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Vibrio parahaemolyticus is a common microflora of marine and estuarine waters known to be associated with gastroenteritis due to consumption of raw seafood, cooked food contaminated through direct contact (cross contamination) or improperly cooked. The aim of this study was to evaluate the effectiveness of two different heat processes in reducing microbial load of V. parahaemolyticus. Data show a 4 Log10/g reduction of bacterial load after 3,30 and 15 minutes at 95 °C.

Herpesvirus (HV) is widely distributed among cetacean populations, with the highest prevalence reported in the Mediterranean Sea. In this study, a comprehensive analysis was conducted, including epidemiological, phylogenetic, and pathological aspects, with particular emphasis on neuropathology, to better understand the impact of HV in these animals. Our results show a higher presence of HV in males compared to females, with males exhibiting a greater number of positive tissues. Additionally, adults were more frequently affected by HV infection than juveniles, with no infections detected in calves or neonates. The affected species were striped (Stenella coeruleoalba) and bottlenose dolphins (Tursiops truncatus). The highest positivity rates were observed in the genital system, cerebrum, and skin tissues. Phylogenetic analysis indicated a higher occurrence of Gammaherpesvirus (GHV) sequences but increased genetic diversity within Alphaherpesvirus (AHV). Key neuropathological features included astro-microgliosis (n = 4) and meningitis with minimal to mild perivascular cuffing (n = 2). The presence of concurrent infections with other pathogens, particularly cetacean morbillivirus (CeMV), underscores the complex nature of infectious diseases in cetaceans. However, the presence of lesions at the Central Nervous System (CNS) with molecular positivity for GHV, excluding the involvement of other potential neurotropic agents, would confirm the potential of this HV subfamily to induce neurological damage. Pathological examination identified lesions in other organs that could potentially be associated with HV, characterized by lymphoid depletion and tissue inflammation. These findings enhance our understanding of HV in odontocetes and highlight the need for ongoing research into the factors driving these infections and their broader implications., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Vargas-Castro et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Photobacterium damselae subsp. damselae (Pdd) is an increasingly common bacterium in post-mortem diagnostics of beached marine mammals, but little is known about its precise etiological responsibility. To estimate the prevalence of Pdd in stranded cetaceans from 2017 to 2022 on the Ligurian coast (Pelagos Sanctuary), we tested tissues from 53 stranded individuals belonging to four cetacean species. DNA extracts from cetacean tissue were screened using a polymerase chain reaction (PCR) assay targeting the Pdd ureC gene. Positive samples were screened by PCR for dly, hlyA pl and hlyA ch hemolysin genes, which were confirmed by sequencing. Twenty-two out of 53 (41.5%) cetaceans analyzed by PCR were confirmed for Pdd DNA in at least one tissue among those analyzed. Five of these cetaceans were positive for at least one of the hemolysin genes tested. In all Pdd-positive cetaceans, other pathogens that were considered responsible for the causa mortis of the animals were also found. The results provide new information on the spread of Pdd in cetaceans and support the thesis that Pdd might be an opportunistic agent that could contribute to worsening health conditions in subjects already compromised by other pathogens. However, further studies are needed to investigate and deepen this hypothesis.

In this study, we evaluated the use of hydrolates, co-products of essential oil distillation, as processing aids to improve the depuration process of Pacific oysters ( Crassostrea gigas ) as a post-harvest method aimed at reducing the norovirus (NoV) viral load. Live oysters were kept in water to which hydrolates of Thymus serpyllum and Citrus limon at 1% were added for 24 h. The concentration of NoV was quantified using the ISO 15216-1 quantitative real-time RT-PCR method in the oyster digestive tissue both before and after the treatment. The results showed a significant reduction of 0.2 log in the NoV GII concentration after 24 h of treatment with 1% C. limon hydrolate. Conversely, treatment with T. serpyllum did not appear to reduce the concentration of NoV compared to the control. Additionally, a sensory analysis was conducted through a blind survey comparing untreated and treated oysters. No changes in the sensory and physical characteristics of the oysters were observed, except for a decrease in the marine flavour intensity, which was positively perceived by consumers. These results indicate that the addition of hydrolates of C. limon at 1% during depuration might represent a promising processing aid for enhancing both the safety and acceptability of live oysters.

