The complete DNA sequence and analysis of the virulence plasmid and of five additional plasmids carried by Shiga toxin-producing Escherichia coli O26:H11 strain H30.

Shiga toxin-producing Escherichia coli (STEC) strains belonging to serogroup O26 have been associated with sporadic cases and outbreaks of hemorrhagic colitis and hemolytic uremic syndrome. In addition to chromosomal virulence genes, STEC strains usually harbor a large plasmid that carries genes associated with pathogenicity. The complete nucleotide sequence and genetic organization of 6 plasmids carried by STEC O26:H11 strain H30 were determined. The large virulence plasmid (pO26-Vir) was approximately 168 kb in size and contained 196 open reading frames (ORFs). pO26-Vir possesses a mosaic structure and shows similarity to the virulence plasmids in locus of enterocyte effacement (LEE)-negative STEC O113:H21 EH41 (pO113), in E. coli clinical strain C1096 (pSERB1), and in E. coli O157:H7 RIMD 0509952 (pO157). Plasmid pO26-Vir shares several highly conserved regions with pO157 and carries important virulence genes, including toxB, katP, espP, and the hly gene cluster. In addition, pO26-Vir possesses genes encoding for type IV pili (pilL-V). The second largest plasmid, pO26-L (73 kb) contains 101 ORFs. pO26-L carries the tetracycline resistance gene and has regions that show similarity to the E. coli conjugative resistance plasmid NR1. The third largest plasmid, pO26-S4 (5.8 kb), is homologous to the ColE2 colicinogenic plasmid that encodes for colicin E2. The remaining 3 plasmids, pO26-S1 (1.5 kb), pO26-S2 (3.1 kb), and pO26-S3 (4.2 kb), carry very little genetic information except for putative proteins involved in plasmid replication and DNA maintenance. The data presented underscore the diversity among the STEC virulence plasmids and provide insights into the evolution of these plasmids in STEC strains that cause serious human illness.

Development of biogenic amines during the ripening of Italian dry sausages.

The effect of modification of different chemical and microbiological parameters and the production of biogenic amines (histamine, cadaverine, putrescine, and tyramine) was examined during ripening of various types of typical Italian dry sausages (salami). Water activity decreased from 0.97 to 0.87, and pH reached the lowest value between the 13th and the 20th day of the ripening period, and then increased. Putrescine (up to 122.7 mg/kg) and tyramine (up to 105.9 mg/kg) mean levels showed dominance in comparison with cadaverine (up to 26.1 mg/kg) and histamine (up to 6.2 mg/kg) mean values in all sausage types. The highest putrescine and tyramine concentrations were observed in salami with the largest diameters. This comparative study suggests a good correlation between microbial behavior and amine evolution, particularly tyramine and putrescine, in dry sausage production.

Detection of Campylobacter from poultry carcass skin samples at slaughter in Southern Italy.

Campylobacter is a major foodborne pathogen responsible for acute gastroenteritis characterized by diarrhea that is sometimes bloody, fever, cramps, and vomiting. Campylobacter species are carried in the intestinal tracts of mammals and birds, and sources of human infection include raw milk, contaminated water, direct contact with pets, and foods, particularly poultry. Campylobacter jejuni and C. coli are the species that account for the majority of human infections. The aim of this work was to determine the prevalence of Campylobacter in 190 poultry carcasses sampled at slaughter and to use a multiplex PCR assay to determine if the isolates were C. jejuni or C. coli. C. coli was not isolated, while C. jejuni was recovered from 52 (37.1%) of 140 carcasses for which pools of four sampling sites (neck, cloaca, breast, and back) were examined. In the remaining 50 carcasses, the four sites were analyzed separately, and C. jejuni was recovered from the samples in the following order: neck (n = 20), cloaca (n = 16), breast (n = 14), and back (n = 11). The results are in agreement with those of other studies, which showed that C. jejuni is more commonly associated with poultry than is C. coli. Control strategies for Campylobacter should include interventions to eliminate C. jejuni in poultry at various stages of production and processing, including at slaughter.

