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:Mastitis is defined as inflammation of the mammary gland and one of the most serious concerns with regards to milk production and animal health in dairy industry. Indeed, mastitis have marked influence on the milk yield, milk constituents, milk quality and nourishment of the neonates. We performed a comparative proteome analysis of the bovine milk obtained from healthy, sub-clinical and clinical mastitis from Indian indigenous cattle Karan Fries.
Project description:Background: S. aureus is one of the main pathogen involved in ruminant mastitis worldwide. The severity of staphylococcal infection is highly variable and ranges from subclinical to gangrenous mastitis. Such variability implies host as well as staphylococcal factors. This work is an in-depth characterization of S. aureus mastitis isolates to identify factors involved in mastitis severity. Methods and findings: We combined three “omic” approaches to comprehensively compare two clonally related S. aureus strains that were isolated from and shown to reproducibly induce severe (strain O11) and milder (strain O46) mastitis in ewes. The genomes of O11 and O46 were sequenced (Illumina technology) to determine their respective gene content and comparative transcriptomic and proteomic analyses were carried out on both strains grown in conditions mimicking mastitis context. High differences were highlighted in mobile genetic elements, iron acquisition and metabolism, transcriptional regulation and exoprotein production. In particular, O11 overproduced exoproteins, including toxins and proteases when compared to O46. This was confirmed in 4 other S. aureus strains isolated from subclinical or clinical mastitis cases. Dose-dependant production of some staphylococcal factors seem to play a role in hypervirulence of strains isolated from severe mastitis. Mobile genetic elements, transcriptional regulators, exoproteins or strain ability to deal with iron starvation constitute good targets for further research to better define the underlying mechanisms of mastitis severity. Conclusions: Differences observed in mastitis severity likely result from the ability of the strains to adapt and to express virulence factors in the mastitis context rather than from deep variations in gene content.
Project description:Analysis of gene expression changes in blood mononuclear cells (BMCs) that occur with Staph Aureus mastitis. We used in house microarrays to indicate the changes that occur in gene expression in the BMCs as a result of mastitis Keywords: Comparison mastitis animal vs control animal
Project description:Background: S. aureus is one of the main pathogen involved in ruminant mastitis worldwide. The severity of staphylococcal infection is highly variable and ranges from subclinical to gangrenous mastitis. Such variability implies host as well as staphylococcal factors. This work is an in-depth characterization of S. aureus mastitis isolates to identify factors involved in mastitis severity. Methods and findings: We combined three “omic” approaches to comprehensively compare two clonally related S. aureus strains that were isolated from and shown to reproducibly induce severe (strain O11) and milder (strain O46) mastitis in ewes. The genomes of O11 and O46 were sequenced (Illumina technology) to determine their respective gene content and comparative transcriptomic and proteomic analyses were carried out on both strains grown in conditions mimicking mastitis context. High differences were highlighted in mobile genetic elements, iron acquisition and metabolism, transcriptional regulation and exoprotein production. In particular, O11 overproduced exoproteins, including toxins and proteases when compared to O46. This was confirmed in 4 other S. aureus strains isolated from subclinical or clinical mastitis cases. Dose-dependant production of some staphylococcal factors seem to play a role in hypervirulence of strains isolated from severe mastitis. Mobile genetic elements, transcriptional regulators, exoproteins or strain ability to deal with iron starvation constitute good targets for further research to better define the underlying mechanisms of mastitis severity. Conclusions: Differences observed in mastitis severity likely result from the ability of the strains to adapt and to express virulence factors in the mastitis context rather than from deep variations in gene content. Expression of S. aureus O46 from subclinical mastitis and O11 from a lethal gangrenous mastitis were compared at two different times
Project description:Mastitis, the inflammation of the mammary gland, is one of the most prevalent diseases in dairy farming worldwide. Unfortunately, the disease is most often present in a subclinical type with no clear symptoms. The sooner the infection is detected, the less opportunities for the disease to progress and the more treatment options remain available. Milk microRNA (miRNA) encapsulated in extracellular vesicles (EV) have been proposed as potential biomarkers of different mammary gland conditions, including subclinical mastitis. However, little is known about the robustness of EV analysis regarding sampling time-point or natural infections. In order to estimate the reliability of EV measurements in raw bovine milk, we first evaluated the changes in EV size, concentration and miRNA cargo during three consecutive days. Then, we compared milk EV differences from natural infected quarters with high somatic cell count (SCC) with their healthy adjacent quarters with low SCC and quarters from uninfected udders. We found that milk EV miRNA cargo is very stable along three days and that infected quarters do not induce relevant changes in milk EV of adjacent healthy quarters, making them suitable controls. We observed cow-individual changes in immunoregulatory miRNA in quarters with chronic subclinical mastitis, pointing towards infection-specific alterations. Finally, we proposed bta-miR-223 as a potential indicator of subclinical mastitis prognosis in raw milk.
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:Bovine mastitis causes changes in the serum exosomal miRNAs expression. Serum samples from healthy dairy cows (n = 7) were compared to those of cows with subclinical (n = 7 ) using small RAN sequencing. Three hundred fifty-five miRNAs (341 known and 14 novel ones) were identified. There were 42 miRNAs up-regulated in serum-derived EVs from cows with subclinical mastitis, including bta-miR-1246, bta-miR-2431-3p, bta-miR-126-3p, bta-miR-29a, etc. The MAPK signaling pathway was the most affected pathway by clinical mastitis. Thus, miRNA alterations in mastitis serum-derived EVs support the potential regulator role of specific miRNAs as exosomal cargo in clinical mastitis physiology.
Project description:We performed a genome-wide transcriptional analysis in the mammary gland in a mouse model of E. coli mastitis using high-density mouse oligonucleotide microarrays. This global transcription analysis revealed that about 7% of tested genes are mobilized in the mouse mammary gland to E. coli endotoxin. We identified 1402 differentially expressed genes that were associated with physiological system development/function and molecular/cellular functions and metabolic/signalling pathways that are highly relevant to host immune-inflammatory defense response against E. coli infection. The mouse differentially expressed genes through the use of comparative mapping/genomics and positional information on reported QTL for bovine mastitis allowed identifying 293 potential candidate genes for bovine mastitis. This study will enable other researchers to combine our mRNA expression data with genetic association studies to discover genomic variation underlying variation of susceptibility to mastitis in dairy cows. Keywords: time course, disease state analysis