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Label-free high-throughput screening using mass spectrometry has the potential to provide rapid large-scale sample analysis at a speed of more than one sample per second. Such speed is important for compound library, assay and future clinical screening of millions of samples within a reasonable time frame. Herein, we present a liquid atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) setup for high-throughput large-scale sample analysis (>5 samples per second) for three substance classes (peptides, antibiotics and lipids). Liquid support matrices (LSM) were used for the analysis of standard substances as well as complex biological fluids (milk). Throughput and analytical robustness were mainly dependent on the complexity of the sample composition and the current limitations of the commercial hardware. However, the ultimate limits of liquid AP-MALDI in sample throughput can be conservatively estimated to be beyond 10-20 samples per second. This level of analytical speed is highly competitive compared with other label-free MS methods, including electrospray ionization and solid state MALDI, as well as MS methods using multiplexing by labelling, which in principle can also be used in combination with liquid AP-MALDI MS.

Background: Bacterial zoonoses in cows, such as leptospirosis and Q fever, usually appear in a subclinical form causing abortion in pregnant cows as the only clinical manifestation following infection. The pathogenesis of both diseases is very complex and still not completely understood. Objective: The aim of this study was to evaluate serum proteome in cows that aborted due to leptospirosis and Q- fever in order to investigate differentially expressed proteins involved in pathophysiological mechanisms of zoonotic infection of cows. Methods: Three groups of cows were included in the study: group I (control, healthy cows, n=5) ; group II (cows infected with Coxiella, n=5) and group III (cows infected with Leptospira, n=5) both comprised cows that have aborted. In collected sera, 2DE and mass spectrometry (MALDI-TOF/TOF, Bruker-Daltonics) were performed. Obtained results were analysed by MASCOT algorithm (www.matrixscience.com) against SwissProt_201407 database. Results: Three proteins (actin, fructose-1, 6-bisphosphatase 1 and transgelin-2) were up-regulated while E3- ubiquitin-protein ligase KCMF1 and autophagy- related protein 13 were down-regulated in both leptospirosis and Q fever compared to the control.. Gap junction alpha-5 protein was down-regulated only in Q fever but not in leptospirosis. Differentially expressed proteins are involved in processes of cell motility, cell adhesion and signal transduction what enable bacterial binding to host cells activating the host innate defense system. Conclusion: The zoonotic pathogens trigger changes in the expression of specific host proteins needed for host-pathogen interaction and bacterial dissemination. The results could contribute to better understanding of mechanisms of host-pathogen interaction and identification of potential biomarker for bacterial infection.

Introduction: Staphylococcus epidermidis GOI1153754-03-14 is able to colonize orthopedic implants and to cause septic non-unions, as validated in a recent in vivo study (Lovati, 2016). To pore over the mechanisms leading to the biofilm formation on metallic implants, in the present study, we carried out the phenotypic and genotypic characterization of the clinical isolate S. epidermidis GOI1153754-03-14. Materials and Methods: The antimicrobial susceptibility and minimum inhibitory concentration (MIC) of the strain were evaluated through the Vitek2 System (Biomerieux), as well as its ability to form biofilm in vitro through a spectrophotometric assay (Stepanovich, 2000). The genomic DNA was extracted by Bacterial Genomic DNA Isolation Kit (Norgen Biotek Corp.). Libraries were prepared with the ThruPLEX DNA-seq (Rubicon Genomics) and then sequenced on the Illumina MiSeq platform through the MiSeq Reagent Kit v3 (600-cycles) to produce 300 bp paired-end reads (Illumina Inc.). Reads were quality-trimmed and gene annotated thanks to the RAST software (Aziz, 2008). Results: The antimicrobial susceptibility along with the MIC values are reported in Table 1. The outputs resulted in 51 contigs (Average = 50,720.6 Mb) with 396X fold average coverage. The total genome is 2,586,753 bp long with a GC content of 31.84% and an N50 value of 7 bp. The whole genome is composed by 2,467 protein-encoding genes and 64 RNAs (55 tRNAs and 9 rRNAs). The entire genome sequence has been deposited in the European Nucleotide Archive (ENA) under the accession no. FWCG01000000 (Bottagisio, 2017). Discussion: The genotypic and phenotypic characterization of the S. epidermidis GOI1153754-03-14 will enable a better comprehension of the mechanisms involved in the biofilm formation on orthopedic implants paving the way for innovative preventative and therapeutic strategies. Moreover, the sequence of this clinical strain is mandatory to develop dedicated proteomics analysis in order to highlight functional mechanism of biofilm formation., International Journal of Health, Animal Science and Food Safety, V. 4, N. 1s (2017)