Fish species identification in surimi-based products.

Whole fish morphologically identified as belonging to Theragra chalcogramma, Merluccius merluccius, Merluccius hubbsi, and Merluccius capensis and 19 fish products commercialized as surimi with different commercial brands and labeled as T. chalcogramma were analyzed by direct sequence analysis of the cytochrome b gene. A phylogenetic analysis of surimi products was performed as well. Results demonstrated that mislabeling is a large-scale phenomenon, since 84.2% of surimi-based fish products sold as T. chalcogramma (16/19) were prepared with species different from the one declared. In fact, only three samples (samples 15-17) were found to belong to T. chalcogramma. In the remaining samples, Merluccidae (samples 4-14), Gadidae (samples 18 and 19), Sparidae (sample 1), and Pomacentridae (samples 2 and 3) families were detected. A phylogenetic tree was constructed, and the bootstrap value was calculated. According to this methodology, 11 samples were grouped in the same clade as Merluccius spp.

Formulation and Shelf-life of Fish Burgers Served to Preschool Children.

Consumer is very careful about healthiness; in this context nutritionists often highlight the importance of fish for human nutrition because of their protein and fatty acid composition. In order to stimulate utilisation and consumption of fish species by unusual target groups such as children, the aim of this research was to formulate and to evaluate shelf-life and nutritional values of fish preparations stored in modified atmosphere packaging (MAP). Fish species used for trail were Trachurus trachurus and Oncorhynchus mykiss fished and farmed in Basilicata region respectively. Fish burgers were made with different ingredients of plant and animal origin and packed in air (control) and in MAP and stored at refrigeration atemperature. Sensory, physical-chemical analysis as pH, aw, total volatile nitrogen (TVN), trimetilammine (TMA), thiobarbituric acid (TBA), free fatty acids (FFA) and microbiological analysis like aerobic plate count, Enterobacteriaceae, Escherichia coli, Pseudomonas spp., sulphite-reducing clostridia, Staphylococci, Salmonella spp. and Listeria monocytogenes were performed at intervals of 0°, 1°, 2°, 5°, 8°, 15°, 22°, day from production. Results showed that fish burgers stored in MAP had a longer shelf-life; protein degradation indexes and spoilage bacterial species showed lower values in the samples packaged in MAP compared with the control. The formulation of the fish burger meets the approval of the target consumers. The mixing of natural ingredients has made possible both the enhancement of the organoleptic characteristics with an excellent balance of nutritional values. The diversification of fish preparations, besides enhancing the fish production of marginal areas would add value to a product with potential and remarkable profit margins.

Preliminary Study on Physicochemical and Biochemical Stress Markers at Poultry Slaughterhouse.

Pre-slaughter stress can result in variations in the glycogen storage and metabolic changes of muscle, responsible for quality poultry meat. Aim of this study was to investigate, as pre-slaughter stress markers and quality meat, physicochemical (pH), biochemical (muscle glycogen content), and chemical (super oxides free radicals) parameters. The carcass quality, as incidence of individual carcass defects, was also evaluated. Twenty broilers were processed with two different electrical stunning: high (250 Hz; 640 mA; 60V) (Lot C or control) and low (150 Hz; 360 mA; 60 V) (Lot A) frequency and intensity, using sinusoidal alternating current. As preliminary results, the use of low frequency and intensity induced faster pH decline post mortem and adequate acidification of pH at 3 hours (6.49 Lot C; 6.37 Lot A), better muscle glycogen reserve (0.770 µL/50 mL Lot C; 1.497 µL/50mL Lot A), and lightly more rapid muscle oxidation (IDF: 0.109 Lot C; 0.122 Lot A), (FOX: 0.131 MeqO2/kg Lot C; 0.140 MeqO2/kg Lot A). The incidence of individual carcass defects sufficient to cause downgrading or rejection, both in Lot C and Lot A, was generally low. In a multidisciplinary approach, to assess animal welfare and quality poultry meat, additional and feasible parameters should be implemented. Monitoring of pH, muscle glycogen reserve and superoxide free radical production measurements might be markers easier to use, routinely, in practice at abattoir. Further studies are needed to evaluate the usefulness of these parameters.

