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, 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.

Project description:Mastitis is a very costly and common disease in the dairy industry. The study of the transcriptome from healthy and mastitic milk somatic cell samples using RNA-Sequencing technology (RNA-Seq) can provide measurements of transcript levels associated with the immune response to the infection. The objective of this study was to characterize the Holstein milk somatic cells transcriptome from 6 cows to determine host response to intramammary infections. RNA-Sequencing was performed on two samples from each cow from two separate quarters, one classified as healthy (n = 6) and one as mastitic (n = 6). In total, 449 genes were differentially expressed between the healthy and mastitic quarters (P-value < 0.01, FDR < 0.05, FC > ±2). Among the differentially expressed genes, the most expressed genes based on Reads Per Kilo base per Million mapped reads (RPKM) in the healthy group were associated with milk components (CSN2 and CSN3), and in the mastitic group they were associated with immunity (B2M and CD74). In-silico functional analysis was performed using the list of 449 differentially expressed genes, which identified 36 significantly enriched metabolic pathways (FDR < 0.01), some of which were associated with the immune system, such as cytokine-cytokine interaction and cell adhesion molecules. Seven functional candidate genes were selected, based on the criteria of being highly expressed and present in significant pathways that are relevant to the inflammatory process (GLYCAM1, B2M, CD74, BoLA DR-Alpha, FCER1G, SDS and NFKBIA). Lastly, we identified the differentially expressed genes that are located in QTL regions previously known to be associated with mastitis, specifically clinical mastitis, somatic cell count and somatic cell score. It was concluded that there are multiple genes within QTL regions that could potentially impact host response to mastitis causing agents, making some cows more susceptible to intramammary infections. The identification of key genes with functional, statistical, biological and positional relevance associated with host defense to infection, will contribute to a better understanding of the underlying genetic architecture associated with mastitis. This in turn will improve the sustainability of agricultural practices, by facilitating the selection of cows with improved host defense leading to increased resistance to mastitis.

Project description:Outcome and resolution of an udder inflammation (mastitis) depends on the type of the invading pathogen. Gram-negative pathogens, such as Escherichia coli mostly trigger strong inflammation and full local activation of the immune defense eventually resulting in pathogen elimination. Staphylococcus aureus and other Gram-positive pathogens elicit only weak immune reactions possibly allowing for pathogen persistence. Little is known about the very early molecular determinants of this pathogen-species specific immune response of the udder. We therefore infected healthy mid-lactating heifers with a high dose of defined E. coli1303 or S. aureus1027 pathogens, sampled udder tissues at 1, 2, and 3 h post infection and globally profiled the transcriptome of samples from the gland cistern. We expectedly found that the E. coli infection elicited a fast and strong immune response. Bioinformatics analyses suggested that pathogen recognizing receptors triggered the activation of the IkB/NF-kB signaling cascade inducing the expression of a wealth of inflammation related genes, but also many immune dampeners. S. aureus infection failed to elicit inflammation and activate IkB/NF-kB signaling. Instead, it triggered immune suppressive mechanisms which were, however not outweighed by strong induction of inflammatory genes. Moreover, it activated the wnt/b-catenin signaling cascade resulting in active suppression of NF-kB signaling and rearrangement of the actin-cytoskeleton through modulating expression of the Rho GTPases. These alterations facilitate invasion of pathogens into host cells. We validated experimentally that pathogenic S. aureus, but not E. coli may invade mammary epithelial cells (MEC). Microscopy revealed an altered structure of the actin-cytoskeleton of primary MEC having internalized live GFP-tagged S. aureus pathogens. Our study reveals for the first time the earliest host responses in the udder differentiating E. coli from S. aureus mastitis. The latter is characterized by eliciting immune suppression rather than inflammation and invasion of S. aureus into the epithelial cells of the host.

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Escherichia coli Bovine mastitis

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