Cetacean morbillivirus (CeMV) has caused several outbreaks, unusual mortality events, and interepidemic single-lethal disease episodes in the Mediterranean Sea. Since 2012, a new strain with a northeast (NE) Atlantic origin has been circulating among Mediterranean cetaceans, causing numerous deaths. The objective of this study was to determine the prevalence of CeMV in cetaceans stranded in Italy between 2018 and 2021 and characterize the strain of CeMV circulating. Out of the 354 stranded cetaceans along the Italian coastlines, 113 were CeMV-positive. This prevalence (31.9%) is one of the highest reported without an associated outbreak. All marine sectors along the Italian coastlines, except for the northern Adriatic coast, reported a positive molecular diagnosis of CeMV. In one-third of the CeMV-positive cetaceans submitted to a histological evaluation, a chronic form of the infection (detectable viral antigen, the absence of associated lesions, and concomitant coinfections) was suspected. Tissues from 24 animals were used to characterize the strain, obtaining 57 sequences from phosphoprotein, nucleocapsid, and fusion protein genes, which were submitted to GenBank. Our sequences showed the highest identity with NE-Atlantic strain sequences, and in the phylogenetic study, they clustered together with them. Regarding age and species, most of these individuals were adults (17/24, 70.83%) and striped dolphins (19/24, 79.16%). This study improves our understanding on the NE-Atlantic CeMV strain in the Italian waters, supporting the hypothesis of an endemic circulation of the virus in this area; however, additional studies are necessary to deeply comprehend the epidemiology of this strain in the Mediterranean Sea., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Vargas-Castro, Peletto, Mattioda, Goria, Serracca, Varello, Sánchez-Vizcaíno, Puleio, Nocera, Lucifora, Acutis, Casalone, Grattarola and Giorda.)

Shellfish sanitary controls are very important to guarantee consumer health because bivalve molluscs (BVM) are filter-feeders so they can accumulate pathogens, environmental contaminants and biotoxins produced by some algae, causing infections and food poisoning in humans after ingestion. The purpose of this work was to analyse with chemometric methods the historical data relating to routine analyses carried out by the competent authority (Liguria Local Health Unit, National Health Service) on the BVM reared in a shellfish farm located in the Gulf of La Spezia (Italy). Chemometric analysis was aimed at identifying any correlations between the variables, as well as any seasonal trends and similarities between the stations, in order to be able to provide further material for a more accurate risk assessment and to improve the monitoring organization for example by reducing sampling stations and/or sampling frequency. The dataset used included 31 variables classified as biotoxicological, microbiological and chemical variables, measured twice a week, monthly or half yearly respectively, for a total of 6 years (from 2015 to 2021), on samples of Mytilus galloprovincialis coming from 7 monitoring stations. The results obtained by the application of principal component analysis have shown positive alga-biotoxin correlations, as well as seasonal trends linked to algae growth, with a greater algal biomass and their toxins during the spring months. In addition, periods characterised by low rainfall were found to affect algal development, promoting especially species such as Dinophysis spp. Considering the microbiological and biotoxicological variables, significant differences between the monitoring stations were not found. However, stations could be distinguished on the basis of the nature of the predominant chemical pollutants., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Valentina Ciccotelli reports article publishing charges was provided by Zooprophylactic Institute of Piemonte Liguria and Valle d’Aosta., (© 2023 The Authors. Published by Elsevier Ltd.)

The use of natural substances with antiviral properties might reduce foodborne viral diseases. In this study, we evaluated the virucidal effect of Citrus limon and Thymus serpyllum essential oils (EOs) and of Citrus Limon , Thymus serpyllum and Thymus vulgaris hydrolates on murine norovirus (MNV), a human norovirus surrogate. To assess the virucidal effect of these natural substances, the reduction in viral infectivity was estimated by comparing the TCID 50 /mL of untreated viral suspension and the viral suspension treated with hydrolates and EOs at different concentrations. The results showed a natural loss of infectivity of the untreated virus after 24 h of approx. 1 log. The EO (1%) of T. serpyllum , and hydrolates (1% and 2%) of T. serpyllum and T. vulgaris immediately caused a reduction in MNV infectivity of about 2 log but did not provide a further significant decrease after 24 h. Instead, the EO (1%) and hydrolate (1% and 2%) of C. limon exerted an immediate reduction in the viral infectivity of about 1.3 log and 1 log, respectively, followed by a further reduction in infectivity of 1 log after 24 h for the hydrolate. These results will allow for the implementation of a depuration treatment based on the use of these natural compounds.

Astroviruses are important human enteric pathogens transmissible with contaminated food and water. Astroviruses have also been identified in mammals, birds, lower vertebrates and invertebrates. The genetic diversity of human and animal astroviruses poses a challenge for diagnostics and taxonomy. As a proof of concept, we used a panastrovirus consensus primer set, able to amplify in a nested RT-PCR protocol a 400-nt-long fragment of the RNA-dependent RNA polymerase of most members of the Astroviridae family, in conjunction with a nanopore sequencing platform, to generate information on the astrovirome in filter-feeding mollusks. Amplicons generated from bivalve samples were used to generate libraries for deep sequencing. In three samples, only one unique RdRp sequence type was obtained. However, in seven samples and in three barcodes with eleven pooled samples, we identified a variety of known and unknown RdRp sequence types, in most cases distantly related to astrovirus sequences available in the databases. In total, 37 different sequence contigs were generated. Avian-origin astrovirus sequences were predominant, likely due to contamination of shellfish harvesting waters by marine birds. Astroviruses of the aquatic eco-system were also identified, whereas human astroviruses were not detected.

