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Antimicrobial resistance is one of the most challenging public health problems worldwide, and integrated surveillance is a key aspect in a One Health control strategy. Additionally

Background The emergence of multidrug resistant (MDR) pathogens has drawn attention to natural antimicrobial compounds, such as essential oils (EOs) Methods The aim of the present study was to investigate the inhibitory activity of three EOs against 15 Salmonella strains resistant to 16 commonly used antimicrobials. EOs of O. vulgare, T. serpyllum, and T. vulgariswere tested in triplicate for 15 bacterial strain by Kirby-Bauer disk diffusion method and broth microdilution method to determine the minimal inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC), according to the Clinical and Laboratory Standards Institute guidelines with minor modifications. Results O. vulgare and T. serpyllum showed high antimicrobial activity compared to T. vulgaris in all tested antibiotic-resistant strains (P-value Conclusions The EOs of O. vulgare and T. serpyllum, which contain carvacrol as the main constituent, showed the greatest antimicrobial effect against MDR Salmonella strains.

During the last decades, plant-based milk has become very appreciated by consumers, becoming a staple ingredient, especially for alternative breakfasts. Milk contains lactose, which is a sugar hydrolysed by the lactase enzyme. Lactose intolerance and lactose malabsorption are very common food intolerances among individuals. However, a lot of consumers consider themselves as lactose intolerant on the basis of self-reported intolerance and start to avoid dairy products, ignoring that plant-based milk alternatives are not nutritionally comparable to animal milk, especially in terms of protein intake. The aim of this study is to grow folder knowledge of the security of plant-based drinks, helping competent authorities to issue a risk assessment and to apply national plans about consumer safety. Results show that proper sanitary practices, such as pasteurization, are necessary in plant-based milk alternatives as well as in dairy milk. Chemical analysis has highlighted that there are no pesticide risks for consumers.

Hard ticks’ geographical distribution and abundance are influenced by wildlife population. This work presents the results of the identification of ticks retrieved from wild animals in the framework of a Regional Plan of Monitoring and Surveillance of Wildlife health. The frequency of distribution of ticks in different hosts and their geographical patterns were also investigated. Ticks were collected from game animals (Sus scrofa, Capreolus capreolus, Dama dama, and Rupicapra rupicapra) during two hunting seasons (2018–2019 and 2019–2020) in the four provinces of the Liguria region in northwest Italy. In the same period, ticks were also collected from carcasses of Vulpes vulpes, Canis lupus, Meles meles, and Asio otus received for necropsy. Tick species were identified according to taxonomic keys. A total of 819 ticks, removed from 259 animals, were found and identified. Overall, Ixodes ricinus was the dominant species (62.6%), followed by Dermacentor marginatus (24.5%), Rhipicephalus sanguineus s.l. (12.5%), Haemaphysalis punctata (0.2%), and Ixodes hexagonus (0.1%). I. ricinus was also the prevalent species in roe deer and in fallow deer and the only species collected from the three wolf carcasses examined. In contrast, D. marginatus was the dominant species in S. scrofa. This last tick species was also more frequent in one province (Imperia), whereas Ixodes spp. were more common in another one (Savona). Wild animals proved to be useful for characterizing and monitoring tick population.

Cadmium (Cd2+) is regarded as one of the most toxic heavy metals, which can enter the food chain through environmental contamination and be bioaccumulated. Its exposure in Ligurian wild boars was monitored between 2016-2020 and revealed high level of this heavy metal in different provinces. In one of these polluted area, 21 wild boars were additionally sampled and the relationship between hepatic and renal Cd2+concentration suggested that majority of these animals presented chronic intoxication. Cd2+exposure of wild boar might lead to an immunosuppression status, thusin vitroexperiments on wild boar monocyte-derived macrophages (moMФ) were carried out. Effects of Cd2+scalar doses were evaluated through viability and adsorption assays, ELISA, qPCR. Moderate doses of this environmental pollutant (20 μM) were absorbed by moMФ, with subsequent reduction of their viability. This heavy metal did not trigger release of either IFN- β, anti-inflammatory or pro-inflammatory cytokines by moMФ, instead 24 h treatment with 20 μM of Cd2+resulted in down-regulated expression of TNF-α, IL-12p40, several TLRs, CD14, MD2, BD2, MyD88, p65, and NOS2. The results of our monitoring activity suggested that wild boar can be useful to monitor environmental exposure of this heavy metal and can help in understanding the type of contamination. In addition,in vitroexperiments on wild boar moMФ revealed that Cd2+exposure negatively affected the immune function of these cells, likely leading to increased susceptibility to infection.

