is responsible for the majority of observed polymorphisms, mostly concentrated in the codons specifying the peptide-binding region. Lactococcus garvieae is the causative agent of lactococcosis, a warm-water bacterial infection pathogenic for cultured freshwater and marine fish. It causes considerable economic losses, limiting the profitability and development of fish industries in general and the intensive production of rainbow trout, Oncorhynchus mykiss (Walbaum), in particular. The disease is currently controlled with vaccines and antibiotics; however, vaccines have short-term efficacy, and increasing concerns regarding antibiotic residues have called for alternative strategies. To explore the involvement of the MHC class II ß-1 domain as a candidate gene for resistance to lactococcosis, we exposed 400 rainbow trout to naturally contaminated water. One single nucleotide polymorphism (SNP) and one haplotype were associated with resistance (P < 0.01). These results are promising for using MHC class IIß as a molecular marker in breeding rainbow trout resistant to lactococcosis.

Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA): emerging contaminants of increasing concern in fish from Lake Varese, Italy.

Perfluoroalkylated substances (PFASs) are highly fluorinated aliphatic compounds with high thermal and chemical stability, used in a range of industrial applications. Extensive screening analyses in biota samples from all over the world have shown the bioaccumulation of PFAS into higher trophic levels in the food chain. Perfluorooctane sulfonic acid (PFOS) and perfluoroctanoic acid (PFOA) are potential reproductive and developmental toxicants and are considered to be emerging endocrine disrupters. Ingestion of fish and other seafood is considered the main source of exposure of these contaminants. Here, we quantified PFOS and PFOA by LC-MS/MS in muscle samples of European perch from Lake Varese, Italy. PFOS was detected in all samples with concentrations of up to 17.2 ng g(-1). Although the reported values were lower than the recommended total daily intake (TDI) proposed by the European Food Safety Authority (EFSA), fish from Lake Varese may be a significant source of dietary PFOS exposure.

Granulomatous lesions in a wild mullet population from the eastern Ligurian Sea (Italy): mycobacteriosis vs. pseudotuberculosis.

Mycobacterium spp. and Photobacterium damselae subsp. piscicida are recognized as the most frequent causative agents of granulomatous lesions in fish. Although frequent episodes of mycobacterial infections have been reported in wild fish worldwide, only sporadic cases have been documented to date in Italy. To investigate for the presence of lesions referable to mycobacteriosis and to identify the mycobacterial species involved, a total of 159 wild mullets were fished from the eastern coast of the Ligurian Sea, killed and necropsied. Liver and spleen samples were collected from all fish for histopathological and microbiological analyses. Molecular investigations for identification of Photobacterium damselae subsp. piscicida were performed. Gross examination revealed granulomatous lesions in one animal; microscopically, 42.14% of fish displayed granulomas with various histological features, 19.50% resulted positive at Ziehl-Neelsen staining, and were confirmed as mycobacterial lesions by culture. The identified colonies were characterized as M. fortuitum, M. abscessus, M. flavescens, M. chelonae, M. septicum and M. nonchromogenicum. In all, 35% of animals resulted positive for Photobacterium damselae subsp. piscicida. These data suggest widespread mycobacterial infection also by Photobacterium damselae subsp. piscicida infections in wild fish. Moreover, the pathogenicity of some mycobacterial species, previously considered as saprophytic, was demonstrated.

Vibrio virulence genes in fishes collected from estuarine waters in Italy.

AIMS: The aim of this study was to investigate the presence of Vibrio vulnificus and potentially pathogenic strains of Vibrio parahaemolyticus in mullets collected from estuarine environment in Italy. METHODS AND RESULTS: Two hundred and ninety-five mullets were analysed by culture using the selective medium thiosulfate citrate bile salt sucrose agar, during a monitoring period of 2 years (2008-2009). Presumptive Vibrio colonies were initially identified by using biochemical tests, and strains identified as V. parahaemolyticus and V. vulnificus were subsequently examined by PCR for the presence of species-specific and virulence genes (toxR, trh, tdh and vvh). V. parahaemolyticus was found in 55% (162/295) of fishes and V. vulnificus in 1% (3/295) with a higher presence in summer months. The trh+/tdh- strains were detected in 16% (47/295) of samples and only one strain resulted trh+/tdh+. One of the V. parahaemolyticus trh+ strains isolated belonged to the O1:KUT (K untypeable), a serotype recently associated to gastroenteritis in Italy. CONCLUSIONS: This is the first report demonstrating a high percentage of potential pathogenic V. parahaemolyticus trh+ strains in estuarine fishes of the Mediterranean area. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings indicate the potential human health risk associated with the presence of pathogenic Vibrio spp. in wild fishes.

Salivary free insulin-like growth factor-i levels: effects of an acute physical exercise in athletes.