Norovirus (NoV) is the most important cause of seafood-borne gastroenteritis worldwide, mainly associated with the consumption of raw oysters. NoV is often present in oysters that comply with existing control standards for shellfish. Therefore, the improvement of post-harvest treatments and practices can represent one of the main strategies to reduce the incidence of viral diseases related to shellfish. This study aimed to investigate long-term relays for the reduction of NoV levels in live oysters, during the high-risk cold months, by transferring the oysters from a more contaminated site to two sites with lower NoV levels. The efficacy of relaying was evaluated by analyzing oyster samples collected at days 0 (T0) and 30 (T30) for NoV levels using a real-time quantitative PCR (RT-qPCR). The NoV level at the relay sites was consistently lower than at the harvest site. The relay process for 30 days in seawater with a lower NoV level resulted in a decrease in the NoV load compared to day 0 with significant reductions depending on the site and genogroup of NoV considered. These results suggest that long-term relaying of oysters to reduce NoV levels is promising and could help growers to improve oyster safety; however, further investigations are needed.

The wild boar is an important natural reservoir for the zoonotic transmission of the hepatitis E virus (HEV) around the world. In particular, HEV genotypes 3 and 4 are an emerging problem in industrialized countries, as the number of wild boars has increased, and their territory is ever closer to farms and populated areas. This study describes the HEV prevalence and geographic circulation among wild boar populations in the Ligurian region (Italy) during the period 2019-2022. Liver samples from 849 wild boars were analyzed for HEV RNA using real-time RT-PCR; positive samples were then subjected to sequencing and phylogenetic analysis. Overall, 6.7% of the wild boars were positive for HEV RNA; however, in the last two years, the percentage of positive animals almost doubled. Phylogenetic analysis showed that wild boar HEV sequences belonged to genotype 3 and clustered within subtypes 3a and 3c, and, for the first time in Italy, subtypes 3b and 3m were identified. Interestingly, 13 sequences could not be assigned to a recognized subtype. Furthermore, the results showed different circulations of identified subtypes across the territory. These findings increase the knowledge of HEV-3 heterogeneity in Italy and describe the role of wild boars in maintaining an active viral circulation in the environment.

Cetacean morbillivirus (CeMV) is responsible for epidemic and endemic fatalities in free-ranging cetaceans. Neuro-inflammation sustained by CeMV is a leading cause of death in stranded cetaceans. A novel dolphin morbillivirus (DMV) strain of Atlantic origin circulating in Italian waters since early 2016 has caused acute/subacute lesions associated with positive immunolabelling of the virus. To date, myelin damage has not been fully documented and investigated in cetaceans. This study describes neuropathological findings in the brain tissue of 31 cetaceans found stranded along the Italian coastline and positive for DMV infection on molecular testing. Cell changes in the areas of myelinopathy were revealed by double indirect immunofluorescence. The most frequent DMV-associated lesions were astro-microgliosis, neuronal necrosis, spongiosis, malacia, and non-suppurative meningoencephalitis. Myelin reduction and areas of demyelination were revealed by means of a specific myelin biomarker. Morbilliviral antigen immunolabelling was mainly observed in neurons and microglial cells, in association with a marked activation of microglia and astrocytes. These findings extend our knowledge of DMV-associated brain lesions and shed light on their pathogenesis.

Depuration is generally the main treatment employed for bivalve mollusks harvested from contaminated sites. Commercial depuration has demonstrated to be effective for removal of bacterial pathogens, although it probably provides only limited efficacy against human enteric viruses. We evaluated the quantitative reduction of norovirus (NoV) genogroups I and II in naturally contaminated oysters after 1, 4, and 9 days of depuration. The process was conducted in an authorized depuration plant, and NoV concentration was determined by RT-qPCR according to ISO 15216-1:2017 method. Regardless of the NoV genogroup, our results showed no significant reduction in NoV concentration after 1 day of depuration. Higher mean reduction (68%) was obtained after 4 days of treatment, while no further increase was observed after 9 days. Overall, reduction was highly variable, and none of the trials showed statistically significant reduction in NoV RNA concentration at the end of each depuration period. Indeed, NoV concentration remained high in 70% of samples even after 9 days of depuration, with values ranging between 4.0 × 10 2 and 2.3 × 10 4 g.c./g. These results indicate that an extension of commercial depuration time does not appear to be effective for reducing or eliminating NoV in oysters.

