Occurrence of hepatitis A and E and norovirus GI and GII in ready-to-eat vegetables in Italy.

Fresh vegetables and their ready-to-eat (RTE) salads have become increasingly recognized as potential vehicles for foodborne diseases. The EU Reg. 1441/2007 establishes microbiological criteria for bacterial pathogens for products placed on the market during their shelf-life (i.e. Salmonella spp., Listeria monocytogenes) for pre-cut fruits and vegetables (RTE) whilst it does not address the problem of contamination by enteric viruses. In this study we investigated the contamination by hepatitis A virus (HAV), hepatitis E virus (HEV) and norovirus (NoV) in 911 ready-to-eat vegetable samples taken from products at retail in Apulia and in Lombardia. The vegetable samples were tested using validated real-time PCR (RT-qPCR) assays, ISO standardized virological methods and ISO culturing methods for bacteriological analysis. The total prevalence of HAV and HEV was 1.9% (18/911) and 0.6% (6/911), respectively. None of the samples analysed in this study was positive for NoV, Salmonella spp. or Listeria monocytogenes. The detection of HAV and HEV in RTE salads highlights a risk to consumers and the need to improve production hygiene. Appropriate implementation of hygiene procedures is required at all the steps of the RTE vegetable production chain and this should include monitoring of emerging viral pathogens.

MALDI-TOF mass spectrometry analysis of proteins and lipids in Escherichia coli exposed to copper ions and nanoparticles.

Escherichia coli (E. coli) is one of the most important foodborne pathogens to the food industry responsible for diseases as bloody diarrhea, hemorrhagic colitis and life-threatening hemolytic-uremic syndrome. For controlling and eliminating E. coli, metal nano-antimicrobials (NAMs) are frequently used as bioactive systems for applications in food treatments. Most NAMs provide controlled release of metal ions, eventually slowing down or completely inhibiting the growth of undesired microorganisms. Nonetheless, their antimicrobial action is not totally unraveled and is strongly dependent on metal properties and environmental conditions. In this work, we propose the use of matrix-assisted laser desorption ionization time-of-flight (MALDI TOF) mass spectrometry as a powerful tool for direct, time efficient, plausible identification of the cell membrane damage in bacterial strains exposed to copper-based antimicrobial agents, such as soluble salts (chosen as simplified AM material) and copper nanoparticles. E. coli ATCC 25922 strain was selected as 'training bacterium' to set up some critical experimental parameters (i.e. cell concentration, selection of the MALDI matrix, optimal solvent composition, sample preparation method) for the MS analyses. The resulting procedure was then used to attain both protein and lipid fingerprints from E. coli after exposure to different loadings of Cu salts and NPs. Interestingly, bacteria exposed to copper showed over-expression of copper binding proteins and degradation of lipids when treated with soluble salt. These findings were completed with other investigations, such as microbiological experiments. Copyright © 2016 John Wiley & Sons, Ltd.

Detection of Vibrio parahaemolyticus in shellfish using polymerase chain reaction-enzyme-linked immunosorbent assay.

AIMS: This study evaluated the application of polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA) for the detection of Vibrio parahaemolyticus in shellfish. METHODS AND RESULTS: The PCRs were selected to amplify a species-specific sequence region. In particular, internal tl biotin-labelled oligonucleotide probe was used to capture the DIG-labelled PCR products. Next, the probe PCR product hybrids, immobilized on a streptavidin-coated microtiter plate, were detected with peroxidase-conjugated anti-digoxigenin antibody (anti-DIG-POD) and the colorimetric peroxidase substrate ABTS [2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)] using an ELISA plate reader. CONCLUSIONS: The PCR-ELISA system described is a feasible, sensitive method for the direct and specific detection of V. parahaemolyticus in shellfish samples. Compared with gel-based detection methods, PCR-ELISA in this study increased sensitivity by 100-fold for V. parahaemolyticus. SIGNIFICANCE AND IMPACT OF THE STUDY: The PCR-ELISA described may be used for potential rapid detection in routine shellfish analysis for the seafood industry. The sector requires simultaneous large-scale sample screenings to monitor contamination levels in processing plants and evaluate the performance of the hazard analysis and critical control point (HACCP) system. PCR-ELISA also proved to be economical, with a cost of about 9 Euros per sample, and the quick assay taking 8 h to complete starting from DNA extraction.