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 × 102 and 2.3 × 104 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.

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.

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 (>104 genome copies per gram, gc/g) and minimum values in summer below the LOQ (

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.

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.

Between May 2011 and February 2014, 205 samples of Crassostrea gigas, farmed on the northwest coast of Italy, were collected and analyzed for OsHV-1, Vibrio parahaemolyticus, V. cholerae, V. alginolyticus, V. aestuarianus, bacteria belonging to V. splendidus clade, E. coli, Salmonella spp., environmental parameters and algae. Statistical analysis was carried out to correlate all the data of the environmental parameters, algae and Vibrio with OsHV-1 in the presence and absence of die-off. Of the samples tested, 46.8 % (96/205) were positive for the detection of OsHV-1 DNA, 0.97 % (2/205) of the oysters were positive for V. harveyi DNA detection and 8.8 % (18/205) were positive for V. aestuarianus. Statistical analysis showed a positive correlation between the presence of V. aestuarianus, OsHV-1 and a die-off occurred in November 2013. Sequencing analysis of the PCR products revealed that the genotype of OsHV-1 is highly related to the variant µVar.

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.

Antimicrobial resistance has become a global threat to public health. There is a critical need to find new antimicrobial substances from natural sources. The aim of this study was to investigate the antimicrobial activity of essential oils (EOs) obtained from Origanum vulgare, Thymus serpyllum, Thymus vulgaris, and Melaleuca alternifolia against multidrug resistant strains of Salmonella isolated from samples of diverse animal origin. The strains were biochemically identified, serotyped, and characterized for their antimicrobial resistance profiles. The antimicrobial activity of the EOs against the strains was evaluated using the Kirby-Bauer diffusion method, followed by determination of the minimal inhibitory concentration and minimum bactericidal concentrations. The EOs of T. serpyllum and O. vulgare, which contain carvacrol as the main compound, show excellent antimicrobial activity.

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.

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).

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.

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 microrganisms are widespread in the population of mullets studied, and this could be a possible cause for outbreaks in favourable environmental conditions.

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.

The school canteens are public catering services of great interest as they provide meals to a high number of consumers who are particularly susceptible to health risks, therefore surveillance and health control are very important to ensure food safety. To this purpose, a convention between the Istituto Zooprofilattico Sperimentale of Piemonte Liguria e Valle d’Aosta, and La Spezia municipality was established for the health control of school canteens. In this article we report the results of analysis performed on food and swab surfaces samples collected during the period 2003-2012 in 22 school canteens and 3 cooking centers. From a total of 1187 samples: 313 raw foods were analyzed for chemical and microbiological parameters to verify compliance with legislation, 71 food preparations were analyzed for bacteria indicators to assess the good manufacturing practices, and 803 surface swabs were tested for total mesophilic count (TMC), Salmonella spp. and Listeria spp. to control cleaning/disinfection conditions. The results show that only 1.3% of raw food samples did not respect the limits imposed by legislation, and 1.4% of food preparations was positive for pathogens. In environmental swabs, pathogenic microorganisms were never isolated and TMC exceeded the limits of acceptability in no more than 27% of cases. The most contaminated surfaces were those in contact with food and the equipment difficult to clean. The results demonstrate that potential hazards are kept to acceptable levels in school canteens and cooking centers investigated. In fact, during the period considered no foodborne diseases were reported among users. However, data obtained may be useful to better define control measures to be adopted to improve the hygienic level production in these structures and to prevent foodborne infections.

Aims: The purpose of this work was to verify whether E. coli is a good indicator of viral contamination in mussels and Adenovirus could represent a better alternative as indicator organism of viral presence to guarantee consumer health protection. Methods and Results: Eighty samples of mussels from La Spezia Gulf were analysed for E. coli, Salmonella, Adenovirus, Norovirus and hepatitis A virus with cultural and biomolecular tests. The results of bacterial parameters showed E. coli within the law’s limits and the absence of Salmonella. Twelve samples were positive for Adenovirus presence, one for Norovirus genogroup II and two for hepatitis A virus. None of these positive mussels was found to be contaminated with more than one virus at the same time. Conclusion: This study showed that there was not a direct correlation between the presence of human pathogenic viruses and bacterial indicators. Significance and Impact of the Study: Both E. coli and Adenovirus cannot be considered valid substitutes for the direct research of human pathogenic viruses in mussels. To improve consumer health protection, the European Commission will provide standardized methods for Norovirus and hepatitis A virus detection as soon as possible.

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.