Salmonella spp. is an important zoonotic agent. Wild boars might host this pathogen in the intestinal tract and might represent a risk for Salmonella spp. transmission to humans. Wild boars are widely spread in Liguria, due to the environmental characteristics of the region. The aim of the study was the isolation, typing, and investigation of antimicrobial susceptibility of the isolated strains of Salmonella spp. During the 2013-2017 hunting seasons, 4335 livers of wild boars were collected and analyzed for the presence of Salmonella spp. A total of 260 strains of Salmonella spp. were isolated and characterized, with a prevalence of 6%. The isolated strains belonged to all six Salmonella enterica subspecies. Most of them were identified as Salmonella enterica subs. enterica of which 31 different serotypes were identified. The dominating serotype identified was S. Enteritidis. The antimicrobial resistance profiles of the isolated strains were analyzed against sixteen molecules. Of the isolated strains, 94.6% were resistant to at least one of the tested antimicrobials. This study showed the circulation of resistant Salmonella spp. strains in the wild boar population living in this area of Italy, underling the potential risk for these animals to disseminate this pathogen and its antimicrobial resistances.

Yersinia enterocolitica ( Ye ) is a very important zoonosis andwild boars play a pivotal role in its transmission. In the last decade, the wild boar population has undergone a strong increase that haspushed them towards urbanized areas, facilitating the human-wildlife interface and the spread of infectious diseases from wildlife to domestic animals and humans. Therefore, it is important to know the serotype, antimicrobial resistance and presence of pathogenicity genes of Yersinia enterocolitica ( Ye ) isolated in species. From 2013 to 2018, we analyzed the liver of 4890 wild boars hunted in Liguria region; we isolated and serotyped 126 Ye positive samples. A decisive role in the pathogenicity is given by the presence of virulence genes; in Ye isolated we found ystB (~70%), ymoA (45.2%), ail (43.6%) and ystA (~20%). Moreover, we evaluated the susceptibility at various antimicrobic agents (Ampicillin, Chloramphenicol, Enrofloxacin, Gentamicin, Kanamycin, Trimethoprim-Sulfamethoxazole, Sulfisoxazole, Ceftiofur and Tetracycline). The antibiotic resistance was analyzed, and we found a time-dependent increase. It is important to shed light on the role of the wild boars as a reserve of potentially dangerous diseases for humans, and also on the antibiotic resistance that represents a public health problem.

The presence of Norovirus (NoV) and Hepatitis E virus (HEV) in non-depurated and depurated oysters raised in the north-west Italian coast was investigated by quantitative real-time RT-PCR. Total and inorganic arsenic (As) and the presence of marine biotoxins (DSP, ASP, PSP) by LC-MS were also investigated. NoV was detected through all the sampling period in non depurated and depurated oysters with highest levels during wintertime (>10 4 genome copies per gram, gc/g) and minimum values in summer below the LOQ (<130/140 gc/g). HEV has never been found as well as biotoxins. Total As concentration was found in oysters in the range 0.45-3.0 mg/kg, while inorganic As was found in all samples in concentrations below the LOQ (<0.020 mg/kg). The study highlights how the 24 h depuration process didn't reduce significantly NoV levels and therefore the high concentration of NoV in oysters could represent a risk for consumers especially during winter and spring months., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Virus detection in food requires appropriate elution and concentration techniques which need to be adapted for different food matrices. ISO/TS-15216-1:2017 and ISO/TS-15216-2:2019 describe standard methods for hepatitis A virus (HAV) research in some food only. Milk-based products containing one or more types of fruit are not covered by ISO procedures, even though they can be contaminated by fruit added to these products or by the food handlers. The aim of this work was to identify an efficient method for the detection of HAV in milk-based products. Four methods were tested to recover HAV from artificially contaminated milk, yoghurt and ice cream containing soft fruits. Results showed that the efficiency of the tested methods depends on the analyzed matrix. In milk we obtained a mean recovery from 13.4% to 1.9%; method based on high speed centrifuge gave the best values. The average recovery in yoghurt was between 3.3% and 114.4%, the latter value achieved by method with beef extract at 3% as eluent. Finally, two methods gave the best results in ice cream with similar recoveries: 29.1% and 27.7% respectively. The first method used glycine as eluent while the other one was based on high speed centrifugation. The ISO method has never proved to be the most efficient in the matrices studied. Therefore, based on the results obtained, a complete rethinking of the ISO method may be necessary to improve its recovery for some products such as milk, while only small changes would be sufficient for other products, such as yoghurt and ice cream., Competing Interests: Declaration of Competing Interest The authors declare there are no conflicts of interest regarding the study., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Shellfish farming is a relevant economic activity in Italy. The Gulf of La Spezia is one of the major production areas for mussels: the area is characterized by the presence of numerous human activities that could harm the quality of seawater. Additionally, the presence of potentially pathogenic microorganisms may influence the health status of animals, which must be constantly monitored. To have a clear view of the health conditions of the mussels ( Mytilus galloprovincialis ) farmed in this area, microbiological, parasitological, and histological analyses were performed. The study was conducted from November 2016 to October 2017. Overall, despite the presence of potentially pathogenic microorganisms for mussels, abnormal mortality rates were not reported during the monitoring period and the histological examination revealed no significant lesions. Our study confirms that studying different aspects together is a useful tool for assessing the health conditions of mussels and points out the importance of adverse environmental conditions for the expression of the pathogenicity of microorganisms.