Ciprofloxacin-modified electrosynthesized hydrogel coatings to prevent titanium-implant-associated infections.

New promising and versatile materials for the development of in situ sustained release systems consisting of thin films of either poly(2-hydroxyethyl methacrylate) or a copolymer based on poly(ethylene-glycol diacrylate) and acrylic acid were investigated. These polymers were electrosynthesized directly on titanium substrates and loaded with ciprofloxacin (CIP) either during or after the synthesis step. X-ray photoelectron spectroscopy was used to check the CIP entrapment efficiency as well as its surface availability in the hydrogel films, while high-performance liquid chromatography was employed to assess the release property of the films and to quantify the amount of CIP released by the coatings. These systems were then tested to evaluate the in vitro inhibition of methicillin-resistant Staphylococcus aureus (MRSA) growth. Moreover, a model equation is proposed which can easily correlate the diameter of the inhibition haloes with the amount of antibiotic released. Finally, MG63 human osteoblast-like cells were employed to assess the biocompatibility of CIP-modified hydrogel coatings.

Survey of the presence of patulin in fruit juices.

Patulin is a mycotoxin produced by fungi belonging to the Penicillium and Aspergillus genera. The occurrence of patulin in fruit juices marketed in Italy in 2008 and purchased from supermarkets and retail shops has been measured. The aim of this study was to assess the presence of patulin in order to evaluate the potential risk for the consumer and, at the end of this food chain, to determine the quality of the raw material used. One hundred and five fruit juices (35 apple juice, 35 mixed-taste juices, 35 pear juices), produced by various Italian and European companies, were analysed using a previously published method. The analytical investigation showed that apple juices had a concentration of patulin ranging between 6 and 30 µg l(-1), with a mean of 18 µg l(-1); mixed fruit juices had a concentration ranging between 1 and 45 µg l(-1), with a mean of 23 µg l(-1). Instead, pear juices had a concentration ranging between 5 and 92 µg l(-1), with a mean of 43 µg l(-1), and 14 samples of the 35 analysed juices showed a patulin level above the highest regulated limit of 50 µg l(-1), imposed by European Commission Regulation 1881/06.

Norovirus in retail shellfish.

Norovirus is a common cause of gastroenteritis outbreaks associated with consumption of raw shellfish. The majority of norovirus infections worldwide are due to genogroup II noroviruses. Bivalve molluscs (mussels, clams and oysters) at the end of the commercial chain, the points of purchase, were sampled between 2005 and 2008 in several retail points in Apulia, Italy, and screened by a semi-nested RT-PCR specific for genogroup II noroviruses. Noroviral RNA was detected in 12.1% of the samples, with lower frequency being observed in samples obtained from hypermarkets (8.1%) rather than in samples from open-air markets and fish shops (17.6% and 16.2%, respectively). By sequence analysis, the strains were characterized as norovirus variants GII.4/2004 and GII.b/Hilversum, which were both circulating in Italy in the same time-span.

Determination of ochratoxin A at part-per-trillion level in Italian salami by immunoaffinity clean-up and high-performance liquid chromatography with fluorescence detection.

A fast high-performance liquid chromatography method has been devised for the determination of ochratoxin A (OTA) in Italian salami in the low part-per-trillion (pg/g) level. The samples were extracted with ethyl acetate and purified by immunoaffinity column (IAC). The IAC eluate could be directly injected or previously concentrated 10-fold. Recovery at 0.5 and 1 ng/g was 77 +/- 4%. The between-day coefficient of variation measured over 5 days on samples spiked at 1 ng/g was 8%. The developed method required a relatively small volume of non-halogenated organic solvent and the whole procedure was simpler and faster compared to other existing procedures. The limit of detection was 0.06 ng/g that could be even lowered using a preconcentration step. A total of 30 salami samples were analysed using this procedure; the most contaminated sample was found to have OTA concentration at 0.4 ng/g level.

