Project description:Mastitis is one of the most prevalent and economically important diseases of dairy animals. The disease is caused by ascending bacterial infection through the teat canal. Among the most common mastitis-causing bacteria are gram-negative coliforms, gram-positive streptococci and staphylococci, and mycoplasma. The most prominent cellular hallmark of acute mammary infection is a massive recruitment of blood neutrophils into the tubular and alveolar milk spaces. The complex biological processes of leukocyte recruitment, activation, adhesion, and migration in the mammary gland remain largely elusive to date. While field research of mastitis in dairy animals contributed a lot to the development of mitigation, control, and even eradication programs, little progress was made toward understanding the molecular mechanisms underlying the pathogenesis of the disease. We report here experimental mastitis model systems in lactating mice challenged with field strains of common udder pathogens in dairy cows. We used these model systems to apply recently developed multiplex gene expression technology (Nanostring nCounter), which enabled us to study the expression of over 700 immune genes. Our analysis revealed a core of 100 genes that are similarly regulated and functionally or physically interacting in E. coli, M. bovis and Strep uberis murine mastitis. Common significantly enriched gene sets include TNFɑ signaling via NFkB, Interferon gamma and alpha response, and IL6-JAK-STAT3 signaling. In addition, we show a significantly enriched expression of genes associated with neutrophil extracellular traps (NET) in glands challenged by the three pathogens. Ligand-receptor analysis revealed interactions shared by the three pathogens, including the interaction of the cytokines IL1β, IL1ɑ, and TNFɑ with their receptors, and proteins involved in immune cell recruitment such as complement C3 and ICAM1 (with CD11b), chemokines CCL3 and CCL4 (with CCR1), and CSF3 (with CSF3R). Taken together, our results show that mammary infection with E. coli, M. bovis and Strep uberis culminated in the activation of a conserved core of immune genes and pathways including NET formation.

Project description:Bovine mastitis remains a primary focus of dairy cattle disease research due to its considerable negative economic impact on the dairy industry. This was the first differential proteomic study aimed to identify milk somatic cell proteins contributing to host defense and their variable responses to naturally occurring mastitis in Sahiwal (Bos indicus) cows. To accomplish this, indigenous Sahiwal cows were categorized based on milk somatic cell counts (SCC)< 2×105cells/mL with no previous history of mastitis as healthy (H) animals. However, cows with sub-clinically mammary infection (SCM) and clinical mastitis (CM) had milk SCC 2-5×105 and > 5×105 cells/mL, respectively. The milk somatic cells were isolated and analysed using a label-free quantitative, liquid chromatography- mass spectrometry (LC-MS/MS) proteomic approach. In total, 3896, 3843 and 3868 proteins showed significant differential abundance between CM vs SCM, CM vs H and SCM vs H groups, respectively (=1 high-quality PSM/s, =1 unique peptide, FDR < 0.01), while 25, 53, 21 proteins were unique to CM, SCM and H groups, respectively. The differentially abundant proteins (DAPs) top significantly (p<0.05) upregulated proteins were annexin A5 (ANXA5), Rho-associated protein kinase 2 (ROCK2) and hepsin (HPN) in CM vs SCM groups, whereas vanin 2 (VNN2), thioredoxin reductase-like selenoprotein T (SELENOT), ceramidase (NAAA), lymphocyte antigen 75 (LY75/CD 205), cysteine-rich PDZ-binding protein (CRIPT), misshapen like kinase 1 (MINK1), macrophage scavenger receptor (MSR1/CD204), leupaxin (LPXN) and lipoamide acyltransferase (DBT) were upregulated common to both CM vs H and SCM vs H groups. These upregulated differentially abundant proteins (DAPs) are mainly associated with host immune functions of the mammary gland during mastitis. Besides this, the top downregulated proteins such as cortactin (CTTN), glutathione S-transferase (GSTM3), G protein-coupled receptor class C group 5 member B (GPRC5B), signal-induced proliferation-associated protein 1 (SPIA1), the exocyst complex component 6 (EXOC3), S100 A16. Additionally, the findings of the GO and KEGG enrichment functional analysis demonstrated Staphylococcus aureus infection, neutrophil extracellular trap formation, extracellular matrix receptor interaction, complement and coagulation cascades, toll-like and NOD-like receptor, PPAR, MAPK signalling, IL-17 signalling, Th1 and Th2 cell differentiation, Fc gamma mediated phagocytosis, cell adhesion molecules, peroxisome, lysosome, endocytosis were the enriched pathways of immune mechanisms regulating during naturally infected mastitis. These findings provide essential information on mammary infection-associated proteomic profiling in milk somatic cells and associated host immune response mechanisms which uncover potential biomarkers for detecting subclinical mastitis in dairy cattle.

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Immune profiling of experimental murine mastitis

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