Between 2015 and the beginning of 2018 (January-March), 30 cetaceans were found stranded along the Ligurian Sea coast of Italy. Necropsies were performed in 22 cases and infectious diseases resulted the most common cause of death. Three striped dolphins, showed a severe coinfection involving the monophasic variant of Salmonella Typhimurium (Salmonella 1,4,[5],12:i:-). The isolates were characterized based on antimicrobial resistance, Multiple-Locus Variable-number tandem-repeat Analysis (MLVA) and whole-genome sequencing (WGS). All isolates demonstrated the same multidrug resistant genotype (ASSuT isolates), showed three different MLVA profiles, two of which closely related, and were identified as Sequence Type 34. Moreover, Single nucleotide polymorphisms (SNP) analysis confirmed strong correlations between two out of the three isolates. To our knowledge, S. 1,4,[5],12:i:-, one of the most common serovars in cases of human infection and food sources worldwide, has not previously been described in marine mammals, and reports of Salmonella-associated disease in free-ranging cetaceans are rare. These results highlight the role of cetaceans as sentinel species for zoonotic and terrestrial pathogens in the marine environment, suggest a potential risk for cetaceans and public health along the North Western Italian coastline and indicate cetaceans as a novel potential reservoir for one of the most widespread Salmonella serovars.

Berries have repeatedly been associated with outbreaks of hepatitis A virus (HAV) infection. The fruits are usually minimally processed in the food industry due to their delicate nature. While washing treatments partially remove enteric viruses, the commonly used chemical additives produce toxic by-products. A valid alternative to preserve the food safety of these products could be the use of essential oils (EOs). EOs exert antimicrobial activity and do not interfere with the nutritional characteristics of food products. We investigated the efficacy of four essential oils, lemon (Citrus limon), sweet orange (Citrus sinensis), grapefruit (Citrus paradisi), and rosemary cineole (Rosmarinus officinalis chemotype 1.8 cineole) in reducing viral loads of HAV in soft fruits. Mixed fruit berries were inoculated with 10 6.74 TCID 50 /ml of HAV, and treated with four different EOs (0.5% lemon, 0.1% sweet orange and grapefruit, and 0.05% rosemary) for 1 h at room temperature. Virus infectivity was then assessed by titration assays for its ability to grow on cell cultures. A statistically significant reduction in HAV titer on the fruit surface was observed after treating the berries with EOs of lemon (2.84 log TCID 50 /ml), grapefruit (2.89 log TCID 50 /ml), and rosemary cineole (2.94 log TCID 50 /ml). Rosemary cineole was the most effective EO in reducing viral titer on berries, followed by grapefruit EO. These results improve our knowledge about the antiviral activity of these EOs and highlight their potential use in fresh produce sanitation.

We summarized the neuropathologic findings in 60 cetaceans stranded along the Italian coastline from 2002 to 2014. The following neuropathologic changes were detected in 45% (27/60) of animals: nonsuppurative meningo-encephalitides (30%, 18/60), nonspecific lesions (12%, 7/60), suppurative encephalitis (2%, 1/60), and neoplasm (2%, 1/60). No histologic lesions were found in 47% (28/60) of the specimens. Five (8%, 5/60) samples were unsuitable for analysis. Analysis with PCR detected Brucella spp., morbillivirus, and Toxoplasma gondii infection in one, six, and seven individuals, respectively. Immunohistochemical analysis confirmed positivity for morbillivirus and for T. gondii infection in three cases each. No evidence of the scrapie-associated prion protein PrPSc was detected. Our findings underscore the importance of an adequate surveillance system for monitoring aquatic mammal pathologies and for protecting both animal and human health.

