Res. from the surface of was induced under low iron conditions, followed by trypsin extraction and separation by 2-dimensional electrophoresis. The separated proteins were blotted with antibodies from mastitic bovine milk and identified by liquid chromatography-mass spectrometry. Thirty-eight unique proteins were identified, of which 8 were predicted to be surface exposed and involved in virulence. Two surface proteins, iron-regulated surface determinant protein C (IsdC) and ESAT-6 secretion system extracellular protein (EsxA), were cloned, expressed, and purified from for confirmation of immune reactivity by ELISA. A PCR of 37 bovine isolates indicated that the presence of is one of the most commonly isolated pathogens in milk, with an estimated 3% of dairy cows worldwide being infected (Schukken et al., 2009). infection often results in chronic, subclinical disease that is highly contagious and difficult or impossible to treat, with cure rates lower than 25% (Rainard, 2005). Despite decades of research, an effective vaccine that can prevent bovine disease is not yet available, likely due to significant strain to strain variability and redundancy of virulence factors. Thus, the strategy to design an effective vaccine for mastitis must include multiple conserved and immunogenic virulence factors that can provide cross-protection. expresses a broad range of virulence factors that include surface proteins covalently attached to the cell wall and secreted proteins expressed during infection (Foster et al., 2014). These exposed proteins are essential for the survival and proliferation of surface adhesin that is also involved in iron sequestration and found to be highly expressed, conserved, and immunogenic during bovine mastitis (Misra et al., 2017). Other adhesins, such as IsdB, ClfA and HlA, also have antigenic properties in models for human infection; however, their role in Rabbit Polyclonal to TAS2R12 bovine disease requires further exploration (Maira-Litrn et al., 2012; Adhikari et al., 2016). Proteomics is an important tool to identify potential vaccine antigens, TH-302 (Evofosfamide) and is especially useful for pathogens such as that express numerous surface exposed proteins (Collado et al., 2016; Dwivedi et al., 2016). Studies have reported the use of 2-dimensional electrophoresis (2DE) for whole or subcellular proteome analysis of for human vaccine development (Sellman et al., 2005; Brady et al., 2006; Solis et al., 2010). Couto et TH-302 (Evofosfamide) al. (2016) recently described the use of immunoproteomics, or specifically the serome proteome analysis technique, to explore and characterize novel vaccine and therapeutic targets for infections in dogs. Immunoproteomics uses a combination of proteomics and immunoblotting from infected hosts to identify antigenic proteins and factors. Mastitic cow milk contains neutrophils, macrophages, lymphocytes, and antibodies as a result of infection and subsequent inflammation (Eisenberg et al., 2016). Secreted antibodies, including IgG and IgM, are present in milk during acute or chronic infection and can be used as tools to identify bacterial antigens that are expressed and immunoreactive during infection. In addition, high concentrations of IgG1 antibody in milk has been found to negatively associate with colony-forming units in experimental infections, indicating these antibodies are important for control of colonization (Boerhout et al., 2016). Serome proteome analysis has been employed to identify antigenic proteins using serum from infected cows with subclinical mastitis (Tedeschi et al., 2009; Xia et al., 2012). The combination of immunoproteomics with bioinformatics tools, such as reverse vaccinology and other in silico approaches, has also been explored to analyze the surface proteome of with a focus on vaccine antigenic targets (Hashmi et al., 2010; Argondizzo et al., 2015; Atshan et al., 2015). Important contributions have been made using these techniques, but more immunologically extensive and disease-specific, as well as host-specific, approaches are needed to develop an effective vaccine against bovine mastitis. Our goal was to use mastitic milk antibodies to promote the identification of relevant antigens for the prevention of bovine mastitis. Here we report that 2DE coupled with immunoblotting and mass spectrometry promoted the identification of immunoreactive candidates from infection of the udder. In addition, sequence analysis and PCR on genomic DNA indicated that EsxA and IsdC are conserved at the AA level and that the presence of TH-302 (Evofosfamide) these antigens is found in the majority of bovine isolates. This is the first known report of the use of immunoproteomics to identify antigens using milk from cows with mastitis. MATERIALS AND METHODS Bacterial Strains, Culture Conditions, and Milk Samples The strains used in 2DE were Newbould 305 (Prasad and Newbould, 1968; Bouchard et al., 2012) and an isolate from clinical mastitis (C1, provided by M. McGuire; Table 1). Thirty-seven additional bovine isolates from at least 5 different, mostly northwestern, US states.