Synthesis, analytical characterization and bioactivity of Ag and Cu nanoparticles embedded in poly-vinyl-methyl-ketone films.

The electrosynthesis of copper and silver core-shell nanoparticles (NPs) by the sacrificial anode technique, employing tetraoctylammonium (TOA) salts as base electrolyte for the first time, is described. These surfactants were selected because they combine high NP stabilizing power with useful disinfecting properties. The resulting colloids were mixed with a solution of an inert dispersing polymer and used to prepare nanostructured composite thin films. The morphologies and chemical compositions of the nanomaterials were characterized by Transmission Electron Microscopy (TEM) and X-ray Photoelectron Spectroscopy (XPS). The TEM reveals that the average core diameter of the metal NPs ranges between 1.7 and 6.3 nm, as a function of the nature of the metal and of the electrosynthesis conditions, and does not change significantly upon inclusion in the polymer matrix. An appreciable concentration of the metal is detected on the nanoparticle surface by XPS. High-resolution XP spectra indicate that both copper and silver are present at zero oxidation state in all of the materials (colloids and composite films). This demonstrates the high efficiency of the surfactant at controlling the morphology and the chemical composition of the nanodispersed metal in both the as-synthesized colloid and in the polymeric dispersion. The nanocoatings are shown to exert a marked inhibitory effect on the growth of eukaryote and prokaryote target microrganisms, and experimental evidence of a synergic disinfecting effect due to the surfactant and the nanodispersed metal is provided. On the basis of these stability and bioactivity results, it is clear that Cu-NPs and Ag-NPs are suitable for application in disinfecting or antifouling paint and coating formulations.

Analytical characterization of bioactive fluoropolymer ultra-thin coatings modified by copper nanoparticles.

Copper-fluoropolymer (Cu-CFx) nano-composite films are deposited by dual ion-beam sputtering. The extensive analytical characterization of these layers reveals that inorganic nanoparticles composed of Cu(II) species are evenly dispersed in a branched fluoropolymer matrix. In particular, X-ray photoelectron spectroscopy has been employed to study the surface chemical composition of the material and to assess how it changes on increasing the copper loading in the composite. Transmission electron microscopy reveals that the copper nanoclusters have a mean diameter of 2-3 nm and are homogeneously in-plane distributed in the composite films. Electrothermal atomic absorption spectroscopy has been used to study the kinetics of copper release in the solutions employed for the biological tests. The Cu-CFx layers are employed as bioactive coatings capable of inhibiting the growth of target microorganisms such as Saccharomyces cerevisiae, Escherichia coli, Staphylococcus aureus, and Lysteria. The results of the analytical characterization enable a strict correlation to be established among the chemical composition of the material surface, the concentration of copper dissolved in the microorganisms broths, and the bioactivity of the nano-structured layer.

Determination of ochratoxin A in meat products by high-performance liquid chromatography coupled to electrospray ionisation sequential mass spectrometry.

A method based on liquid chromatography with electrospray ionisation ion trap mass spectrometry, for the determination of ochratoxin A (OTA) in meat products using ochratoxin B (OTB) as an internal standard, is described. Fragmentation patterns of OTA and OTB were studied by sequential mass spectrometry. Trace determination was then accomplished by consecutive reaction monitoring (CRM) of a fragment obtained by MS(3) experiments. This led to a better signal-to-noise ratio and to a higher specificity of the technique. The response to OTA was linear over at least one concentration decade with a limit of detection of 0.6 ng/g. The method was applied to pig tissue samples naturally contaminated by OTA.

Updated perspectives on emerging vibrios associated with human infections.

This review describes the ecological, clinical and epidemiological features of emerging vibrios and discusses what laboratory methods are being used for the detection of pathogenic vibrios in clinical, environmental and food samples. After selecting articles illustrative of the current scientific research on pathogenic vibrios, the review focuses on the need for better insight into the risk factors of emerging infections to establish adequate prevention procedures.