The Gulf of La Spezia (northern Tyrrhenian Sea, Italy) is a commercially important area both as a shipping port and for mussel farming. Recently, there has been increased concern over environmental disturbances caused by anthropogenic activities such as ship traffic and dredging and the effects they have on the health of farmed mussels. This paper reports the results of microbiological and histological analyses, as well as of measurement of several biomarkers which were performed to assess the health status of mussels (Mytilus galloprovincialis) from four rearing sites in the Gulf of La Spezia. Mussels were collected between October 2015 and September 2016 and histological analyses (including gonadal maturation stage), as well as the presence of pathogenic bacteria (Vibrio splendidus clade, V. aestuarianus and V. harveyi), viruses (Herpes virus and ostreid Herpes virus 1) and protozoa (Marteilia spp., in the summer season only) were carried out on a monthly basis. Conversely, biomarker responses in haemocyte/haemolymph (total haemocyte count, haemocyte diameter and volume, lysozyme and lactate dehydrogenase activities in cell-free haemolymph, and micronuclei frequency) and in gills and digestive gland (cortisol-like steroids and lipid peroxidation levels), were evaluated bimonthly. Microbiological data indicated that mussels contain a reservoir of potentially pathogenic bacteria, viruses and protozoa that in certain environmental conditions may cause a weakening of the immune system of animals leading to mortality episodes. The percentage of parasites detected in the mussels was generally low (9.6% for Steinhausia mytilovum, that is 17 samples out of 177 examined females; 3.4% for Proctoeces maculatus; 0.9% for Mytilicola intestinalis and 2% for ciliated protozoa), while symbiont loads were higher (31% for Eugymnanthea inquilina and Urastoma cyprinae). Interestingly, a previously undescribed haplosporidian was detected in a single mussel sample (0.2%) and was confirmed by in situ hybridization. Cells morphologically similar to Perkinsus sp. trophozoites were observed in 0.7% of the mussels analysed; however, infection with Perkinsus spp. could neither be confirmed by ISH nor by PCR. Different pathological aspects, such as host defence responses and regressive/progressive changes were detected in the gills, digestive glands, gonads and mantle. Only one single case of disseminated neoplasia (0.2%) was observed. As for the biomarker evaluation, the MANOVA analysis revealed the statistically significant effect that the variable "sampling site" had on the biological parameter measured, thus suggesting that the multibiomarker approach was able to differentiate the rearing sites., (Copyright © 2018 Elsevier Inc. All rights reserved.)

A juvenile female striped dolphin Stenella coeruleoalba live stranded on 4 March 2016 at Alassio, western Ligurian Sea coast, Italy. The dolphin died shortly after stranding, and a complete postmortem examination was performed. Necropsy revealed severe tracheal occlusion and unilateral bronchial stenosis with luminal accumulation of abundant green-yellow mucous-gelatinous material. Histological features suggestive of tracheobronchial aspergillosis were observed. Cultures of lung tissue and tracheo-bronchial exudate isolated Aspergillus fumigatus, identified by a Microseq D2 LSUrDNA fungal sequencing kit. A pan-Herpesvirus nested-PCR assay on frozen samples obtained from multiple organs was positive. Phylogenetic analysis on the partial DNA polymerase gene revealed that the striped dolphin isolate was closely related to known cetacean Alphaherpesvirus sequences from the same host species. Attempted virus isolation was unsuccessful. The tissue levels of different persistent organic pollutants and the toxicological stress, evaluated using a theoretical model, showed a severely impaired immune response. This study reports the first case of occlusive mycotic tracheobronchitis in a free-living cetacean and the first molecular identification of an Alphaherpesvirus in a free-ranging striped dolphin stranded on the coast of Italy.

Between 2007 and 2014, 83 cetaceans were found stranded along the Ligurian coast of Italy, in the Pelagos Sanctuary, the largest marine protected area in the Mediterranean basin. Forty-nine (59%) were submitted to complete or partial necropsy, depending on the conservation status of the carcass. Based on gross and histological pathology and ancillary testing, the cause of death was determined and categorized as anthropogenic or natural (i.e., nonanthropogenic) in origin for 33 animals (67%) and of undetermined origin in the remaining 16 (33%). Natural causes of death, accompanied by either poor or good nutritional status, were attributed to 29 animals (59%), whereas four (8%) were diagnosed with an anthropogenic cause of death, consisting of interaction with fishing activities. Infectious and noninfectious disease was the most common cause of death, involving 29 cetaceans (59%). These data are valuable for understanding health and mortality trends in cetacean populations and can provide information for establishing policies for cetacean conservation and management in such an important protected area of the Mediterranean basin.

Opisthorchis felineus (family Opisthorchiidae) is a parasitic flatworm representing a serious threat to humans in some countries. Opisthorchiasis occurs after consumption of raw or undercooked cyprinid fish infected by the metacercarial stage of the parasite. Due to its small size, detection of the parasite in fish fillet is time-consuming and difficult. Furthermore, isolated metacercariae can be identified to genus but not to species level using morphological features and molecular techniques are necessary. In this work, we describe the development of primers for a diagnostic PCR amplification of a 254-bp fragment of the cytochrome c oxidase I in the mitochondrion of Opisthorchis felineus metacercariae isolated from fish fillet, together with a validation protocol for this method., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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)., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Meat products from HEV-infected reservoir animal species are capable of transmitting HEV to humans and represent a public health concern. Human HEV cases have been linked to the consumption of raw or undercooked pig liver sausages, pork, and game meats, such as wild boars and deer worldwide. Direct exposure to swine or wild game species might also represent a source of HEV transmission especially for veterinarians, hunters, or butchers. A limited amount of data is available on HEV prevalence in wild boars in Italy and no data are available for other wild game species intended for human consumption. In this study, the circulation of HEV in four different animal species hunted in north-western Italy was evaluated to gain insight into the infection levels and the genetic diversity of the virus in such animal populations. Liver samples of 372 wild boars, 30 roe deer, 47 European hares and 38 coypus were analyzed for HEV RNA by real-time RT-PCR; positive samples were then sequenced and submitted to phylogenetic analysis. HEV RNA was detected in the livers of 7/372 (1.9%) wild boars tested, while no sample was positive for roe deer, European hare, and coypu. Phylogenetic analysis showed that wild boar HEV sequences belonged to HEV subtypes 3e, 3c, and 3f. Our results indicate that HEV is circulating only in wild boar among the considered game species in north-western Italy and suggest a potential zoonotic risk related to handling and/or consumption of raw or undercooked meat and products made of the liver from this species.