BACKGROUND/AIMS: The offer of human saliva IGF-I (sIGF-I) measurement in athletes investigation is a new proposal. The aim was to investigate the physical exercise effect on sIGF-I and explore plasma free IGF-I relation. MATERIALS AND METHODS: Saliva and blood were collected from well-trained athletes, investigated immediately before and at the end of a physical exercise test. RESULTS: sIGF-I was significantly increased at the end of the physical exercise. The plasma free IGF-I concentrations did not demonstrate any difference. The saliva total protein level (sTP) was also significantly increased. A positive correlation between sTP and sIGF-I, was observed, both before and after physical exercise, and between salivary and plasma free IGF-I only after physical exercise. The salivary free IGF-I level significantly increased after physical exercise, moreover a correlation with the plasma levels exists in post-exercise condition. CONCLUSION: The physical exercise affects sIGF-I as well as the sTP. The correlation between plasma and salivary free IGF-I levels only in post-exercise condition suggests further studies to investigate the effects of different type and duration of physical exercise. The comparison with other salivary biochemical parameter investigation would also further increase comprehension on the role of salivary IGF-I.

Dioxin-like Compounds in Lake Fish Species: Evaluation by DR-CALUX Bioassay.

Fish consumption is the principal source of intake of organochlorinated compounds in humans. Compared with other types of foods of animal origin, fish contain the highest levels of polychlorinated biphenyls (PCBs), polychlorinated dibenzo- p-dioxins, and polychlorinated dibenzofurans, all of which are classified as highly toxic organochlorine compounds. Currently, lakes and fish farms in northern Italy are not regularly monitored for PCBs and dioxins in areas contaminated by industrial sources, partially because of the high costs of traditional analytical methods that limit the number of samples to be analyzed. The DR-CALUX cell bioassay is based on the uptake of the cellular aryl hydrocarbon receptor (AhR) for dioxins and dioxin-like compounds. The aim of this study was to assess the levels of dioxins and dioxin-like PCB contamination in Lake Maggiore and Lake Como, two lakes in northwestern Italy, and in nearby areas. The levels were quantified using the cell bioassay DR-CALUX and reference controls in two wild fish species, perch ( Perca fluviatilis) and roach ( Rutilus rutilus), and in a farmed species, rainbow trout ( Oncorhynchus mykiss). Tissue samples collected from the farmed rainbow trout were also submitted to immunohistochemical analysis of CYP1A expression as a marker for environmental pollutant-induced liver damage. The levels of dioxins, furans, and dioxin-like PCBs were all below the maximum levels and action limits set by European Union Regulation, suggesting no risk for human health associated with the consumption of the fish species caught or farmed in these areas.

Complete genome sequence of Ostreid herpesvirus type 1 µVar isolated during mortality events in the Pacific oyster Crassostrea gigas in France and Ireland.

Infections with Ostreid herpesvirus 1 (OsHV-1) microvariants in young Pacific oysters are associated with massive mortality events and significant economic losses. Previous studies, focusing on few regions of the genome, have revealed the genomic diversity of these genotypes with respect to the reference type. We used a NGS process to sequence the whole genome of the OsHV-1 µVar in infected individuals, collected during mortality events in France and Ireland. The final genome length of OsHV-1 µVar was approximately 205kbp, shorter than the reference genotype and the overall genome organisation resembled herpes simplex viruses. 94.4% similarity was observed with the OsHV-1 reference genotype. Large indels, including five deletions and three insertions were found to induce the loss and the addition of several ORFs, summed with codon substitutions in 64% of genes shared with the reference type. This diversity raises the question of the exact origin and evolution of OsHV-1 µVar.

Trace element occurrence in the Pacific oyster Crassostrea gigas from coastal marine ecosystems in Italy.

The Pacific oyster is one of the world's most widespread bivalves and a suitable species for biomonitoring trace elements in marine environments thanks to its bioaccumulation ability. As it is also an edible mollusc, concentrations of harmful elements in its tissues must be monitored. For these purposes, 464 wild individuals were collected from 12 sites along the Italian coasts. The concentration of fourteen trace elements (Al, As, Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Se, Sn, Tl, and Zn) in their tissues was quantified. Among the three heavy metals, cadmium, lead, and mercury, none exceeded the maximum limit for in food set by European Union regulations but Cd in one sample from the Varano Lagoon resulted extremely close to this value. Contamination by Hg of the northern Adriatic and Orbetello Lagoons was also observed. Moreover, there was a positive association between the lagoon's environmental conditions and the bioaccumulation of this element in oysters. Despite the ban instituted 15 years ago on the use of Sn in antifouling paints, this element is still present in several marine environments, as demonstrated in the oysters sampled from harbour areas. Samples collected from harbours also showed very high concentrations of Cu and Zn due to the ability of oysters to accumulate these elements, which have replaced Sn in antifouling paints. Analysis of the samples from most sites indicated a low risk of human exposure to harmful elements through oyster consumption; nonetheless, chemical sanitary controls should focus primarily on Cd, Cu, and Zn.

Evaluation of the suitability of a partially defatted black soldier fly (Hermetia illucens L.) larvae meal as ingredient for rainbow trout (Oncorhynchus mykiss Walbaum) diets.

