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.

Project description:In dairy cows, administration of high dosages of niacin (NA) was found to cause anti-lipolytic effects, which are mediated by the NA receptor hydroxyl-carboxylic acid receptor 2 (HCAR2) in white adipose tissue (WAT), and thereby to an altered hepatic lipid metabolism. However, almost no attention has been paid to possible direct effects of NA in cattle liver, despite showing that HCAR2 is expressed also in the liver of cattle and is even more abundant than in WAT. Due to this, we hypothesized that feeding of rumen-protected NA to dairy cows influences critical metabolic and/or signaling pathways in the liver through inducing changes in the hepatic transcriptome. In order to identify these pathways, we applied genome-wide transcript profiling in liver biopsies obtained at 1 wk postpartum (p.p.) from dairy cows of a recent study (Zeitz et al., 2018) which were fed a total mixed ration without (control group) or with rumen-protected NA from 21 d before calving until 3 wk p.p. Hepatic transcript profiling revealed that a total of 487 transcripts were differentially expressed [filter criteria fold change (FC) > 1.2 or FC < -1.2 and P < 0.05] in the liver at 1 wk p.p. between cows fed NA and control cows. Substantially more transcripts were down-regulated (n = 338), while only 149 transcripts were up-regulated by NA in the liver of cows. Gene set enrichment analysis (GSEA) for the up-regulated transcripts revealed that the most enriched gene ontology (GO) biological process terms were exclusively related to immune processes, such as leukocyte differentiation, immune system process, leukocyte differentiation, activation of immune response and acute inflammatory response. In line with this, the plasma concentration of the acute phase protein haptoglobin tended to be increased in dairy cows fed rumen-protected NA compared to control cows (P < 0.1). GSEA of the down-regulated transcripts showed that the most enriched biological process terms were related to metabolic processes, such as cellular metabolic process, small molecule metabolic process, lipid catabolic process, organic cyclic compound metabolic process, small molecule biosynthetic process and cellular lipid catabolic process. In conclusion, hepatic transcriptome analysis shows that rumen-protected NA induces genes which are involved mainly in immune processes including acute phase response and stress response in dairy cows at wk 1 p.p. These findings indicate that supplementation of rumen-protected NA to dairy cows in the periparturient period may induce or amplify the systemic inflammation-like condition which is typically observed in the liver of high-yielding dairy cows in the p.p. period.

Project description:Bovine mastitis, the infection of the mammary gland which leads to great health and economic challenges for dairy farmers is accompanied by dramatic changes in the milk proteome. In this study of naturally occurring mastitis not only have the changes in the milk proteome been quantified in subclinical and clinical mastitis but simultaneous changes in the serum proteome have also been characterised and quantified. Milk and serum samples from healthy dairy cows (n=12) were compared to those of cows with subclinical (n=10) and clinical mastitis (n=112) using TMT label-based proteomic approach. The study included the milk and serum samples taken from thirty-two dairy cows ( kept on private farms located in Croatia. All cows were checked by physical examination. Somatic cells count (SCC) and mastitis test in milk samples were performed. According to the results, cows were assigned into three groups: Group I (control, n=10) consisted of healthy cows with SCC below 400,000 cells/ml on the monthly check-up and a negative mastitis test and without any clinical sign of mastitis. Group II (subclinical mastitis, n=12) comprised cows without clinical signs of mastitis but with SCC above 400,000 cells/ml on the monthly basis and a positive mastitis test at the time of sampling. Group III (clinical mastitis, n=10) consisted of cows with clinical signs of mastitis which include changes in milk appearance (flakes and clots in milk), different stages of udder inflammation (hyperemia, edema, pain, udder enlargement and elevated udder temperature) and disturbance of general health (depression, relaxed cold ears, dehydration, elevated body temperature, increased heart and respiratory rate, decreased ruminal contraction and decreased appetite). Blood samples were taken from v. coccygea and centrifuged at 3000 g for 15 min after clotting for two hours at room temperature. Serum samples were stored at -80°C until analysis. Milk samples were taken aseptically before the morning milking. First few streams were discarded. Teat ends were disinfected with cotton swabs soaked with 70% ethanol. Samples were taken into sterile tubes and transported to laboratory on ice within a few hours.

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