A comparison of RT-PCR-based assays for the detection of HAV from shellfish.

The hepatitis A virus (HAV) is the most common cause of viral infection linked to shellfish consumption. The lack of correlation between the fecal coliform indicators and the presence of enteric viruses in shellfish and their harvesting waters points to the need for molecular methods to detect viruses. We compared two RT-PCR based techniques currently available for the detection of the hepatitis A virus (HAV) in shellfish. Both approaches involve extraction of viral particles by glycine buffer and concentration of virus particles by one or two PEG precipitation steps. One procedure involves as RNA extraction method the use of oligo (dT) cellulose to select poly (A) RNA, and the other uses a system in which total RNA is bound on silica membrane. Comparison of the two RT-PCR based methods highlighted the efficiency of the first approach which is less time-consuming and technically demanding than the second.

Determination of ochratoxin A in pig tissues by liquid-liquid extraction and clean-up and high-performance liquid chromatography.

A fast, simple, and sensitive HPLC-FD method is described for determination of ochratoxin A (OTA) in pig kidney and muscle; a small mass (<2.5 g) of sample and a relatively small volume (<15 mL) of a non-halogenated extraction solvent are required. Ochratoxin B, systematically absent from all the samples investigated, was used as internal standard. Liquid-liquid partition was used for sample clean-up. Recoveries at the 1 ng x g(-1) level were 86+/-15% and 74+/-8% for kidney and muscle, respectively, and detection limits were 0.14 and 0.15 ng x g(-1). Clean-up by solid-phase extraction (SPE) is required for pig liver. A survey of the OTA content of tissues of pigs slaughtered in southern Italy revealed that 52 out of 54 analysed samples were contaminated; the OTA concentration in kidney ranged between 0.26 and 3.05 ng x g(-1). The effect of measurement precision on compliance with legal limits is also discussed.

Detection of hepatitis A virus in shellfish (Mytilus galloprovincialis) with RT-PCR.

A PCR assay for the detection of hepatitis A virus (HAV) in shellfish is described. The procedure involves the concentration of viral particles with the use of polyethylene glycol (PEG), followed by viral RNA extraction and purification with oligo(dT) cellulose. Reverse transcriptase-PCR detection was accomplished in a single step with the use of primers specific for the VP3-VP1 region of the genome. The procedure detected one 50% tissue culture infective dose (0.6 PFU) per 25 g of shellfish homogenate. Heminested PCR was then carried out to verify the specificity of the PCR products. The method was used to detect HAV in shellfish samples from EU categories B and C and to evaluate the quality of shellfish in routine monitoring for HAV in view of the relevant public health implications of this foodborne disease.

Polymerase chain reaction for the direct detection of Brucella spp. in milk and cheese.

A polymerase chain reaction test was developed to detect Brucella spp. directly in milk and cheese and optimized using primers for the BSCP-31 gene. A total of 46 cheese samples produced with sheep and goats milk were assayed, and Brucella spp. was detected in 46% of them, especially in cheese made from sheep milk. This method is of remarkable epidemiologic interest because it is an indirect test indicating the sanitary quality of milk used in dairy industries. The method showed good sensitivity and specificity. It is faster and less expensive than the conventional bacteriological assays.

Flavonoids from leaves of Olea europaea L. cultivars.

We isolated and identified the following flavonoid compounds from the dried leaves of some blooming cultivars of Olea europaea L.: hesperidin, rutin, luteolin-7-O-glucoside, apigenin, apigenin-7-O-glucoside, quercetin, kaempferol. The structure of the isolated flavonoids was determined by UV, 1H-NMR, 13C-NMR, HPLC.

HLA-B8,DR3 haplotype affects lymphocyte blood levels.