BACKGROUND: Two trials were performed to evaluate a partially defatted Hermetia illucens (HI) larvae meal as potential feed ingredient in rainbow trout (Oncorhynchus mykiss Walbaum) diets. In the first trial, 360 trout (178.9 ± 9.8 g of mean initial body weight) were randomly divided into three experimental groups (4 tanks/treatment, 30 fish/tank). The fish were fed for 78 days with isonitrogenous, isolipidic and isoenergetic diets containing increasing levels of HI, on as fed basis: 0% (HI0, control diet), 25% (HI25) and 50% (HI50) of fish meal substitution, corresponding to dietary inclusion levels of 0, 20% and 40%. In the second trial, 36 trout (4 tanks/treatment, 3 fish/tank) were used to evaluate the in vivo apparent digestibility coefficients (ADC) of the same diets used in the first trial. RESULTS: Survival, growth performance, condition factor, somatic indexes, and dorsal fillet physical quality parameters were not affected by diet. The highest dietary inclusion of HI larvae meal increased dry matter and ether extract contents of trout dorsal fillet. The use of HI larvae meal induced a decrease of valuable polyunsaturated fatty acids (PUFA) even if differences were only reported at the highest level of HI inclusion. The insect meal worsened the lipids health indexes of the same muscle. Dietary inclusion of insect meal did not alter the villus height of the fish. No differences were found among treatments in relation to ADC of ether extract and gross energy, while ADC of dry matter and crude protein were higher in HI25 if compared to HI50. CONCLUSIONS: The obtained results showed that a partially defatted HI larvae meal can be used as feed ingredient in trout diets up to 40% of inclusion level without impacting survival, growth performance, condition factor, somatic indexes, dorsal fillet physical quality parameters, and intestinal morphology of the fish. However, further investigations on specific feeding strategies and diet formulations are needed to limit the observed negative effects of the insect meal on the FA composition of dorsal muscle.

Presence of trace metals in aquaculture marine ecosystems of the northwestern Mediterranean Sea (Italy).

Information regarding chemical pollutant levels in farmed fish and shellfish, along with the risks associated with their consumption is still scarce. This study was designed to assess levels of exposure to 21 trace elements in fish (Dicentrarchus labrax), mussels (Mytilus galloprovincialis) and oysters (Crassostrea gigas) collected from aquaculture marine ecosystems of the northwestern Mediterranean Sea. Metal concentrations showed great variability in the three species; the highest values of the nonessential elements As and Cd were found in oysters while the highest levels of Al, Pb and V were found in mussels. The essential elements Cu, Mn and Zn were highest in oysters, but Fe, Cr, Ni, Se, Co and Mo levels were highest in mussels. Fish had the lowest concentrations for all trace elements, which were at least one order of magnitude lower than in bivalves. The rare earth elements cerium and lanthanum were found at higher levels in mussels than in oysters, but undetectable in fish. The maximum values set by European regulations for Hg, Cd and Pb were never exceeded in the examined samples. However, comparing the estimated human daily intakes (EHDIs) with the suggested tolerable copper and zinc intakes suggested a potential risk for frequent consumers of oysters. Similarly, people who consume high quantities of mussels could be exposed to concentrations of Al that exceed the proposed TWI (tolerable weekly intake).

Human exposure to metals due to consumption of fish from an artificial lake basin close to an active mining area in Katanga (D.R. Congo).

The concentrations of 14 essential and nonessential trace elements were determined in fish from Lake Tshangalele, Katanga province, Democratic Republic of Congo. This province has been a place of intensive mining activities for centuries, which have increased in recent years, due to the use of metals such as copper and cobalt for the industries of fast-growing countries. Lake Tshangalele, which receives effluents from metallurgical and mining plants in Likasi, is home to several fish species that are an important part of the diet of the local population, and, therefore, it constitutes a relevant site for documenting the human exposure to metals as a result of a fish diet. The highest concentrations (median levels, dry weight) of cobalt (7.25mgkg(-1)), copper (88.1mgkg(-1)), iron (197.5mgkg(-1)), manganese (65.35mgkg(-1)), zinc (122.9mgkg(-1)) and aluminum (135.4mgkg(-1)) were found in fish collected closest to the copper mining plant, with decreasing concentrations along the lake, up to the dam. In the most contaminated fish samples, values of up to 270.1mgkg(-1) for Al, 173.1mgkg(-1) for Cu, 220.9mgkg(-1) for Zn, 211.0mgkg(-1) for Mn, 324.2mgkg(-1) for Fe, 15.1mgkg(-1) for Co, 4.2mgkg(-1) for Cr, 1.6mgkg(-1) for Cd, 1.9mgkg(-1) for Pb, and 1.8mgkg(-1) for Ni were found. Metal contamination from mining activity resulted in being of great concern because of potential health risks to the local inhabitants due to the consumption of heavily contaminated fish. CAPSULE: High levels of metals, especially cobalt, aluminum, iron, manganese, zinc and cadmium were found in fish from Tshangalele water system.