Engraulis encrasicolus and Sardina pilchardus are pelagic fishes of notable economic and gastronomic importance in the northwest Mediterranean (Ligurian Sea, Italy). The consumption of thermally unprocessed or lightly processed, marinated or salted anchovies and sardines presents a potential risk to acquire anisakiasis, a fish-borne parasitic disease in humans. Prevalence and abundance of Anisakis larvae in Engraulis encrasicolus and Sardina pilchardus from the Monterosso fishing grounds (Cinque Terre National Park, Ligurian Sea, Italy) were assessed, and the larvae were identified by morphological and PCR-RFLP methods. Anisakis larvae, all belonging to Anisakis pegreffii spp. were found in the visceral mass of 1050 anchovies (0.8% overall prevalence), whereas no Anisakis larvae were found in the 750 sardines examined. According to these data, the risk of acquiring anisakiasis from the consumption of raw or undercooked anchovies and sardines caught in the fishing area we investigated is very low., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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., (© 2013 John Wiley & Sons Ltd.)

This study aims to investigate the occurrence of Anisakidae larvae (genera Anisakis and Hysterothylacium) in fishes and squids used for human nutrition and increase the knowledge of the distribution of Anisakidae larvae in this area of Mediterranean Sea. Distribution and correct identification of Anisakidae larvae in fish species is important for the assessment of their relative epidemiological role; especially when the fishes are of high commercial value and largely used in human nutrition. The study investigated the occurrence of Anisakidae larvae (genera Anisakis and Hysterothylacium) in 195 fishes owing to 22 different species and 60 squids (Illex coindetii) sampled in northern Ligurian Sea. A combination of morphological and PCR-RFLP methods have been used. A total of 177 anisakid larvae were isolated in 42/195 (21.5%) fish of 11 species and only one larva in one squid. These larvae were identified morphologically as belonging to the genera Anisakis (36/177) or Hysterothylacium (142/177). All Anisakis larvae were isolated from Trachurus trachurus and were identified at species level by PCR-RFLP as belonging to Anisakis pegreffii. Hysterothylacium sp. in short, larvae presence occurred in most of examined fish species with a higher density in Mullus barbatus (mean intensity 5.6 larvae) and Serranus scriba (MI 5.3)., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Norovirus (NoV) are increasingly important as etiological agents of gastrointestinal infections. Consumption of bivalve molluscs and ready-to-eat fishery products is one of the most common ways of acquiring NoV foodborne infections, and the rise of outbreaks of viral gastroenteritis represents an important health problem that is also responsible for economic losses. The aim of this work was to define the prevalence of NoV contamination in preserved fishery products and in shellfish commercialized in Italy, taking into account the results obtained during 9 years of survey (2003-2011) and paying special attention to the regions more involved in national production. A total of 4463 samples were examined (2310 mussels, 1517 clams, 510 oysters, 22 other shellfish species, 104 preserved seafood products) and the average positivity rate for NoV presence was 4.1% and ranged from 0.6% in 2007 to 9.8% in 2003 and from 1.9% in preserved seafood products to 4.7% in mussels. Genetic characterization of circulating strains showed a prevalence of genogroup II genotypes, including GII.b and GII.e polymerase types and different GII.4 variants. This information could contribute to the optimization of risk-based sampling strategies for NoV contamination in seafood, taking into account variability in different species and from year to year.

