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A high-performance liquid chromatography – fluorescence detection (HPLC-FLD) method was established for the determination of piperazine residues in whole eggs, albumen, and yolk employing pre-colum...

An ultra-performance liquid chromatography-electrospray ionization tandem triple quadrupole mass spectrometry (UPLC-ESI/MS/MS) method was developed for the detection of piperazine in chicken muscle. Following extraction and purification, the chicken muscle extract was injected into the UPLC system and analyzed. Piperazine detection was performed on a triple-quadrupole mass spectrometer with the ESI interface operating in positive mode. The most sensitive mass transition from the precursor ion to the product ion was 87.1 → 44.1 for piperazine. The coefficient (R 2) of piperazine was 0.9995 in the range of 1–200 μg/kg. The recovery and relative standard deviation (RSD) ranged from 102.93 to 111.46% and from 4.57 to 5.28% at 50.0, 100.0 and 200.0 μg/kg adding levels, respectively. Limits of detection (LODs) and limits of quantification (LOQs) were 0.4 and 1.0 μg/kg, respectively. This method was fully validated based on its specificity, sensitivity, linearity, precision, accuracy, matrix effect, and stability results. Compared to other researches, this new method not only omitted the derivative process, which simplify the pre-processing, but also proved a better sensitivity based on its low LOD and LOQ values.

Several previous studies have shown that bacteriophages can significantly affect the production of various cytokines. The aim of this present study was to investigate the inflammatory effects and mechanisms of bacteriophage vB_SauM_JS25 in stimulated MAC-T bovine mammary epithelial cells by real-time polymerase chain reaction (PCR) and Western blotting. Experiments show that vB_SauM_JS25 reduces Staphylococcus aureus- or lipopolysaccharide (LPS)-induced levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, IL-8, IL-10, and regulated on activation, normal T cell expressed and secreted (RANTES) mRNA in MAC-T cells, in a manner expected to be unrelated to its antibacterial action. Moreover, S. aureus bacteriophage vB_SauM_JS25 suppressed the LPS-induced phosphorylation of nuclear factor (NF)-κB p65, which may represent an important mechanism mediating these effects. A carefully regulated balance between activation and inhibition by bacteriophages must be kept avoiding inappropriate inflammatory responses. The ability of vB_SauM_JS25 to influence the immune response highlights the potential development and application of bacteriophage-based therapies and may represent a novel anti-inflammatory therapeutic strategy.

Primary porcine bronchial epithelial cells (PBECs) are an ideal model to study the molecular and pathogenic mechanisms of various porcine respiratory pathogens. However, the short lifespan of primary PBECs greatly limit their application. Here, we isolated and cultured primary PBECs and established immortalized PBECs by transfecting primary PBECs with the pEGFP-hTERT recombinant plasmid containing human telomerase reverse transcriptase (hTERT). Immortalized PBECs (hTERT-PBECs) retained the morphological and functional features of primary PBECs as indicated by cytokeratin 18 expression, telomerase activity assay, proliferation assays, karyotype analysis, and quantitative reverse-transcriptase polymerase chain reaction. Compared to primary PBECs, hTERT-PBECs had higher telomerase activity, extended replicative lifespan, and displayed enhanced proliferative activity. Moreover, this cell line is not transformed in vitro and does not exhibit a malignant phenotype in vivo, suggesting that it can be safely used in further studies. Besides, hTERT-PBECs were susceptible to swine influenza virus of H3N2 subtype and porcine circovirus type 2. In conclusion, the immortalized hTERT-PBECs represent a valuable in vitro model, which can be widely used in the study of porcine respiratory pathogenic infections.

This paper describes a novel method that combines acetic anhydride derivatization with gas chromatography–electron ionization tandem mass spectrometry (GC–EI/MS/MS) for the sensitive and selective determination of piperazine in chicken and pig tissues. Samples were extracted using an accelerated solvent extraction (ASE) apparatus, purified by solid-phase extraction (SPE) and derivatized with acetic anhydride. This optimized method was validated according to the requirements defined by the European Union and the Food and Drug Administration. At the limit of quantification (LOQ) spiked levels of 50.0, 100.0, 500.0, 1000.0 and 2000.0 μg/kg, the average recoveries of piperazine in chicken and pig tissues were 77.46–96.26%, with relative standard deviations (RSDs) of 1.55–6.64%. The intra-day RSDs were 1.39–5.92%, and the inter-day RSDs were 2.24–8.39%. The limits of detection (LODs) and the LOQs were 1.4–1.6 μg/kg and 4.8–5.2 μg/kg, respectively. The decision limits (CCα) were 102.02–105.17 μg/kg, and the detection capabilities (CCβ) were 104.03–109.09 μg/kg. Finally, the new approach was verified for the quantitative determination of piperazine in 30 commercial chicken and pig tissues from local supermarkets.

A widely applicable method involving liquid chromatography-electrospray ionization tandem triple quadrupole mass spectrometry (LC-ESI/MS/MS) was developed for the simultaneous determination of chloramphenicol, thiamphenicol, florfenicol, and florfenicol amine in eggs. Samples were extracted with ethyl acetate-acetonitrile-ammonium hydroxide (49:49:2, v/v) and defatted with hexane saturated with acetonitrile. The analysis was carried out on a mass spectrometer via an electrospray interface operated in the positive and negative ionization modes using deuterated chloramphenicol-d5 as the internal standard. The limits of detection and limits of quantification were 0.04–0.5 μg/kg and 0.1–1.5 μg/kg in eggs, respectively. The average recoveries of the four analytes from egg samples were 90.84–108.23%, with relative standard deviations of less than 9.61%. The corresponding intra-day and inter-day variations were found to be less than 8.11% and 11.30%, respectively. Finally, the new approach was successfully applied to the quantitative determination of these analytes in 50 commercial eggs from local supermarkets.

The occurrence and prevalence of mcr gene in Gram-negative bacteria from animals was firstly reported in 2015, which greatly threat the values of the human last-resort antimicrobial-colistin. Here we have isolated a lytic bacteriophage, designated vB_EcoM_mcr1, from sewage effluent of a chicken farm in Jiangsu, Mid-east of China, which could infect mcr-positive Escherichia coli strains. Electron microscopy showed Phage vB_EcoM_mcr1 does not resemble to any known family of the phage, with an isometric head (110 nm) and a short contractile tail (35 nm). The phage showed a narrow host ranges against E. coli isolates of chicken origin, lysing 3 of 34 (8.8%) mcr-positive strains. Its latent period and burst size were approximately 5 min and 34 PFU/cell, respectively. The genome of phage vB_KpnM_mcr1 was double stranded DNA and estimated to be about 60 kb. The bacteriolytic activity of this phage at a multiplicity infection (MOI) of ≥0.001 indicted its efficiency for reducing bacterial growth. It is the first time to identify the phage, vB_EcoM_mcr1,which could infect mcr-positive E. coli. These findings suggest that phage vB_EcoM_mcr1 could be considered as a potential therapeutic or prophylactic candidate against mcr-positive E. coli infection in chicken., Journal of Targeting Infectiuos Diseases, Vol 1, No 1 (2016)