Multiplex PCR to detect bacteriophages from natural whey cultures of buffalo milk and characterisation of two phages active against Lactococcus lactis, ΦApr-1 and ΦApr-2.

This work investigated bacteriophage induced starter failures in artisanal buffalo Mozzarella production plants in Southern Italy. Two hundred and ten samples of whey starter cultures were screened for bacteriophage infection. Multiplex polymerase chain reaction (PCR) revealed phage infection in 28.56% of samples, all showing acidification problems during cheese making. Based on DNA sequences, bacteriophages for Lactococcus lactis (L. lactis), Lactobacillus delbruekii (L. delbruekii) and Streptococcus thermophilus (S. thermophilus) were detected. Two phages active against L. lactis, ΦApr-1 and ΦApr-2, were isolated and characterised. The genomes, approximately 31.4 kb and 31 kb for ΦApr-1 and ΦApr-2 respectively, consisted of double-stranded linear DNA with pac-type system. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS­PAGE) showed one major structural protein of approximately 32.5 kDa and several minor proteins. This is the first report of phage isolation in buffalo milk and of the use of multiplex PCR to screen and study the diversity of phages against Lactic Acid Bacteria (LAB) strains in artisanal Water Buffalo Mozzarella starters.

Soybean and Lactose in Meat Products and Preparations Sampled at Retail.

Food allergies and intolerances have increased during the last decades and regulatory authorities have taken different measures to prevent and manage consumers' adverse reactions, including correct labelling of foods. Aim of this work was to search for soybean and lactose in meat products and meat preparations taken from retail in some provinces of Campania Region (Southern Italy) and to evaluate the food labels compliance with Regulation (EU) n.1169/2011. Soybean and lactose were searched using commercial kits in n. 58 samples of meat products produced in or distributed by 19 establishments, and in n. 55 samples of meat products and n. 8 of meat preparations produced in 21 plants. All samples were selected on the basis of the absence of any information on the labels about the presence of the two searched allergens, with the exception of n. 5 samples tested for lactose. Traces of soybean were detected in 50 out of the 58 examined samples, at concentrations up to 0.93 mg kg-1. Only two samples contained levels above the detection limit of 0.31 mg kg-1. Lactose levels ranging from 0.11 to 2.95 g/100 g, i.e. above the detection limit, were found in all the tested samples (n. 63). The results of the present research underline the need for careful controls and planning by operators as part of the self-control plans, and deserve attention from the competent authorities considering not only the consumers' health but also the great attention media pay to regulations providing consumers with information on food.

Occurrence and distribution of polycyclic aromatic hydrocarbons in mussels from the gulf of Naples, Tyrrhenian Sea, Italy.

To assess the potential impact of the industrial activity on food safety and risk for consumers, the aim of the study was to evaluate the levels of 14 polycyclic aromatic hydrocarbons (PAH) in 69 samples of wild and farm Mytilus galloprovincialis, collected in sites of coast of Gulf of Naples, Tyrrhenian Sea. All hydrocarbons were found in samples. Higher levels of pyrolytic PAHs were in wild than in farm mussels. Benzo(a)pyrene exceeded the Regulation (EC) n.835/11 levels of 1 µg/kg in 15 samples (71.42%) of wild and 25 samples (65.79%) of farm mussels. System of sum of 4 hydrocarbons exceeded the law level in 15 samples (71.42%) of wild and 21 samples (55.26%) of farm mussels. Wild mussel levels showed a potential impact of pyrolytic sources of PAH on food safety. Occurrence of carcinogenic PAHs should be a cause for concern, in areas where the mussels are being farmed for human consumption.