Listeria monocytogenes (LM) is a foodborne pathogen responsible of listeriosis. In the spreading of this pathology, milk and dairy products are key reservoir for this pathogen1. Food processing represents one of the major steps that could be linked to LM growth. Inhibition of LM growth through competition of Lactococcus lactis (LAC) could represent a solution to this problem. Exoproteome of LM and two different strains of Lactic Acid Bacteria in co-culture have been studied in order to highlight mechanisms of bacterial competition useful to improve food safety. Two different strains of LAC and one strain of LM were cultivated in appropriate medium cultures (BHI), also in competition. Filtrated cultures (SECRETOME) were lyophilized and resuspended for proteomics analysis. Shotgun analysis on each secretome was performed on nano UPLC-MS system. Obtained data reveal, during competition, the higher production by LM of moonlighting protein Enolase and Glucose 6 Phosphate isomerase, of Septation ring formation regulator EzrA, involved into cell replication and the lower secretion of Endopeptidase P60. In parallel, L. lactis produced higher amounts of Secreted 45 kDa protein and switched from lantibiotic Nisin A production to Nisin Z production. In competition with LM, LAC strain investigated produce higher amounts of Secreted 45 kDa protein with peptidoglycan lytic activity and the selective secretion of Nisin Z probably to improve lantibiotic solubility in less acidic environment. Next step will be validation of obtained results in dairy products. These results are of interesting to design new strategies of fighting LM as contaminant in food from animal origin. Work supported by Ministry of Health-CCM “Milano EXPO 2015 Project: Garantire la sicurezza alimentare- Valorizzare le produzioni”, International Journal of Health, Animal Science and Food Safety, Vol 2, No 1s (2015): Proceeding of Veterinary and Animal Science Days

Abortion in cows represents a serious burden for dairy industry with a great economic impact. Most abortions are due to infectious diseases, several of them are zoonoses. Bacterial zoonoses such as leptospirosis, Q fever and brucellosis are important cattle infections associated with intrauterine death and reduced fertility. The aim of this study was to explore differential proteome profile in serum of cows that aborted due to subclinical infection of leptospirosis and Q fever in order to uncover the pathophysiological molecular mechanisms involved in abortion caused by infectious diseases.The study was conducted on 15 dairy cows divided into three groups: Group I (control, healthy cows, n=5) ; Group II (cows infected with Coxiella, n=5) and Group III (cows infected with Leptospira, n=5). Group II and Group III comprised cows that have aborted. In collected sera, 1D- and 2D-electrophoresis were carried out and the mass spectra were acquired with an Ultraflex III MALDI-TOF/TOF spectrometer (Bruker-Daltonics).Six proteins were found to be (p≤0.05) either up-regulated or down-regulated in bovine sera with Q fever and leptospirosis in comparison to the control. This homology between the two pathologies might suggest that they share some common features that can cause abortion.

Research for biomarkers discovery of bovine mastitis aims to identify reliable biomarkers for early detection and drug efficacy. Most of proteomic studies on mastitis have been performed on milk and somatic cells. Although proteomic profile of bovine milk whey proteins have been well characterized still poor information have been provided on serum and plasma proteomics of bovine mastitis. Aim of this work is to extend the current knowledge on molecular circulating biomarkers of mastitis including both subclinical and clinical animal group. Collected evidences showed complementary data between oxidative stress response, lipid metabolism and the differential protein expression.

Sheep gastrointestinal nematode (GIN) infestation represents a limiting factor for sheep farming and milk production in Italy. The development of anthelmintic resistance to conventionally used drugs suggests the path towards the use of natural remedies as a possible alternative. The purpose of this study is to evaluate the in vitro anthelmintic efficacy of the hydroalcoholic extracts of basal leaves (It-BL), cauline leaves (It-CL) and flowers (It-F) of Isatis tinctoria (Brassicaceae), a spontaneous Sicilian species renowned as an important source of bioactive compounds. The dry extracts of the different parts of the plant were tested using the egg hatch test (EHT) in vitro to verify the efficacy against ovine GIN at different concentrations (1.00, 0.5, 0.25, 0.125 mg/mL). Thiabendazole and deionized water were used as positive and negative controls, respectively. The results obtained from EHT indicated that all the I. tinctoria extracts were highly effective (p < 0.0001) in inhibiting egg hatching within 48 h of exposure. The in vitro inhibitory effect was never less than 84% in all doses tested, and it was only slightly lower than the standard drug thiabendazole (95.6%). The current study documents the anthelmintic activity of I. tinctoria against sheep’s GIN, suggesting its application as alternative natural method to limit the use of antiparasitic drugs.