Project description:Our objectives were to compare gene expression profiles in neutrophils (PMN) during a Streptococcus uberis mastitis challenge between lactating cows subjected to feed restriction to induce negative energy balance (NEB; n = 5) and cows fed ad libitum to maintain positive energy balance (PEB; n = 5). The NEB cows were feed-restricted to 60% of calculated net energy for lactation requirements for 7 d, whereas PEB cows were fed the same diet for ad libitum intake. After 5 d of feed restriction, one rear mammary quarter of each cow was inoculated with 5,000 cfu of Streptococcus uberis (strain O140J). Blood PMN were isolated at 24 h post-inoculation from all cows for RNA extraction and microarray analysis. NEB resulted in 94 differentially expressed genes compared with PEB. Of these, 51 genes were down-regulated, including genes involved with antigen presentation (HLADRA and HLAA), respiratory burst (SOD1), and the pro-inflammatory response (TNFA and IRAK-1). The most affected genes up-regulated by NEB (n = 43) included IL1R2 and IL6, toll-like receptors (TLR2 and TLR4), and THY1. Network analysis by Ingenuity Pathway Analysis Â® revealed that TNFA was associated with the expression of numerous differentially expressed genes involved with immune response in NEB cows compared with PEB cows. Energy balance alters PMN expression of several genes involved with immune response, which provides new information on transcriptomic mechanisms associated with post-partal NEB and immune response during early lactation. Briefly, 10 multiparous Holstein cows in PEB and past peak lactation were used for this study. The NEB cows were feed-restricted to 60% of calculated NEL requirements for 7 d, whereas PEB cows were fed the same diet for ad libitum intake. After 5 d of feed restriction, one rear mammary quarter of each cow was inoculated with 5,000 cfu of Streptococcus uberis (strain O140J). Blood PMN were isolated at 24 h post-inoculation from all cows. A 13,257-oligonucleotide (70-mers) array was used for transcript profiling. Cy3- and Cy5 labelled cDNA from PMN and a reference standard were used for hybridizations. All samples were hybridized to duplicate slides with reverse labeling (20 microarrays total).

Project description:Background Our objective was to compare mammary tissue gene expression profiles during a Streptococcus uberis (S. uberis) mastitis challenge between lactating cows subjected to dietary-induced negative energy balance (NEB; n = 5) and cows fed ad libitum to maintain positive energy balance (PEB; n = 5). The NEB cows were feed-restricted to 60% of calculated net energy for lactation requirements for 7 d, and cows assigned to PEB were fed the same diet for ad libitum intake. Five days after feed restriction, one rear mammary quarter of each cow was inoculated with 5,000 cfu of S. uberis (O140J). At 20 h post-inoculation, both the S. uberis-infected mammary quarters (i.e., YES) and non-infected rear quarters (i.e., NO) from all cows were biopsied for RNA extraction. Effect of S. uberis Intramammary Infection (IMI; YES vs NO) S. uberis IMI resulted in 2,102 oligos (1,939 annotated genes) differentially expressed genes (DEG; YES versus NO). Of these, 1,082 genes were up-regulated during IMI and were primarily involved with the immune response, e.g., IL6, TNF, IL8, IL10, SELL, LYZ, and SAA1. Genes down-regulated (1,020) included those associated with milk fat synthesis, e.g., LPIN1, LPL, CD36, and BTN1A1. Pathway analysis of all DEG indicated that IL-10 Signaling, IL-6 Signaling, and Glucocorticoid Receptor Signaling were among the top canonical pathways affected by infection. Interestingly, LXR/RXR Signaling was also a primary affected pathway, indicating a relationship between immune system response and metabolism during an IMI. Network analysis indicated that TNF had positive relationships (i.e. up-regulation) with genes involved with immune system function (e.g., CD14, IL8, IL1B, and TLR2) and negative relationships (i.e., down-regulation) with genes involved with lipid metabolism (e.g., GPAM, SCD, FABP4, CD36, and LPL) and antioxidant activity (SOD1). Results provided information into the early response factors associated with the innate immune response to S. uberis infection. Our study indicated that IMI challenge with S. uberis (strain O140J) elicited a strong transcriptomic response, leading to an overall up-regulation of genes involved in the innate immune response and a down-regulation of genes involved with lipid metabolism. Effect of NEB Within Infected Quarters (YES) Energy balance (NEB vs. PEB) resulted in 285 DEG (FDR ≤ 0.05), with 85 DEG up-regulated and 200 DEG down-regulated. Canonical pathways most affected by NEB were IL-8 Signaling (10 genes), Glucocorticoid Receptor Signaling (13), and NRF2-mediated Oxidative Stress Response (10). Among genes differentially expressed by NEB, Cell Growth and Proliferation (48) and Cellular Development (36) were the most enriched functions. Up-regulated genes included ANXA1, HMOX1, and HLAA; down-regulated genes included NOTCH1 and CCND1. Regarding immune response, HLAA was up-regulated due to NEB, whereas the majority of genes involved in immune response were down-regulated (e.g., AKT1, IRAK1, MAPK9, and TRAF6). Results helped identify mechanisms affected by dietary-induced NEB that may be associated with the impairment of immune function and increased risk of mastitis in cows experiencing postpartal NEB. Effect of NEB Within Non-Infected Quarters (NO) Energy balance (NEB vs. PEB) resulted in 278 DEG within non-infected rear mammary quarters. Up-regulated DEG most affected by NEB (genes = 180) included genes involved in lipid metabolism and amino acid metabolism. The down-regulated DEG most affected by NEB (n = 98) included genes involved in carbohydrate metabolism, anti-inflammatory response, and apoptosis. Among genes up-regulated, Ingenuity Pathway Analysis® identified Lipid Metabolism (8), Molecular Transport (14), and Small Molecule Biochemistry (15) as some of the most enriched molecular functions. Genes down-regulated by NEB were associated with Cell Growth and Proliferation (21) and Cell Death (18). Results indicate that dietary induced-NEB alters genes primarily associated with cellular metabolism, proliferation, and tumorigenesis but no candidate genes were identified as being associated with risk of disease.

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