Assessing the health status of farmed mussels (Mytilus galloprovincialis) through histological, microbiological and biomarker analyses.

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.

Noroviruses in seafood: a 9-year monitoring in Italy.

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.

Occurrence of both subspecies of Photobacterium damselae in mullets collected in the river Magra (Italy).

The natural reservoirs and biological characteristics of pathogenic populations of both subspecies of Photobacterium damselae in aquatic habitants remain unclear because of difficulties in obtaining pathogenic strains from the environment. In the present study, we assessed the occurrence of Photobacterium damselae subsp. piscicida and Photobacterium damselae subsp. damselae , considered to be the causative agent of past epizootic outbreaks in mullets collected in the river Magra, Italy. Two hundred and seventy-eight mullets were collected during a period of two years (2008-2009) and analyzed using multiplex PCR. During this period, 57% of fishes were positive for Photobacterium damselae subsp. piscicida and 37% for Photobacterium damselae subsp. damselae, with an higher presence in summer months although none of PCR-positive mullets showed clinical signs of disease. Our results indicate that the two micro-organisms are widespread in the population of mullets studied, and this could be a possible cause for outbreaks in favourable environmental conditions.

HAV detection from milk-based products containing soft fruits: Comparison between four different extraction methods.

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.

Antiviral Activity of Essential Oils Against Hepatitis A Virus in Soft Fruits.

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 106.74 TCID50/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 TCID50/ml), grapefruit (2.89 log TCID50/ml), and rosemary cineole (2.94 log TCID50/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.

Development and validation of species-specific molecular diagnostic tool for Opisthorchis felineus (Digenea, Opisthorchiidae) metacercariae.

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.

Detection of West Nile and Usutu Viruses in Italian Free Areas: Entomological Surveillance in Piemonte and Liguria Regions, 2014.

West Nile virus and Usutu virus have established in different parts of Italy over the past 10 years. Piemonte and Liguria Regions (Northwestern Italy) are known to be nonendemic areas, despite the presence of competent vectors and environmental conditions conducive to maintaining infection. This work evidences for the first time, through an entomological surveillance implemented on the basis of risk factor approach, the presence of West Nile and Usutu viruses in Piemonte and Liguria Regions (Northwestern Italy).

Molecular Investigation on the Presence of Hepatitis E Virus (HEV) in Wild Game in North-Western Italy.

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.

Food safety considerations in relation to Anisakis pegreffii in anchovies (Engraulis encrasicolus) and sardines (Sardina pilchardus) fished off the Ligurian Coast (Cinque Terre National Park, NW Mediterranean).

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.

Survey on the presence of Anisakis and Hysterothylacium larvae in fishes and squids caught in Ligurian Sea.

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).

Potential risk of norovirus infection due to the consumption of "ready to eat" food.

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.