Efficient HPLC method for the determination of nicarbazin, as dinitrocarbanilide in broiler liver.

A simple, fast and reliable HPLC-UV method has been developed for the determination of dinitrocarbanilide residues in broiler liver. Liver samples (2 g) were extracted with two portions of acetonitrile (10 and 5 ml), defatted with hexane and evaporated to dryness under nitrogen. Extracts were reconstituted in acetonitrile-water (70/30, v/v, 500 microl), loaded onto C18 solid phase (SPE) cartridges and eluted with acetonitrile-water (70/30, v/v, 2.5 ml) into clean test-tubes. Extracts were evaporated to dryness and reconstituted in acetonitrile-water (80/20, v/v, 500 microl). An aliquot of the extract was assayed by high performance liquid chromatography (HPLC) with UV detection at 350 nm. The method was validated according to EU guidelines using liver tissues fortified at levels of 100, 200 and 300 microg/kg, with dinitrocarbanilide. The decision limit (CC(alpha)) and the detection capability (CC(beta)) were calculated from the within laboratory repeatability data to be 228 and 266 microg/kg, respectively. The mean recovery was typically >70% and the limits of quantitation was 12.5 microg/kg (based on the lowest standard on the calibration curve).

Mitochondrial cytochrome b DNA sequence variations: an approach to fish species identification in processed fish products.

The identification of fish species in food products is problematic because morphological features of the fish are partially or completely lost during processing. It is important to determine fish origin because of the increasing international seafood trade and because European Community Regulation 104/2000 requires that the products be labeled correctly. Sequence analysis of PCR products from a conserved region of the cytochrome b gene was used to identity fish species belonging to the families Gadidae and Merluccidae in 18 different processed fish products. This method allowed the identification of fish species in all samples. Fish in all of the examined products belonged to these two families, with the exception of one sample of smoked baccalà (salt cod), which was not included in the Gadidae cluster.

Fate of eprinomectin in goat milk and cheeses with different ripening times following pour-on administration.

The distribution of eprinomectin in goat milk and cheeses (cacioricotta, caciotta, caprilisco) with different ripening times following a pour-on administration at a single dose rate (500 microg/kg of body weight) and a double dose rate (1,000 microg/kg of body weight) to goats with naturally occurring infections of gastrointestinal nematodes was studied. Milk residues of eprinomectin reached a maximum of 0.55+/-0.18 microg/kg and 1.70+/-0.31 microg/kg at the single and double doses, respectively. The drug concentrations decreased progressively until the fifth day after treatment, when they were less than the detection limit at both dose rates. The eprinomectin levels measured in all cheese types (both treatments) were higher than those recovered in milk at all the sampling times. In caciotta cheeses, the eprinomectin residues levels were constantly higher than other cheeses. With the exception of cheeses made with milk the first day after treatment, eprinomectin concentrations were nearly constant up to the fourth day then decreased by the fifth and sixth days after treatment. In all cases, at both the single and double dosages, the maximum level of eprinomectin residues in goat milk and cheeses remained below the maximum residual level of 20 microg/liter permitted for lactating cattle.

Optimization of Stunning Electrical Parameters to Improve Animal Welfare in a Poultry Slaughterhouse.

Animal killing for food production and the related operations are events that may induce pain, stress, fear and other forms of suffering to the animals. To face this problem and guarantee the animal welfare, the EU has adopted the Regulation (EC) N. 1099/2009 on the protection of animals at the time of killing. Electrical water bath stunning is one of the methods used in commercial slaughterhouses to protect poultry welfare. In particular, this method induces unconsciousness into the birds due to run of electrical current through the head and body. The aim of the present work was to find an optimal setting of electrical parameters to obtain an effective water bath stunning in a commercial poultry slaughterhouse. Moreover, the influence of the tested electrical parameters on meat quality was evaluated. All the experiments confirmed that high stunning frequencies induce a lower occurrence of lesions on carcasses but, on the other hand, require greater current intensities to be effective. A frequency of 750 Hz and an average current intensity of 200 mA for each bird in the water bath resulted as the best combination of electrical parameters to obtain a proper stunning without any consequence on the meat quality.