The relationship between Vibrio parahaemolyticus strains isolated from the aquatic environment and those isolated from cases of infection in humans is poorly understood due to the low prevalence of tdh- and/or trh-positive strains in the environment. To address this concern, it would be useful to analyse the genetic relationships among environmental and food strains and with reference to clinical isolates, also applying molecular typing methods. The aim of this study was to evaluate the prevalence of toxigenic V.parahaemolyticus in Italian coastal waters and seafood, to examine intra-species variability and to identify, using serotyping and pulsed-field gel electrophoresis (PFGE), relationships among strains from different sources, geographical origin and period of isolation. Of the 192 V.parahaemolyticus strains isolated in different Italian areas and examined in this study, 25 (13.0%) proved to carry the trh gene while none of the strains proved positive to the search by PCR for tdh and Group-Specific-toxRS genes. The prevalence of toxigenic strains in the Tyrrhenian Sea was significantly lower than that calculated for the Ligurian coasts. Regarding the sources of isolation, the higher prevalence of trh-positive V.parahaemolyticus was revealed in fish, followed by clams, plankton, oysters, mussels and lastly seawater. Within the toxigenic strains, 16 serotypes and 20 distinct PFGE patterns were identified. Two clusters, which included a total of 8 V.parahaemolyticus strains, were specifically associated with the North Adriatic Sea area and were stable over time. Our results demonstrate that trh-positive V.parahaemolyticus strains circulated in Italy in the period 2002-2009 with a prevalence higher than that reported from other European and extra-European countries, confirming that toxigenic V.parahaemolyticus is an emerging public health concern in Italy, regardless of its pandemic potential., (© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.)

We investigated the virulence properties of four Vibrio parahaemolyticus strains causing acute gastroenteritis following consumption of indigenous mussels in Italy. The isolated strains were cytotoxic and adhesive but, surprisingly, lacked tdh, trh, and type three secretion system 2 (T3SS2) genes. We emphasize that nontoxigenic V. parahaemolyticus can induce acute gastroenteritis, highlighting the need for more investigation of the pathogenicity of this microorganism.

In this study, we investigated the presence of enteric viruses such as norovirus (NoV), hepatitis A virus (HAV), hepatitis E virus (HEV), and adenovirus (HAdV), in vegetables available on the Italian markets. For this aim, 110 national and international "ready to eat" samples were collected and analyzed by biomolecular tests and positive samples were confirmed by sequencing. All samples (100 %) were negative for HAV, HEV, and HAdV, while 13.6 % (15/110) were positive for NoV. Actually there is not a formal surveillance system for NoV infections in Italy but we clearly demonstrated a potential risk associated with the consumption of "ready to eat" vegetables. This study confirmed for the first time in Italy the presence of norovirus in semi-dried tomatoes by PCR technique.

Objective: celiac disease (CD) is an immune-mediated chronic inflammatory disease associated with HLA-DQ2 and DQ8 molecules. We evaluated the role of HLA in the CD diagnostic algorithm in order to contribute to the development of practical indications for the use of HLA typing., Material and Methods: we selected 317 subjects typed for DR-DQ genes. CD was present in 123 patients, and 89 were included in the study; a control sample of 70 healthy individuals was recruited., Results: 64% of patients with CD carried DQ2 heterodimer (α5β2), 13.5% carried DQ8 heterodimer without DQ2, 21.4% only showed β2 chain and 1.1% were positive for DQ2 α5 chain. The only presence of α5 chain did not predispose to CD, while DQB1*02 allele resulted more frequent than in other reports, pointing out the intrinsic correlation between β2 chain and CD. In the case-control study we observed a progression of increased risk, ranging from 1:7 for HLA-DQ2 homozygous to 1:85 for DQ8 heterozygous subjects. Overall, 8,6% of first degree family members were affected, exclusively in presence of HLA-DQ2, -DQ8 or DQB1*02, and CD was significantly more frequent among siblings than parents. Finally, considering the different patterns of clinical presentation among the HLA-DQ risk classes identified we found no relationship between CD clinical presentation and HLA-DQ risk categories., Conclusions: our results strengthen the evidence that HLA-DQ status strongly influences the development of CD and demonstrate that knowledge of a patient's HLA-DQ genotype allows to establish clinically relevant genetic risk profiles.

Background: Although Morbillivirus and Toxoplasma gondii have emerged as important pathogens for several cetaceans populations over the last 20 years, they have never been identified together in a Mysticete. In particular, morbilliviral infection has been never described in the Mediterranean fin whale population., Case Presentation: On January 2011 an adult male of fin whale (Balaenoptera physalus) stranded along the Tyrrhenian coastline of Italy. During necropsy, tissue samples from heart, skeletal muscle, mesenteric lymph nodes, liver, spleen, lung, and kidney were collected and subsequently analyzed for Morbillivirus and Toxoplasma gondii by microscopic and molecular methods. Following the detailed necropsy carried out on this whale, molecular analysis revealed, for the first time, the simultaneous presence of a Dolphin Morbillivirus (DMV) and T. gondii infection coexisting with each other, along with high organochlorine pollutant concentrations, with special reference to DDT., Conclusion: This report, besides confirming the possibility for Mysticetes to be infected with DMV, highlights the risk of toxoplasmosis in sea water for mammals, already immunodepressed by concurrent factors as infections and environmental contaminants.

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., (© 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.)