Aniello Anastasio

Benzimidazole carbamate residues in milk: Detection by Surface Plasmon Resonance-biosensor, using a modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) method for extraction

A surface plasmon resonance (SPR) biosensor screening assay was developed and validated to detect 11 benzimidazole carbamate (BZT) veterinary drug residues in milk. The polyclonal antibody used was raised in sheep against a methyl 5(6)-[(carboxypentyl)-thio]-2-benzimidazole carbamate protein conjugate. A sample preparation procedure was developed using a modified QuEChERS method. BZT residues were extracted from milk using liquid extraction/partition with a dispersive solid phase extraction clean-up step. The assay was validated in accordance with the performance criteria described in 2002/657/EC. The limit of detection of the assay was calculated from the analysis of 20 known negative milk samples to be 2.7mugkg(-1). The detection capability (CCbeta) of the assay was determined to be 5mugkg(-1) for 11 benzimidazole residues and the mean recovery of analytes was in the range 81-116%. A comparison was made between the SPR-biosensor and UPLC-MS/MS analyses of milk samples (n=26) taken from cows treated different benzimidazole products, demonstrating the SPR-biosensor assay to be fit for purpose.

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.

Genetic identification and distribution of the parasitic larvae of Anisakis pegreffii and Anisakis simplex (s. s.) in European hake Merluccius merluccius from the Tyrrhenian Sea and Spanish Atlantic coast: implications for food safety.

The consumption of the hake Merluccius merluccius is widespread in European countries, where this fish has a high commercial value. To date, different larval species of Anisakis have been identified as parasites in M. merluccius from European waters, Anisakis pegreffii and Anisakis simplex (s. s.) being the two most common. The aim of the study is to present data on the occurrence of Anisakis spp. larvae in the viscera and flesh of M. merluccius. Consequently, the distribution and infection rates of different species of Anisakis in different sites (viscera, and dorsal and ventral fillets) were investigated in hake caught in the central Tyrrhenian Sea (FAO 37.1.3) and the NE Atlantic Ocean (FAO 27 IXa). A sample of N=65 fish individuals (length>26 cm) was examined parasitologically from each fishing ground. The fillets were examined using the pepsin digestion method. A large number (1310) of Anisakis specimens were identified by multilocus allozyme electrophoresis (MAE) and mtDNA cox2 sequence analysis; among these, 814 larvae corresponded to A. simplex (s. s.) and 476 to A. pegreffii. They were found to infect both the flesh and the viscera. The two species co-infected the same individual fish (both in the viscera and in the flesh) from the FAO 27 area, whereas only A. pegreffii was found in hake from the Tyrrhenian Sea. The average parasite burden of A. pegreffii in hake from the Tyrrhenian Sea was significantly lower to that observed from hake off the Atlantic coast of Spain, both in prevalence and in abundance. In addition, whereas no significant difference in overall prevalence values was recorded between the two Anisakis species in the viscera of the FAO 27 sample, significant differences were found in the abundance levels observed between these species in the flesh, with A. simplex (s. s.) exhibiting significantly higher levels than that observed for A. pegreffii (p<0.001). Given that the pathogenic role in relation to man is known for these two species of Anisakis, both the flesh inspection and the infection rates of the different anisakid species assume particular importance in terms of assessing the risk they pose to humans.

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.

Characterization of Shiga toxin subtypes and virulence genes in porcine Shiga toxin-producing Escherichia coli

Similar to ruminants, swine have been shown to be a reservoir for Shiga toxin-producing Escherichia coli (STEC), and pork products have been linked with outbreaks associated with STEC O157 and O111:H-. STEC strains, isolated in a previous study from fecal samples of late-finisher pigs, belonged to a total of 56 serotypes, including O15:H27, O91:H14, and other serogroups previously associated with human illness. The isolates were tested by polymerase chain reaction (PCR) and a high-throughput real-time PCR system to determine the Shiga toxin (Stx) subtype and virulence-associated and putative virulence-associated genes they carried. Select STEC strains were further analyzed using a Minimal Signature E. coli Array Strip. As expected, stx2e (81%) was the most common Stx variant, followed by stx1a (14%), stx2d (3%), and stx1c (1%). The STEC serogroups that carried stx2d were O15:H27, O159:H16 and O159:H-. Similar to stx2a and stx2c, the stx2d variant is associated with development of hemorrhagic colitis and hemolytic uremic syndrome, and reports on the presence of this variant in STEC strains isolated from swine are lacking. Moreover, the genes encoding heat stable toxin (estIa) and enteroaggregative E. coli heat stable enterotoxin-1 (astA) were commonly found in 50 and 44% of isolates, respectively. The hemolysin genes, hlyA and ehxA, were both detected in 7% of the swine STEC strains. Although the eae gene was not found, other genes involved in host cell adhesion, including lpfAO113 and paa were detected in more than 50% of swine STEC strains, and a number of strains also carried iha, lpfAO26, lpfAO157, fedA, orfA, and orfB. The present work provides new insights on the distribution of virulence factors among swine STEC strains and shows that swine may carry Stx1a-, Stx2e-, or Stx2d-producing E. coli with virulence gene profiles associated with human infections.

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.

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.

Heavy metals in canned tuna from Italian markets.

Fish is a good source of nutrients for humans but can pose a risk to human health because of the possible presence of some xenobiotics such as heavy metals and persistent organic contaminants. Constant monitoring is needed to minimize health risks and ensure product quality and consumer safety. The aim of the present study was to use atomic absorption spectrometry to determine the concentrations of some heavy metals (Hg, Pb, and Cd) in tuna packaged in different kinds of packages (cans or glass) in various countries (Italy and elsewhere). Concentrations of Cd and Hg were within the limits set by European Commission Regulation (EC) No 1881/2006 and in many samples were below the detection limit. Pb concentrations exceeded European limits in 9.8% of the analyzed samples. These results are reassuring in terms of food safety but highlighted the need to constantly monitor the concentrations of heavy metals in fish products that could endanger consumer health.

Proteomics analysis for the identification of three species of Thunnus

The genus Thunnus comprises many species, some of higher quality and commercial value for their excellent organoleptic features, while others of lower quality and value. Consequently, these species are subjected to frequent fraudulent substitution. Increasing trade in fillet and minced fish makes the identification of external anatomical and morphological features of fish impossible. Proteomics was used for the identification of three Thunnus species. Muscle extracts were evaluated by both mono- and two-dimensional electrophoresis and mass spectrometric techniques. Preliminary results demonstrate that the tested species displays a high degree of polymorphism, making possible an accurate identification.

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.