Claudia Chirollo

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.

Evaluation and comparison of four protein extraction protocols for mono- and two-dimensional electrophoresis in Mytilus galloprovincialis

In this study, four protein extraction protocols from Mytilus galloprovincialis were evaluated with the aim to identify the most practical, efficient and reproducible method. Four extraction protocols frequently used for mussels and organic matrices were selected and compared. The methods were based on the use of: i) TRIzol reagent; ii) Lysis buffer; iii) phenylmethanesulfonyl fluoride; iv) trichloroacetic acid-acetone. Protein concentration was measured by the Bradford method. Three specimens of mussels were studied and the analysis was conducted in triplicate for each of the four protocols. Results indicated that the four methods could extract significantly different protein profiles. The highest number of protein spots resolved in 2DE gels and the best reproducibility was obtained using trichloroacetic acid-acetone protocol. Results afforded the selection of a suitable extraction protocol to be used for ecotoxicoproteomics studies from mussels and for other proteomic studies conducted by particularly complex tissues such as Mytilus galloprovincialis.

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.

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.

Investigation of the Persistence of Penicillin G and Dihydrostreptomycin Residues in Milk of Lactating Buffaloes (Bubalus bubalis) Using Ultra-High-Performance Liquid Chromatography and Tandem Mass Spectrometry

The purpose of this research was to evaluate the persistence of penicillin G and dihydrostreptomycin in milk of lactating buffaloes following intramuscular injection of procaine penicillin G (200000 IU/mL) and dihydrostreptomycin sulfate (250 mg/mL) every 24 h for 3 days. Milk samples were collected twice daily up to the 13th milking post-treatment and analyzed by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry. The analytical method has been validated according to Commission Decision 2002/657/EC. The highest concentrations of penicillin G (275 μg kg-1) and dihydrostreptomycin (220.5 μg kg-1) were detected in the milk of the first milkings post-treatment, and levels were below the maximum residue limit of 4 and 200 μg kg-1 in all treated buffaloes at milkings 12 and 2, respectively. The results of this study demonstrate that a nine-milking withdrawal time set for bovine milk was not adequate for depletion of penicillin G in lactating buffaloes.

Persistence of α-cypermethrin residues in milk of lactating donkeys (Equus asinus) using UHPLC-MS/MS

The aim of this study was to measure the persistence of residues of the pyrethroid insecticide α-cypermethrin (ACYP) in the milk of lactating donkeys following pour-on treatment. Milk was collected from animals (n = 7) before the treatment and at 12, 24, 36, 48, 60, 72 and 84 h post-treatment. The last sampling was taken 7 days post-treatment (168 h). Milk samples were analysed by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). The analytical method was validated following requirements of Commission Decision 2002/657/EC. All samples showed levels of ACYP below the maximum residue limit (MRL) of 20 μg kg−1 established for bovine milk (Commission Regulation (EU) No. 37/2010). The results demonstrate that there is minimal partitioning of ACYP into milk in lactating donkeys from pour-on treatment. Graphical Abstract