Levels of benzo[a]pyrene (BaP) in "mozzarella di bufala campana" cheese smoked according to different procedures.

The content of benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon, was determined by HPLC-FL in "mozzarella di bufala campana" cheese, a stretched cooked cheese, either experimentally smoked according to traditional procedures, using straw, cardboard, and wood shavings or aromatized with smoke flavoring. The BaP residues, researched also in cheese samples sold at retail, were detected in the rind, in the core, and in the slice (outer and inner parts). In the cheeses experimentally smoked with straw and cardboard the BaP levels, ranging from 0.38 to 2.12 microg kg(-1) and from 0.46 to 2.40 microg kg(-1), respectively, were statistically higher than those of the cheeses smoked with wood shavings and aromatized with liquid smoke (from 0.19 to 0.80 microg kg(-1) and from 0.18 to 0.50 microg kg(-1), respectively). However the cheeses treated with liquid smoke flavor showed a BaP content exceeding the level allowed by the European Union. In the samples sold at retail, smoked with straw, values were lower than those obtained from samples smoked experimentally with the same combustible. This is probably due to different smoking technologies among the several provinces of the Protected Designation of Origin (PDO) area. PDO is a term used to characterize foodstuffs produced and prepared in a given geographical region by the means of a recognized process. A standardization of the traditional smoking procedures and an improvement of liquid smoke purification treatments are recommended for mozzarella cheese.

Residue study of ivermectin in plasma, milk, and mozzarella cheese following subcutaneous administration to buffalo (Bubalus bubalis).

The distribution of ivermectin in buffalo plasma and milk after administration of a single subcutaneous dose (0.2 mg kg(-)(1) b.w.) was studied. Ivermectin reached the maximal concentration in plasma (28.5 +/- 1.7 ng mL(-)(1)) and milk (23.6 +/- 2.6 ng mL(-)(1)) after 2.4 +/- 0.32 and 2.8 +/- 0.44 days, respectively. The drug showed a parallel disposition in milk and plasma, with a ratio of 1.12 +/- 0.16. Ivermectin concentrations were detected in mozzarella cheese obtained from milk collected on days 1, 3, 4, and 20 following administration. The highest values (81.4 +/- 3.26 ng g(-)(1)) were found in the cheese produced on day 3 and were 4-fold higher than those present in the milk.

The Capacity of Listeria Monocytogenes Mutants with In-Frame Deletions in Putative ATP-Binding Cassette Transporters to form Biofilms and Comparison with the Wild Type.

Listeria monocytogenes (Lm) is a food-borne pathogen responsible for human listeriosis, an invasive infection with high mortality rates. Lm has developed efficient strategies for survival under stress conditions such as starvation and wide variations in temperature, pH, and osmolarity. Therefore, Lm can survive in food under multiple stress conditions. Detailed studies to determine the mode of action of this pathogen for survival under stress conditions are important to control Lm in food. It has been shown that genes encoding for ATP-binding cassette (ABC) transporters are induced in Lm in food, in particular under stress conditions. Previous studies showed that these genes are involved in sensitivity to nisin, acids, and salt. The aim of this study was to determine the involvement of some ABC transporters in biofilm formation. Therefore, deletion mutants of ABC transporter genes (LMOf2365_1875 and LMOf2365_1877) were created in Lm F2365, and then were compared to the wild type for their capacity to form biofilms. Lm strain F2365 was chosen as reference since the genome is fully sequenced and furthermore this strain is particularly involved in food-borne outbreaks of listeriosis. Our results showed that ΔLMOf2365_1875 had an increased capacity to form biofilms compared to the wild type, indicating that LMOf2365_1875 negatively regulates biofilm formation. A deeper knowledge on the ability to form biofilms in these mutants may help in the development of intervention strategies to control Lm in food and in the environment.