The number of lymphocytes in the blood is constant, pointing to an effective control of circulating lymphocyte values. The mechanisms of this regulation are uncertain, although it is likely that the number of blood lymphocytes is conditioned by hormones, homing factors and cytokines whose production is at least partly restrained by genetic factors. Particularly genetic factors linked to major histocompatibility complex (MHC) appear to be involved. In human beings a decreased number of blood lymphocytes has been described in healthy subjects carrying the Human Leucocyte Antigens (HLA) haplotype HLA-B8,DR3. In the present study, to inquire into the mechanisms of this lymphocyte decreased number, we have performed an analysis of blood subset values in these subjects. When the absolute values of lymphocytes were analysed according to HLA phenotype, HLA-B8,DR3 positive subjects (N = 26) displayed significantly lower values as compared to HLA-B8,DR3 negative ones (N = 282). The analysis of lymphocyte subpopulations performed by flow cytometry in 72 subjects did not show significant changes in lymphocyte subset percentages between HLA-B8,DR3 positive subjects and negative ones. Thus, the decrease of circulating lymphocytes seems to be due to a reduction of cell number affecting all lymphocyte subsets rather than a single cell subpopulation. The analysis of in vitro spontaneous apoptosis performed by flow cytometry in a smaller sample of subjects showed a significant increase of spontaneous apoptosis in lymphocytes from HLA-B8,DR3 positive individuals suggesting a possible explanation for the deviation from normal lymphocyte count observed in these subjects. However it is intriguing that a decreased number of blood lymphocytes can be observed in healthy HLA-B8,DR3 positive subjects but also in autoimmune diseases linked to this haplotype like systemic lupus erythematosus and insulin-dependent diabetes. Furthermore, in our opinion, this finding is to be kept in mind in evaluating hematological parameters in healthy subjects.

Microbial detection by a glucose biosensor coupled to a microdialysis fibre.

The use of a glucose biosensor coupled to microdialysis sampling in a flow injection analysis system is described to follow the growth of Escherichia coli in a glucose-containing liquid culture medium. The experimental set-up permitted a throughput rate of 25 samples h-1. Growth curves were modelled by a modified Gompertz equation, which permitted the determination of lag time and maximum specific growth rate. The time required to produce an appreciable variation in the biosensor response (minimum detection time, MDT) was determined. A plot of MDT versus microbial concentration was found to be linear in the range 10(6)-10(10) colony forming units (cfu) ml-1. A microbial concentration of 10(6) cfu ml-1 can be detected after about 5 h.

Pharmacological and histopathological aspects of 1-methyl-4-phenyl-4-hydroxy-piperidine (MPIP).

Increasing dosages of 1-methyl-4-phenyl-4-hydroxypiperidine (MPIP) were administered i.p. to rats. Histopathological examination of these animals revealed effects on the central nervous system.

Dominant V beta 8 gene usage in response to TNP: failure to use other V beta chains following removal of V beta 8+ T cells by monoclonal antibody in vivo.

This paper investigates the V beta usage of lymph node cells from mice immunized with TNP and of cell lines made from them. In cell lines stimulated weekly with TNP in vitro for 1 month, about 87% of the cells were V beta 8+ and further analysis showed that these cells were actually V beta 8.2+. This was also true for the cells that proliferated in lymph nodes in response to TNP 4 days after primary immunization, i.e. proliferation occurred mainly in the V beta 8+, and in particular in the V beta 8.2+, population while much less proliferation occurred when the V beta 8- or V beta 8.2- T-cell populations are used. This was not due to non-specific damage during separation, as the response to concanavalin A and alloantigen was intact. In a separate series of experiments, mice were acutely depleted of V beta 8+ T cells by treatment with F23.1 or a control monoclonal antibody (mAb) in vivo given before immunization. Treatment with the relevant mAb virtually abolished the response to TNP. In contrast, SJL mice, which lack the gene segment coding for the V beta 8 family and several other V beta chains, made a normal proliferative and delayed-type hypersensitivity (DTH) response to TNP. This poses the problem, which may be important in the study of the T-cell repertoire, of why acute removal of V beta 8+ T cells, which are dominantly used in the response to TNP, does not allow T cells using other chains to substitute in the response, while the absence of this population over a long period of time, because of a deletion in the genome, allows the use of T cells bearing other V beta chains.