Bovine mastitis is the most frequent disease on dairy farms, which leads to a decrease in the health welfare of the animals and great economic losses. This study was aimed at determining the quantitative variations in the milk proteome caused by natural infection by Staphylococcus and Streptococcus species in order to gain further understanding of any discrepancies in pathophysiology and host immune responses, independent of the mastitis level. After identification of Staphylococcus (N = 51) and Streptococcus (N = 67) spp., tandem mass tag (TMT)-labeled quantitative proteomic and liquid chromatography-mass spectrometry (LC-MS/MS) techniques on a modular Ultimate 3000 RSLCnano system coupled to a Q Exactive Plus was applied on aseptically sampled milk from Holstein cows. Proteome Discoverer was used for protein identification and quantitation through the SEQUEST algorithm. Statistical analysis employing R was used to identify differentially abundant proteins between the groups. Protein classes, functions and functional-association networks were determined using the PANTHER and STRING tools and pathway over-representation using the REACTOME. In total, 156 master bovine proteins were identified (two unique peptides, p < 0.05 and FDR < 0.001), and 20 proteins showed significantly discrepant abundance between the genera (p < 0.05 and FDR < 0.5). The most discriminatory proteins per group were odorant-binding protein (higher in staphylococci) and fibrinogen beta chain protein (higher in streptococci). The receiver operating characteristic (ROC) curve showed that protein kinase C-binding protein NELL2, thrombospondin-1, and complement factor I have diagnostic potential for differentiating staphylococci and streptococci intramammary infection and inflammation. Improved understanding of the host response mechanisms and recognition of potential biomarkers of specific-pathogen mastitis, which may aid prompt diagnosis for control implementation, are potential benefits of this study.

Mycobacterium avium subsp. paratuberculosis (MAP) is a causative agent of Johne’s disease in all ruminants worldwide. Economic problems in dairy cattle and sheep industries, public health concern, persistence of MAP in the environment and lack of effective vaccines mentioned necessity of research about various antigens to introduce as vaccine candidates. Based on MAP pathogenesis, it seems that research about the production of new recombinant proteins to stimulate cell-mediated immunity is helpful. This study describes successful expression and purification of a chimeric fusion protein which consists of Heparin-Binding Hemagglutinin Adhesin (HBHA) and high antigenic region of Fibronectin Attachment Protein (FAP-P). Triggered antigen-specific IFN-γ response of isolated PBMCs from immunized goats to rHBHA-FAP and all crude proteins of MAP (PPD), was measured by ELISA. Significant increases were observed in the IFN-γ production level of peripheral blood mononuclear cells (PBMCs) stimulated by constructed chimeric protein from rHBHA-FAP and PPD vaccinated goats. Antigen-specific gamma interferon (IFN-γ) secretion in positive group (immunized by PPD) against rHBHA-FAP and test group (immunized by rHBHA-FAP) against PPD, also statistically insignificant rises between stimulation with rHBHA-FAP and PPD, suggested the potential and specificity of our chimeric protein to stimulate cell mediated immunity against MAP. Collectively, these results demonstrate that rHBHA-FAP elicits a strong IFN-γ production in PBMC culture. Therefore, further studies of the present product as a candidate vaccine in naturally infected animals should be conducted, to analyze its potential.