Polychlorodibenzodioxin and -furan and dioxin-like polychlorobiphenyl distribution in tissues and dairy products of dairy buffaloes.

A pilot study was performed on three different dairy buffalo herds exposed without exposure control conditions to Polychlorodibenzodioxins and -furans (PCDDs, PCDFs) and Dioxin-like Polychlorobiphenyls (DL-PCBs). This study dealt with the relationship between the contamination levels (pg WHO2005-TE/g fat) in individual raw milk and those in edible tissues and with the contamination transfer from farm bulk milk to dairy products. On a cumulative basis, kidney (41, 67, and 21 pg WHO-TE/g fat) resulted more in equilibrium with milk (48, 42, and 20) than did muscle (25, 31, and 9), while liver showed a large bioaccumulation (221, 304, and 75), with marked differences of the congener profile. Mozzarella cheese contamination (23, 42, and 29 pg WHO-TE/g fat) was higher than that of bulk milk (20, 36, and 21), which suggested a role of casein precipitation in congener transfer. The above information could improve the effectiveness of risk management during a "dioxin" crisis.

Norovirus monitoring in bivalve molluscs harvested and commercialized in southern Italy.

Norovirus (NoV) is the main cause of human nonbacterial gastroenteritis throughout the world. NoVs are classified into five genogroups: GI, GII, GIII, GIV, and GV. NoVs from GI and GII are the most commonly reported NoVs associated with human infections, and raw or undercooked shellfish have been identified as the main potential infection vehicle. European Commission Regulation 2073/2005 defines only bacteriological parameters for use as safety criteria for shellfish because reference methods for detection of viruses are lacking. From July 2007 to April 2010, 163 shellfish samples were collected in southern Italy from harvesting areas, authorized or nonauthorized retailers, and a restaurant after an outbreak of human gastroenteritis. The shellfish were analyzed for the presence of NoVs from GI and GII using the one-step real-time reverse transcription PCR protocol. A total of 94 shellfish samples (57.7%) were positive for the presence of NoV, and GII was the most frequently identified genogroup.

Investigation of the persistence of levamisole and oxyclozanide in milk and fate in cheese.

In this study, dairy cows (n = six) were treated with an oral combination product containing levamisole (5 mg/kg body weight, (bw)) and oxyclozanide (10 mg/kg bw). Animals were milked twice daily up to day 16 post-treatment. Milk samples were subsequently analyzed by ultraperformance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). The highest levels of levamisole (<600 µg/kg) and oxyclozanide (<25 µg/kg) were determined at first and third milking, respectively. Residues of levamisole and oxyclozanide were typically below reporting limits of 0.83 and 1 µg/kg respectively at the 11th and 13th milking, respectively. Soft (3 days ripening), hard (35 days ripening) and whey cheeses were produced from the milk samples collected from the first two milkings. Levamisole residues were found to concentrate in all cheese types. There was a 3-fold concentration effect for levamisole in mature cheese. Oxyclozanide residues were found to occur at lower levels in soft and hard cheese than milk with a 10-fold concentration in whey cheese compared to milk. The results of this study demonstrate that levamisole and oxyclozanide residues are rapidly excreted in dairy cows and milk is compliant after a few days. Oxyclozanide and levamisole residues were shown to be stable during the fermentation process and the whey heat treatment to persist in cheese.

FCS-based sensing for the detection of ochratoxin and neomycin in food.

In this work, we present an advanced fluorescence assay for the detection of traces of ocratoxin A and neomycin in food. The described assay is based on measurement of the fluctuations of the fluorescein-labeled analytes by a focused laser beam in the absence and in the presence of the specific antibodies anti-analytes. A competitive assay based on the utilization of unlabeled analytes was developed. The obtained results indicated that the combination of high-avidity IgG antibodies together with an innovative fluorescence immunoassay strategy resulted in the detection limit of 0.0078 ng and 0.0156 ng for ochratoxin A and neomycin, respectively.